thesis karthika revised - Murdoch University...Karthika Krishnasamy , Richard Bell and Qifu Ma (2013), Low to moderate sodium is beneficial to wheat genotypes grown under potassium

SODIUM AND CULTIVAR EFFECTS ON POTASSIUM NUTRITION OF WHEAT

THIS THESIS IS PRESENTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF MURDOCH UNIVERSITY

By

KARTHIKA KRISHNASAMY

Bachelor of Technology in Horticulture;

Honours in Environmental Science

School of Veterinary and Life Sciences

Murdoch University

2015

ii

DECLARATION

I declare that this thesis is my own account of my research and contains as its

main content work which has not previously been submitted for a degree at any

tertiary education institution.

........................

Karthika Krishnasamy

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Papers and Presentations from this research

Journal Paper

1. Krishnasamy, K., Bell, R and Ma, Q (2014). Wheat responses to sodium vary with

potassium use efficiency of cultivars. Frontiers in Plant Science, 5: 631.

doi: 10.3389/fpls.2014.00631.

http://journal.frontiersin.org/Journal/10.3389/fpls .2014.00631/abstract#

International Conference Presentations

1. Richard Bell, Qifu Ma and Karthika Krishnasamy (2013) Wheat and barley genotypes

differ in growth response to soil potassium supply under low to moderate sodium supply, in:

XVII. International Plant Nutrition Colloquium (IPNC) held on 19- 22 August, 2013 at the

Istanbul Convention and Exhibition Centre (ICEC), Istanbul, Turkey.

2. Karthika Krishnasamy , Richard Bell and Qifu Ma (2013), Moderate sodium has positive

effects on wheat grown in a potassium deficient split-root system, in: XVII. International

Plant Nutrition Colloquium (IPNC) held on 19- 22 August, 2013 at the Istanbul Convention

and Exhibition Centre (ICEC), Istanbul, Turkey.

3. Karthika Krishnasamy , Richard Bell and Qifu Ma (2013), Low to moderate sodium is

beneficial to wheat genotypes grown under potassium deficient conditions, in: Combio, 2013

held on 29 September to 3rd October at Perth Convention and Exhibition Centre, Perth,

Western Australia.

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ABSTRACT

In arid and semiarid regions, soil salinity is largely due to excessive sodium chloride (NaCl)

which, apart from osmotic and specific Na+ and Cl- ion effects, has a detrimental effect on

potassium (K) uptake and nutrition of most crops. However, in K deficient soils, Na+ can

substitute for some functions of K+, provided that plants have the ability to take up,

translocate, and compartmentalise Na+ into the vacuoles where it mainly replaces the

biophysical functions of K+ in maintaining cell turgor, ionic balance, regulating osmotic

potential and improving water balance via stomatal conductance. Potassium deficiency and

soil salinity stress have become increasingly common in agricultural lands of Western

Australia (WA) and many parts of the world, but the role of Na in K nutrition of wheat

(Triticum aestivum L.) is not well understood. The interaction between K and Na in wheat

genotypes differing in K-use efficiency has not been researched previously. This research

focussed mainly on low to moderate concentrations of Na in wheat K nutrition and less

emphasis is placed on Na toxicity effects as there is a large body of research available on Na

toxicity effects. A series of glasshouse experiments were designed for both soil and solution

culture where Na was supplied at a range of concentrations at low and adequate K levels. The

responses of K-efficient and K-inefficient Australian wheat cultivars were examined. Plant

responses were assessed by measuring plant growth, leaf gas exchange, shoot and root K and

Na concentrations and their contents. High soil Na levels (100 and 200 mg Na/kg) greatly

reduced the plant growth in wheat cultivars predominantly at low soil K (40 mg K/kg). By

contrast, low to moderate Na levels (25 and 50 mg Na/kg in soil culture and 2 mM Na in

solution culture) stimulated wheat growth at low K supply, particularly in K-efficient

cultivars compared with K-inefficient cultivars. Roots were more responsive to low

concentrations of Na than shoots in experiments where growth stimulation was observed.

Low to moderate Na supply also increased leaf net photosynthesis and stomatal conductance

at low K supply, with the measured values similar to those observed under adequate K

condition both in soil and solution culture. In the split-root experiment, the positive effects of

moderate soil Na on growth and K uptake of low K plants were evident when K and Na were

supplied in the same or different parts of the root system. In low K soil, low to moderate Na

levels increased plant K content, particularly shoot K content, which may account for the

increased leaf net photosynthesis rate, stomatal conductance, and plant dry weight. In contrast

to previous reports, which attributed Na stimulation of plant growth at low K to increased

Na+ uptake and utilisation in place of K+, in wheat, Na+ increased K+ uptake in soil culture,

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and it increased Rb+ uptake (as a tracer of K+) in solution culture experiments. Hence we

attribute most of the benefits of low to moderate Na application in wheat to improved K

uptake and K nutrition. The main mechanism for Na+-stimulated K+ uptake under limited K

availability with low external Na supply in wheat is likely the effect of Na+ on K+

transporters, both on high-affinity and low-affinity K+ uptake transport systems. In this study,

K-use efficiency among wheat cultivars showed varied responses to Na supply at low K, with

increased stimulation in root growth, shoot K concentrations, K uptake and leaf

photosynthesis in K-efficient cultivar relative to K-inefficient cultivar. Genotypic differences

in K-use efficiency also influenced Na uptake and salt tolerance with K-efficient cultivars

being more salt tolerant than K-inefficient cultivars. The current research on K+ substitution

by Na+ in wheat physiological processes is of great importance in fertiliser management

strategies. The application of expensive K fertilisers is limited by poor farmers especially in

developing countries, and partial substitution of K by Na in plant nutrition can decrease the

cost of production. Based on this study, when K-efficient wheat cultivars are grown under

low to moderately saline conditions, the substitution of K by Na was not strong enough to

recommend Na-based fertilisers in place of K in wheat. Nevertheless, the alleviation of K

deficiency symptoms in wheat by addition of moderate Na provides a trigger for conducting

further studies. The present research based on glasshouse experiments needs to be evaluated

under field conditions with further studies under varying soil and agro-climatic conditions to

define critical soil levels of Na that stimulate wheat growth.

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TABLE OF CONTENTS

Page numbers

DECLARATION……………………………………………………………………. ii

ABSTRACT…………………………………………………………………………. iv

TABLE OF CONTENTS……………………………………………………………. vi

LIST OF FIGURES………………………………………………………………….. xii

LIST OF TABLES…………………………………………………………………… xv

LIST OF ABBREVIATIONS AND SYMBOLS…………………………………… xx

ACKNOWLEDGEMENTS………………………………………………………….. xxiii

Chapter 1. Introduction………………………………………………………………. 1

1.1 Potassium nutrition in plants…………………………………………….... 1

1.2 Sodium nutrition in plants……………………………………………….... 1

1.3 Interaction between potassium and sodium………………………………. 2

1.4 K deficiency in soils………………………………………………………. 3

1.5 Salinity issues of WA……………………………………………………... 3

1.6 Research aim and scope of the study……………………………………… 4

1.7 Layout of the thesis………………………………………………………... 4

Chapter 2. Literature review…………………………………………………………... 6

2.1 Introduction………………………………………………………………... 6

2.2 K functions in plants………………………………………………………. 6

2.2.1 K and enzyme activation………………………………………… 7

2.2.2 K and protein synthesis………………………………………….. 7

2.2.3 K and stomatal activity………………………………………….. 7

2.2.4 K and photosynthesis…………………………………………..... 8

2.2.5 K and stress tolerance in plants………………………………….. 8

2.2.5.1 Anti-oxidant activity…………………………………... 9

2.2.5.2 Drought and heat stress……………………………….. 10

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2.2.5.3 Low temperature and frost stress…………………….. 11

2.2.5.4 Disease and pest resistance……………………………. 12

2.3 K deficiency in plants……………………………………………………... 12

2.4 K uptake and accumulation by plants…………………………………….. 13

2.5 Forms of K in soil………………………………………………………..... 14

2.5.1 Unavailable/mineral K…………………………………………... 15

2.5.2 Slowly available/non-exchangeable/fixed-K………………….... 15

2.5.3 Readily available/ exchangeable K……………………………… 15

2.5.4 Soil solution K…………………………………………………... 15

2.6 Removal of K from soil…………………………………………………… 16

2.7 Na functions in plants…………………………………………………….. 17

2.8 Interaction between K and Na……………………………………………. 18

2.8.1 High Na………………………………………………………….. 19

2.8.1.1 Na toxicity effects…………………………………….. 19

2.8.1.2 Imbalance in K/Na ratios…………………………….... 19

2.8.2 Low to moderate Na…………………………………………….. 20

2.9 Functions of K replaced by Na…………………………………………… 20

2.10 Plant responses to Na at low K………………………………………….. 22

2.11 K and Na transporters…………………………………………………… 30

2.12 Genotypic variation in Na substitution of K……………………………. 30

2.13 Salinity and duplex soils…………………………………………………. 31

2.14 Research scope, aim and research questions……………………………. 32

2.15 Conclusion………………………………………………………............. 33

Chapter 3. Wheat responses to sodium vary with potassium use efficiency of cultivars………………………………………………………………………………..

34

3.1 Introduction……………………………………………………………..... 34

3.2 Materials and methods……………………………………………………. 35

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3.2.1 Potassium and sodium treatments………………………………. 36

3.2.2 Measurements…………………………………………………… 36

3.2.3 Statistical analysis………………………………………………. 37

3.3 Results…………………………………………………………………….. 37

3.3.1 Plant growth…………………………………………………….. 37

3.3.2 Leaf gas exchange……………………………………………….. 43

3.3.3 K and Na concentrations in shoots and roots……………………. 43

3.3.4 Soil exchangeable cations after K and Na addition……………... 49

3.4 Discussion…………………………………………………………………. 50

3.5 Conclusion………………………………………………………………… 55

Chapter 4. Split-root experiment

Moderate sodium increased K uptake, leaf gas exchange and plant growth of wheat cv. Wyalkatchem grown in a K-deficient split-root system……………………………

56

4.1 Introduction……………………………………………………………...... 56



4.2.2 Measurements…………………………………………………… 58

4.2.3 Statistical analysis……………………………………………….. 59

4.3 Results……………………………………………………………………... 59

4.3.1 Plant growth……………………………………………………... 59

4.3.2 Leaf gas exchange……………………………………………….. 62

4.3.3 K and Na concentrations and accumulation…………………….. 63

4.4 Discussion…………………………………………………………………. 68

4.5 Conclusion………………………………………………………………… 74

Chapter 5. Column experiment

Potassium response of wheat grown in columns with drying topsoil and varying subsoil K and Na levels………………………………………………………………..

75

5.1 Introduction………………………………………………………………. 75

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5.2 Materials and methods…………………………………………………… 76

5.2.1 Treatments………………………………………………………. 77

5.2.2 Measurements…………………………………………………… 79


5.3 Results…………………………………………………………………….. 79

5.3.1 Plant growth…………………………………………………….. 79

5.3.2 Leaf gas exchange………………………………………………. 86

5.3.3 K and Na concentrations………………………………………… 91

5.4 Discussion…………………………………………………………………. 99

5.5 Conclusion………………………………………………………………… 102

Chapter 6. Solution culture short-term response

Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium (Na+) supply using rubidium (Rb+) tracer in solution culture experiments: short-term responses……………………………………………………………………………..

103

6.1 Introduction………………………………………………………………. 103

6.2 Materials and methods…………………………………………………… 104

6.2.1 Plant culture……………………………………………………. 104

6.2.2 Basal nutrient solution…………………………………………. 105

6.2.3 Potassium and sodium treatments……………………………… 105

6.2.4 Measurements………………………………………………….. 105

6.2.5 Statistical analysis……………………………………………… 106

6.3 Results……………………………………………………………………. 106

6.3.1 Experiment 1…………………………………………………… 106

6.3.1.1 Plant growth………………………………………….. 106

6.3.1.2 Leaf gas exchange……………………………………. 106

6.3.1.3 K, Na and Rb concentrations in shoot and root………. 108

6.3.1.4 K, Na and Rb contents in shoot and root…………….. 110

6.3.2 Experiment 2…………………………………………………… 112

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6.3.2.1 Plant growth …………………………………………. 112

6.3.2.2 Leaf gas exchange…………………………………… 112

6.3.2.3 K, Na and Rb concentrations in shoot and root……… 114

6.3.2.4 K, Na and Rb contents in shoot and root……………. 117

6.4 Discussion……………………………………………………………….. 119

6.5 Conclusion……………………………………………………………….. 122

Chapter 7. Solution culture experiment long-term response

Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium (Na+) supply using rubidium (Rb+) tracer in a solution culture experiment: long-term responses………………………………………………………………………………

123

7.1 Introduction………………………………………………………………. 123


7.2.1 Plant culture…………………………………………………….. 124

7.2.2 Basal nutrient solution………………………………………….. 124


7.2.4 Measurements…………………………………………………… 125


7.3 Results……………………………………………………………………. 126

7.3.1 Plant growth…………………………………………………….. 126

7.3.2 Leaf gas exchange………………………………………………. 130

7.3.3 K, Na and Rb concentrations……………………………………. 132

7.3.4 K, Na and Rb contents………………………………………….. 137

7.4 Discussion…………………………………………………………………. 141

7.5 Conclusion………………………………………………………………… 144

Chapter 8. General discussion and conclusions………………………………………. 145

8.1 Introduction………………………………………………………………. 145

8.2 Growth stimulation by Na………………………………………………… 145

8.3 Na effects on K deficient wheat………………………………………….. 147

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8.4 Stimulation of K uptake by Na……………………………………………. 148

8.5 Possible mechanisms of Na+-induced K+ uptake………………………… 150

8.6 Toxicity effects of Na…………………………………………………… 152

8.7 Na effects on cultivars differing in K-use efficiency…………………… 153

8.8 Implications of low to moderate Na for plant K nutrition……………… 153

8.9 Conclusions and recommendations……………………………………… 154

8.9.1 Conclusions……………………………………………………. 154

8.9.2 Further research recommendations…………………………….. 156

References…………………………………………………………………………….. 157

Appendices……………………………………………………………………………. 166

Appendix 1

1.1 K and Na concentrations in leaves, spikes and stem……………………… 166

1.2 Ca and Mg concentrations in young and old leaves………………………. 170

Appendix 2

Root: Shoot ratios of wheat grown in columns harvested at 5 WAS………… 173

Appendix 3

Experimental setup used in solution culture experiments…………………….. 174

Appendix 4

4.1 Plant growth (Experiment- 1) …………………………………………….. 175

4.2 Pre-treatment leaf gas exchange measurements (Experiment- 1)…………. 178

4.3 Plant growth (Experiment- 2)……………………………………………... 179

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LIST OF FIGURES

Figure 2.1 Role of potassium in resisting plant stresses (Wang et al., 2013)………….. 9

Figure 2.2 Role of K in drought stress (Wang et al., 2013)…………………………… 11

Figure 2.3 Schematic representations of different forms of soil K [modified figure from Department of Environment and Primary Industries report, 2014]………………

14

Figure 3.1 Wyalkatchem (K-efficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg) under soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks…………………………………………………………………………………...

38

Figure 3.2 Gutha (K-inefficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg) under soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks…………………………………………………………………………………...

38

Figure 3.3 Shoot dry weight (g/plant) (upper sub-figures), and tillers/plant (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1 for analysis of variance results……………………….

39

Figure 3.4 Root dry weight (g/plant) (upper sub-figures) and root: shoot ratio (n=3) (lower sub-figures) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1 for analysis of variance results…………………….

41

Figure 3.5 Leaf photosynthesis (upper sub-figures) and stomatal conductance (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1 for analysis of variance results…………………….

42

Figure 3.6 K concentration (mg/g, dry weight) in shoot (upper sub-figures) and root (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.2 for analysis of variance results………………..

44

Figure 3.7 K content (mg/plant) in shoot (upper sub-figures) and root (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.2 for analysis of variance results……………………………

45

Figure 4.1 Potassium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems (±SE, n=4)……………………….

65

Figure 4.2 Sodium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems (±SE, n=4)……………………….

67

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Figure 4.3 Wheat (cv. Wyalkatchem) at six weeks after transplanting grown in a split-root system treated with 40 mg K/kg and nil, 50 mg Na/kg. The picture shows the growth difference with and without Na addition (50 mg Na/kg)……………………..

70

Figure 4.4 Correlation between shoot dry weight/plant (g) harvested at 6 weeks after transplanting and the shoot K concentration (mg K/g, dry weight) measured in low soil K (40 mg K/kg) split-root treatments……………………………………………...

72

Figure 5.1 Column experiment of wheat cv. Wyalkatchem at 3 weeks after sowing (left). Column set-up with plastic tubes used for subsoil watering, commencing at 5 weeks after sowing (right)……………………………………………………………...

78

Figure 5.2 Shoot dry weight (g) and tiller number per plant at 5 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1. See Table 5.2 for statistical analysis………………………………………………………………………………….

80

Figure 5.3 Columns were supplied with low K (40 mg K/kg) in the whole profile with varying subsoil Na levels: a) nil Na, b) 50 mg Na/kg, and c) 200 mg Na/kg. Shoot growth and tillering was depressed by 200 mg Na/kg at 5 weeks after sowing………..

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Figure 5.4 Root dry weight (g/plant) of wheat cv. Wyalkatchem in different sections of column (0- 20, 20- 40 and 40- 60 cm) at 5 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1. See Table 5.2 for statistical analysis………..

82

Figure 5.5 Shoot dry weight (g) and tillers per plant at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1 and see Table 5.2 for statistical analysis……………………………………………………………………………….....

84

Figure 5.6 Root dry weight (g/plant) in different sections of column (0- 20, 20- 40 and 40- 60 cm) at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1……………………………………………………………………………..

85

Figure 5.7 Root: shoot ratios of wheat at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1……………………………………………….

86

Figure 5.8 Leaf photosynthesis, stomatal conductance, and transpiration at 5 weeks after sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and for statistical analysis refer Table 5.3…………………………………………………………………

88

Figure 5.9 Leaf photosynthesis, stomatal conductance, and transpiration at 7 weeks after sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and for statistical analysis refer Table 5.3…………………………………………………………………

89

Figure 5.10 Leaf photosynthesis, stomatal conductance, and transpiration at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and Table 5.3 for statistical analysis……………………………………………………………………..

90

Figure 5.11 Correlation between leaf net photosynthesis rates and shoot Na concentrations (mg Na/g, dry weight) at final harvest at 11 weeks after sowing…………………………………………………………………………………

101

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Figure 6.1 Leaf net photosynthesis, stomatal conductance, and transpiration of wheat cultivars Wyalkatchem and Gutha treated with low K (0.2 mM K) for two weeks, followed by two K levels (0.2 and 2 mM) and three Na levels (0, 10 and 20 mM) (±SE, n=4) and measured 42 hours after the treatments (Experiment 1)………………

108

Figure 6.2 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for two weeks, followed by two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) (±SE, n=4) and measured 42 hours after the treatments (Experiment 2)……………………………………………………………………….....

114

Figure 7.1 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (harvested 32 days after transplanting, pre-rubidium addition) (±SE, n=4)…………………………………………………………………………………….

127

Figure 7.2 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium harvest), and after Rb treatment for 48 hours (post-rubidium harvest, 35 days after transplanting) (±SE, n=4)…………………………….

129

Figure 7.3 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (±SE, n=4)……………………………….......

131

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LIST OF TABLES

Table 2.1 Removal of K through grain and hay harvest of different crops……………. 17

Table 2.2 Functions of K replaced and not replaced by Na……………………………. 21

Table 2.3 Sodium response at low K in various crop species………………………….. 24

Table 3.1 Statistical summary of plant growth and leaf gas exchange in four wheat cultivars (Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks……………………………………………………………………………………

40

Table 3.2 Statistical summary of K and Na concentrations and contents in four wheat cultivars (Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two levels of soil K (40, 100 mg K/kg) and five levels of Na (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks……………………………………………………………………………………

46

Table 3.3 Shoot and root Na concentrations (mg/g, dry weight) and contents (mg/plant) of four wheat cultivars treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks (n=3). See Table 3.2 for statistical summary of main effects and interactions of the treatments………………..

48

Table 3.4 Shoot and root K/Na ratios of four wheat cultivars treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks (n=3). See Table 3.2 for statistical summary of main effects and interactions of the treatments………………………………………………………………………………...

49

Table 3.5 Concentrations of exchangeable cations in non-planted soils (n=3) with or without 50 mg Na/kg at two K levels (40, 100 mg K/kg) after one week of incubation. Means with different letters differ at P≤0.05…………………………………………..

50

Table 4.1 Split- root treatments experimental design………………………………….. 58

Table 4.2 Shoot dry weight (g) and number of tillers per plant of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems. Means (n=4) in a column with different letters differ at P≤0.05………………………………………

60

Table 4.3 Total root dry weight (g) per plant and their root: shoot ratios of wheat cv. Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). One-way analysis of variance was conducted to assess the effects of split-root treatments. Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in total root dry weight and root: shoot ratios between the 11 split-root treatments and the specific root responses between the two compartments were compared within each split-root treatment. Means (n=4) with different letters differ at P≤0.05…………………………………………….

61

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Table 4.4 Leaf net photosynthesis rate (Pn), stomatal conductance (Gs) and transpiration (E) of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems. Means (n=4) with different letters differ at P≤0.05………………..

62

Table 4.5 Shoot and root K concentrations (mg K/g) of wheat cv. Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in shoot K concentrations between the 11 split-root treatments and the specific root K concentrations between the two compartments were compared within each split-root treatment. Means (n=4) with different letters in a column differ at P≤0.05..………………………………………………………………

64

Table 4.6 Shoot and root Na concentrations (mg Na/g) of wheat cv. Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in shoot Na concentrations between the 11 split-root treatments and the specific root Na concentrations between the two compartments were compared within each split-root treatment. Means (n=4) with different letters in a column differ at P≤0.05………………………………………………………………

66

Table 4.7 K/Na ratios of wheat (whole plant) cv. Wyalkatchem were compared between the split-root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………………………

68

Table 5.1 Experiment design showing topsoil watering, topsoil K (mg K/kg) and subsoil K (mg K/kg) and Na (mg Na/kg) treatments harvested at 5 and 11 weeks after sowing ………………………………………………………………………………….

78

Table 5.2 Statistical summary of plant growth at 5 and 11 weeks after sowing treated with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table 5.1…………………………...

81

Table 5.3 Statistical summary of leaf gas exchange at 5, 7 and 10 weeks after sowing treated with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table 5.1……………………

91

Table 5.4 Shoot K and Na concentrations and accumulation in wheat cv. Wyalkatchem harvested at 5 weeks after sowing. Means (n=3) with different letters differ at P≤0.05…………………………………………………………………………

92

Table 5.5 Statistical summary of shoot K and Na concentrations and content at 5 and 11 weeks after sowing in wheat plants treated with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table 5.1. Note only whole shoots and roots were analysed at 5 weeks……………………………………………………………………………………

93

Table 5.6 K concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Means (n=3) with different letters differ at P≤0.05……………

95

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Table 5.7 Shoot K and Na accumulation in wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Values are means of 3 replicates. Means (n=3) with different letters differ at P≤0.05. …………………………………………………………………

96

Table 5.8 Na concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Means (n=3) with different letters differ at P≤0.05……………

97

Table 5.9 Shoot K/Na ratios in wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Means (n=3) with different letters differ at P≤0.05…………………………..

98

Table 6.1 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for a further 48 hours (Experiment 1)…………….………………………….

107

Table 6.2 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5mM) for a further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters differ at P≤0.05…………………………………..

109

Table 6.3 Statistical summary of shoot and root K, Na, Rb concentrations and contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for 48 hours (Experiment 1; n=4)…………………………………………………………………………………….

110

Table 6.4 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters differ at P≤0.05…………………………………..

111

Table 6.5 The whole plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters differ at P≤0.05…………………………………..

112

Table 6.6 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM K), two Na levels (0, 2 and 10 mM Na) and Rb (0.5mM) (n=4) added for 48 hours (Experiment 2)…………………………………….

113

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Table 6.7 Statistical summary of shoot and root K, Na, Rb concentrations and contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2; n=4).…………………………………………………….

115

Table 6.8 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05…………………………………..

116

Table 6.9 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05…………………………………..

118

Table 6.10 Plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………

119

Table 7.1 Statistical summary of plant growth in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium harvest), and after Rb treatment for 48 hours (post-rubidium or final harvest).…………………………………………………………………………………

128

Table 7.2 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3), number of tips and forks in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested after Rb treatment for 48 hours (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05…………………………………………………

130

Table 7.3 Shoot and root K and Na concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………

133

Table 7.4 Statistical summary of shoot and root K, Na and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks and then treated with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-rubidium harvest), and harvested after Rb treatment for 48 hours (Post-rubidium harvest)……………………………………….

134

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Table 7.5 Young leaf, old leaf, and the rest of shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested 48 hours after Rb addition (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………………

136

Table 7.6 Shoot and root K and Na contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, and harvested after treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05………………………………………………………………….

138

Table 7.7 Statistical summary of shoot and root K, Na and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb harvest) and harvested 48 hours after Rb treatment (post-rubidium or final harvest)……………………………………………..

139

Table 7.8 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested 48 hours after Rb addition (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05……………………………………

140

xx

LIST OF ABBREVIATIONS AND SYMBOLS

µ Micro

µM Micromolar

˚C Degree Celsius

AAS Atomic absorption spectrophotometer

Al Aluminium

ANOVA Analysis of Variance

B Boron

Ca Calcium

CAM Crassulacean acid metabolism

CAT Catalase

CEC Cation exchange capacity

CO2 Carbon dioxide

DAT Days after transplanting

DI De-ionized water

E Transpiration

FC Field capacity

GRDC Grains Research and Development Corporation

GRS Grains Research Scholarship

Gs Stomatal conductance

HAK/HKT High affinity potassium transporter

H2O2 Hydrogen peroxide

hsd honest significant difference

ICP Inductively coupled plasma

K Potassium

KCl Potassium chloride

M Molar

xxi

Mg Magnesium

min Minute

mM Millimolar

Na Sodium

NaCl Sodium chloride

NAD(P)H Nicotinamide adenine dinucleotide phosphate

nmol Nanomoles

NO3 Nitrate

n.s Not significant

O2 Oxygen

O2- Superoxide radical

OH- Hydroxyl radical

Pn Photosynthesis

PVC Poly vinyl chloride

ROS Reactive oxygen species

SE Standard error

SOD Superoxide dismutase

SOPIB Sulphate of Potash Information Board

SWA South Western Australia

vs Versus

WA Western Australia

WAS Weeks after sowing

WAT Weeks after transplanting

WUE Water use efficiency

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ACKNOWLEDGEMENTS

This study was supported by the Grains Research and Development Corporation (GRDC

Project UMU00035), and a Grain Research Scholarship (GRS- 10268), and the Sulphate of

Potash Information Board (SOPIB).

I am deeply grateful to my supervisor, Professor Richard Bell for his constant guidance,

encouragement and valuable suggestions in the course of experiment and writing of thesis.

His excellent supervision and support motivated to publish journal and present conference

papers. I am truly thankful to have conducted this research under such an expert and

knowledgeable person.

I extend my greatest thanks and appreciation to Dr. Qifu Ma, my co-supervisor for his ideas,

motivation and valuable advice. He was easy to approach and his suggestions were helpful in

setting experiments and thesis writing, and his guidance during field visit helped to gain

practical exposure and made this whole research journey an enjoyable one.

I would like to extend my sincere thanks to Professor Giles Hardy, Sonia Aghighi for helping

with WinRhizo root scanning, Andrew Foreman for providing training on flame photometer

and also, to Wendy Vance and other fellow post-graduate students and friends of ‘Land

management group’ for their generous help and support during the research. Special thanks to

people from DAFWA, in particular Craig Scanlan for helping in soil collection, and farmers

of Dowerin for letting to collect soil for glasshouse experiments.

I am grateful to my family and friends for their support and motivation throughout this

journey. In particular to my husband, Pradeep for his unending support and understanding

which made the completion of this study and to my little person Eeshva for his valuable

distractions and being good to let me do final stages of writing and revisions.

1

CHAPTER 1

INTRODUCTION

1.1 Potassium nutrition in plants

Potassium (K) is an essential element for higher plants since plants are unable to complete

their life cycle in its absence, and its function cannot be fully replaced by any other element.

Potassium, unlike other nutrients (apart from chloride) does not become a part of the

chemical structure of compounds, but, plays regulatory roles within the plant as a cation.

Potassium is taken up in large quantities in plant tissues (Wakeel et al., 2011). It is a

dominant cation in the cytoplasm of plant cells constituting up to 100 g/kg of plant dry

weight (Very & Sentenac, 2003). Plant cytoplasm concentrations are tightly regulated and

maintained at ~100 mM, however, vacuolar K concentrations are highly variable reflecting

plant K status (Marschner, 1995).

Potassium plays an important role in photosynthesis, protein synthesis, enzyme activation,

osmoregulation, stomatal movement, phloem loading and transport and stress tolerance in

plants (Mengel & Kirkby, 2001; Römheld & Kirkby, 2010). It is highly mobile in plants and

is readily re-translocated from source to sink organs. When plant K concentration is lower

than 10 g/kg dry weight, most species will show deficiency symptoms with interveinal

chlorosis in older leaves. With the progression of deficiency, necrosis and death of tips and

margins of leaves may occur (Gierth & Mäser, 2007). Plant K status is dependent upon soil K

availability and K uptake by roots.

1.2 Sodium nutrition in plants

The role of sodium (Na) in plant nutrition and its status as an essential element is still being

debated. Plant species are characterized as natrophilic or natrophobic depending on their

growth response to Na and their capacity for uptake and transport. Natrophilic plants absorb

Na+ but translocation to shoots is slow and Na+ is compartmentalised in root vacuoles

(Wakeel et al., 2011). In contrast to K, Na is only beneficial for certain plant species

characterized by C4 and CAM photosynthetic pathways (Marschner, 1995), but Na is

beneficial in relatively low concentrations for many plants and it is toxic to the majority of

plants at high concentrations (Mäser et al., 2002). In some plant species where Na is

beneficial, the main functions are in growth stimulation, osmotic regulation, better water

balance and some other non-specific functions (Kronzucker et al., 2013; Wakeel et al., 2011).

2

Under abundant Na availability, growth of many plants is limited due to water stress and

specific Na+ ion toxicity when plant cytoplasmic Na+ concentrations are above 20 mM

(Benito et al., 2014). However, other studies reported no negative or even positive effects of

Na typically at low Na concentrations with partial substitution of K by Na when K supply

was low and plants suffered at least partial K deprivation (Kronzucker et al., 2013).

1.3 Interaction between potassium and sodium

Potassium and Na ions are similar in ionic radius and ionic hydration energies (Marschner,

1995), and because of this chemical similarity, it is assumed that the both ions compete with

each other (Subbarao et al., 2003). Since K+ and Na+ exhibit homologous behaviour they

share some physiological functions (Almeida et al., 2010). Potassium is required in high

concentrations for plant growth and development, whereas Na is beneficial to certain

halophytes at relatively low concentrations (Greenway & Munns, 1980; Mäser et al., 2002).

In halophytes, presence of Na in the environment and its uptake can reduce the plant K

requirement to meet the plants metabolic requirements (Benito et al., 2014).

Sodium can have either negative effects on plant growth at high levels of supply in soil or

positive effects at low to moderate supply in low K soil, but both these effects vary with plant

tolerance to salinity and with soil K and Na levels (Kronzucker et al., 2013). In arid and

semiarid regions, soil salinity is largely due to excessive NaCl which, apart from osmotic and

specific Na+ and Cl- ion effects, has a detrimental effect on potassium (K+) uptake and

nutrition of most crops (Römheld & Kirkby, 2010). Soil salinity, mainly associated with

sodium chloride (NaCl) is a major environmental stress that affects K+ uptake and transport

by plants (Szczerba et al., 2009). In saline soils, plant physiological functions such as enzyme

activation and protein synthesis are inhibited due to depression in K+ uptake by competing

Na+ ions (Al-Rawahy et al., 1992; Blumwald et al., 2000).

It has been widely reported that K+ counteracts Na+ stress in plants while there are few

reports that Na+ can in turn, alleviate K+ deficiency symptoms (Ali et al., 2009). Although the

complete role of Na+ in plant metabolism still awaits resolution, it is commonly assumed that

Na+ can substitute biophysical functions of K+ in non-halophytic plants, given that the plants

have the ability to take up Na+, translocate it to the shoot, and compartmentalise it in the

vacuoles (Subbarao et al., 2003). Sodium can replace K+ in the vacuole as an alterative

inorganic osmoticum under K-limited conditions, and the released K+ is then available for

more K-specific processes (Benito et al., 2014). Sodium can alleviate K deficiencies in some

3

species such as sugar beet, lettuce, cotton, ryegrass, spinach, marigold, tomato, celery, carrot

(Benito et al., 2014; Idowu & Aduayi, 2007; Marschner, 1995; Mundy, 1983; Tahal et al.,

2000) and barley (Ma et al., 2011). Sodium is beneficial to plant growth when available K is

deficient, but the degree of this beneficial effect varies between crop species, and even

between genotypes of the same plant species (Marschner, 1995).

1.4 K deficiency in soils

Large areas of agricultural soil in the world are reported to be K-deficient and unbalanced K

fertilization may result in significant K depletion from soil reserves and decreased soil

fertility (Zörb et al., 2014). Potassium concentration of top soils is usually > 1 % (10 g K/kg)

in most soils of the world, whereas the top soils of WA contain < 0.1 % (1 g K/kg) and

concentrations > 1 % are relatively rare reflecting the highly weathered state of these soils

(Pal, 1999). Moreover K deficiency is further worsened due to continued removal of grain,

hay/straw, without adequate K replacement and therefore K fertilisation management is

required for profitable cropping (Ma et al., 2011).

1.5 Salinity issues of WA

Soil salinity in arid and semi arid areas is a major constraint of crop productivity in many

parts of the world. The global estimate for agricultural land threatened by or already lost to

salinity exceeds 900 million ha (Kaya, 2002). The Land Monitor method has estimated that

the current area affected by salinity in Western Australia is about 1 million ha and the annual

rate of increase is about 14,000 ha (McFarlane et al., 2004). Sodic soils and duplex soils are

also major soil constraints for crop production in Australia. Sodic soils are common in

Western Australia, particularly in south-west agricultural area (Cochrane et al., 1994).

Duplex/ texture-contrast soils account for about 12 % of the land area of Australia (Dracup et

al., 1992) and half to 2/3rd of the cultivated area in south-Western Australia (Belford, 2005).

The duplex soils have varying concentrations of nutrients due to differences in clay content

and mineralogy. However, considering the interactive effects of K and Na, there is a

possibility that K requirements in moderately saline and in sodic soils may be decreased due

to the presence of Na. This thesis had an emphasis on moderately saline-sodic soils and

interactions between K and Na under such conditions are investigated.

4

1.6 Research aim and scope of study

In saline/sodic soils and low fertility soils, the partial substitution of K+ by Na+ in

physiological processes of wheat would have substantial practical implications for K fertiliser

management.

This study is a part of two research projects funded by the Grains Research and Development

Corporation (GRDC) and the Sulphate of Potash Information Board (SOPIB) to examine K

nutrient management in low K and saline/sodic soils in the drought-prone environments of

south-west Australia. As this research looks into important soil constraints prevailing, the

research findings of this study with further field studies would help in decision making for

nutrient management to improve crop productivity in saline/sodic soils and drought-prone

environments.

In K deficient soils, Na can to a certain degree substitute the role of K in some plants

(Marschner, 1995; Subbarao et al., 2003). In the Chenopodiaceae family, crops like spinach,

beet and sugar beet have received detailed attention in terms of K and Na interactions

(Kronzucker et al., 2013). Wheat (Triticum aestivum L.) is a major cereal grown worldwide

and it is cultivated in semi-arid regions of Western Australia. However, little is known about

K requirements of wheat cultivars grown under saline and sodic soils (Ma et al., 2011).

Understanding the Na and cultivar effects on K nutrition in wheat is the main aim of this

thesis. The potential of K substitution by Na in wheat nutrition may offset the requirement of

expensive K fertiliser and therefore may help in profitable as well as sustainable cropping in

agriculture.

1.7 Layout of the thesis

A review of K and Na nutrition and functions in plants, and of K deficiency effects is

presented in Chapter 2 along with a review on Na effects on K nutrition and their

interactions. Previous research on K substitution by Na in wheat and other plant species will

also be reviewed. The main objectives and research questions of the present thesis are

identified.

Chapter 3 reports on an experiment with wheat cultivars treated with soil K and Na levels. In

this chapter, Na effects on four wheat cultivars differing in K-use efficiency were examined

under low and adequate K supply in terms of growth behaviour, leaf gas exchange

measurements, ion concentrations and content. Sodium levels that are beneficial to wheat

cultivars along with the cultivar effect will be identified. The effects of external Na supply on

5

soil exchangeable K availability was also investigated in a short-term soil incubation

experiment.

In Chapter 4, the split-root experiment consisted of 2 K and 3 Na levels to examine whether

K replacement by Na would depend on both these cations being present in the same or

different parts of the root system.

A column experiment with varying subsoil K and Na levels under topsoil water deficit is

reported in Chapter 5. This experiment aimed to understand the relationship between K

responses and subsoil Na because low K soils commonly contain significant exchangeable Na

in the subsoil.

Following the detailed experiments in soil-based systems, a series of solution culture

experiments were conducted (Chapters 6 and 7). These experiments aimed to investigate

whether supply of low external Na conditions would alter wheat K uptake using Rb as a

tracer. Wheat cultivars differing in K-use efficiency and short-term versus long-term

responses were compared. The experiments gathered evidence on whether Na induced

increased K uptake by wheat cultivars and on cultivar effects on K uptake.

A general discussion is undertaken in Chapter 8 where the main issues are discussed with

other published findings. Also the main findings and conclusions are brought together in

Chapter 8 with further research recommendations outlined.

6

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

To provide food for an expanding global population, a massive increase in crop production is

required in a more resource-efficient way. In this context, K is an important macronutrient

that plays a critical role in a number of physiological and biochemical processes required for

growth and yield of plants. Potassium constitutes about 2.1 to 2.3 % of the earth’s crust, and

is the seventh or eighth most abundant element (Zörb et al., 2014). Plants can only acquire K+

from solution and its availability in soils is dependent on nutrient dynamics and total K

content. Although soil K reserves may be large, extensive areas of the world are reported to

be deficient in K availability for plants. A proper understanding and management of K

nutrition and its interactions is needed to counter the declining soil fertility and improve food

security.

This review focuses on the role of K in plants, including physiological functions and

deficiency effects in plants. Potassium availability for plant growth, forms of K in soil, and K

uptake by plants are also discussed. This review also discusses Na nutrition in plants,

interaction between K and Na in various plants, and the potential for partial substitution of K

by Na. The main aim and research questions are identified at the end of the literature review.

2.2 K functions in plants

Potassium is the most abundant inorganic cation in plant cells and is vital for plant growth. It

is a highly mobile element in plants, and highest concentrations are found in young and

developing tissues indicative of its role in cell metabolism and growth. Potassium plays a

major role in physiology and biochemistry of plants. Regulation of stomatal opening and

closing, leaf movements, and also other plant tropisms are driven by K+-generated turgor

pressure in cells (Maathuis & Sanders, 1996; Zhao et al., 2001). It acts as an osmoticum in

maintaining turgor pressure, and influencing solute transport and water balance in plants. The

maintenance of turgor pressure is essential for continued cell expansion, and growth of plant

cells (Römheld & Kirkby, 2010). Potassium is essential in activating numerous enzymes,

including those involved in photosynthesis, energy metabolism, protein synthesis and starch

synthesis (Mengel & Kirkby, 2001). It is also essential in maintenance of transmembrane

voltage gradients for cytoplasmic pH homeostasis (Römheld & Kirkby, 2010). The important

K functions in plants are reviewed briefly.

7

2.2.1 K and enzyme activation

The activation of enzymes is a major, critically important, irreplaceable role of K in plant

growth and development. Concentrations of K+ in the cytosol are maintained relatively

constant at around 100 mM which is optimal for the function of cytosolic enzymes (Ashley et

al., 2005). Potassium is important in activation of a large number of enzymes involved in

energy metabolism, protein synthesis and solute transport (Römheld & Kirkby, 2010) by

inducing conformational changes in the enzymic proteins (Marschner, 1995). It is known that

more than 70 important enzymes involved in plant growth are activated by K+ (Anschütz et

al., 2014). Some of the enzymes activated by K+ include pyruvate kinase and

phosphofructokinase involved in carbohydrate metabolism, asparaginase involved in N

metabolism, starch synthase, and membrane-bound proton- pumping ATPases (Blevins,

1985; Marschner, 1995).

2.2.2 K and protein synthesis

Potassium is believed to regulate every major step of protein synthesis, including the

synthesis of ribosomes, and aminoacyl-tRNA binding to ribosomes, peptidyl transfer,

guanosine-5’- triphosphate (GTP) utilization, protein synthesis from charged tRNA transfer,

and messenger RNA turnover (Blevins, 1985). It is claimed that protein synthesis requires

higher concentrations of K+ than for enzyme activation and the “reading” of genetic code in

plant cells to produce proteins is not possible without adequate K concentrations (Marschner,

1995). A probable function of K is in polypeptide synthesis in the ribosomes, since that

process requires a high K+ concentration (Wyn Jones & Pollard, 1983). In K-sufficient plants

high-molecular weight compounds like proteins are increased, whereas, in K-deficient plants

low-molecular weight compounds like amino acids, amides and nitrate accumulate instead of

proteins (Wang et al., 2013).

2.2.3 K and stomatal activity

Potassium plays an important role in opening and closing of stomates, the pores through

which the leaves exchange CO2, O2 and water vapour with the atmosphere. When K+ moves

into the guard cells around the stomata, the cells accumulate water and swell, causing the

pores to open, allowing gases to move freely in and out. When water supply is short, K+ is

pumped out of the guard cells. The pores close tightly to prevent loss of water and minimize

drought stress to the plant. This stomatal movement is essential for transpiration, and CO2

uptake for photosynthesis (Humble & Raschke, 1971). Under K deficiency, stomatal closure

8

may take longer and be incomplete, and plants are more susceptible to water stress (Mäser et

al., 2002). Potassium deficient plants show decrease in turgor pressure and become flaccid

due to impaired stomatal functioning (Römheld & Kirkby, 2010).

2.2.4 K and photosynthesis

Potassium plays an essential but complex role in regulating the rate of photosynthesis of

higher plants. It is the dominant counter-ion to the light-induced H+ flux across the thylakoid

membranes and is also required for the establishment of the transmembrane pH gradient

necessary for the synthesis of adenosine triphosphate (ATP) and activation of enzymes

involved in photosynthesis (Marschner, 1995). The primary effect of K in photosynthesis is

in maintaining the stomal K concentration of the chloroplast to allow CO2 fixation. Potassium

deficiency reduces ATP production, photosynthetic activity, chlorophyll content and

translocation of fixed carbon in plants (Zhao et al., 2001). This depression in photosynthesis

causes an excessive accumulation of light energy and photo reductants in the chloroplasts

which results in the formation of reactive oxygen species (ROS), and chloroplast damage

(Cakmak, 2005). This is discussed in more detail below.

2.2.5 K and stress tolerance in plants

Potassium plays a major role in protecting plants against environmental stresses such as

drought, frost, heat, salinity, high light intensity, and nutrient limitations. It is also claimed

that high K status in crops decreases the incidence of diseases and pests (Römheld & Kirkby,

2010). There is evidence that K also plays a regulatory role in plant stress responses as

discussed briefly below (Fig. 2.1).

Plants exposed to environmental stresses suffer from oxidative damage catalyzed by ROS

which impairs cellular function and causes plant growth depression (Cakmak, 2005).

Reactive oxygen species are extremely cytotoxic and can seriously disrupt normal

metabolism through oxidative damage to lipids, nucleic acids and proteins (Heidari &

Jamshidi, 2011). Examples of ROS are hydroxyl radical (OH-), singlet oxygen/superoxide

radical (O2-), and hydrogen peroxide (H2O2). In plants, ROS are predominantly produced

during photosynthetic electron transport and activation of membrane bound NAD(P)H

oxidases (Römheld & Kirkby, 2010). When K is deficient there is a severe reduction in

photosynthetic CO2 fixation, impairment in partitioning of fixed carbon to sink organs, and

decreased utilisation of photosynthates. According to Cakmak (2005), there is a decrease in

net photosynthesis under K deficiency due to reduced stomatal conductance, increased

9

mesophyll resistance, and lowered ribulose bisphosphate carboxylase activity. Such

disturbances result in excessive photosynthetically-produced electrons and thus increased

ROS production by intensified transfer of electrons to O2. Also there is an increase in

NADPH oxidation under K deficiency, up to 8-fold when compared with K-sufficient plants

(Cakmak, 2005).

Fig. 2.1 Role of potassium in resisting plant stresses (Wang et al., 2013)

2.2.5.1 Anti-oxidant activity

Under stress conditions, plants have evolved molecular defence systems that limit the

formation of ROS and promote their removal. Chloroplasts are the major organelles

producing ROS when plants are exposed to environmental stress conditions, and the

consequences include membrane damage, chlorophyll degradation, and development of leaf

chlorosis and necrosis (Cakmak, 2005). The plant enzymatic defences include production of

antioxidant enzymes such as phenol peroxidases (POX), superoxide dismutase (SOD),

catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), which together

with other enzymes of the ascorbate-glutathione cycle promote the scavenging of ROS

(Heidari & Jamshidi, 2011). Catalase (CAT) activity decreased when K levels increased

under salt stress, in millet (Heidari & Jamshidi, 2011).

10

2.2.5.2 Drought and heat stress

Potassium is an important mineral nutrient contributing to osmotic adjustment under drought

stress in many plant species (Damon et al., 2011). Drought stress causes stomatal closure and

therefore decreases CO2 fixation in plants. The formation of ROS is intensified due to

inhibited CO2 reduction by drought stress with consequent oxidative damage to chloroplasts

(Cakmak, 2005). Plants suffering from drought have a larger internal requirement for K, and

adequate K supply will enhance plant adaptation to drought by ensuring improved control of

stomatal opening and closing. For example, the increase in K supply in external solution from

0.2 to 6 mM K alleviated the drought stress in wheat (Gupta et al., 1989). Under drought

stress, root growth and rate of K+ diffusion are restricted, limiting K+ acquisition (Wang et

al., 2013) and increasing K application reduces the damage by significantly increasing the

depth of root penetration, root surface exposed to soil and K absorption (Valadabadi &

Farahani, 2010). However, increasing root penetration when there is limited sub-soil water

available is of questionable value.

Plants exhibit several resistance mechanisms for survival during mild to severe water stress.

Fig. 2.2 summarises the role of K in plants under drought stress (Wang et al., 2013). One

such mechanism is by active solute accumulation in plant organs subjected to prolonged

stress, referred to as ‘osmotic adjustment’, which maintains turgor in plants, or an increased

water potential gradient from soil to leaf resulting in water uptake (Gebre & Tschaplinski,

2000). Different plant species accumulate different solutes for osmotic adjustment (Gebre &

Tschaplinski, 2000). Potassium is the most common solute in wheat and other species

(Damon et al., 2011). In an experiment by Damon et al. (2011), to study the osmotic

adjustment under drought stress, among 5 wheat genotypes with differing K-use efficiency, K

fertiliser application accounted for 38 % (Wyalkatchem) to 51 % (Nyabing) of leaf osmotic

adjustment with drought stress imposed by withholding water from 35 days after sowing.

Fig. 2.2 Role of K in drought stress

2.2.5.3 Low temperature and frost stress

Under chilling and frost stresses, plant metabolic reactions are inhibited due to cold

osmotic, oxidative and other stresses

lipids and membrane structure are altered. Reactive oxygen species are formed under low

temperature stress because absorbed light energy exceeds the chloroplast capacity to use it in

CO2 fixation (Römheld & Kirkby, 2010)

electron transport, stomatal conductance, and CO

Increasing K supply helps in alleviating low

secondary metabolite transcripts associated with cold tolerance

acts as an osmoticum in maintaining high concentration of K in cell sap thus lowering its

freezing point (Römheld & Kirkby, 2010)

Role of K in drought stress (Wang et al., 2013)

2.2.5.3 Low temperature and frost stress

Under chilling and frost stresses, plant metabolic reactions are inhibited due to cold

osmotic, oxidative and other stresses (Wang et al., 2013). Also the fluidity of membrane



(Römheld & Kirkby, 2010). Moreover low temperature impairs photosynthetic

electron transport, stomatal conductance, and CO2 fixation in plants

Increasing K supply helps in alleviating low temperature stress in plants by producing

secondary metabolite transcripts associated with cold tolerance (Wang et al., 2013)

in maintaining high concentration of K in cell sap thus lowering its

(Römheld & Kirkby, 2010).

11

(Wang et al., 2013)

Under chilling and frost stresses, plant metabolic reactions are inhibited due to cold-induced

Also the fluidity of membrane



. Moreover low temperature impairs photosynthetic

fixation in plants (Cakmak, 2005).

temperature stress in plants by producing

(Wang et al., 2013), and K

in maintaining high concentration of K in cell sap thus lowering its

12

2.2.5.4 Disease and pest resistance

Potassium fertiliser is widely reported to decrease disease and pest symptoms in many host

plants (Wang et al., 2013). As a mobile regulator of enzyme activity, K is involved in

essentially all cellular functions that influence disease severity (Huber & Arny, 1985). High

K status in plants favours the synthesis of high molecular weight compounds like proteins,

starch and cellulose, thereby depressing the synthesis of low molecular weight compounds

like soluble sugars, organic acids, amino acids and amides in plant tissues. The low molecular

weight compounds are necessary for feeding pathogens and insects. Therefore, K deficient

plants are more vulnerable for pest and disease attack (Marschner, 1995).

The generally inverse relationship of available K in soil to disease severity has made it a

common practice to fertilise with K to reduce certain diseases. Potassium significantly

decreased the incidence of fungal diseases by 70 %, bacteria by 69 %, insects and mites by 63

%, viruses by 41 % and nematodes by 33 % (Wang et al., 2013). It is believed that high levels

of K could directly inhibit the growth and zoospore release of pathogens in crop production

(Sugimoto et al., 2009).

Although there is a large volume of literature on the relationship between K and plant

disease, there is a very little quantitative information available on the concentrations of K in

soil or plant tissues that result in changed disease expression (Huber & Arny, 1985). Higher

levels of K, relative to other nutrients, decreased the severity of yellow disease (Fusarium

oxysporum) of cabbage, Fusarium wilt of tomato, Fusarium wilt of pea, Stewart’s wilt

(Erwinia stewartii) of maize, and downy mildew (Perenospora tabacina) of tobacco (Huber

& Arny, 1985). The application of 20- 30 mM potassium nitrate significantly reduced the

infections caused by Phytophthora sojae (stem rot) in soybean (Sugimoto et al., 2009). High

rates of K application reduced population density of homopterous pests on cereals, legumes

and maize plants (El-Gindy et al., 2009). Also, K fertilisers promotes thicker epidermal walls

which promotes vigorous plant growth by inducing disease resistance (Sugimoto et al., 2009),

and an increase in the thickness of epidermal leaves suppressed the infestation of piercing and

sucking pests (El-Gindy et al., 2009).

2.3 K deficiency in plants

The reason for wide spread K deficiency includes a gradual decline in soil K levels due to

land clearing, increased cropping in marginal K soils, introduction of crops with a high K

requirement, increased demand for K due to improved agronomic practices including

13

increased use of N fertilisers (Edwards, 1997). Potassium deficiency is extensive across

Western Australia (Brennan et al., 2004). Potassium concentration of top soils is usually > 1

% (10 g K/kg) in most of the areas of the world, whereas, the top soils of WA are K-deficient

and concentrations of > 1 % are relatively rare which reflects the highly weathered state of

these soils (Pal, 1999). Soil properties arising from parent material, degree of weathering,

texture, clay mineralogy as well as land use and rainfall pattern influence the development of

K deficiency (Moore, 2004).

When plant K concentration is lower than 10 mg K/g dry weight, most species will show

deficiency symptoms with interveinal chlorosis in older leaves, and with the progression of

deficiency, necrosis and death of tips and margins of old leaves occur in extreme cases

(Gierth & Mäser, 2007). Since K is highly mobile in plants, mild deficiency does not result in

visible symptoms immediately as K is withdrawn from old leaves and retranslocated to

growing tissues. In early stages of K deficiency, there is a reduction in growth rate (without

visible symptoms- called hidden hunger), and later on, chlorosis and necrosis develop in

mature leaves (Römheld & Kirkby, 2010).

2.4 K uptake and accumulation by plants

The availability of soil K to plants is influenced by many factors, which include clay

mineralogy, particle size, water content, acidity, aeration, and organic matter level (Moore,

2004). It is also dependent on the levels of other cations, especially Ca, Mg and Na, in soil

solution (Jalali, 2008). Plant K+ acquisition from soil is dependent on factors influencing root

development such as root structure, root density, rooting depth and root hair length, root

distribution in the soil and the ability of roots to absorb mineral nutrients. Factors like

salinity, drought, soil compaction, Al toxicity in acid soils, and B deficiency can inhibit root

growth and hence lower K uptake from the soil (Römheld & Kirkby, 2010). Potassium

retention in the soil in a plant-available form is achieved by cation exchange. Plant roots take

up K+ from a wide range of external concentrations which typically vary from 0.1 to 10 mM

(Szczerba et al., 2009).

Plants accumulate considerable quantities of K which constitutes between 20 and 100 mg K/g

of plant dry weight (Ashley et al., 2005). The critical K concentration for many crop species

is in the range of 5 to 20 mg K/g, plant dry matter (Zörb et al., 2014). For effective

biochemical functions in plants, K concentrations of 100- 150 mM must be present in

metabolically active compartments like the cytosol, nucleus, stroma of chloroplast, and

14

matrix of mitochondria (Britto & Kronzucker, 2008; White, 2013). In plants, the cytosolic K+

pool appears to be relatively stable, around 100 mM (Gierth & Mäser, 2007), which is

considered optimal for the function of cytosolic functions (Ashley et al., 2005). In contrast,

vacuolar K+ concentrations vary greatly, between 10 and 500 mM (Marschner, 1995;

Szczerba et al., 2009), reflecting K status of the plant (Gierth & Mäser, 2007). When K

supply is sufficient the vacuolar K+ pool is increased, but when K is deficient vacuolar K+

storage is depleted to sustain a constant concentration in the cytoplasm (Gierth & Mäser,

2007). An optimal cytosolic K+ concentration is considered necessary for optimal enzyme

activity and photosynthetic activity (Cuin et al., 2003; Szczerba et al., 2009).

2.5 Forms of K in soil

Potassium occurs in primary minerals, clay minerals and also in crop and microbial residues;

it may be in soluble or insoluble forms. Soils usually have > 2 % (20 g K/kg) of total K but of

this generally < 0.1 % (1 g K/kg) is available to plants (Schulte & Kelling, 2009). There are

dynamic reactions between different forms of K: the four different forms of K and their

relative availabilities are illustrated in figure (Fig. 2.3) and discussed below.

Fig. 2.3 Schematic representation of different forms of soil K (Department of Environment

and Primary Industries, 2014)]

15

2.5.1 Unavailable/ Mineral K

This large reservoir of K is present in the crystalline structure of minerals such as feldspars

and micas and it is slowly available (Malvi, 2011; Schulte & Kelling, 2009). Mineral K

becomes available when primary minerals such as micas (biotite, muscovite) and feldspars

(KAlSi 3O8 orthoclase, microcline) weather or decompose. Soils formed on rocks weathered in

situ (i.e. acid and basic igneous or metamorphic rocks) may have adequate reserves of K.

Also in alluvial soils, feldspars weather more readily than micas and thus are an important

source of K. Feldspars are abundant in acid igneous rocks such as granite which underlie

much of the south-western Australia, but in this landscape they are highly weathered and so

in most soils the only unweathered primary mineral left is quartz due to its high resistance to

weathering (Moore, 2004). The weathering process is far too slow to supply the required K

for field crops in any given year, while trees and long-term perennials are benefited by K

released by weathering (Schulte & Kelling, 2009).

2.5.2 Slowly available/ non-exchangeable/fixed K

Non-exchangeable K (unlike mineral K) is associated with clay minerals, but is not bonded

covalently with soil minerals. It is held between adjacent tetrahedral layers of micas,

vermiculites, and 2: 1 clay minerals such as illite (Moore, 2004; Schulte & Kelling, 2009).

The non-exchangeable K acts as a reserve source of K in the soil, and is released as these

minerals expand when wet and is slowly available to plants (Malvi, 2011). Some is released

to become exchangeable when the levels of exchangeable and soil solution K are decreased

by crop uptake, removal and leaching (Peterburgsky & Yanishevsky, 1961). Soils of south-

western Australia have limited non-exchangeable K due to the low content of 2: 1 clay

minerals (Pal et al., 2001).

2.5.3 Readily available/ Exchangeable K

Exchangeable K is held by the negative electrostatic charges on the surfaces of organic matter

and clay minerals and is in rapid equilibrium with soil solution K (Römheld & Kirkby, 2010).

It is easily exchanged with other cations and readily available to plants (Moore, 2004).

Exchangeable K is the form that is extracted in the routine soil analysis of soil samples to

generate a recommendation for K fertiliser use (Malvi, 2011).

2.5.4 Soil solution K

Soil solution K is found in the thin film of water in pores and around soil particles and is

easily absorbed by plants. The levels of dissolved K in soil solution usually range between

16

0.2 to 10 mM (Schulte & Kelling, 2009). Soluble K is easily available to plants and microbes,

but it is subjected to leaching (Zörb et al., 2014). As the readily available K is absorbed, soil

solution K is replenished from both readily exchangeable K and non-exchangeable K held in

clay particles (Moore, 2004; Römheld & Kirkby, 2010).

2.6 Removal of K from soil

There are several mechanisms by which K is removed from soil, including leaching of K

below the root zone, loss of K by erosion, and removal of K in harvested produce and hay.

The continued export of K in primary produce is regarded as the ‘mining’ of K from soil

reserves (Pal et al., 2001). There is a potential for rapid K depletion in soil, if K removed is

not balanced by regular K fertilisation either with mineral K fertilisers or crop residue

recycling (Römheld & Kirkby, 2010). Leaching of K is dependent on the amount of applied

K, form of K, concentration of other cations in the soil solution, soil organic matter and clay

contents (Kolahchi & Jalali, 2007). Also the amount of rainfall and the porosity of soil will

influence K leaching. For example, in sandy soils, K does not interact strongly with the soil

matrix and is subsequently leached by rainfall or irrigation, because of low clay content and

low sorption capacity (Kolahchi & Jalali, 2007). The forms of K in soil, in order of their

availability for leaching are solution > exchangeable > non-exchangeable > mineral

(Kolahchi & Jalali, 2007).

Large quantities of K are removed from soil with the harvest of plants. It is reported that

globally the annual above-ground plant biomass contains 60 million tonnes of K (Römheld &

Kirkby, 2010). The amounts of K present in common crops and removed at harvests are listed

in Table 2.1.

The vegetative parts of plants contain higher concentrations of K than the grains/reproductive

part (Table 2.1). The removal of animal dung and crop residues from farmland as a source of

animal feed, bio-energy for heating and cooking considerably lowers the soil K status

(Römheld & Kirkby, 2010).

17

Table 2.1 Removal of K through grain and hay harvest of different crops

Crop Grain kg K/t Mature shoots kg K/t

Rice 2 -

Wheat/barley/oats 4 12

Maize 5 3

Sorghum 5 5

Chick pea 8 15

Lupin 8 -

Soybean 16 14

Canola 9 -

Sunflower 8 -

Peanut 7 20

Millet 16 -

Source: Australian Soil Fertility Manual, 2000

2.7 Na functions in plants

In contrast to K, sodium (Na) is only beneficial for certain halophytes and plant species

characterized by C4 and CAM photosynthetic pathways at relatively low concentrations

(Mäser et al., 2002). It may be classified as an essential mineral nutrient for some plant

species in the families of Amaranthaceae, Chenopodiaceae, and Cyperaceae and a supply of

around 100 µM Na+ enhanced the growth and alleviated visual K deficiency symptoms in

these plants (Marschner, 1995). For the role of Na in mineral nutrition of plants, three aspects

have to be considered: a) its essentiality for that plant species, b) the extent to which it can

replace K functions in plants, and c) its additional growth enhancement effect (Marschner,

1995). Non-halophytic plants from non-saline environments, while expressing genetic

variation for salt tolerance within a particular species, are generally effective in excluding

Na+ and preferential absorption of K+ ions (Schachtman & Liu, 1999).

According to Marschner (1995), the application of Na fertilisers has beneficial effects: a) in

natrophilic plant species, b) when soil levels of available K or Na or both are low, and c) in

areas with irregular rainfall or transient drought during the growing season, or both. Sodium

18

caused growth stimulation in plants by its effect on generation of turgor, osmotic adjustment,

cell expansion and water balance of plants (Kronzucker et al., 2013; Wakeel et al., 2011). The

application of Na fertilisers also results in an increase in the leaf area index early in the

growing season and a corresponding increase in light interception, thus improving the water

use efficiency of leaves under moderate stress conditions during the growing season (Durrant

et al., 1978).

In C4 plants, Na increases the efficiency of CO2 utilization between mesophyll and bundle

sheath cells in the photosynthetic pathway, enhances NO3 uptake by the roots and its

assimilation in the leaves, and is also required for chlorophyll synthesis (Subbarao et al.,

2003). Under Na deficiency in C4 plants, the conversion of pyruvate into phosphoenol

pyruvate (PEP), which takes place in mesophyll chloroplasts, is impaired. Furthermore, C3

metabolites like alanine and pyruvate were found to accumulate, whereas C4 metabolites,

PEP, malate, and asparate, decreased under Na deficiency. Also in Na-deficient plants (C4,

CAM, or C3), nitrate reductase activity is very low but can be rectified within 2 days when

Na is supplied (Marschner, 1995).

2.8 Interaction between K and Na

Potassium and Na ions are similar in ionic radius and ionic hydration energies. The hydrated

Na ion (Na+) has a radius of 0.358 nm, and the radius of K ion (K+) is 0.331 nm (Marschner,

1995). Because of the chemical similarity between K and Na, it is assumed that K and Na

compete for common adsorption sites in the roots (Subbarao et al., 2003). Generally an

excess of one cation in the nutrient medium reduces the net uptake of other cations, but the

sum of all cations in the plant tissue often remains constant (Pervez et al., 2006). However, it

has been argued that many plants have high degree of selectivity and there is some evidence

that concentration of cations like Ca, Mg and Na in plants can be reduced as a result of

inhibition by the uptake of K (Mundy, 1983). Potassium substitution and growth stimulation

by Na are of great interest for agronomic production, and better use of fertilisers (Mäser et

al., 2002).

Larson and Pierre (1953) suggested that plants can be classified according to their Na/K

response as follows: a) plants that respond to Na with an adequate supply of K; b) plants that

respond to Na when K is deficient, indicating that Na partly replaces K in its functions; and c)

plants that respond slightly, if at all, to Na under any conditions. Marschner (1995) proposed

four groups according to the differences in their growth response to Na as follows: a) plant

19

species, where a high proportion of K can be replaced by Na and additional growth

stimulation occurs which is not achieved by increased K content of plants, e.g., sugar beet,

table beet, turnip, Swiss chard and many C4 grasses (wheat grass, Rhodes grass etc.); b)

specific growth responses in plants to Na are observed but are much less distinct and a

smaller proportion of K can be replaced without a decrease in growth, e.g., cabbage, radish,

cotton, pea, flax, wheat and spinach; c) substitution can only take place to a very limited

extent and Na has no specific effect on growth, e.g., barley, millet, rice, oat, tomato, potato

and ryegrass; d) no substitution of K by Na is possible, e.g., maize, rye, soybean. However,

this classification should be treated with caution, since the differences among cultivars in the

same species are not taken into account.

2.8.1 High Na

2.8.1.1 Na toxicity effects

In arid and semiarid regions, the presence of excessive Na as NaCl has a detrimental effect on

the growth of most of crop plants (Marschner, 1995). In highly saline soil, plants will take up

Na+ in place of K+. Excessive Na+ interferes with the shoot transport or long distance

transport and cytosolic functions of K+, and greatly inhibits plant growth and development.

Even in the case of halophytes that accumulate larger quantities of Na+ inside the cell, their

cytosolic enzymes are as sensitive to Na+ as those of glycophytes (Malvi, 2011). High Na+

accumulation under hot temperatures may lead to cell wall rupturing and slow programmed

death of plants (Malvi, 2011).

Replacement of a high proportion of K+ by Na+ inhibits the activity of many enzymes that

specifically or more sensitively respond to K+. It was reported that plant growth was inhibited

in many species grown on saline soils due to depression in K+ uptake by competing Na+ ions

(Al-Rawahy et al., 1992). This inhibition is dependent on levels of Na and K; the higher the

Na+/K+ ratio, the greater the damage (Malvi, 2011).

2.8.1.2 Imbalance in K/Na ratios

The ability of plants to maintain a high K+/Na+ ratio in the cytosol plays an important role in

stress tolerance. The influx of Na+ ions through K+ pathways alters the ion ratios in plants.

The cytosolic enzymes in plants are not adapted to high Na+ levels and hence, plants respond

to elevated Na+ concentrations by maintaining low cytosolic Na+ concentrations and high

K+/Na+ concentrations by Na+ extrusion and/or the intracellular compartmentalisation of Na+

(predominantly in plant vacuole) (Blumwald, 2000). In saline soils, high Na+/K+ ratio reduces

20

plant growth and eventually becomes toxic to plants (Schachtman & Liu, 1999). Therefore,

increase in K uptake in saline soils will increase the K+/Na+ soil ratio, and potentially

minimise the salt stress in plants (Wu et al., 1996). It is critical to understand how plant roots

distinguish K+ and Na+ ions in saline soils for proper nutrient management (Schachtman &

Liu, 1999).

2.8.2 Low to moderate Na

Even though high concentrations of Na have a large depressive effect on plant yield, K and

Na share various physiological functions (Almeida et al., 2010). Sodium can substitute

biophysical functions of K, provided that the plants have the ability to take up Na, translocate

it to the shoot, and compartmentalise it in their vacuoles (Subbarao et al., 2003). There are

studies on beneficial effects of Na in various plant species that focussed on partial to near-

complete replacement of K by Na. Substantial positive effects on plant growth have been

reported in plants, particularly at deficit K supply and in plants suffering K+ deprivation

(Kronzucker et al., 2013). The functions of K replaced by Na and studies where Na was

found to be beneficial are reviewed briefly in the following sections.

2.9 Functions of K replaced by Na

Sodium improves the water balance of plants when the water supply is limited via effects on

stomatal conductance. Under drought stress, when there is a sudden decrease in plant

available water, the stomata of the plants supplied with Na close more rapidly than the plants

supplied with K alone, and after stress release, exhibit a delay in opening (Mäser et al., 2002).

It is claimed that in natrophilic species, Na+ replaces K+ in its role in stomatal opening, as for

example, in sugar beet, Na+ substituted K+ in stomatal functions (Marschner, 1995). The

addition of Na to the culture medium lessened the effects of K deficiency on photosynthetic

or respiratory CO2 exchange (Terry & Ulrich, 1973), and increased leaf net photosynthesis of

Theobroma cacao (Gattward et al., 2012). A list of the key functions of K substituted and not

substituted by Na is provided in Table 2.2.

21

Table 2.2 Functions of K replaced and not replaced by Na

Functions of K replaced by Na Source

Growth stimulation (Wakeel et al., 2011)

Improved water balance via stomatal conductance

(Marschner, 1995)

Maintenance of ionic balance (Subbarao et al., 2003)

Regulation of osmotic pressure (Marschner, 1995)

Vacuolar functions (Ali et al., 2006; Mäser et al., 2002)

Photosynthesis/CO2 exchange (Gattward et al., 2012; Terry & Ulrich, 1973)

Functions of K not replaced by Na Source

Enzyme activity (Subbarao et al., 2003)

Cytoplasmic functions (Benito et al., 2014)

Protein synthesis (Kronzucker et al., 2013)

Oxidative phosphorylation (Kronzucker et al., 2013)

Biochemically, enzyme activation requires relatively low concentrations of K (about 10-50

mM) for maximum activity, and this K requirement may be further reduced by the

substitution of Na and other monovalent cations like Cs and Rb (Subbarao et al., 2003). In K

deficient soils, Na assumes the role of K in maintaining ionic balance, electroneutrality and

regulating osmotic pressure (Marschner, 1995). Sodium can alleviate or even eliminate K

deficiencies in some species such as barley, sugar beet, lettuce, cotton, turnip, pangola grass,

Italian ryegrass and Rhodes grass by partly replacing the role of K in the plant (Marschner,

1995; Mundy, 1983). Some crops like marigold, spinach, and sugar beet showed a yield

increase to added Na even in the presence of adequate amounts of K (Idowu & Aduayi,

2007). It was found in an experiment that high yields occurred in ryegrass at optimal and also

suboptimal K concentrations when Na was included in the nutrient solution with the highest

yield for plant tops containing 4-8 % K without Na, and 1-4 % K with Na (Mundy, 1983).

There was poor growth, chlorosis and necrosis in Atriplex vesicaria when the basic nutrient

solution had less than 0.1 µM Na+, despite a high K+ concentration (Marschner, 1995).

Sodium ion can replace K+ to a certain degree in the vacuole, particularly in osmotic

functions. It is reported that in red beet Na+ can replace K+ in vacuolar functions, thus

22

offsetting 95 % of the plant’s K requirement (Subbarao et al., 2003). According to Marschner

(1995), when Na contents in the leaves are high, the K content required for optimal growth

decreased from 3.5 to 0.8 % of the leaf dry weight in Italian ryegrass, from 2.7 to 0.5 % in

Rhodes grass, and from 4.3 to 1.0 % in lettuce. The grain yield of rice grown at 25 µM K

more than doubled in the presence of 43 mM Na. Addition of Na also increased dry weight of

Arabidopsis at 10 µM K. Sodium ion can exert beneficial effects on certain plant species

even when K+ supply is not limiting. In sugar beet, replacement of 5 mM K in the nutrient

substrate with 2.5 mM each of K and Na increased plant dry weight and sucrose content in

the storage root. Also, Na may improve the plant’s water balance in sugar beet (Mäser et al.,

2002).

2.10 Plant responses to Na at low K

The presence of low to moderate Na may stimulate plant growth under K depleted conditions.

The studies in different plant species where Na was found to have beneficial effects are

summarised in Table 2.3 together with the K and Na concentrations used and key results from

the study. There are numerous studies that have reported beneficial effects of Na on growth

and yield, plant physiology and on visible improvement with less pronounced K deficiency

symptoms. Among the species examined, the Chenopodiaceae family including crops like

spinach, beet and sugar beet had received detailed attention, and studies in different crop

species have been listed in a recent review by Kronzucker et al. (2013).

In some plants, supplementation of Na in reduced amounts was found to eliminate K

deficiency symptoms (Marschner, 1995; Wakeel et al., 2011). This may offset the

requirement of K fertiliser and therefore may help in profitable cropping. Wheat is a major

cereal grown in saline, semi-arid regions of Western Australia. But there is little research on

potential replacement of K by Na in wheat. Box and Schachtman (2000) investigated whether

Na can benefit wheat growth under low K by stimulating K+ uptake through the Na+

energised HKT1 symporter and they found that low concentrations of Na+ stimulated wheat

growth at low external K in only one of their experiments when light levels were low. By

contrast, Ma et al. (2011) reported no growth stimulation in wheat with Na addition at low K

supply even though barley growth was stimulated under the same conditions. Rubio et al.

(1995) showed an increase in K+ uptake by wheat with 1 mM Na+ addition.

There are differences in accumulation of K+ and Na+ among different species. Wheat shows

distinctive behaviour relative to other species like barley with very high K+ selectivity. In the

23

study of Ma et al. (2011), shoot K+ /Na+ in wheat cv. Wyalkatchem was 10-15 times higher

than the ratio in shoots of two barley cultivars grown under the same conditions. Further

detailed research on K and Na interactions in wheat cultivars are essential since the findings

could have practical implications in nutrient management.

24

Table 2.3 Sodium response at low K

Crop species K level Na level What improved with Na?

Type and duration of experiment

Key results Reference

1. Cacao tree (Theobroma cacao)

2.5 and 4.0 mM K.

Six replacement ratios of K+ by Na+ (0, 10, 20, 30, 40, 50% replacement, mol/mol)

Photosynthetic rate, WUE, and mineral nutrition

Soil culture in glasshouse and harvested at 180 DAT

Results show that Na+ can partially replace K+, with significant beneficial effects on photosynthesis, WUE and mineral nutrition.

(Gattward et al., 2012)

2. Rice (Oryza sativa)

1, 2, 5, 10, 50, 100 and 200 ppm (as KCl) or

0.02, 0.05, 0.13, 0.26, 1.28, 2.56 and 5.13 mM K

1000 ppm (as NaCl) or 43.5 mM Na

Leaf turgidity, shoot dry wt (panicle wt. and straw)

Solution culture and maintained until harvest of rice.

Shoot dry weight increased with addition of 1000 ppm (43.5 mM) Na in 1, 2, 5 and 10 ppm (0.02, 0.05, 0.13 and 0.26 mM, respectively) K treatments.

Application of Na+ altered the leaf habit, from ‘flaccid’ to ‘erect’, in K deficient plants.

High % of unfilled grains in low K without Na treatments.

Relationship between shoot dry weight and K content showed that, within the deficiency range, higher dry weights were obtained at the same K content in the presence of Na.

(Yoshida & Castaneda, 1969)

25

3. Eucalyptus (Eucalyptus grandis)

1.5 (58kg K/ha), 3.0 (116kg K/ha), 4.5 kmol K/ha (174 kg K/ha) as KCl

3.0 kmol K/ha (116kg K/ha) applied as K2SO4

3.0 kmol Na/ha

(68 kg Na/ha) and 1.5 kmol K/ha + 1.5 kmol Na/ha (58 kg K/ha as KCl and 34 kg Na/ha as NaCl). Treatments were compared with control with no K, Na application

Growth (height and basal area)/ above- ground biomass

Field study for 4 years from planting

Application of 3.0 kmol Na/ha increased tree height and basal area by 14 % and 32 %, respectively, in comparison with control. Combined K and Na fertilization (K 1.5 + Na 1.5) led to a growth in height and basal area that was intermediary between treatments K 1.5 and K 4.5.

(Almeida et al., 2010)

4. Tomato (Lycopersicon esculentum)

Growth experiment

0.5 and 4.5 mM K+ (as KCl)

1, 5, 15 and 30 mM Na+ (as NaCl)

Plant growth and root growth stimulation.

Solution culture and harvested on day 28

In K+ -deficient plants, external NaCl concentrations of 1 and 5 mM were optimal, restoring growth to levels not significantly different from the maximal (4.5 mM) level.

The presence of Na+ increased ability to direct K+ to growing leaves which is crucial for maximization of growth under K+ -deprivation.

(Walker et al., 2000)

26

5. Tomato (Lycopersicon lycopersicum)

0, 32, 64 and 128 mg/kg (as KCl)

0, 2, 4, 8, 16 and 32 mg/kg soil (as NaCl)

Number of leaves, Number of flowers, and fruit yield

Soil culture experiment

The interaction of 2-4 mg Na/kg soil with 32-64 mg K/kg soil resulted in vigorous and healthy plant growth with bright green leaves, broad, succulent and well-spaced foliage.

The results indicated an efficient nutrient combination of Na and K at Na: K ratio of 1:8 to 1:32, which resulted in balanced tomato nutrition when compared with plants that received high doses of K.

(Idowu & Aduayi, 2007)

6. Tomato

(Lycopersicon esculentum and L. pennelIii)

K-free solution and 5 mM K

5 mM and 100 mM NaCl

Plant dry weight Solution culture experiment

2 varieties: Lycopersicon esculentum Mill. cv.M82 and its wild salt-tolerant relative species L. pennellii

Addition of 5 mM Na+ to K-free medium increased plant dry weight in both species.

L. pennellii plants (wild sp.) were more efficient in substitution of K+ function by Na +, as there was a greater increase of dry weight in the wild species; a higher retranslocation of K+ from old to young leaves was noticed, and consequently a higher K-efficiency (dry weight/K+) ratio.

In wild species, Na+ can be

(Tahal et al., 2000)

27

used as a cheap osmoticum in the vacuole and, possibly, as a partial substitute for K+ in some of its functions.

7. Wheat

(Triticum aestivum)

growth experiment

a) 20, 100 and 2000 µM KCl

b) 20 µM KCl

c) 20, 100 and 2000 µM KCl

0 and 500 µM NaCl

Plant dry weight Solution culture and harvested at 32 days (exp- a), 42 days (exp- b) and 40 days (exp- c)

Sodium stimulated the wheat growth significantly only at low (20 µM) external K+ in one of the long term experiments, but not in two other experiments.

(Box & Schachtman, 2000)

8. Wheat

(Triticum aestivum)

Wheat root high-affinity K uptake transporter HKT1 was shown to function as a high-affinity K+-Na+ cotransporter. High-affinity K+ uptake was activated by micromolar Na+ concentrations, however, at physiologically detrimental concentrations of Na+, K+ accumulation mediated by HKT1 was blocked and low-affinity Na+ uptake occurred (approximately 16 mM Na+), which correlated to Na+ toxicity in plants.

(Rubio et al., 1995)

28

9. Barley (Hordeum vulgare L.)

30 mg K/kg and 75 mg K/kg (all plants were grown in for 4 weeks to create mild K deficiency)

0, 100 and 300 mg Na/kg

Leaf development, tiller numbers, shoot dry weight

Soil culture experiment in glasshouse for 7 weeks

Moderate Na treatment enhanced shoot growth by 42 % in the K deficient plants.

(Ma et al., 2011)

10. Red beet (Beta vulgaris)

5.0, 1.25, 0.25, and 0.10 mM

50 mM Na replaced K in osmotic functions without negatively affecting the plant water status, or growth

Nutrient film technique for 42 days with two cultivars

Leaf relative water content and osmotic potential were significantly higher for both cultivars with Na at low-K treatments.

(Subbarao et al., 2000)

11. Cotton

(Gossypium hirsutum)

a) glasshouse experiment- 6 mM K (as KNO3) K: Na ratios- 1:0, 2/3:0, 1/3:0, 2/3:1/3, 1/3:2/3, 0:1

(Na as NaNO3 and NaH2PO4)

b) field experiment- 5 combinations of KCl and NaCl (mg/kg): 1) K 80, Na 35; 2) K 115, Na 35; 3) K 80, Na 65; 4) K 80, Na 90; 5) K 115, Na 65

Dry matter accumulation and cotton seed yield

a) Sand culture in GH watered with modified Hoagland solution, harvested @ 30 days

b)field experiment

Replacing 1/3rd K with Na increased cotton seedling development; replacing 2/3rd or completely with Na- reduced germination rate, restrained cotton seedling growth and nutrition uptake.

Highest seed cotton yield was obtained when K and Na were added at rates of 115 and 65 mg kg-1, respectively in the top 20 cm of soil.

(Zhang et al., 2006)

29

12. Cotton


3 combinations of K and Na

1) 3:1 (2.25 mM K: 0.75 mM Na),

2)1:1 (1.5 mM K: 1.5 mM Na)

3) 1:3 (0.75 mM K:

2.25 mM Na)

Main aim of this study was to look at genotypic difference.

Solution culture with 4 genotypes, harvested at 35 and 42 days after transplanting

The growth was better when K and Na were added in ratio of 3:1. There was significant effect on biomass production, K- use efficiency and K: Na ratios among genotypes.

(Ali et al., 2009)

13. Cotton


Different levels of

K and Na with K: Na ratios of 3.5: 1 (control), 3.75: 1, 4: 1, 4.25: 1, 4.5: 1, 2.8: 1, 3: 1, 3.2: 1, 3.4: 1, 3.6: 1. K+ Na in kg ha-1 with K: Na ratios were as: 210+60 (3.5: 1) i.e. control, 225+60 (3.75: 1), 240+60 (4:1), 255+60

(4.25: 1), 270+60 (4.5: 1), 210+75 (2.8: 1), 225+75 (3: 1), 240+75 (3.2: 1), 255+75 (3.4: 1) and 270+75 (3.6: 1)

Number of bolls/plant and seed cotton yield

Field study.

Varieties differing in K-use efficiency used.

Harvest at 80 DAS to measure ionic ratios

Maximum seed cotton yield was obtained at K: Na ratio of 3.4:1 followed by 3.6:1 in both cotton varieties.

Enhanced number of bolls/plant was produced with at ratio 4.25:1 followed by 3.4:1. Variety difference: NIBGE-2 showed relative high Na substitution capacity for K and yielded better than MNH-786. The variation in K/Na selectivity of xylem transport from roots to the leaves proved to be one important cause of inter-specific differences in cotton varieties.

(Ali et al., 2013)

30

2.11 K and Na transporters

Epidermal and cortical cells of roots are involved in K+ uptake from soil solution. Plants use

low and high affinity transporters to take up K+ from extracellular medium (Britto &

Kronzucker, 2008). The pathways of Na+ entry into plants are not definitively known, despite

years of intensive research, however, evidence mostly supports a primary role of K+

transporters and transporters from the HKT family for Na+ influx into plant roots

(Kronzucker & Britto, 2011). High external Na+ concentrations may upset K+ equilibrium by

increased efflux, reduced influx and decreased cytoplasmic K+ concentrations (Wakeel,

2013).

Potassium transporters and channels are located at the plasma membrane of root cells (Wang

& Wu, 2010), and a large number of genes encoding for plant K+ transport have been

identified in many species (Very & Sentenac, 2003). For example, a total of 71 K+ channels

and transporters have been identified in Arabidopsis sp. so far (Wang & Wu, 2010). External

K+ concentration influences the activities of K+ channels and transporters (Wang & Wu,

2010). Potassium acquisition from low external concentrations is usually considered to be an

energy-demanding process, while that from high K concentrations in solution is energetically

passive (Britto & Kronzucker, 2008).

Low-affinity K+ transport system works at high external K+ concentrations (> 1 mM K). The

three important low-affinity K+ channels are: 1. Inward rectifying K+ channels (KIRC), such

as AKT1 that show high K+/Na+ selectivity and activate K+ influx upon plasma membrane

hyperpolarization; 2. Outward rectifying K+ channels (KORC) which play a role in Na+

influx into plant cells, and these channels unlike KIRC, open upon plasma membrane

depolarization; 3.Voltage-independent cation channels (VIC) in plant plasma membranes that

have relatively high Na+/K+ selectivity (Blumwald, 2000; Szczerba et al., 2009).

The high-affinity K+ uptake (HAK) mechanism operates at low external K+ concentrations (<

1 mM K) and the high affinity K+ transporter (HKT1) is highly selective for K+ but could also

be a low-affinity Na+ transporter or Na+/K+ symporter (Britto & Kronzucker, 2008; Szczerba

et al., 2009).

2.12 Genotypic variation in Na substitution of K

It is well known that varieties within the same species differ widely in their ability to take up

and utilize mineral nutrients. The extent to which Na+ can replace K+ varies among different

plant species, different cultivars of the same species, and even between different leaves of the

31

same plant with younger leaves relying more on K than older ones (Mäser et al., 2002). Also,

the extent of substitution differs between individual organs and between cell compartments,

being very large in the vacuoles, but very limited in the cytoplasm (Marschner, 1995).

Variation in K uptake and utilization was noticed among varieties of sweet potato (George et

al., 2002), cotton (Ali et al., 2006), and wheat (Damon & Rengel, 2007). Genotypic

differences are also reported in terms of salt tolerance and Na substitution of K functions (Ali

et al., 2006; Mäser et al., 2002). Ali et al. (2006) studied 30 cotton genotypes in hydroponics

and found that the genotypes differed significantly in growth responses, K uptake, K use

efficiency and the extent K substitution by Na, and they confirmed that screening of

genotypes is an effective approach for enhancing growth and yield under K deficient

conditions. There is evidence that genotypes can vary in Na substitution of K functions in

plants illustrating that Na resistance is a quantitative trait (Ali et al., 2006; Mäser et al.,

2002). In addition there are variations in Na/salt tolerance among genotypes. There is lack of

research on Na substitution of K functions among wheat cultivars.

The variation in Na absorption and the extent of K substitution by Na in plant species may

explain their differential response to Na and K applications. The increased capacity to

substitute Na+ for K+ was suggested by Rengel and Damon (2008) to be one of the possible

mechanisms underlying K utilization efficiency. However, in their previous studies to rank

wheat genotypes according to K-use efficiency there was no examination of the role of low to

moderate Na supply Na on plant K nutrition. Also the salt tolerance among wheat genotypes

differing in K use efficiency has not been studied.

2.13 Salinity and duplex soils

Soil salinity in arid and semi arid areas is a major constraint affecting crop productivity in

many parts of the world (Alhagdow et al., 1999). Sodic soils and duplex soils are also major

soil constraint affecting crop production in Australia. Duplex soils are described as the soils

that have an abrupt textural difference between the surface soil horizons and the subsurface,

and they exhibit great diversity in their properties, particularly genesis and mode of

development (Chittleborough, 1992) and they are classified under order ‘Sodosols’ according

to the Australian soil classification (Isbell, 2002). Duplex soils account for about 12 % of the

land area of Australia (Dracup et al., 1992) and, are widespread in Western Australia,

accounting for half to 2/3rd of the cultivated area in south-Western Australia (Belford, 2005).

The distribution of nutrients with depth in duplex soils is different from that of uniform soil

32

profiles. The duplex soils of western Australia often have low topsoil K but varying

concentrations of K in the heavier textured subsoils due to differences in clay content and

mineralogy (Wong et al., 2000). For example, the duplex soils of the western wheatbelt (with

clay mineralogy- kaolinite, sesquioxides) have uniformly low Colwell K concentration in

sandy horizon A and clay horizon B. In contrast, duplex soils in eastern and south-eastern

wheatbelt usually hold higher concentration of potassium in the clay horizon B compared to

the sandy A horizon (McArthur, 1991; Wong et al., 2000). The clayey subsoils are sodic

when about 10 % of exchangeable ions are Na and this may interfere with crop production

(Belford, 2005). In case of duplex soils, sampling only the top soil gives an incomplete

estimate of nutrient profile and misjudgement in nutrient application.

The distributions of Na and K down the profile may vary in duplex soils, but the implications

of this for crop K nutrition have not been examined. While topsoils are commonly low in Na

and K, variable levels of Na and K can occur in sub-soil. It is not known for example if Na in

the sub-soil is able to alleviate the effects of low topsoil K. Whether Na substitution is

effective in alleviating K deficiency may depend on the plant species, the levels of Na and K

in the sub-soil, the length of time it takes for roots to reach the sub-soil (which depends on

depth of the sub-soil) and the root length density in the sub-soil. In sodic duplex soils, Na+

may assume the role of K+ in some of the physiological functions as discussed above, but the

variable distribution of both K and Na with depth in the root zone adds uncertainty to the

effectiveness of Na as a replacement for K requirements in such soils.

2.14 Research scope, aim and research questions

The spread of high-yielding varieties and increases in cropping intensity to fulfil increasing

food demands globally are depleting soil K (Wakeel et al., 2011). Application of K fertilisers

to increase crop productivity is relatively expensive especially for resource-poor farmers in

developing countries. The partial substitution of K+ by Na+ in physiological processes of

plants can have substantial implications for K management and could reduce costs of crop

production if its value to crop grain yield was better understood.

The main aim of this research is to study the role of Na and cultivar effects on K nutrition of

wheat in the context of drought-prone environments and soils that are moderately saline

and/or sodic. To understand the interaction between K and Na in wheat, the present study was

conducted with the following research questions:

• Is Na beneficial to wheat and if so what levels of soil Na are beneficial?

33

• Can Na substitute for K requirements in wheat when available soil K is deficient?

• Do wheat genotypic differences in K-use efficiency alter the extent of K substitution

by Na?

• Is there a relationship between K responses and sub-soil Na?

• Is there an impact of Na supply on wheat K nutrition when K uptake from the topsoil

is restricted by water deficit conditions?

• What happens when K and Na are present at different root sections? Does K

replacement by Na depend on both these cations being present in the same part of the

root system?

• Is there an increase in wheat K+ and/or Na+ uptake with addition of external Na at

deficient K supply, and are there any differences due to K-use efficiency of wheat

cultivars and their uptake?

2.15 Conclusion

The literature review above explains in detail the role of K in plants, K deficiency effects,

forms of K in soil, importance of Na, interactions between K and Na, functions of K that can

be replaced by Na, and then relates the proposed research to sodicity and duplex soil

conditions prevailing in Western Australia. The beneficial effect of Na ion has been

established for some C4 plants, while for many higher plants Na can have beneficial effects

under specific circumstances without being an essential element. The present thesis aims to

identify whether Na is beneficial to wheat growth and the circumstances under which this

occurs. In addition to varying K and Na supply and placement in the root zone, variation in K

use efficiency of wheat cultivars is used to explore the research questions. The research

questions will be tested with a series of pot and solution culture experiments described in the

following chapters (Chapter 3 to 7).

34

CHAPTER 3

Wheat responses to sodium vary with potassium use efficiency of cultivars1

3.1 Introduction

Key physiological roles for K are in stomatal regulation and in photosynthesis (Römheld &

Kirkby, 2010). Sodium can substitute for non-specific biophysical functions of K+, especially

where plants have the ability to take up, translocate, and compartmentalise Na in their

vacuoles where it can replace functions of K in maintaining cell turgor (Gattward et al., 2012;

Subbarao et al., 2003). In K-deficient soils, Na can play the role of K in maintaining ionic

balance (Subbarao et al., 2003), regulating osmotic pressure (Marschner, 1995), provide

partial K-substitution in protein synthesis (Flowers & Dalmond, 1992), contribute to vacuolar

functions (Mäser et al., 2002), and improve water balance via regulation of stomatal

conductance (Gattward et al., 2012; Marschner, 1995). Under K deficiency, the addition of

Na replaced K in stomatal functions of sugar beet and reduced the effects of K deficiency on

photosynthetic or respiratory CO2 exchange (Terry & Ulrich, 1973), and in net photosynthetic

rate of Theobroma cacao (Gattward et al., 2012). Also under water deficit, stomata of sugar

beet leaves supplied with Na closed more rapidly but exhibited a delay in opening compared

to supply of K only (Mäser et al., 2002).

Sodium is reported to alleviate effects of K deficiency on plant growth in sugar beet, lettuce,

cotton, ryegrass, spinach, marigold, tomato (Idowu & Aduayi, 2007; Marschner, 1995;

Mundy, 1983; Pi et al., 2014; Tahal et al., 2000), and barley (Ma et al., 2011). However, in

wheat which maintains a high selectivity of K+ uptake relative to Na+ uptake, there are few

reports of Na partially substituting for K. Box and Schachtman (2000) investigated whether

Na supply can benefit wheat growth under low K by stimulating K+ uptake through the Na+

energised HKT1 symporter and found that low concentrations of Na+ did not increase K+

uptake to a large extent and while Na+ stimulated wheat growth at low external K it was only

when light levels were low. By contrast, Marschner (1995) classified wheat as having

moderate response to low Na at low K. Hence further investigation is needed to clarify the

response of wheat to low Na levels especially under low K.

Varieties of the same species can vary in K accumulation and utilization, e.g. sweet potato

(George et al., 2002), cotton (Ali et al., 2006), and wheat (Damon & Rengel, 2007).

1 This Chapter is a slightly modified version of Krishnasamy et al. (2014). Wheat responses to sodium vary with potassium use efficiency of cultivars. Frontiers in Plant Science, 5: 631. doi: 10.3389/fpls.2014.00631.

35

Genotypic differences in cotton (Ali et al., 2006) and sugar beet (Marschner et al., 1981) are

also reported in terms of Na substitution of K functions. Ali et al. (2006) studied 30 cotton

genotypes in hydroponics and found that the genotypes differed significantly in growth

responses, K uptake, K-use efficiency and the extent of K substitution by Na. However, there

is a shortage of information about how cultivar variation in K-use efficiency alters effects of

low to moderate soil Na on plant K nutrition. Understanding K and Na interactions among

wheat cultivars that vary in K-use efficiency would improve management of K fertiliser in

sodic and K-deficient soils.

We hypothesised that if high K efficiency in wheat was related to higher K uptake, K

efficient cultivars would exhibit not only reduced salinity effects but also a reduced response

to Na-substitution of K in plants. Alternatively, if greater Na substitution of K was the main

mechanisms for greater K-use efficiency such cultivars could be more susceptible to salinity

and demonstrate greater response to low to moderate Na levels in low soil. We examined the

effect of Na levels on K uptake, the K+/Na+ ratios, leaf gas exchange, and plant growth of

wheat cultivars differing in K-use efficiency. Supply of NaCl ranged from low to moderate

levels, designed for substitution of K by Na at low K supply, ranging to toxic levels for wheat

at high Na.

3.2 Materials and methods

Wheat (Triticum aestivum L.) cvv Wyalkatchem, Cranbrook, Gutha, and Gamenya were

grown in a naturally-lit glasshouse at Murdoch University, Perth (32°04′S, 115°50′E) from

late winter to mid spring. The average minimum and maximum temperatures during the

experiment were 8.4 and 26 ˚C, respectively. Cultivars Wyalkatchem and Cranbrook are K-

efficient, whereas Gutha and Gamenya are K-inefficient in terms of K uptake and use

(Damon & Rengel, 2007). The sandy soil (classified as ‘Chromosols’ according to the

Australian soil classification) was collected from an unfertilised field, 150 km northeast to

Perth, and had the following properties: pH 4.9 (0.01 M CaCl2), EC1:5 0.03 dS/m, 7 mg NH4-

N/kg and 9 mg NO3-N/kg (Searle, 1984), <15 mg K/kg and 29 mg P/kg (Colwell, 1963) and

organic C 0.17% (Walkley & Black, 1934).

Sieved soils (< 2 mm) were well mixed with basal nutrients and individual treatments of K

and Na, and filled into undrained plastic pots (diameter 190 mm, depth 190 mm) at 6 kg/pot.

Basal nutrients were applied at the following rates (mg/kg): 103 (NH4)2HPO4, 237

Ca(NO3)2.4H2O, 80 MgSO4.7H2O, 18 FeSO4.7H2O, 14 MnSO4.H2O, 9 ZnSO4.7H2O, 8.3

36

CuSO4.5H2O, 0.33 H3BO3, 0.3 CoSO4.7H2O, 0.33 Na2MoO4.2H2O. Seeds were washed with

5% (w/w) hypochlorite solution for 1 minute, thoroughly rinsed and soaked in de-ionised

(DI) water for 2 hours, and then placed in a refrigerator at 4 ˚C overnight. The sprouted seeds

were transferred to trays containing 0.05 mM CaCl2 solution, and covered for 2 days. Five

germinated seeds per pot were transplanted, and 10 days later the seedlings were thinned to 3

plants per pot. During the experiment, the pot soils were watered daily to field capacity (15 %

w/w) with DI water. The plants were supplied with 0.5 mM urea solution fortnightly to

maintain adequate N supply. The pots were re-arranged every week to reduce positional

effects on plant growth.

3.2.1 Potassium and sodium treatments

Two soil K levels were applied: 40 mg K/kg (low) and 100 mg K/kg (adequate) based on

earlier trials (Ma et al., 2011). Muriate of potash (KCl) was used as it is the dominant K

fertiliser (Moore, 2004). Each soil K level also included 5 Na levels: nil, 25, 50, 100 and 200

mg Na/kg supplied as NaCl. Equivalent Na concentrations in soil solution were 0, 7.25, 14.5,

29.1, and 58.2 mM, respectively, while ECe (electrical conductivity of saturated soil extract)

values for Na treatments at 40 K were 0.85, 1.26, 2.66, 5.18 and 10.9 dS/m and at 100 K were

1.23, 1.82, 3.78, 7.28 and 13.7 dS/m. Therefore, the experiment comprised a factorial

combination of 4 wheat cultivars, 2 K levels, and 5 Na levels. All the treatments were

replicated three times. At potting, individual treatments with various K and Na levels were

mixed thoroughly with basal nutrients using a rotary mixer.

3.2.2 Measurements

Plants were grown for 8 weeks and during that period the number of leaves and tillers was

recorded weekly. Leaf net photosynthesis, transpiration and stomatal conductance were

measured using the LCpro+ advanced photosynthesis system (ADC Bioscientific, UK) at 7

weeks after transplanting. The measurements were made in fully expanded young leaves at

ambient relative humidity of 50 %, leaf temperature of 25 ˚C, reference CO2 of 380

µmol/mol, and photosynthetically active radiation of 1500 µmol/m2·s1.

At harvest, the shoot was cut at the soil surface, and the fresh weight was recorded

immediately. Roots were collected after washing in tap water and rinsing in de-ionised (DI)

water. The shoot and root samples were dried in a forced-draught oven at 60˚C for 48 hours

and dry weight was recorded. About 0.2 g of each milled sample was weighed into centrifuge

tubes and digested in 5 mL 70 % (w/w) HNO3 at 75 ˚C for 10 min, and then at 109 ˚C for 15

37

min. After the samples were cooled, 1 mL of 30 % (w/w) H2O2 was added and further

digested at 109 ˚C for 15 min. The digestion was made in a micro-wave oven (CEM Mars 5,

CEM Corp., USA) based on method of Huang et al. (2004) for cation analysis. The digested

samples were diluted with milli-Q water and concentrations of K, Na, Ca and Mg were

determined by inductively coupled plasma-atomic emission spectroscopy (VISTA

Simultaneous ICP-AES, Varian). The K+/Na+ ratios in shoots and roots were calculated based

on their content.

A supplementary study was conducted to determine whether the extractable cation levels,

particularly K, were influenced by different soil Na levels. Two kilograms of soils were

thoroughly mixed with basal nutrients and two K levels (40, 100 mg K/kg). Each soil K level

was treated with 2 levels of Na: nil, 50 mg Na/kg. The pots were watered with DI water to

field capacity, and allowed to equilibrate for a week, while mixing daily. The soil samples

were then analysed for bicarbonate-extractable (Colwell) K (Colwell, 1963) and

exchangeable cations. Soils were extracted at a ratio of 1: 10 with 0.1 M NH4Cl for 2 hours

and exchangeable cation concentrations were determined by ICP (Rayment & Lyons, 2010).

3.2.3 Statistical analysis

Statistical analyses were conducted using the SPSS statistical package (IBM SPSS statistics,

vs 18). Three-way analysis of variance was conducted to assess the effects of soil K and Na

supply, genotype and their interactions. Tukey’s HSD was computed at P ≤ 0.05 to test for

differences among the treatment means.

3.3 Results

3.3.1 Plant growth

Shoot growth

Low K supply (40 mg K/kg) induced K-deficiency symptoms after 6 weeks (Fig. 3.1 and 3.2)

and significantly reduced shoot dry weight at 8 weeks, but the reduction was greater in K-

inefficient cultivars Gutha and Gamenya (32 % lower) than K-efficient cultivars

Wyalkatchem and Cranbrook (17-18 % lower). When K supply was low, the addition of low

to moderate Na (25, 50 mg Na/kg) alleviated K-deficiency symptoms in old leaves (Fig. 3.1

and 3.2) but had no significant effects on shoot dry weight (Fig. 3.3). Similarly, at adequate K

supply, addition of 25 - 50 mg Na/kg had no effect on shoot dry weight. High soil Na levels

(100, 200 mg Na/kg) reduced shoot dry weight especially in K-inefficient cultivars (Fig. 3.3).

When compared with nil Na, high Na reduced shoot dry weight by 44 % in Gamenya, 38 %

38

in Gutha, 31 % in Wyalkatchem and 22 % in Cranbrook. The interactions between K, Na and

cultivars on shoot dry weight were significant (P ≤0.05) (Table 3.1).

Fig. 3.1 Wyalkatchem (K-efficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg)

under soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks.

Fig. 3.2 Gutha (K-inefficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg) under

soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks.

39

Fig. 3.3 Shoot dry weight (g/plant) (upper sub-figures), and tillers/plant (lower sub-figures)

(n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open

circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1

for analysis of variance results.

Gutha

0 50 100 150 2000

2

4

6

8

10 Gamenya

Soil Na levels (mg/ kg)

0 50 100 150 200

Wyalkatchem

No.

of t

ille

rs

0

2

4

6

8

10Cranbrook

Gutha

0

2

4

6

8 Gamenya

WyalkatchemS

hoot

Dry

we

ight

(g)

0

2

4

6

8

10Cranbrook

K*Na*cv P= 0.02HSD= 1.40

K*Na*cv P= 0.008HSD= 1.60

40

Adequate soil K produced the same number or more tillers than low K at all soil Na levels,

except in Gamenya that had fewer tillers at 100 mg Na/kg. Compared with Wyalkatchem,

fewer tillers per plant were produced in cvv Gutha and Gamenya (Fig. 3.3). Plants treated

with low to moderate soil Na (25, 50 mg Na/kg) had similar tiller number as those of nil Na

plants. However, high Na reduced tillers significantly in all four cultivars (P ≤0.05; Table

3.1).

Table 3.1 Statistical summary of plant growth and leaf gas exchange in four wheat cultivars

(Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two K levels (40, 100 mg

K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks.

*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant

Parameters Soil K Soil Na

Cultivar K×Na K×cv Na×cv K×Na×cv

Shoot dry weight *** *** * ** *** *** *

Tiller number *** *** *** * *** *** **

Root dry weight *** *** *** n.s * *** n.s

Root: shoot ratio ** *** *** n.s * *** n.s

Photosynthesis *** *** *** *** n.s * ***

Stomatal conductance

*** *** ** * n.s n.s *

Root growth

Root dry weight of all four cultivars was greater at adequate K than at low K supply,

regardless of soil Na levels (Fig. 3.4). Low to moderate soil Na had positive effects on root

dry weight in all four cultivars when soil K was low, and even at adequate K supply low soil

Na was beneficial to root dry weight except in Gamenya (Fig. 3.4). The Na-induced root

stimulation was greater in K-efficient cultivars. High soil Na levels suppressed root dry

weight in all four cultivars at both soil K levels, with greater reduction of root dry weight in

K-inefficient cultivars (55 % in Gutha and 66 % in Gamenya) than in K-efficient cultivars

(33 % in Wyalkatchem, 50 % in Cranbrook). In general, low K plants had lower root: shoot

ratios compared with K adequate plants, except Cranbrook at low Na (Fig. 3.4), however, the

interactions between K and Na for root: shoot ratio was not significant (Table 3.1).

41

Gutha

0 50 100 150 2000.0

0.2

0.4

0.6

0.8 Gamenya


0 50 100 150 200

Wyalkatchem

root

: sh

oot r

atio

0.0

0.2

0.4

0.6

0.8Cranbrook

Gutha

0

1

2

3Gamenya

Wyalkatchem

Roo

t Dry

we

ight

(g)

0

1

2

3

4Cranbrook

K*Na*cv P= 0.09HSD= 0.82

K*Na P=n.sK*Na*cv P= n.s

Fig. 3.4 Root dry weight (g/plant) (upper sub-figures) and root: shoot ratio (n=3) (lower sub-

figures) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg

(open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See

Table 3.1 for analysis of variance results.

42

Gutha

0 50 100 150 2000

100

200

300

400 Gamenya


0 50 100 150 200

Wyalkatchem

Sto

mat

al c

ondu

ctan

ce (

mm

ol H 2

O /m

2 s)

0

100

200

300

400Cranbrook

Gutha

0

5

10

15

20

25

Wyalkatchem

Leaf

ne

t pho

tosy

nthe

sis

(µm

ol C

O 2/m

2 s)

0

5

10

15

20

25

30

Gamenya

CranbrookK*Na*cv P= 0.001HSD= 5.2

K*Na*cv P= 0.04HSD= 167

Fig. 3.5 Leaf photosynthesis (upper sub-figures) and stomatal conductance (lower sub-

figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg

K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks.

See Table 3.1 for analysis of variance results.

43

3.3.2 Leaf gas exchange

At 4 weeks, low K depressed net photosynthesis of the youngest fully expanded leaves but

there was no effect of low to moderate Na on gas exchange. At 8 weeks, consistent with the

shoot dry weight responses, net photosynthesis of the youngest fully expanded leaves was

higher in plants with adequate K than low K supply, except in Gutha at 50 mg Na/kg (Fig.

3.5). The addition of 25 and 50 mg Na/kg increased leaf photosynthesis in all cultivars at low

soil K and also in the K-inefficient cultivars at adequate K supply, whereas higher soil Na

suppressed leaf photosynthetic rate relative to addition of 25 and 50 mg Na/kg. The increase

in leaf net photosynthesis induced by 25 to 50 mg Na/kg at low K was almost equal to that at

100 mg K/kg and nil Na. There were significant interactions between soil K, Na and cultivars

for leaf photosynthesis (P ≤0.05) (Table 3.1). Similarly, stomatal conductance of low K

plants increased with the addition of 25 mg Na/kg in all cultivars (Fig. 3.5). Higher soil Na

reduced stomatal conductance in the K-efficient cultivars but was less so in the K-inefficient

cultivars. At low K supply, the addition of low to moderate soil Na increased transpiration

rate of K- efficient cultivars by 54 %, whereas in K-inefficient cultivars the increase was only

by 35 % relative to nil Na (data not presented).

3.3.3 K and Na concentrations in shoots and roots

Potassium concentration in leaves and stems of all four cultivars was significantly higher

with adequate K than low K supply when soil Na levels ranged from nil to moderate, whereas

spikes had similar K concentrations irrespective of K and Na treatments (see Appendix 1.2

for K and Na concentrations in leaves, spikes and stem). At low K supply, plants with nil Na

treatment had the lowest shoot K concentration in all cultivars (Fig. 3.6). Low to moderate Na

supply increased shoot K concentrations of the four cultivars on average by 25 % relative nil

Na at low K supply (Table 3.2). High soil Na also increased shoot K concentrations with both

low and adequate soil K supply in all cultivars, but probably due to a concentration effect as a

result of growth suppression.

Although shoot K content was much greater in the adequate K soil than in the low K soil, it

showed little difference or declined across soil Na levels at the high level of soil K (Fig. 3.7).

At low soil K supply (40 mg K/kg), shoot K contents increased significantly with low to

moderate soil Na addition in K-efficient cultivars but not in K-inefficient cultivars (Fig. 3.7).

There were significant interactions of soil K, Na supply and cultivars on shoot K contents

(P≤0.05) (Table 3.2).

44

Fig. 3.6 K concentration (mg/g, dry weight) in shoot (upper sub-figures) and root (lower sub-

figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg

K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks.

See Table 3.2 for analysis of variance results.

Gutha

0 50 100 150 2000

2

4

6

8

10 Gamenya

Soil Na levels (mg/kg)

0 50 100 150 200

Wyalkatchem

Roo

t K c

once

ntra

tion

(m

g/g,

dry

we

ight

)

0

2

4

6

8

10Cranbrook

Gutha

0

10

20

30

Wyalkatchem

Sho

ot K

con

cent

ratio

n (

mg/

g, d

ry w

eig

ht)

0

10

20

30

40

Gamenya

Cranbrook K*Na P=0.000K*Na*cv P= n.sHSD= 6.2

K*Na*cv P= 0.01HSD= 3.04

45

Fig. 3.7 K content (mg/plant) in shoot (upper sub-figures) and root (lower sub-figures) (n=3)

of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open

circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.2

for analysis of variance results.

Gutha

0 50 100 150 2000

50

100

150

200Gamenya


0 50 100 150 200

Wyalkatchem

Sho

ot K

con

tent

(m

g/pl

ant)

0

50

100

150

200

K 40 K 100

Cranbrook

Gutha

0 50 100 150 2000

5

10

15

20 Gamenya


0 50 100 150 200

Wyalkatchem

Roo

t K c

onte

nt (

mg/

plan

t)

0

5

10

15

20 Cranbrook

K*Na*cv P= 0.03HSD= 18.4

K*Na*cv P= 0.00HSD= 4.0

46

Wheat roots accumulated considerably less K than shoots. Root K concentration and content

of all cultivars was significantly higher at adequate K supply than with low K supply (Fig. 3.6

and Fig. 3.7). At low K supply, soil Na addition had no significant effect on root K content

(Fig. 3.7). At adequate K supply, there was decrease in root K content with addition of soil

Na, except in Gutha at 25 mg Na/kg which showed a significant increase, and the decrease

due to Na was more obvious in K-inefficient cv. Gamenya. The three way interaction

between soil K and Na levels and cultivars was significant (P≤0.05, Table 3.2) for root K

concentrations and contents.

Table 3.2 Statistical summary of K and Na concentrations and contents in four wheat

cultivars (Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two levels of soil K

(40, 100 mg K/kg) and five levels of Na (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks.


Parameters Soil K Soil Na Cultivar K×Na K×cv Na×cv K×Na×cv

K conc. shoot *** *** n.s ** n.s n.s n.s

K conc. Root *** *** *** *** *** *** **

Shoot K content *** *** *** *** *** * **

Root K content *** *** *** *** *** *** ***

Na conc. shoot ** *** n.s n.s n.s n.s n.s

Na conc. root ** *** *** n.s *** *** ***

Shoot Na content n.s *** n.s n.s n.s n.s n.s

Root Na content *** *** *** *** n.s *** n.s

Shoot K+/Na+ *** *** ** *** * n.s n.s

Root K+/Na+ *** *** n.s *** n.s n.s n.s

In all cultivars, shoot Na concentrations were closely associated with soil Na levels (Table

3.3). Old leaves and stem concentrated at least four times more Na than young leaves.

Sodium concentration in spikes was least influenced by soil Na irrespective of soil K and

cultivars, and there were only negligible concentrations of Na measured in spikes (data in

Appendix 1.1). Soil K levels did not influence shoot Na concentration and content. There

47

were no significant interactions observed between K and Na supply for shoot Na

concentrations and contents, nor among the cultivars (Table 3.2).

In contrast to K, Na concentration and content in roots were much higher than in shoots

(Table 3.3). Root Na concentrations rose with increase in soil Na levels. Similar to shoot, soil

K levels had no influence on root Na concentration. However, root Na content was higher in

plants grown at adequate K except at nil Na. The interactions between K and Na and cultivars

were significant for root Na concentrations but not for Na contents (Table 3.2).

Shoot K+/Na+ ratios noticeably decreased with increase in soil Na levels in all four cultivars

regardless of soil K levels (Table 3.4). At low soil K, Gamenya and Wyalkatchem had the

lowest and highest shoot K+/Na+ ratios, respectively. However, at adequate K, Cranbrook had

highest shoot K+/Na+ ratio at most of the soil Na levels. Roots had considerably lower K+/Na+

ratios than shoots. They decreased further with increasing soil Na levels but there was no

particular trend observed among the cultivars. The interaction between K and Na supply on

shoot and root K+/Na+ ratio was significant but not among the cultivars (Table 3.2).

48

Table 3.3 Shoot and root Na concentrations (mg/g, dry weight) and contents (mg/plant) of

four wheat cultivars treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25,

50, 100, 200 mg Na/kg) for 8 weeks (n=3). See Table 3.2 for statistical summary of main

effects and interactions of the treatments.

Measured parameters

cvv Wyalkatchem Cranbrook Gutha Gamenya

Na K40 K100 K40 K100 K40 K100 K40 K100

Shoot Na concentration

HSD0.05= 1.5

Nil Na 0.09 0.15 0.11 0.08 0.12 0.13 0.11 0.12

Na25 0.18 0.23 0.33 0.18 0.26 0.33 0.29 0.33

Na50 0.61 0.49 0.82 0.42 0.82 0.54 1.07 0.49

Na100 1.16 1.51 2.11 1.27 1.48 2.36 2.55 1.61

Na200 2.76 2.72 3.71 2.81 4.00 2.89 3.34 2.71

Root Na concentration

HSD0.05= 3.57

Nil Na 2.74 1.56 1.76 1.32 3.07 1.32 2.92 1.32

Na25 4.23 4.01 4.9 4.94 7.73 6.23 5.22 4.62

Na50 7.72 8.47 7.14 8.79 9.57 7.48 9.21 8.93

Na100 10.1 10.03 12.1 12.3 10.6 12.8 10.9 8.65

Na200 16.5 12.9 15.4 18.5 16.5 14.6 18.4 15.6

Shoot Na content

HSD0.05= 6.2

Nil Na 0.42 0.91 0.52 0.49 0.52 0.99 0.47 0.83

Na25 0.90 1.30 1.53 1.11 1.28 2.41 1.38 2.10

Na50 3.05 2.92 4.51 2.33 3.82 3.93 4.91 3.21

Na100 5.31 7.94 9.48 6.94 5.43 10.9 10.5 8.93

Na200 8.77 10.9 12.6 13.5 12.1 13.1 8.04 11.0

Root Na content

HSD0.05= 6.3

Nil Na 4.13 3.18 3.44 3.22 2.68 2.62 2.89 2.95

Na25 9.86 11.6 12.8 14.9 8.87 13.5 6.34 7.62

Na50 15.9 24.1 17.9 22.4 11.0 14.5 9.50 15.2

Na100 16.9 22.0 15.7 22.3 7.60 14.1 9.50 12.6

Na200 14.1 19.4 16.0 21.6 9.19 10.6 6.79 10.9

49

Table 3.4 Shoot and root K/Na ratios of four wheat cultivars treated with two K levels (40,

100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks (n=3). See

Table 3.2 for statistical summary of main effects and interactions of the treatments.

Measured parameter

cvv Wyalkatchem Cranbrook Gutha Gamenya

Treatment K40 K100 K40 K100 K40 K100 K40 K100

Shoot K+/Na+

HSD0.05= 81

Nil Na 123 196 115 279 90.5 177 104 196

Na25 86.7 100 46.1 137 54.5 68.3 44.7 68.4

Na50 24.5 48.9 17.1 56.5 17.7 39.3 12.9 47.2

Na100 12.9 17.9 8.05 21.9 10.3 13.4 5.73 17.8

Na200 6.63 10.04 5.93 9.71 4.55 8.78 4.98 10.4

Root K+/Na+

HSD0.05= 2.5

Nil Na 0.36 7.03 0.67 5.49 0.72 5.56 0.72 7.53

Na25 0.45 1.67 0.14 0.94 0.35 1.34 0.38 1.38

Na50 0.19 0.81 0.04 0.57 0.11 0.69 0.24 0.72

Na100 0.15 0.77 0.11 0.45 0.23 0.69 0.27 0.70

Na200 0.17 0.78 0.12 0.39 0.21 0.62 0.25 0.62

Leaf Ca and Mg concentrations measured were lower in K adequate plants when compared

with K deficient plants. Leaf Ca concentrations in both soil K levels decreased with increase

in soil Na in all four cultivars. The interaction between K and Na levels among the genotypes

was not significant for leaf Ca and Mg concentrations (see Appendix 1.2).

3.3.4 Soil exchangeable cations after K and Na addition

The soil incubation experiment did not show any significant effects of Na addition on

exchangeable soil K levels or exchangeable Ca, Mg and Al levels. Colwell-extractable K and

exchangeable K measured were slightly higher when soil had Na applied, compared with nil

soil Na, but the increase was not statistically significant (Table 3.5).

50

Table 3.5 Concentrations of exchangeable cations in non-planted soils (n=3) with or without

50 mg Na/kg at two K levels (40, 100 mg K/kg) after one week of incubation. Means with

different letters differ at P≤0.05.

Measured parameters

Soil treatment

K 40 K 40 K 100 K 100

Nil Na 50 Na Nil Na 50 Na

Colwell K (mg/kg) 48.3b 51.0b 96.0a 102a

Exc. K (cmol/kg) 0.13a 0.14a 0.24b 0.27b

Exc. Na (cmol/kg) 0.02b 0.19a 0.02b 0.18a

Exc. Ca (cmol/kg) 0.68a 0.71a 0.44b 0.61ab

Exc. Mg (cmol/kg) 0.09a 0.10a 0.10a 0.10a

Exc. Al (cmol/kg) 0.02a 0.03a 0.03a 0.03a

3.4 Discussion

Wheat response to soil NaCl supply in the present study varied with soil Na and K levels: 1)

root growth was stimulated by low to moderate soil Na levels with low soil K; 2) shoot and

root dry weight were suppressed with high Na regardless of soil K levels. However, the Na

effect varied with K-use efficiency of wheat cultivars with K-efficient cultivars being more

responsive to root dry weight stimulation by low to moderate Na under K deficiency along

with greater increases in shoot K uptake and stomatal conductance than K-inefficient

cultivars. Genotypic differences in K-use efficiency also influenced Na uptake and salt

tolerance: K-efficient cultivars were more tolerant of high salt levels than K-inefficient

cultivars.

The growth stimulation at low to moderate Na (25, 50 mg/kg) supply under K deficiency was

clearly evident in wheat roots but in shoots only through the alleviation of old leaf K

deficiency symptoms. The shoot dry weight and fresh weights were not significantly affected

by low to moderate Na in K deficient plants. However, Na at 100-200 mg/kg negatively

affected both root and shoot dry weight of wheat with both low and adequate soil K due to

salt toxicity. The present results in wheat were in contrast to salt-tolerant barley where the

addition of 100 mg Na/kg to K-deficient soil stimulated significant shoot growth increase but

51

not in root growth (Ma et al., 2011). Moreover, Ma et al. (2011) found, similar to the present

study, no significant benefit after 20 days of 100 mg Na/kg supply on K deficient (30 mg

K/kg) wheat growth. Indeed, 100 mg Na/kg (equivalent to about 30 mM Na in soil solution)

for 8 weeks had negative effects on wheat growth in the current study. Clearly there were

contrasting effects of low to moderate Na on wheat and barley. Barley responded positively

to moderate Na (100 mg Na/kg) supplied to K-deficient plants, but the response was

restricted to the shoots and not the roots. Wheat on the other hand only responded to Na at

lower levels (25-50 mg Na/kg but not 100 mg Na/kg) with the strong response in roots but

not in shoots.

Differences in accumulation of K+ and Na+ between barley and wheat may explain their

contrasting responses to low to moderate Na supply. Wheat roots accumulated significantly

higher Na than in shoots. Indeed the low to moderate level of Na that stimulated root growth,

increased root Na from 3 mg Na/kg (at nil Na) to 9.5 and 14 mg Na/kg at 25 and 50 mg

Na/kg respectively, while shoot Na only increased from 0.48 mg Na/kg to 1.3 and 4 mg

Na/kg, at 25 and 50 mg Na/kg respectively. In the study of Ma et al. (2011), shoot K+ /Na+ in

wheat cv Wyalkatchem was 10-15 times higher than the ratio in shoots of two barley cultivars

grown under the same conditions. By contrast, there were no differences among barley

cultivars and wheat in K+ /Na+ ratio in roots. Hence, the high accumulation of Na in barley

shoots produced a K+ /Na+ ratio of 0.5 in barley cv. CM72 but 10 in wheat. Under these

conditions, the partial substitution of K by Na is feasible in barley since there is sufficient

Na+ to provide equivalent osmotic effects to those of K+. In barley the accumulation of Na+

by low-K plants was limited to shoots as was the shoot growth response. By contrast, the low

shoot Na+ concentration in wheat shoots relative to K+, provides too little Na for the

replacement of K functions in the shoot to be feasible. For example in the low K plants, shoot

K concentration was 13.9 mg K/g, dry weight (equivalent to 89.5 mM K in tissue water) at 25

mg Na/kg of soil, whereas, depending on cultivars there was only 0.18-0.33 mg Na/g, dry

weight (mean tissue water Na concentration of 2.8 mM). Hence, there may be other processes

in wheat that led to growth stimulation at 25-50 mg Na/kg.

In both the present study, and that of Ma et al. (2011), low to moderate Na supply to low-K

wheat plants increased shoot K concentrations and this should have stimulated growth. The

average shoot K concentrations in low and adequate soil K treatments were 14 g/kg and 24

g/kg, which are in the deficient and sufficient ranges, respectively, for wheat growth at the

boot to heading stage (Reuter et al., 1997). The increases in photosynthesis rate and stomatal

52

conductance also evident with low to moderate Na supply to K-deficient are both expected

responses in the shoot to increased K concentration (Zhao et al., 2001). The increase in root

growth is another expected plant response to increased shoot K concentration because

increased photosynthesis results in greater assimilate supply to roots and increases root: shoot

ratio of cereals (e.g. (Degl'Innocenti et al., 2009; Ma et al., 2011; Ma et al., 2013)). Hence a

possible explanation for the Na stimulation of growth in wheat is that Na increases K supply

to the shoot which in turn stimulates photosynthesis and the greater supply of assimilate

allows for increased root growth. With only a single harvest it is not possible to definitively

piece together this chain of events. However, clearly the evidence in support of the first

response, the increase in K uptake leading to greater shoot K is pivotal.

Increased shoot K content could arise from several mechanisms. Firstly, increase of root Na

concentration at low to moderate Na may release vacuolar K+ that is made available for

cytoplasmic functions in the root cells or for translocation to the shoot (Walker et al., 2000).

The increase in root Na concentration at 25 -50 mg Na/kg of soil was substantial, while root

K contents remained unchanged. The effects of 25-50 mg Na/kg of soil on root K

concentrations varied among cultivars. By contrast, shoot K content increased by about 40 %

with the supply of 25-50 mg Na/kg of soil. Hence the low to moderate Na supply appeared to

favour K partitioning to the shoot of wheat.

A second possible mechanism for increased K uptake is Na activation of K+ symporters in

roots. At low external Na+ and K+ concentrations, high-affinity K+ uptake transporters

function as Na+-K+ symporters, as demonstrated by Na+-stimulated K+ uptake and K+-

stimulated Na+ uptake, however, at high external Na+ concentrations, some of these

transporters become Na+ uniporters, no longer transporting K+ (Benito et al., 2014; Rubio et

al., 1995). However, in an experiment by Box and Schachtman (2000), there was no evidence

of enhanced K+ uptake in wheat due to Na supply, even though there was an increase in

wheat growth due to external Na+ i.e., according to them the positive effect of Na at low soil

K can be largely attributed to substitution of Na+ in wheat K functions and direct effect of

Na+ on growth. Box and Schachtman (2000) investigated the Na+ activated (activation of K+

symporters) K+ uptake only under low light conditions in wheat and concluded that it was

functionally a minor process for K+ uptake by wheat, indicating there may be effects of Na on

transporters not identified by them. Other mechanism for increased K+ uptake could be by a

low-affinity K+ uptake system (such as AKT) which at moderate salinity (20 mM NaCl in

53

barley) hyperpolarized the plasma membrane and increased K+ uptake via inward-rectifying

hyperpolarized-activated K+ channels (Chen et al., 2005; Shabala & Cuin, 2008).

A part but not the entire increase of K content could be attributed to the increase in

extractable soil K by soil Na supply. In the incubation experiment, soil exchangeable and

Colwell K showed a non-significant increase with addition of Na. However, for a pot with 6

kg of soil, the change in Colwell K was equivalent to around 18 mg in the 50 mg Na/kg

treatment and could have provided 6 mg of extra K+ to each plant in the 3-plant pots, which

would account for part of the increased shoot K content in the Na-added plants. From the

present results, it would be premature to conclude that Na stimulation of wheat growth in K-

deficient plants is unrelated to the increased K availability in soil. Interestingly, previous

studies on Na stimulation of plant growth in K-deficient plants did not consider increased K

uptake from soil as an explanation for the response: they focussed on Na substitution of K

functions.

As explained above, Na substitution of K in shoots of wheat in the present experiment was

unlikely because the shoot Na concentrations were too low to provide any significant

replacement of the osmotic effects of K in vacuoles or in other functions of K+. By contrast,

the increase in root Na+ was more than sufficient to replace osmotic functions of K+ in roots.

The increase in root Na concentration may stimulate root elongation of K-deficient plants

(Ali et al., 2009) by turgor effects on cell expansion. Whether an increase in root elongation

could contribute to increase root K uptake is unclear and there is no direct evidence in the

present study to address this question. Such an effect is more likely to be expressed in soil

where root elongation has a major role in determining nutrient uptake by providing access to

additional nutrient supply (Barber & Silberbush, 1984). It is unlikely the Na substitution of K

in roots would directly increase root growth because their dry matter increase would be

limited by inadequate assimilate supply to roots under low K supply. Hence, it is proposed

that the stimulation of root growth by low to moderate Na is mediated in shoots, probably by

increased photosynthesis leading to greater assimilate supply to roots. The increase in root

growth in turn could allow for increased K uptake by roots.

Potassium efficient cultivars were more salt tolerant than K-inefficient cultivars in the order:

Cranbrook> Wyalkatchem> Gutha> Gamenya, in terms of shoot dry weight (Genc et al.,

2007). However, K-efficient cultivars mostly had similar K+/Na+ ratio as K-inefficient

cultivars. This is consistent with an earlier study where K+/Na+ ratio did not explain the

variation in salt tolerance among wheat cultivars (Genc et al., 2007). In contrast, the ability of

54

plants to maintain a high K+/Na+ ratio was positively correlated with salt tolerance in other

studies (Chen et al., 2007; Cuin et al., 2009; Shabala & Cuin, 2008; Wu et al., 1996). Cuin et

al. (2008) emphasized that Na+ exclusion is not a sufficient tool for salt tolerance but the

ability of roots to retain K+ correlated better with salt tolerance in wheat. Moreover, a recent

study in wheat suggests that salt-tolerant cultivars have an enhanced ability to sequester Na+

into vacuoles of root cells, whereas in sensitive cultivars large quantities of Na+ are located in

the root cell cytosol (Cuin et al., 2011). In this study, cv. Cranbrook was least effective in

retaining root K under increasing Na supply among the cultivars. Cultivars may differ in the

extent of Na translocation to shoots. The substitution of K+ by Na+ in cereals is likely to be

influenced not only by plant K status, but also by the potential of the cultivar to accumulate

significant Na concentrations in their shoots, as emphasised for the salt tolerant barley cv.

CM72 (Ma et al., 2011), or in roots as with wheat in the present study.

In the present study, the K-use efficiency of wheat cultivars studied across a range of Na

levels from no added Na up to toxic levels was consistent with the ranking of cultivars for K-

use efficiency by Damon and Rengel (2007). However, there has been little information

reported on the role of Na supply in K-use efficiency in wheat. According to this study, K-

efficient cultivars Wyalkatchem and Cranbrook had higher response to low to moderate Na

supply relative to K-inefficient cultivars Gutha and Gamenya. In contrast to the suggestion by

Rengel and Damon (2008) that increased capacity to substitute Na+ for K+ may be a

mechanism underlying K-use efficiency in wheat, we found that Na stimulated greater K

uptake in K-efficient cultivars. The main mechanism identified by Damon and Rengel (2007)

for K efficiency in wheat cultivars like Wyalkatchem was greater utilization efficiency of

shoot K rather than greater K uptake. According to them K efficiency is a measure of

genotypic tolerance in low K soils and can be quantified as K efficiency ratio (ratio of growth

at deficient and adequate K supply). In the present study, there was greater K uptake by K-

efficient cultivars or greater K content in shoots with low to moderate Na supply. The

stimulation of photosynthesis, stomatal conductance and transpiration efficiency and root dry

weight were greater in the K-efficient cultivars. This is consistent with greater utilization

efficiency of shoot K in the K-efficient cultivars leading to greater photosynthesis and hence

roots dry weight response to shoot K. Given this explanation the weak responses in shoot dry

weight to low to moderate Na are surprising. There was alleviation of K deficiency symptoms

in old leaves by low to moderate Na. However, since the symptoms only appeared at 6 weeks

55

after sowing and the shoots were harvested at 8 weeks, it is possible that the shoot response

lagged behind that of roots and given more time would have been more substantial.

The stimulation of root growth to a greater extent than shoot growth in wheat by low to

moderate Na in low K plants may have greater significance when the crop is under stress in

the field than in the present well-watered pot experiment. There should be a direct benefit

from an increased root mass under drought stress particularly in K-deficient wheat for which

depressed root growth is a characteristic symptom (Ma et al., 2011; Ma et al., 2013).

3.5 Conclusion

In this experiment, wheat cultivars differing in K-use efficiency varied in response to soil K

and Na supply. When supplied with low to moderate Na under K deficiency, positive

responses in K uptake, leaf photosynthesis, stomatal conductance and root dry weight were

observed in all four cultivars, particularly in K-efficient cultivars. In contrast to previous

findings, we conclude that low to moderate Na stimulated increase in shoot K uptake by

wheat, which particularly in K-efficient cultivars promoted photosynthesis and root growth

and further access to soil K. In the present study, the shoot Na concentrations at low to

moderate Na supply to soil were too low to feasibly substitute for biophysical functions of K

in the shoot. Four mechanisms are proposed to explain the increased K uptake in shoots of

wheat by low to moderate Na supply, but further studies are needed to clarify the relative

contribution of each mechanism to the growth stimulation.

56

CHAPTER 4

SPLIT-ROOT EXPERIMENT

Moderate sodium increased K uptake, leaf gas exchange and plant growth of wheat cv.

Wyalkatchem grown in a K-deficient split-root system

4.1 Introduction

In saline soils, excessive NaCl has a detrimental effect on nutrition of most crops (Römheld

& Kirkby, 2010), and plant physiological functions are inhibited due to depression in

potassium (K+) uptake by competing sodium (Na+) ions (Blumwald et al., 2000). Potassium

and Na ions are similar in ionic radius and ionic hydration energies (Marschner, 1995), and

because of this chemical similarity, it is assumed that both these ions compete for common

adsorption sites in the roots (Subbarao et al., 2003). Potassium is required in high

concentrations for plant growth and development, whereas Na is beneficial to many

glycophytes as well as to certain halophytes in relatively low concentrations (Greenway &

Munns, 1980; Mäser et al., 2002).

Despite the fact that high Na is detrimental to plant growth and K nutrition, low to moderate

Na was reported to have beneficial effects in some plant species, especially when K is present

at suboptimal concentrations (Ma et al., 2011). Plants can utilize Na+ ions in several key

cellular processes as long as the concentrations remain less than osmotically challenging

levels (Kronzucker et al., 2013). Sodium can help maintain cell turgor, ionic balance, regulate

osmotic pressure, and improve water relations via stomatal conductance (Subbarao et al.,

2003). It has been shown that under K deficiency, low external Na+ could substitute as an

enzyme activator or as a vacuolar solute by releasing vacuolar K+ to fulfil its functions in

cytoplasm (Walker et al., 2000).

The beneficial effects of Na on some natrophilic plant species are well documented

(Marschner, 1995), but there is only limited research available on cereal crops especially in

soil-based systems (Ma et al., 2011; Miyamoto et al., 2012). Growth stimulation due to added

Na at deficient soil K supply was observed in rice (Miyamoto et al., 2012; Yoshida &

Castaneda, 1969), barley (Ma et al., 2011) through solution/sand culture studies under

controlled conditions or in field studies. Studies on wheat reported insignificant or limited

effects on growth due to low to moderate Na supply under K deficiency in a soil-based

system (Ma et al., 2011) but significant growth stimulation when low concentrations of Na

57

(500 µM Na+) were added to K-deficient nutrient solution (20 µM external K) (Box &

Schachtman, 2000).

An earlier pot experiment with four wheat cultivars (Chapter 3) suggested that addition of

low to moderate Na (25 to 50 mg Na/kg) to low K soil (40 mg K/kg) had beneficial effects on

plant growth (especially roots), leaf gas exchange and plant K uptake. The implications of

varying K and Na distribution in a soil profile for wheat K nutrition, and the extent of Na

substitution in K functions have not been examined previously. The main objective of this

experiment was to study whether the beneficial effect of Na depends on both K and Na being

present at same part of the root system in wheat. A split-root experiment was conducted,

where the root system was divided into two halves and treated with different combinations of

K and Na supply, to investigate how shoot and root growth, and concentrations and uptake of

K and Na in wheat were affected by varying combinations of K and Na between the

compartments.


Wheat (Triticum aestivum L.) cv. Wyalkatchem was grown in a naturally-lit glasshouse at

Murdoch University, Perth (32°04′S, 115°50′E) from mid winter to early spring. Soil

(classified as ‘Chromosols’) was collected from an unfertilised paddock east of Dowerin, and

had the following properties: pH (CaCl2) 4.9; EC1:5 0.03 dS/m; 7 mg NH4-N/kg and 8.7 mg

NO3-N/kg (Searle, 1984); < 15 mg K/kg and 29 mg P/kg (Colwell, 1963); organic C 0.17%

(Walkley & Black, 1934). A split root system was used by joining two undrained plastic pots

(length and breadth 110 mm, depth 140 mm) together, filling each with sieved soils (< 2 mm)

with a mixture of basal nutrients and individual treatments of K and Na into the two adjoining

compartments containing 2 kg soils/compartment. Two notches were made in the adjoining

walls to facilitate planting of two plants per split root system. The level of soil was kept 2 cm

below the top edge of the compartments to avoid soil contamination between the two

compartments. Basal nutrients were applied at the following rates (mg/kg): 237

Ca(NO3)2.4H2O, 103 (NH4)2HPO4, 80 MgSO4.7H2O, 18 FeSO4.7H2O, 14 MnSO4.H2O, 9

ZnSO4.7H2O, 8.3 CuSO4.5H2O, 0.33 H3BO3, 0.3 CoSO4.7H2O, 0.33 Na2MoO4.2H2O. Wheat

seeds were first sown in potting mix in germination trays and after 10 days, two uniform

seedlings were transplanted into each split-root system with the seminal roots being evenly

divided between the two compartments. The pots were watered to field capacity (15% w/w)

with de-ionised water throughout the experiment and were re-arranged every week to reduce

positional effects on the plant growth.

58


Two levels of soil K were used in this experiment: 40 mg K/kg (low) and 100 mg K/kg (high)

using KCl. Each soil K level was combined with three soil Na levels applied to one or two of

the compartments: 0, 50 and 200 mg Na/kg in a total of 11 split-root treatments, as listed in

Table 4.1. When potting, K and Na treatments were thoroughly mixed with basal nutrients in

a rotary mixer, and each split-root treatment was replicated four times.

Table 4.1 Split- root treatments experimental design

Treatment Compartment/ Side A (mg/kg) Compartment/ Side B (mg/kg)

soil K soil Na soil K soil Na

1 40 0 40 0

2 40 0 40 50

3 40 50 40 50

4 40 0 40 200

5 40 200 40 200

6 40 0 100 0

7 100 0 100 0

8 100 0 100 50

9 100 50 100 50

10 100 0 100 200

11 100 200 100 200

4.2.2 Measurements

The plants were harvested 6 weeks after being transplanted into the split-root system. The

development of leaves and tillers was recorded weekly throughout the experiment. Before

harvest, leaf net photosynthesis, stomatal conductance and transpiration rate were measured

using an LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The

measurements were made in fully expanded young leaves at ambient relative humidity of 50

%, leaf temperature of 25˚C, reference CO2 of 380 µmol/mol, and photosynthetically active

radiation of 1500 µmol/m2·s1.

59

At harvest, the shoots were cut at the soil surface and shoot fresh weight was recorded

immediately. Roots were collected from individual compartments of the split-root system

after being washed in tap water and rinsed with DI water. The shoot and root samples were

dried in a forced-draught oven at 60˚C for 48 hours and the dry weights were recorded. The

dried samples were milled for K and Na analysis. Samples of 50- 200 mg were weighed into

50 mL centrifuge tubes and digested in 5 mL of 70% (w/w) HNO3 at 75˚C for 10 min, and

then at 109˚C for 15 min, and after samples were cooled, 1 mL of 30% (w/w) H2O2 was

added and further digested at 109˚C for 15 min. The digestion was made in a micro-wave

(CEM Mars 5, manufactured by CEM Corp., USA) for cation analysis (Huang et al., 2004).

The samples were then diluted with milli-Q water and the concentrations of K and Na were

determined by flame photometer (Model 410, Sherwood Scientific Ltd, UK).


Statistical analyses were conducted using the statistical program SPSS 18.0. One-way

analysis of variance was conducted to assess the effects of split-root treatments. Tukey’s

HSD was computed at P ≤ 0.05 for comparing the means of the 11 split-root treatments and

the specific responses between the two compartments were compared within each split-root

treatment.

4.3 Results

4.3.1 Plant growth

Low soil K (40 mg K/kg) in combination with nil soil Na in both compartments had the

lowest shoot growth. Severely restricted growth was also observed in treatments which had

low soil K and high Na (200 mg Na/kg) either in one or both compartments (Table 4.2). Low

soil K in combination with moderate Na (50 mg Na/kg) in one compartment significantly

increased shoot growth and tiller production relative to low K in combination with nil Na. At

low soil K, the addition of moderate Na to either one or both compartments increased the

shoot dry weight on average by 8.2 and 11 times, respectively, compared to low K treatment

without Na in both compartments. Interestingly, at low K supply, the presence of moderate

Na in both compartments resulted in significantly higher shoot dry weight than adequate K

combined with nil Na in one compartment only (Table 4.2). The effect of adequate K without

Na in both compartments was similar to low K with moderate Na in both compartments. At

high soil K supply (100 mg K/kg), the presence of moderate Na in both compartments

60

reduced shoot dry weight, with further reduction in shoot growth and tiller production at high

Na (Table 4.2).

Table 4.2 Shoot dry weight (g), and number of tillers per plant of wheat cv. Wyalkatchem

treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg

Na/kg) combined in 11 different split-root systems. Means (n=4) in a column with different

letters differ at P≤0.05.

Treatment Side A (mg/kg) Side B (mg/kg) Shoot dry weight

(g/plant) Tillers

soil K, Na soil K, Na

1 40, 0 40, 0 0.10f 1.00e

2 40, 0 40, 50 0.86abc 4.87bc

3 40, 50 40, 50 1.16a 6.00ab

4 40, 0 40, 200 0.15f 1.75de

5 40, 200 40, 200 0.28ef 1.75de

6 40, 0 100, 0 0.61cde 3.62bcde

7 100, 0 100, 0 1.05ab 8.00a

8 100, 0 100, 50 0.95abc 6.12ab

9 100, 50 100, 50 0.75bcd 4.75bc

10 100, 0 100, 200 0.70bcd 4.12bcd

11 100, 200 100, 200 0.40def 2.87cde

Root growth showed similar response to K and Na treatments as shoot growth. Low soil K in

combination with moderate soil Na in both compartments resulted in the highest root dry

weight among all the treatments, followed by high soil K with moderate Na in both

compartments (Table 4.3). Low K supply when combined with moderate Na in one

compartment stimulated root growth significantly relative to root growth in the adjoining

compartment with nil soil Na. At nil soil Na, root growth was significantly greater in the

adequate K compartment than the adjoining low K compartment (Treatment 6; Table 4.3),

but root growth was largely uniform between the two compartments when K supply was the

61

same. Unlike shoot growth, the presence of moderate Na in both compartments stimulated

root growth at high soil K. However, the presence of high Na (200 mg Na/kg) in both

compartments reduced root growth regardless of soil K levels.

Root: shoot ratios did not show a particular pattern among the treatments (Table 4.3). The

ratio was higher when there was moderate soil Na in both compartments regardless of soil K

levels. Presence of high soil Na in both compartments also increased root: shoot ratios,

mainly due to poor shoot growth.

Table 4.3 Total root dry weight (g) per plant and their root: shoot ratios of wheat cv.

Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and

three Na levels (0, 50 and 200 mg Na/kg). One-way analysis of variance was conducted to

assess the effects of split-root treatments. Tukey’s HSD was computed at P ≤ 0.05 for

comparing the differences in total root dry weight and root: shoot ratios between the 11 split-

root treatments and the specific root responses between the two compartments were

compared within each split-root treatment. Means (n=4) with different letters differ at

P≤0.05.

Treatment Side A (mg/kg) Side B (mg/kg) Root dry weight (g) Root: Shoot

soil K, Na soil K, Na Side A Side B Total

1 40, 0 40, 0 0.02x 0.02x 0.04e 0.41bc

2 40, 0 40, 50 0.10y 0.19x 0.29cd 0.34bc

3 40, 50 40, 50 0.34x 0.32x 0.66a 0.57abc

4 40, 0 40, 200 0.03x 0.01y 0.04e 0.30c

5 40, 200 40, 200 0.06x 0.11x 0.17de 0.59abc

6 40, 0 100, 0 0.06y 0.11x 0.18de 0.30c

7 100, 0 100, 0 0.16x 0.17x 0.33cd 0.32c

8 100, 0 100, 50 0.26x 0.23x 0.49abc 0.51abc

9 100, 50 100, 50 0.26x 0.31x 0.57ab 0.77a

10 100, 0 100, 200 0.20x 0.15x 0.35bcd 0.50abc

11 100, 200 100, 200 0.12x 0.12x 0.25de 0.64ab

62


Low K in combination with nil soil Na in both compartments had the lowest leaf net

photosynthesis among the treatments (Table 4.4). Leaf net photosynthesis increased when

moderate Na was added at least in one compartment with low K supply and the increase was

almost similar to nil Na with adequate soil K (100 mg K/kg) in one compartment. Regardless

of soil K levels, an addition of high Na (200 mg Na/kg) in both compartments reduced

photosynthesis rate considerably. The response of stomatal conductance (Gs) in the leaves

showed similar trends as net photosynthesis rate (Pn). Stomatal conductance and transpiration

rate of wheat supplied with adequate soil K were higher than low soil K plants. The addition

of moderate Na in one compartment of low soil K split-root system increased transpiration

rate significantly compared to low K treatment without soil Na.

Table 4.4 Leaf net photosynthesis rate (Pn), stomatal conductance (Gs) and transpiration (E)

of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three

levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems. Means

(n=4) with different letters differ at P≤0.05.

Treatment Side A (mg/kg)

Side B (mg/kg)

Pn Gs E

soil K, Na soil K, Na µmol CO2/m2.s mmolH2O/m2.s mmolH2O/m2.s

1 40, 0 40, 0 4.49d 59.3c 1.15b

2 40, 0 40, 50 14.6ab 169ab 3.14a

3 40, 50 40, 50 16.3a 167ab 3.14a

4 40, 0 40, 200 11.2bc 198a 2.74ab

5 40, 200 40, 200 6.09d 96.9bc 2.11ab

6 40, 0 100, 0 15.2ab 219ab 3.47a

7 100, 0 100, 0 17.1a 224a 3.70a

8 100, 0 100, 50 16.7a 220a 3.40a

9 100, 50 100, 50 16.6a 218a 3.25a

10 100, 0 100, 200 12.0bc 177ab 2.57ab

11 100, 200 100, 200 8.37cd 154ab 2.58ab

63

4.3.3 K and Na concentrations and accumulation

Shoot K concentrations did not correlate well with soil K supply alone. At low soil K, plants

with nil soil Na in both compartments had highest shoot K concentration, followed by low K

with high soil Na in one or both compartments (Table 4.5). With adequate soil K supply,

moderate Na significantly reduced shoot K concentration, but high Na even in one

compartment increased shoot K concentration mainly due to a concentration- effect.

In contrast to shoot K concentrations, root K concentrations correlated better with soil K

levels. Root K concentrations were higher in adequate soil K treatments than low K. At low

soil K, root K concentration was lowered in wheat grown with high soil Na in one

compartment and nil Na in both compartments (Table 4.5). The root K concentrations were

similar between the two compartments when there was low soil K in both compartments

irrespective of soil Na levels. At low soil K, addition of moderate Na in one compartments

increased root K concentration in the adjoining compartment as well. However, the

compartment with adequate K and nil Na had significantly higher root K concentration than

adjoining compartment of low K without Na or adequate K with high Na (Table 4.5).

Plants grown in adequate K without Na in both compartments accumulated a significantly

higher amount of K than other treatments (Fig. 4.1). At low soil K, addition of moderate Na

to one or both compartments increased plant K content significantly compared to low K

treatments with nil or high soil Na. At adequate K level, addition of moderate Na decreased K

accumulation with further reductions at high soil Na.

Shoot and root Na concentrations in wheat increased with increase in soil Na concentrations

(Table 4.6). Low K in combination with 200 mg Na/kg in both compartments had the highest

shoot and root Na concentrations. Adequate K in combination with nil Na in both

compartments had the lowest shoot and root Na concentrations. The addition of moderate and

high Na, irrespective of soil K levels increased the shoot Na concentrations. There was

significant difference in root Na concentrations between the compartments of the split-root

system, and the roots grown in the compartment with high soil Na had higher Na

concentration than the adjoining compartment (Table 4.6).

64

Table 4.5 Shoot and root K concentrations (mg K/g) of wheat cv. Wyalkatchem in the split-

root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and

200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in

shoot K concentrations between the 11 split-root treatments and the specific root K

concentrations between the two compartments were compared within each split-root

treatment. Means (n=4) with different letters in a column differ at P≤0.05.


Side B (mg/kg)

Shoot K

(mg K/g, dry wt) Root K (mg K/g, dry wt)

Soil K, Na Soil K, Na Side A Side B

1 40, 0 40, 0 51.8a 2.40x 3.12x

2 40, 0 40, 50 28.4d 11.8x 11.5x

3 40, 50 40, 50 16.1e 10.8x 11.9x

4 40, 0 40, 200 45.8ab 3.61x 1.42x

5 40, 200 40, 200 34.8cd 7.00x 7.78x

6 40, 0 100, 0 41.4bc 16.4y 22.8x

7 100, 0 100, 0 40.3bc 22.9x 19.5x

8 100, 0 100, 50 32.0d 24.6x 22.4x

9 100, 50 100, 50 30.3d 22.5x 21.7x

10 100, 0 100, 200 34.1cd 20.8x 16.8y

11 100, 200 100, 200 43.7ab 16.6x 15.4x

65

Potassium uptake per plantK

up

take

(m

g/p

lant

)

0

20

40

60

80

100

120

140

K uptake

side A: K- 40 40 40 40 40 40 100 100 100 100 100 Na- 0 0 50 0 200 0 0 0 50 0 200

side B: K- 40 40 40 40 40 100 100 100 100 100 100 Na- 0 50 50 200 200 0 0 50 50 200 200

P=0.000HSD0.05=43

Fig. 4.1 Potassium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two

levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined

in 11 different split-root systems. (±SE, n=4).

66

Table 4.6 Shoot and root Na concentrations (mg Na/g) of wheat cv. Wyalkatchem in the

split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0,

50 and 200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the

differences in shoot Na concentrations between the 11 split-root treatments and the specific

root Na concentrations between the two compartments were compared within each split-root

treatment. Means (n=4) with different letters differ at P≤0.05.


Side B (mg/kg)

Shoot Na

(mg Na/g, dry wt) Root Na (mg Na/g, dry

wt)

Soil K, Na Soil K, Na Side A Side B

1 40, 0 40, 0 1.29c 0.502x 0.50x

2 40, 0 40, 50 1.96c 5.15y 8.58x

3 40, 50 40, 50 2.39c 10.6x 10.5x

4 40, 0 40, 200 17.3ab 5.16y 24.4x

5 40, 200 40, 200 19.4a 17.82x 15.7x

6 40, 0 100, 0 0.15c 1.03x 0.69x

7 100, 0 100, 0 0.08c 0.42x 0.38x

8 100, 0 100, 50 1.69c 2.69y 9.80x

9 100, 50 100, 50 4.74c 11.1x 10.5x

10 100, 0 100, 200 5.12c 3.40y 14.0x

11 100, 200 100, 200 12.7b 12.03x 12.6x

Wheat accumulated the highest Na when grown in the treatment with low K and moderate Na

(50 mg Na/kg) in both compartments, followed by adequate soil K and 50 mg Na/kg in both

compartments (Fig. 4.2). There was no significant difference in plant Na content between the

treatments with low and adequate soil K.

67

Sodium uptake per plantN

a up

take

(m

g/p

lant

)

0

20

40

60

80

100Na uptake

side A: K- 40 40 40 40 40 40 100 100 100 100 100 Na- 0 0 50 0 200 0 0 0 50 0 200

side B: K- 40 40 40 40 40 100 100 100 100 100 100 Na- 0 50 50 200 200 0 0 50 50 200 200

P=0.000HSD0.05=12.6

Fig. 4.2 Sodium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two

levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined

in 11 different split-root systems. (±SE, n=4).

The K+/Na+ ratios of whole plants decreased with increase in soil Na concentrations. The

treatments with nil Na added to either of the compartments had high K+/Na+ ratios. Wheat

grown in adequate K without Na in both compartments had significantly higher K+/Na+ ratio

than the other treatments (Table 4.7). With addition of 200 mg Na/kg, there was more than 50

fold reduction in K+/Na+ ratio.

68

Table 4.7 K/Na ratios of wheat (whole plant) cv. Wyalkatchem were compared between the

split-root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50

and 200 mg Na/kg). Means (n=4) with different letters differ at P≤0.05.

Treatment Side A (mg/kg) Side B (mg/kg) K/Na ratio

soil K, Na soil K, Na

1 40, 0 40, 0 26.3bc

2 40, 0 40, 50 3.33c

3 40, 50 40, 50 1.66c

4 40, 0 40, 200 1.09c

5 40, 200 40, 200 0.98c

6 40, 0 100, 0 53.0b

7 100, 0 100, 0 109a

8 100, 0 100, 50 5.70c

9 100, 50 100, 50 2.80c

10 100, 0 100, 200 3.30c

11 100, 200 100, 200 2.03c

4.4 Discussion

Sodium can be beneficial or toxic, depending on plant species, cultivar and levels of Na+ and

K+ in the root medium. The effects of Na toxicity in saline soils have received much more

attention than the benefit from low to moderate Na. In some C4 plant species, Na is

considered as a ‘functional-nutrient’ (Subbarao et al., 2003) where it can stimulate

photosynthesis and Na+-coupled trans-membrane transport (Murata & Sekiya, 1992). In non-

C4 plants, Na can have beneficial effects in some plant species, even though it is not essential

for growth (Gattward et al., 2012; Terry & Ulrich, 1973), and there is evidence that Na+ can

replace some K+ functions under K deficiency (Ali et al., 2006; Subbarao et al., 2003; Walker

et al., 2000). The previous pot experiment showed Na was beneficial to wheat cultivars

grown under K deficient conditions (Chapter 3). The role of K+ in minimising the effects of

Na+ toxicity has been extensively researched in wheat (El-Lethy et al., 2013; Zheng et al.,

69

2008). However, the possibility of Na+ in substituting for K+ functions in wheat grown under

K deficiency has not been thoroughly examined. This study demonstrated moderate Na (50

mg/kg)- induced increased plant K uptake, increased leaf gas exchange, and increased growth

of K deficient wheat cv. Wyalkatchem in a split-root set up. Relative to the stimulation of

growth in wheat cv. Wyalkatchem in Chapter 3, the growth response here was much more

striking and included a strong shoot response in contrast to the effects reported in Chapter 3.

In this experiment, plants grown under low soil K without Na in both compartments showed

severe K deficiency symptoms with old leaf necrosis, stunting and drooping of plants (Gierth

& Mäser, 2007). With moderate NaCl addition, the leaves were erect, healthier and did not

show any deficiency symptoms (Fig. 4.3). The observations suggest that Na can be a partial

replacement for K responsible for maintaining turgidity in wheat cells particularly when K

supply is limited. Similar beneficial effect due to added Na was noticed in rice grown at low

K concentrations (Yoshida & Castaneda, 1969). In this experiment, shoot Na concentrations

with 50 mg Na/kg addition to one or both low K compartments did not exceed 2.5 mg Na/kg

suggesting there was not enough shoot Na to replace K functions while root Na

concentrations increased significantly with moderate Na. However, shoot Na content

increased significantly from 0.14 mg Na/plant at nil soil Na to 1.8 mg Na/plant and 3.0 mg

Na/plant with addition of 50 mg Na/kg to one and both the compartments, respectively at low

soil K (40 mg K/kg).

The response of wheat in tiller production and shoot growth to addition of moderate Na (50

mg Na/kg) in either one or both compartments of the split-root system was comparable to that

at 100 mg K/kg supply. This finding suggests that Na addition to K deficient medium

stimulated shoot growth in wheat regardless of the presence of K and Na in same or different

parts of the root system.

70

Fig. 4.3 Wheat (cv. Wyalkatchem) at six weeks after transplanting grown in a split-root

system treated with 40 mg K/kg and nil, or 50 mg Na/kg. The image shows the growth

difference with and without Na addition (50 mg Na/kg).

At low soil K, addition of moderate Na even to one of the compartments stimulated root

growth and also boosted root growth in the adjoining compartment with nil Na. Interestingly,

moderate Na addition to both compartments stimulated root growth at adequate soil K by 70

% when compared with nil Na treatment, and root: shoot ratio was also significantly higher.

Moderate salt (50 mg Na/kg) was beneficial to root development in wheat even when there

was adequate soil K. The stimulation of root growth by 50 mg Na/kg was in contrast to a

recent study by Ma et al. (2011) who reported adverse effects of soil 100 mg Na/kg on root

growth in wheat (cv. Wyalkatchem) at either deficient or adequate K supply. Soil Na

concentration at which Na stimulated wheat root growth in this experiment was in range of

Na levels (25 to 50 mg Na/kg) found to stimulate root growth in the earlier (Chapter 3)

experiment. Interestingly, NaCl induced growth stimulation in the split-root system of this

study was considerably higher than that previously measured in wheat (Box & Schachtman,

2000; Ma et al., 2011). In the previous pot experiment, the shoot dry weight showed no

71

significant increase while root dry weight of K-efficient cultivars in particular increased

significantly with low to moderate Na added to K-deficient soils (Chapter 3). Although,

similar low K treatment of 40 mg K/kg was used in both the experiments, and root to soil

mass ratio was almost the same (1: 2.1 in pot and 1: 2.0 in split-root), the K deficiency

symptoms were seen earlier (3- 4 weeks after transplanting) and severe in the split-root

experiment with K-efficient Wyalkatchem, and hence there appeared to be a stronger shoot

and root growth stimulation due to Na.

There was a significant increase in leaf gas exchange in the presence of moderate Na in one

or both compartments at low soil K supply, compared with nil Na plants which showed

obvious K deficiency symptoms, e.g. yellow and droopy leaves. At low K, moderate Na

increased net photosynthesis, stomatal conductance and transpiration rate in leaves by more

than three times, and the results was comparable to that by adequate K. Researchers suggest

that Na is beneficial in non-specific ionic roles in cell vacuoles, as an osmoticum at deficient

K (Marschner, 1995; Subbarao et al., 2003). Moreover, the presence of moderate Na at one or

both compartments at low K would be beneficial to physiological properties like stomatal

conductance which could improve plant water relations and growth (Ma et al., 2011).

However, non-specific ionic roles of Na in cell vacuoles would not explain increased net

photosynthesis with added Na at low K supply.

High shoot K concentrations at low K in combination with nil Na at both compartments and

high Na were mainly due to the ‘concentration- effect’ as a result of growth reduction since in

these treatments plant K accumulation was considerably lower than other treatments. Shoot

dry weight at low soil K supply was inversely correlated to shoot K concentration (Fig. 4.4).

Wheat grown in low K and nil Na compartments had adequate shoot K, however, there was

very low K in roots (~0.3 %) which would have restricted root growth and hence access to

nutrients and water due to poorly developed root systems.

72

Fig. 4.4 Correlation between shoot dry weight/plant (g) harvested at 6 weeks after

transplanting and the shoot K concentration (mg K/g, dry weight) measured in low soil K (40

mg K/kg) split-root treatments.

The results showed that K uptake at low soil K was significantly stimulated by addition of

moderate Na (50 mg Na/kg equivalent to ~15 mM Na in soil solution) either to one or both

compartments. A similar increase in K uptake due to Na was noticed by other researchers

(Idowu & Aduayi, 2007; Walker et al., 2000; Zhang et al., 2006). In tomato, however, shoot

K uptake was increased at much lower soil Na concentration of 4 to 16 mg Na/kg, and it

decreased at 32 mg Na/kg (Idowu & Aduayi, 2007), and in solution culture experiment, there

was increase in plant K uptake with 1 or 5 mM Na addition (Walker et al., 2000) and in

cotton field experiment, there was increase in K uptake from soil when topsoil Na

concentration was 65 mg Na/kg (Zhang et al., 2006). Increased K uptake in wheat cv.

Wyalkatchem was found to be consistent with previous experiment where there was

significant increase in shoot K uptake in K-efficient cultivars Wyalkatchem and Cranbrook

with low to moderate Na addition (Chapter 3).

The increase in K uptake in this study could be due to two main possibilities: firstly, an

increase in plant-availability of soil K due to added soil Na and secondly, increased K uptake

due to K uptake transporters. A soil incubation study with the present soil showed the change

in exchangeable K was not significant (Chapter 3). For a split-pot with total 4 kg of soil, an

y = -26.498x + 48.923R=-0.89

R² = 0.7896

0

10

20

30

40

50

60

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Sho

ot K

con

cent

ratio

n (m

g/g)

Shoot dry weight/plant (g)

Correlation made in low K (40 mg K/kg) and three Na (0, 50 and 200 mg Na/kg) split-root treatments

Shoot dry weight vs Shoot K concentrations

73

increase of 12 mg Colwell K with 50 mg Na/kg could provide an extra 6 mg K per plant

which could at best explain only a part of increase in K uptake due to soil Na.

One of the high affinity K+ uptake mechanisms is a Na+- energized high- affinity K+ symport

HKT1 (Schachtman & Liu, 1999), and it may play an important role in K+ acquisition when

external Na+ concentrations are low (Box & Schachtman, 2000; Rubio et al., 1995). The

capacity to take up Na+ in soils at low K supply may be an evolutionary advantage that plants

have developed, and molecular approaches have identified high-affinity K+ and Na+ transport

systems for various species including rice, barley and wheat (Rodriguez-Navarro & Rubio,

2006). It was claimed that high-affinity K+ uptake was activated at micromolar Na+

concentrations, while at physiologically detrimental concentrations of Na+, K+ uptake

mediated by HKT1 was blocked and low-affinity Na+ uptake occurred, which correlated to

Na+ toxicity in plants (Rubio et al., 1995). However, recent evidence suggests that HKT

select Na+ over K+ and functions as a Na+ uniporter (Benito et al., 2014). The role of low-

affinity K+ uptake mechanism is also been suggested to increase K+ uptake at low external

Na+ concentrations, however detailed studies to directly demonstrate this mechanism are

lacking (Shabala & Cuin, 2008).

Wheat roots concentrated more Na than shoots in this experiment with Na addition which

was consistent with an earlier experiment (Chapter 3). In the split-root experiment, there was

translocation of Na to the nil Na compartment from adjoining compartments with Na, as the

roots in nil Na had significantly higher Na concentration when there was Na in adjoining

compartment than roots grown in nil Na in both compartments (Table 4.6). However, the

roots grown in the Na compartment had significantly higher Na concentration than those in

the adjoining compartment without Na. Since Na is classified as a mobile element in plant

phloem (Marschner, 1995), the presence of Na+ could activate K+ uptake channels and

transporters even in the adjoining compartments without Na. In plants, the pathways of Na

entry into plants are not widely researched (Kronzucker & Britto, 2011) and highly Na+-

selective channels have not been found (Schachtman & Liu, 1999). It is believed that because

of thermodynamics and interactions between K and Na uptake, it is possible that Na+ enters

the cell cytoplasm through K+ channels, and transport proteins like HKT1, KUP or HAK,

NSC (Non-selective cation channels), LCT (low-affinity cation transporter) transports both

K+ and Na+ into the plants (Kronzucker & Britto, 2011; Schachtman & Liu, 1999).

In this experiment, there was inhibition of wheat growth at low soil K without soil Na. The

beneficial role of Na+ in cv. Wyalkatchem was evident with significant increase in plant K+

74

uptake, leaf gas exchange measurements and plant growth. Although, moderate soil Na

addition did not increase shoot Na concentration to levels that could replace K+ functions,

there was significant increase in wheat K+ uptake due to added Na+. This increased shoot K

uptake may explain the increased leaf net photosynthesis rate and stomatal conductance with

Na addition and could promote carbon assimilate supply to roots which in turn could

stimulate root growth, and access to more soil K, water and other nutrients from soil.

Moreover, the addition of moderate rates of Na+ at deficient soil K supply has stimulatory

effects on roots and shoots, irrespective of whether K and Na are present in the same or

different parts of the roots.

Further detailed investigation in solution culture is warranted on whether the presence of low

external concentrations of Na+ increases K+ uptake in wheat under K deficiency. Such

information would be of greater use to understand K and Na interactions and uptake

particularly because the effect of Na on exchangeable K availability in growing media would

be eliminated and using a tracer like Rb+ in nutrient solution could help test effects of

external Na on K+ uptake more accurately.

4.5 Conclusion

Moderate Na level (50 mg Na/kg) was beneficial for wheat cv. Wyalkatchem grown under K

deficiency (40 mg K/kg) and Na stimulation of growth by low-K plants occurred regardless

of the supply of K and Na in the same or different parts of the root system. Moderate Na

eliminated the effects of low soil K supply on shoot and root growth and produced dry matter

similar to adequate K supply. There was significant increase in leaf photosynthesis, stomatal

conductance and plant K uptake due to moderate Na addition, but probably mediated by

increased shoot K uptake. A detailed solution culture based study using tracers can help to

validate Na+-induced K+ uptake in wheat cultivars more directly (Chapters 6 and 7).

75

CHAPTER 5

COLUMN EXPERIMENT

Potassium response of wheat grown in columns with drying topsoil and varying subsoil

K and Na levels

5.1 Introduction

Large agricultural areas of the world are reported to be K deficient and the problem is

exacerbated by continued removal of grain and straw from crop fields and this is limiting

cereal production (Ma et al., 2011). Potassium concentrations measured in topsoils of south-

west Western Australia (SWA) showed 55 % of samples had Colwell K < 100 mg/kg, 26 %

had < 40 mg/kg and 11 % had < 20 mg/kg (McArthur, 1991). Moreover, there is an

increasing demand for K fertiliser usage due to the expansion of intensive agriculture

(Römheld & Kirkby, 2010; Zörb et al., 2014). The annual growth rate of the global demand

for K fertilisers is estimated to be 3.8 % for the period 2011- 2014 and, the cost for K

fertilisers is increasing as the reserves are concentrated in a few countries (Miyamoto et al.,

2012). It is therefore important to increase the efficiency of K fertiliser use by the crops

without decreasing crop yields.

Sodium is not an essential element for all higher plants, however, application of Na+ was

found to be beneficial in some cellular functions when K is present at suboptimal

concentrations (Ma et al., 2011; Miyamoto et al., 2012; Subbarao et al., 2003). It is believed

that Na+ could substitute for K+ in its role as an enzyme activator in vacuoles, and its

accumulation could release K+ to fulfil other more specific biochemical functions, reducing

K+ requirements (Rodriguez-Navarro & Rubio, 2006; Walker et al., 2000). The extent of K

substitution and growth stimulation by Na are of great interest for crop production and K

fertiliser management (Mäser et al., 2002). The use of Na by plants may be particularly

important in saline-sodic soils where K+ and Na+ compete for uptake (Box & Schachtman,

2000). My previous glasshouse experiments (Chapter 3 and 4) suggest that Na at low to

moderate concentrations are beneficial to wheat root growth under K deficient conditions and

alleviated leaf symptoms of K deficiency.

Duplex/ texture-contrast soils account for about 12 % of the land area of Australia (Dracup et

al., 1992) and are a major soil constraint for crop production. The nutrient distribution in

duplex soils varies with depth in contrast to that of uniform soil profiles. The duplex soils of

western Australia (WA) have varying concentrations of K with depth due to differences in

76

clay content and mineralogy (Wong et al., 2000). For many duplex soils in WA, both surface

and subsoil layers are low in extractable K, as the dominant clay mineral is kaolinite, which

naturally has a low K content (Brennan & Bell, 2013).

In the no-till farming systems, K can be stratified within the fertilised topsoil. It is common

also for profiles with low K to contain significant exchangeable Na in the subsoil (Q. Ma and

R. Bell, unpublished data). In SWA, varying levels of subsoil K and Na from that of topsoil

are common in the duplex soil, but the influence of such heterogeneity on crop K nutrition

has not been examined. Topsoil drying/ drought are also common occurrences during the

growing season in rain-fed conditions of SWA. The presence of shallow and localised K may

limit plant K uptake in such water-limited environments since the topsoil is prone to drying,

because water deficit reduces not only root growth but also K diffusion. Understanding K

response under such conditions can help to manage K application strategies and can increase

grain productivity in rainfed cropping systems.

A column experiment was conducted to investigate the effect of subsoil Na and K on K

substitution by Na and crop K nutrition when K uptake from the topsoil is restricted by water

deficit. In this study, wheat was grown in soil columns with different K levels in the topsoil

but low to moderate Na supply in the subsoil. The potential interaction of subsoil Na and K

was assessed under the condition of dry topsoil. Potassium deficiency has detrimental effects

on wheat root development when compared to shoots (e.g. Ma et al. (2011)). In previous

experiments, addition of moderate levels of Na (25 and 50 mg K/kg) stimulated root growth

significantly under K deficient conditions (Chapters 3), and the beneficial effect was evident

even when K and Na were present at different parts of the root system (Chapter 4). It was

hypothesised based on previous experiments (Chapters 3 and 4) that subsoil Na may make a

positive contribution to growth (root growth) when plants have limited K supply. The

hypothesis was investigated under well-watered and topsoil dry conditions.


Wheat (Triticum aestivum L.) cv. Wyalkatchem was grown in a naturally-lit glasshouse at

Murdoch University, Perth, (32°04′S, 115°50′E) from mid winter to early spring. Soil was

collected from an unfertilised paddock at Northam (classified as Chromosols), and had the

following properties: pH (CaCl2) 5.2; EC1:5 0.03 dS/m; 3.56 mg NH4-N/kg and 4.8 mg NO3-

N/kg (Searle, 1984); 20 mg K/kg, 2.5 mg S/kg and 1.2 mg Al/kg; 0.37 % organic C (Walkley

& Black, 1934). The air-dried soil was thoroughly mixed with basal nutrients and treatments

77

of K and Na and then filled into specially structured PVC columns (about 10 kg/column).

Each column (80 cm depth and 10 cm diameter) was formed of two halves of a PVC cylinder

that was split vertically and held together with adhesive tape. Basal nutrients were applied at

the following rates (mg/kg): 237 Ca(NO3)2.4H2O, 103 (NH4)2HPO4, 80 MgSO4.7H2O, 18

FeSO4.7H2O, 14 MnSO4.H2O, 9 ZnSO4.7H2O, 8.3 CuSO4.5H2O, 0.33 H3BO3, 0.3

CoSO4.7H2O, 0.33 Na2MoO4.2H2O.

5.2.1 Treatments

Two levels of K (40, 120 mg K/kg) in the topsoil were combined with three levels of subsoil

Na (0, 50 and 200 mg Na/kg). Half of the columns were well-watered throughout, but the

topsoil of the remaining columns was dried by withholding watering from 5 weeks after

sowing. All treatments were replicated three times, totalling 72 columns (Table 5.1). In the

columns, topsoil (0- 15 cm of the column) had a mixture of basal nutrients and two K

treatments (40 and 120 mg K/kg) with top 3- 4 cm left free for watering. The buffer layer

(15- 20 cm) was filled with basal nutrients, and the subsoil (20- 80 cm) with basal nutrients,

three Na treatments, two levels of K in topsoil dry columns (40 and 120 mg K/kg) or one

level of K in topsoil wet columns (40 mg K/kg). A plastic tube (1 cm diameter) with holes

made at 20, 25, 30, 35 and 40 cm depth and closed at the bottom was inserted into the

columns to water the subsoil only (Fig. 5.1). This was mainly to prevent wetting of topsoil

from subsoil watering alone and to prevent leaching of nutrients, particularly K, from the

topsoil. During the course of experiment (including initial 5 weeks), the subsoil was always

watered using the plastic tube inserted and care was taken to add just enough water to wet

topsoil and over watering was avoided to minimise leaching to subsoil.

Six seeds were sown per column, and after emergence the seedlings were thinned to three per

column and one week later the seedlings were further thinned to two per column. The plants

were harvested at either 5 or 11 weeks after sowing. Among the 72 columns, 18 columns

were harvested at 5 weeks after sowing before topsoil drying. The remainder of columns

were split into topsoil wet (18) and topsoil dry (36) columns (Table 5.1). Topsoil wet

columns were continuously watered with DI water to field capacity (14 % w/w), while topsoil

dry columns had water withheld from 5 weeks after sowing. The subsoils of all columns

received DI water to FC through the inserted tubes. Final harvest was made at 11 weeks after

sowing.

78

Table 5.1 Experiment design showing topsoil watering, topsoil K (mg K/kg) and subsoil K

(mg K/kg) and Na (mg Na/kg) treatments harvested at 5 and 11 weeks after sowing.

Topsoil watering and harvest Topsoil K

mg K/kg

Subsoil K

mg K/kg

Subsoil Na

mg Na/kg

No. of columns

Well-watered

(harvest at 5 weeks after sowing) 40, 120

40

0, 50 and 200

18

Well-watered

(harvest at 11 weeks after sowing)

40, 120

40

0, 50 and 200

18

Dry

(watering withheld from 5 weeks after sowing to harvest at 11 weeks

after sowing)

40, 120

40 and 120

0, 50 and 200

36

Fig. 5.1 Column experiment of wheat cv. Wyalkatchem at 3 weeks after sowing (left).

Column set-up with plastic tubes used for subsoil watering, commencing at 5 weeks after

sowing (right)

79

5.2.2 Measurements

Plant phenology was recorded weekly throughout the experiment. Leaf net photosynthesis,

stomatal conductance and transpiration rate were measured at 5, 7 and 10 weeks after sowing,

using the LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The

corresponding growth stages at measurements were Z 16, 27 (5 weeks), Z 19, 29, 36 (7

weeks) and Z 19, 29, 53 (10 weeks) identified using Zadoks growth scale for cereals. The

measurements were made on fully expanded young leaves at ambient relative humidity of 50


radiation of 1500 µmol/m2·s. Shoot fresh weight was recorded immediately after harvest, and

the shoot were separated into young leaves (top 2-3 leaves in each tiller), old leaves (basal 3-

5 on main tiller), rest of the shoot and ears. The growth stage at the time of harvest was Z 19,

29, 61. Then the column was split open to observe root development at various depths in the

column by separating roots at 0- 20, 20- 40 and 40- 80 cm. The roots were collected on a 2

mm sieve after washing in tap water and rinsing with DI water. The shoot and root samples

were dried in an oven for 48 hours at 60˚C and dry weights were recorded. The samples were

then milled for K and Na analysis following methods described in the previous chapter

(Chapter 4).


Statistical analyses were conducted using SPSS 18.0. Two-way analysis of variance was

conducted to assess the effects of soil K and Na supply and their interactions at 5 weeks and

11 weeks harvest. Repeated measures ANOVA was used to analyse leaf gas exchange

parameters as there were three measurements made in columns at 5, 7 and 10 weeks after

sowing. Tukey’s HSD was computed at P ≤ 0.05 for pair-wise comparison of means.

5.3 Results

5.3.1 Plant growth

Initial harvest- 5 weeks after sowing

Shoot dry weight at the initial harvest (5 weeks) was unaffected by treatments except for a

significant reduction with addition of high Na in the subsoil (200 mg Na/kg) when low soil K

was applied throughout the soil profile. There was no significant change in shoot dry weight

with the addition of 50 mg Na/kg in the subsoil (P > 0.05; Table 5.2). More tillers were

produced with high topsoil K and nil subsoil Na. There was a significant reduction (P ≤ 0.05)

in tiller number with high subsoil Na and low subsoil K (Fig. 5.2 and 5.3). The potassium

80

effect was significant for shoot dry weight and tiller numbers harvested at 5 weeks after

sowing (P ≤ 0.05), while the interaction between K and subsoil Na was not significant for

shoot dry weight (Table 5.2).

Soil K

40/40(W) 120/40(W)

Tille

rs/ p

lant

0

2

4

6

0 Na 50 Na 200 Na

Shoot Dry weight and tillers- harvest @ 5 WASsh

oot d

ry w

t/ pl

ant (

g)

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 5.2 Shoot dry weight (g) and tiller number per plant at 5 weeks after sowing (±SE, n=3).

For treatment descriptions refer to Table 5.1. See Table 5.2 for statistical analysis.

81

Fig. 5.3 Columns were supplied with low K (40 mg K/kg) in the whole profile with varying

subsoil Na levels: a) nil Na, b) 50 mg Na/kg, and c) 200 mg Na/kg. Shoot growth and

tillering was depressed by 200 mg Na/kg at 5 weeks after sowing.

Table 5.2 Statistical summary of plant growth at 5 and 11 weeks after sowing treated with

two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg

Na/kg). For treatment details refer to Table 5.1.


5 weeks after sowing 11 weeks after sowing

Parameters Na K Na*K Na Treatment

(combination of K and water)

Na*treatment

Shoot dry wt/plant

* ** n.s *** ** n.s

No of tillers/plant *** *** * *** ** n.s

Root dry wt/plant n.s * n.s *** *** *

Root: shoot ratio n.s n.s n.s *** n.s n.s

The main effect of K treatment was significant for root dry weight harvested at 5 weeks after

sowing (Table 5.2) but presence of subsoil Na did not had an effect on root dry weight (Fig.

a b c

82

5.4; Table 5.2). In the column, the top section (0-20 cm) had more root dry weight than other

sections. Root: shoot ratios ranged from 0.37 to 0.4 with no significant difference among the

treatments (Appendix 2).

0 Na

0.00

0.05

0.10

0.15

0.20

0.25

0.30

50 Na

40/40(W) 120/40(W)

Roo

t dry

wei

ght/

plan

t(g)

0.00

0.05

0.10

0.15

0.20

0.25

200 Na

Soil K

40/40(W) 120/40(W)

0.00

0.05

0.10

0.15

0.20

0.25

40- 60 cm20- 40 cm0- 20 cm

Fig. 5.4 Root dry weight (g/plant) of wheat cv. Wyalkatchem in different sections of column

(0- 20, 20- 40 and 40- 60 cm) at 5 weeks after sowing. For treatment descriptions refer to

Table 5.1. See Table 5.2 for statistical analysis.

Final harvest- 11 weeks after sowing

Shoot dry weight measured 11 weeks after sowing did not show significant difference

between 40 and 120 mg K/kg treatments, and ranged from 12.2 to 13.7 g/plant (averaged

across Na levels and water treatments). However, low soil K throughout the profile in

combination with dry topsoil resulted in the lowest shoot biomass among all the treatments.

The plants grown in high subsoil K had higher biomass than high soil K in topsoil only when

topsoil was kept dry. In this experiment, the combination of K and water treatments had a

significant effect but its interaction with subsoil Na was not significant for shoot dry weight

83

at 11 weeks harvest (P > 0.05) (Table 5.2). The addition of moderate subsoil Na to low K

profile did not change shoot dry weight significantly either in topsoil dry and wet treatments.

Tiller production increased when there was moderate subsoil Na with low soil K in the whole

profile regardless of wet or dry topsoil (Fig. 5.5). However, high subsoil Na decreased the

number of tillers in all treatments. There was no difference in tillering between well-watered

and dry topsoil profiles (Table 5.2).

Total root dry weight was relatively high when there was high soil K throughout the profile

and reduced in the dry topsoil columns in presence of low soil K throughout the soil profile

(Fig. 5.6). There was significant reduction in root dry weight when there was high subsoil Na

(200 mg Na/kg) with low soil K throughout the profile. Among the sections, the top 0- 20 cm

of the soil column had almost 65 % of the total root distribution. The root dry matter in 40-

80 cm section of the column decreased due to topsoil drying with low soil K throughout the

profile, especially with addition of subsoil Na. The presence of high subsoil Na reduced root

dry weight when there was 120 mg K/kg either in topsoil or subsoil of the soil profile with

significant interaction between Na and treatments (P ≤ 0.05) (Table 5.2). Root: shoot ratios in

the nil and moderate subsoil Na columns were almost the same irrespective of difference in

soil K levels and watering (Fig. 5.7). However, at high subsoil Na, the ratio was lowered

significantly.

84

Shoot Dry weightsh

oot d

ry w

t/ pl

ant (

g)

0

2

4

6

8

10

12

14

16

whole profile wet Top soil dry, sub soil wet

No. of tillers

Soil K mg/kg

40/40(W) 120/40(W) 40/40 120/40 40/120 120/120

Tille

rs/ p

lant

0

2

4

6

8

0 Na 50 Na 200 Na

Fig. 5.5 Shoot dry weight (g) and tillers per plant at 11 weeks after sowing (±SE, n=3). For

treatment descriptions refer to Table 5.1 and see Table 5.2 for statistical analysis.

85

0 Na

0

2

4

6

8

50 Na

Roo

t dry

wei

ght/

plan

t (g)

0

2

4

6

200 Na

Soil K levels

40/40(W) 120/40(W) 40/40 120/40 40/120 120/120

0

2

4

6

40-80 20-40 0-20

whole profile wet topsoil dry, subsoil wet

Root dry weight

Fig. 5.6 Root dry weight (g/plant) in different sections of column (0- 20, 20- 40 and 40- 60

cm) at 11 weeks after sowing. For treatment descriptions refer to Table 5.1.

86

Root: Shoot

Soil K

40/40(W) 120/40(W) 40/40 120/40 40/120 120/120

root

: sho

ot ra

tio

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 Na 50 Na 200 Na


Fig. 5.7 Root: shoot ratios of wheat at 11 weeks after sowing (±SE, n=3). For treatment

descriptions refer to Table 5.1 and Table 5.2 for statistical analysis.


Leaf gas exchange parameters were measured at 5, 7 and 10 weeks after sowing (WAS).

Photosynthesis and stomatal conductance at 5 WAS showed a significant reduction in

presence of high subsoil Na (200 mg Na/kg) compared to nil and moderate Na levels in

subsoil (Fig. 5.8). The main effect of K and watering treatments for leaf net photosynthesis

was not significant (P > 0.05), and neither was the interaction of subsoil Na with K and

watering treatments (Table 5.3). However, stomatal conductance showed a significant

interaction (P ≤ 0.05). Stomatal conductance at 5 WAS reduced considerably with dry topsoil

and low soil K throughout the profile with all levels of subsoil Na when compared with well-

watered high K topsoil without Na. Low K and wet soil profile with high subsoil Na had the

low stomatal conductance among all treatments. The interaction of Na with K and watering

treatments was not significant for transpiration (Table 5.3).

87

At 7 WAS, high soil K throughout the profile had significantly higher photosynthesis rate,

stomatal conductance and transpiration compared with low soil K throughout the profile,

especially when the profile had dry topsoil (Fig. 5.9). Addition of high subsoil Na lowered

photosynthesis, especially when there was low K throughout the profile and the interaction

between subsoil Na and treatment was significant for photosynthesis measured at 7 WAS (P

≤ 0.05; Table 5.3). Although the Na and treatment effect was significant for photosynthesis,

there was no interaction for stomatal conductance and transpiration (Table 5.3). Leaf gas

exchange measured at 10 WAS had a similar pattern to that observed at 7 WAS, except there

was significant interaction between subsoil Na and treatment for transpiration rate measured

at 10 WAS (Fig. 5.10 & Table 5.3). Irrespective of the treatments, the rate of photosynthesis,

stomatal conductance, and transpiration decreased considerably in later growth stages.

88

Transpiration -5 weeks

Soil K

40/40(W) 120/40(W) 40/40 120/40 40/120 120/1200

2

4

6

0 Na 50 Na 200 Na

Photosynthesis -5 WAS

Pho

tosy

nthe

sis

µmol

CO

2/m

2 .s

0

5

10

15

20

25

30

Transpiration -5 weeksStomatal conductance -5 WAS

Sto

mat

al c

ondu

ctan

ce

mm

olH

2O/m

2 .s

0

200

400

600

Transpiration -5 WAS

Tran

spira

tion

mm

olH

2O/m

2 .s

whole profile wet Topsoil dry, subsoil wet

Fig. 5.8 Leaf photosynthesis, stomatal conductance, and transpiration at 5 weeks after sowing

(±SE, n=3). For treatment descriptions refer Table 5.1 and for statistical analysis refer Table

5.3.

89

Photosynthesis- 7 WAS

Pho

tosy

nthe

sis

µmol

CO

2/m

2 .s

0

5

10

15

20

25

30

Stomatal conductance- 7 WAS

Sto

mat

al c

ondu

ctan

ce

mm

olH

2O/m

2 .s

0

200

400

600

Transpiration- 7 WAS

Soil K

40/40(W) 120/40(W) 40/40 120/40 40/120 120/120

Tran

spira

tion

mm

olH

2O/m

2 .s

0

2

4

6

0 Na 50 Na 200 Na

whole profile wet Topsoil dry, subsoil wet

Fig. 5.9 Leaf photosynthesis, stomatal conductance, and transpiration at 7 weeks after sowing

(±SE, n=3). For treatment descriptions refer Table 5.1 and Table 5.3 for statistical analysis.

90

Photosynthesis- 10 WAS

Pho

tosy

nthe

sis

µm

ol C

O2/

m2 .s

0

5

10

15

20

25

30

Stomatal conductance- 10 WAS

Sto

mat

al c

ondu

ctan

ce

mm

olH

2O/m

2 .s

0

200

400

600

Transpiration- 10 WAS

Soil K

40/40(W) 120/40(W) 40/40 120/40 40/120 120/120

Tran

spira

tion

mm

olH

2O/m

2 .s

0

2

4

6

0 Na 50 Na 200 Na


Fig. 5.10 Leaf photosynthesis, stomatal conductance, and transpiration at 10 weeks after

sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and Table 5.3 for statistical

analysis.

91

Table 5.3 Statistical summary of leaf gas exchange at 5, 7 and 10 weeks after sowing treated

with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200

mg Na/kg). For treatment details refer to Table 5.1.


Parameters Na Treatment

(combination of K and water treatments)

Na*Treatment

Gas exchange- 5 WAS

Photosynthesis * n.s n.s

Stomatal conductance *** *** *

Transpiration n.s * n.s

Gas exchange- 7 WAS

Photosynthesis n.s * **

Stomatal conductance ** *** n.s

Transpiration n.s *** n.s

Gas exchange- 10 WAS

Photosynthesis *** ** *

Stomatal conductance * *** n.s

Transpiration ** *** *

5.3.3 K and Na concentrations

Shoot K concentrations at 5 weeks after sowing were similar (ranging from 37 to 42 mg K/g,

dry weight) among the treatments (Table 5.4).

92

Table 5.4 Shoot K and Na concentrations and accumulation in wheat cv. Wyalkatchem

harvested at 5 weeks after sowing. Means (n=3) with different letters differ at P≤0.05.

Topsoil Soil K (mg/kg) Subsoil Na

(mg/kg)

Concentration (mg/g, plant dry wt)

Accumulation (mg/plant, dry wt basis)

Watering Topsoil K

Subsoil K

Shoot K Shoot Na

Shoot K Shoot Na

Wet 40 40 0 40.3a 0.13c 25.7ab 0.08c

50 39.7a 0.52bc 26.0ab 0.34bc

200 37.2a 1.34a 18.2b 0.65a

Wet 120 40 0 41.9a 0.12c 31.4a 0.09c

50 40.5a 0.39c 26.1ab 0.25bc

200 40.3a 0.89ab 24.1ab 0.53ab

At 5 weeks, wheat grown at low soil K throughout the profile with high subsoil Na

accumulated less K than high topsoil K column with nil Na (Table 5.4). However, addition of

subsoil Na did not have a significant effect on shoot K concentration (P > 0.05, Table 5.5).

Shoot Na concentrations were dependent on subsoil Na levels and the highest concentration

was with high subsoil Na in a low soil K column among the treatments. Similarly, shoot Na

accumulation was much higher with high subsoil Na (200 mg Na/kg) in low K soil profile

than nil and 50 mg Na/kg subsoil Na (Table 5.4).

93

Table 5.5 Statistical summary of shoot K and Na concentrations and content at 5 and 11

weeks after sowing in wheat plants treated with two levels of soil K (40 and 120 mg K/kg)

and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table

5.1. Note only whole shoots and roots were analysed at 5 weeks.


5 weeks after sowing 11 weeks after sowing

Parameters Na Treatment


Na × treatment

Na Treatment


Na × treatment

Shoot K concentration

n.s n.s n.s

Shoot Na concentration

* *** n.s

K concentration- Ears

** *** n.s

K concentration- young leaves

*** *** **

K concentration- old leaves

*** *** *

Na concentration- Ears

*** *** *

Na concentration- young leaves

*** *** ***

Na concentration- old leaves

*** *** ***

Shoot K accumulation

* n.s n.s *** *** **

Shoot Na accumulation

*** n.s n.s *** *** ***

Potassium concentration in ears/spikes was significantly decreased by Na (P ≤ 0.05) (Table

5.5 and 5.6). High soil K throughout the profile without subsoil Na had significantly higher K

94

concentration in spikes than low soil K throughout the profile irrespective of topsoil drying

and subsoil Na levels (Table 5.6). Soil K supply increased K concentrations in young and old

leaves. Under topsoil dry conditions, wheat grown in high soil K (120 mg K/kg) throughout

the profile irrespective of subsoil Na, and those grown in high subsoil K with nil and

moderate subsoil Na had significantly higher K concentrations in young and old leaves than

low K throughout the profile (Table 5.6). Potassium concentration in old leaves was

significantly lower when there were low soil K throughout the soil profile at all Na levels

than high K profiles in both topsoil dry and wet conditions. Old leaves had considerably

lower K compared with young leaves particularly at low K supply throughout the profile. The

addition of moderate subsoil Na did not alter K concentrations in any of the treatments.

Wheat grown in low soil K throughout the profile irrespective of subsoil Na accumulated

significantly less shoot K than treatments that had high subsoil K. The interaction between

subsoil Na and treatment was significant for shoot K content in columns harvested at 11

WAS (P ≤ 0.05, Table 5.4), and the shoot K accumulation in treatments with high subsoil K

and nil and 50 mg Na/kg subsoil Na was significantly higher than other treatments (Table

5.7).

Subsoil Na levels largely determined the shoot Na concentration and accumulation (Table 5.7

and 5.8), and the interaction between subsoil Na and treatment was significant for Na

concentration in ears and leaves, and shoot content harvested at 11 WAS (P ≤ 0.05, Table

5.5). Shoot Na accumulation increased significantly with addition of moderate or high subsoil

Na in the low subsoil K treatments (40 mg K/kg) but was significantly lowered by high

subsoil K even with moderate and high subsoil Na (Table 5.7). In contrast to K

concentrations, old leaves concentrated considerably more Na than ears and young leaves.

95

Table 5.6 K concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11

weeks after sowing. Means (n=3) with different letters differ at P≤0.05.

Topsoil Soil K (mg/kg) Subsoil Na (mg/kg)

K concentration (mg/g, plant dry weight)

Watering Topsoil K Subsoil K ears old leaves young leaves

Wet 40 40 0 12.8bcde 2.66e 9.86defg

50 12.4cde 2.64e 9.73defg

200 12.2cde 2.29e 9.22efg

Wet 120 40 0 13.5abcde 9.18cd 13.7cd

50 13.8abcd 9.85cd 13.9cd

200 12.6cde 8.33d 12.5cdef

Dry 40 40 0 12.7bcde 2.58e 9.71defg

50 12.1de 2.75e 8.06g

200 11.8e 2.40e 8.59fg

Dry 120 40 0 12.7bcde 8.49d 12.6cdef

50 13.1bcde 9.79cd 13.5cde

200 12.4cde 8.19d 12.2cdefg

Dry 40 120 0 14.7ab 17.9a 19.4b

50 14.0abcd 18.4a 19.2b

200 14.1abc 12.8bc 16.5bc

Dry 120 120 0 15.1a 19.4a 25.0a

50 14.7ab 18.5a 24.0a

200 13.8abcd 15.7ab 18.9b

96

Table 5.7 Shoot K and Na accumulation in wheat cv. Wyalkatchem harvested at 11 weeks

after sowing. Values are means of 3 replicates. Means (n=3) with different letters differ at

P≤0.05.


Accumulation (mg/plant, dry wt basis)

Watering Topsoil K Subsoil K Shoot K Shoot Na

Wet 40 40 0 130fgh 3.45e

50 124gh 14.3bcd

200 110h 21.1ab

Wet 120 40 0 175de 2.01e

50 177de 15.9bc

200 150defg 17.2abc

Dry 40 40 0 115gh 5.66e

50 116gh 17.7abc

200 110h 24.5a

Dry 120 40 0 152defg 3.17e

50 164def 13.3cd

200 139efgh 15.2bc

Dry 40 120 0 224bc 0.96e

50 226bc 3.55e

200 174de 7.44de

Dry 120 120 0 266a 0.88e

50 243ab 1.55e

200 188cd 4.40e

97

Table 5.8 Na concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11

weeks after sowing. Means (n=3) with different letters differ at P≤0.05.


Na concentration (mg/g, plant dry weight)

Watering Topsoil K Subsoil K ears old leaves young leaves

Wet 40 40 0 0.08cd 1.13fg 0.06e

50 0.12cd 4.04cd 0.26de

200 0.32ab 6.06b 0.72ab

Wet 120 40 0 0.06d 0.39g 0.08e

50 0.10cd 2.79de 0.39bcde

200 0.24bcd 4.61c 0.63abc

Dry 40 40 0 0.10cd 1.39efg 0.08e

50 0.24bcd 4.94bc 0.47bcd

200 0.43a 7.99a 0.88a

Dry 120 40 0 0.05d 0.91g 0.08e

50 0.08cd 4.11cd 0.20de

200 0.24bcd 5.44bc 0.74ab

Dry 40 120 0 0.08cd 0.07g 0.05e

50 0.10cd 0.55g 0.23de

200 0.10cd 2.38ef 0.27cde

Dry 120 120 0 0.05d 0.07g 0.06e

50 0.08cd 0.14g 0.18de

200 0.15bcd 1.05fg 0.21de

Shoot K+/Na+ ratios were influenced by soil K and Na levels (Table 5.9). There was no

significant difference in ratios between topsoil wet and dry columns. The ratios were

increased by K supply, especially in subsoil, and reduced with the increase in subsoil Na

levels.

98

Table 5.9 Shoot K/Na ratios in wheat cv. Wyalkatchem harvested at 11 weeks after sowing.

Means (n=3) with different letters differ at P≤0.05.

Topsoil Soil K (mg/kg) Subsoil Na (mg/kg) Shoot K/Na

Watering Topsoil K Subsoil K

Wet 40 40 0 39.3efg

50 8.90fg

200 5.50g

Wet 120 40 0 90.0d

50 11.9fg

200 9.00fg

Dry 40 40 0 21.4fg

50 6.80g

200 4.50g

Dry 120 40 0 48.2ef

50 12.5fg

200 9.20fg

Dry 40 120 0 240b

50 65.7de

200 23.4fg

Dry 120 120 0 300a

50 160c

200 44.0efg

99

5.4 Discussion

Potassium and Na co-exist on the soil exchange complex and in soil solution, and they may

exert antagonistic or synergistic effects on absorption and translocation of each other within

plants, particularly under saline and sodic conditions (Hussain et al., 2013). Potassium is an

essential nutrient for plant physiology, and higher plants require large amounts of K which

cannot be totally replaced by Na addition. However the capacity of wheat to respond

positively to NaCl applications might have important consequences in wheat K management

in saline and sodic soils. The addition of low to moderate Na (25 to 50 mg Na/kg) to K-

deficient soil enhanced wheat growth, leaf gas exchange measurements and K uptake in

previous experiments (Chapter 3 and 4). In the current experiment, the effect of varied

subsoil K and Na which commonly occur in duplex soils was examined under both well-

watered and topsoil dry conditions.

Wheat growth stimulation due to moderate subsoil (50 mg Na/kg) Na when soil K supply was

limited throughout the profile was insignificant in this experiment which was in contrast to

the previous experiment where the presence of 50 mg Na/kg in one or both the compartments

in split-root increased the shoot and root dry weight significantly (Chapter 4). The negative

effect of Na was evident at 200 mg Na/kg, particularly when there was low K throughout the

profile. In a comparable study by Ma et al. (2011), the addition of 100 mg Na/kg to a K

deficient soil caused a slight but not significant decrease in shoot and root dry weight of cv.

Wyalkatchem while 300 mg Na/kg addition caused a significant reduction in wheat growth.

Potassium deficiency had greater effect on root growth than shoot growth in previous

experiments for wheat (Chapter 3) and also in barley (Degl'Innocenti et al., 2009; Ma et al.,

2011) where root/shoot ratios were generally higher at the adequate soil K levels than low K

levels. In the current column experiment, there was comparatively poor root development

when columns had low K, and the presence of moderate subsoil Na (50 mg Na/kg) did not

affect roots, whereas, high subsoil Na (200 mg Na/kg) further intensified the K-deficiency

effect on roots. In field studies, drought often increase root length and density especially, at

depths in soil in search of available water (Comas et al., 2013) and increases root/shoot ratios.

However, in this experiment there was no detailed scanning of roots to measure diameter,

density, length etc., and the presence of dry topsoil did not influence root dry weight mainly

because subsoil was watered regularly from 5 WAS which was sufficient for plants and there

was no significant water stress observed in the columns unlike topsoil dry field conditions.

100

Also the effect on root growth or root/shoot ratio depends on which occurs first, either K

deficiency or drought.

In this experiment, moderate subsoil salinity (50 mg Na/kg) did not alter leaf gas exchange

measurements. Similar to growth results discussed, this was in contrast to previous

glasshouse experiments (Chapter 3 and 4) where there was significant increase in the

measurements comparable to adequate K supply. The leaf gas exchange measurements were

not significantly altered in barley cultivars and wheat cv. Wyalkatchem with 100 mg Na/kg

addition to K deficient soil (Ma et al., 2011). High subsoil salinity reduced the leaf net

photosynthesis rate, stomatal conductance and transpiration rate of wheat. Leaf net

photosynthesis rate before 11 weeks after sowing was negatively correlated (R= -0.686) with

shoot Na concentrations (Fig. 5.11). Salinity can have negative effect on photosynthetic

process by an effect on stomatal closure that limits the CO2 diffusion to the chloroplasts

(Degl'Innocenti et al., 2009). Moreover, when utilization of absorbed light energy in CO2

fixation was restricted by salinity, the electron flux to O2 increased, resulting in an

accumulation of reactive oxygen species (ROS) in chloroplasts (Shabala et al., 1998).

Stomatal conductance of wheat grown in wet topsoil conditions was higher than with topsoil

dry. Stomatal conductance decreased with increase in water stress as leaf dehydration can

lead to turgor lose causing passive stomata closure and hence carbon entry, and consequently

the supply of CO2 to fixation site is reduced (Khakwani et al., 2012).

Higher K concentration in young leaves than old leaves in treatments with low soil K

throughout the profile suggests that there was effective translocation of K+ from old to young

leaves. However, there was no significant effect of added subsoil Na on K concentrations.

The presence of moderate subsoil Na (50 mg/kg) did not affect K+ uptake which was in

contrast to the previous split-root experiment where K+ uptake at low soil K was significantly

increased due to added Na+. Similar to results of this study, the presence of Na+ did not

increase K+ uptake of rice (Yoshida & Castaneda, 1969), wheat (Box & Schachtman, 2000),

and tomato (Walker et al., 2000) grown under solution culture in various other experiments

although an increase in dry weight due to added NaCl at deficient K levels was noticed. At

high subsoil Na (200 mg/kg), an antagonistic relationship developed between K and Na, and

Na+ inhibited plant K+ uptake.

101

Fig. 5.11 Correlation between leaf net photosynthesis rates and shoot Na concentrations (mg

Na/g, dry weight) at final harvest at 11 weeks after sowing.

Compared to the previous experiments, the columns of this experiment contained more soil

(10 kg/column) and hence more available K for plants. The available K per column with two

wheat plants at low K (40 mg/kg) soil treatments was 400 mg. The supply of 200 mg K/plant

was 2.5 times greater than in the previous pot and split-root experiments (available K/plant=

80 mg) (Chapters 3 and 4). This may explain why the K deficiency symptoms at low K

treatments were absent as were substitution effects of Na at low K in this experiment.

Sodium-enhanced plant growth was earlier considered accidental or under weak

physiological regulation, however, recent studies on characterisation of high-affinity Na+

uptake in several plants when K+ is exhausted strongly suggest there is physiologically

programmed role of Na+ in some plants under insufficient K+ supply (Subbarao et al., 2003;

Wakeel et al., 2011). In this study to explore the possibility of K substitution of Na in wheat,

the beneficial effects of low to moderate Na was considerably greater in previous

experiments when growth was limited due to deficient K supply and wheat showed moderate

to severe K deficiency symptoms. This makes it clear that the synergistic or antagonistic

effect between K and Na depends on the amount of K and Na present in the soil (Ali et al.,

2013). In the presence of sufficient K already in the soil, the addition of subsoil Na had little

effect on wheat growth or the substitution of K functions.

R² = 0.4702

y = -2.638x + 17.696

8

10

12

14

16

18

20

22

0 0.5 1 1.5 2 2.5

Pho

tosy

nthe

sis

(µm

ol C

O 2/m

2 .s)

Shoot Na concentration (mg/g, plant dry weight)

Photosynthesis vs Shoot Na concentration

102

5.5 Conclusion

The presence of moderate levels of subsoil Na (50 mg Na/kg) did not have a significant effect

on growth, leaf gas exchange and K accumulation of wheat cv. Wyalkatchem grown in

columns with low profile K supply under both topsoil dry and wet conditions (P=0.05). The

findings of this experiment differed from the previous pot and split-root experiments (Chapter

3 and 4) probably due to larger soil volume and thus relatively more available K per plant

which largely prevented K deficiency. This emphasises that the beneficial role of Na in K

nutrition may occur only when wheat is grown under K-limited conditions with apparent K

deficiency symptoms. It can be concluded that the extent of Na stimulation of growth by

wheat was determined by the amount of soil available K as well as Na.

103

CHAPTER 6

SHORT-TERM SOLUTION CULTURE EXPERIMENT

Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium

(Na+) supply using rubidium (Rb+) tracer in solution culture experiments: short-term

responses

6.1 Introduction

Low Na was found to be beneficial for wheat shoot and root growth when soil K was

deficient (Chapter 4), and the response varied between K-efficient and K-inefficient cultivars

(Chapter 3). Under low K supply (example 40 mg K/kg), four wheat genotypes differing in

K-use efficiency responded to low to moderate Na (25 to 50 mg Na/kg) by increasing leaf net

photosynthesis, root growth and shoot K+ uptake especially in K-efficient cultivars compared

with nil Na treatment (Chapter 3). Also, in the split-root experiment with cv. Wyalkatchem

(Chapter 5), the presence of Na in just one of the two compartments was able to increase

plant growth and K uptake significantly under low K supply.

Generally the Na+ stimulation of plant growth in low K plants has been attributed to the

uptake of Na which then substitutes for non-specific functions of K in cells. However, in

wheat the shoot Na concentrations were too low to substitute for K in plants. By contrast,

with 25 or 50 mg Na/kg of soil, there was an increase in shoot K concentration and content

(Chapters 3 and 4). This raises questions whether the presence of Na+ as an energising ion

may result in increased K+ acquisition (Box & Schachtman, 2000) or the enhanced K+ uptake

is due to added Na that releases soil K and makes it available to plants. The presence of Na

may also stimulate root growth (Ali et al., 2009) which in turn increases K uptake.

In the split-root experiment, leaf K concentration decreased but root K concentration

increased with addition of moderate Na (50 mg Na/kg) to one or both the root compartments

at low soil K. Box and Schachtman (2000) reported that the beneficial effect of Na on wheat

grown in low external K was due to the direct effect of Na+ on growth in one of their

experiments, but not in two other experiments. They also found that Na+ did not stimulate

Rb+ uptake, but K+ stimulated Na+ uptake in short-term tracer flux experiments. A further

investigation on whether low concentrations of Na+ increase K+ uptake in wheat with low K

supply would help understand K and Na interactions particularly at low soil concentrations of

K and Na.

104

My preliminary soil incubation experiment (an experiment in Chapter 3) showed that adding

Na had a slight but non-significant effect on exchangeable soil K levels. This suggests that

enhanced K availability in soil by Na supply cannot explain the entire increased K uptake in

the experiments reported in Chapters 3 and 4. However, by testing the effect of Na on K

uptake in nutrient solution, any Na-induced increase in K availability in the soil medium can

be avoided. It was hypothesised that low concentrations of external Na+ would increase K+

uptake in wheat under deficient soil K conditions through the effect of Na+ on K+ transporters

(Box & Schachtman, 2000; Rubio et al., 1995) or the effect of Na+ in increasing membrane

hyperpolarisation (Shabala & Cuin, 2008). Short-term tracer flux experiments were

conducted using rubidium (Rb+) as a tracer for K+ uptake to determine whether low

concentrations of Na+ stimulated Rb+ uptake in wheat. Rubidium has almost an identical

hydrated ion radius as K+ and these two ions behave similarly with regard to plant absorption

(Drobner & Tyler, 1998). In this experiment, two wheat genotypes differing in K-use

efficiency (Damon & Rengel, 2007) were used to further probe the effects of Na+ on rate of

K+ uptake.


6.2.1 Plant culture

Wheat (Triticum aestivum L.) cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient)

were grown in a naturally-lit glasshouse at Murdoch University, Perth, Western Australia

(32°04′S, 115°50′E). Two short-term experiments with different K and Na levels were

conducted during winter (Experiment 1) and early spring (Experiment 2) in a nutrient

solution culture. Wheat seeds were surface sterilised by washing with 5 % sodium

hypochlorite solution for 1 minute and then thoroughly rinsed with DI water. The seeds were

soaked in DI water for 2 hours and germinated on paper towels moistened with 0.05 mM

CaCl2 in the dark for 2 days at 25˚C. The germinated seedlings were then transplanted to 4 L

pots with ¼ strength modified Hoagland’s solution at six seedlings per pot. The seedlings

were held in plastic lids of the pots supported by polystyrene foam. In the pots, the nutrient

solution was continuously bubbled with compressed air (see Appendix 3 for experimental

setup). The nutrient solution was changed every 3 days throughout the experiment. The

experiments were a factorial combination of two wheat cultivars, two K levels and three Na

levels (see below) and each treatment was replicated four times.

105

6.2.2 Basal nutrient solution

A modified Hoagland’s solution (equivalent to ½ strength) was used in the experiment,

including 2 mM Ca(NO3)2.H2O, 0.5 mM NH4H2PO4, 1 mM MgSO4.7H2O, 200 µM Fe-citrate

(FeC6H5O7.5H2O), 9.2 µM H3BO3, 1 µM MnCl2.4H2O, 0.4 µM ZnSO4.7H2O, 0.2 µM

(NH4)6Mo7O24.4H2O, and 0.4 µM CuSO4.5H2O. The pH of the nutrient solution was

maintained at 5.5 throughout the experiment and was adjusted using either 1M HNO3 or 0.5

M Ca(OH)2. All chemicals used were of analytical grade.


Two short-term experiments with various K and Na concentrations were conducted in K+

uptake studies. In the first experiment, two K concentrations at 0.2 mM (low) and 2 mM K

(adequate) as KCl and three Na concentrations at 0, 10 and 20 mM Na as NaCl were used. In

the second experiment, two K concentrations of 0.05 mM (low) and 2 mM K (adequate) and

three Na concentrations of 0, 2 and 10 mM Na were used. The plants were initially grown in

low K (0.2 mM in first experiment or 0.05 mM K in second experiment) plus basal nutrient

solution without Na for two weeks, followed by a harvest of two plants per pot to analyse

initial ion concentrations of the shoot and root. Then half of the pots were continuously

supplied with low K, while the remaining pots were supplemented with extra K to raise the

concentrations to 2 mM K (adequate). Both the low and adequate K pots were treated with

the three Na levels. Rubidium chloride was added as a tracer (0.5 mM Rb+) together with the

K and Na treatments, and the plants were harvested after 48 hours to measure the uptake of

K, Na and Rb ions in root and shoot.

6.2.4 Measurements

There were two harvests: initial harvest before treatment addition (at 15 days after

transplanting) and final harvest (at 17 days after transplanting in first experiment, and 19 days

after transplanting in second experiment). The final harvests in both experiments were made

48 hours after the K and Na treatments and Rb addition. Plant phenology (leaf numbers, tiller

numbers) was recorded throughout the experiment. Leaf net photosynthesis, stomatal

conductance and transpiration rate were measured before and 1.5 days after treatment

application using the LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The

measurements were made in fully expanded young leaves at ambient relative humidity of 50


radiation of 1500 µmol/m2·s.

106

At harvest, shoot fresh and dry weights, and root dry weight were determined. The roots of

two plants per pot were measured for root length, surface area, average diameter, root volume

using a root scanner controlled by WinRhizo software (WinRhizo Pro 2007, Regent

Instruments Quebec, Canada). The shoot and root samples were dried in a forced-draught

oven at 60˚C for 48 hours and their dry weights were recorded. The samples were then

milled, digested as described previously (Chapter 3) and concentrations of K, Na and Rb

were measured using inductively-coupled plasma-atomic emission spectroscopy.


Statistical analyses were conducted using the statistical program SPSS 18.0. Three-way

analysis of variance was conducted to assess the effects of soil K and Na supply and their

interactions with cultivars. Tukey’s HSD was computed at P ≤ 0.05 for pair-wise comparison

of means.

6.3 Results

6.3.1 Experiment 1

6.3.1.1 Plant growth

Pre-treatment shoot dry weight and root dry weight were 0.03 and 0.02 g/plant, respectively

in cv. Wyalkatchem, and 0.11 and 0.05 g/plant, respectively in cv. Gutha. Forty eight hours

after K, Na treatments and Rb addition there was no significant treatment effect on shoot or

root biomass in both cvv Wyalkatchem and Gutha (Appendix 4).

6.3.1.2 Leaf gas exchange

Pre-treatment measurements did not show any difference in leaf gas exchange among

cultivars (Appendix 4). Consistent with the responses of plant growth parameters, leaf

photosynthesis did not change after short-term treatments (measurements taken 42 hours after

treatment addition) (Fig. 6.1). By comparison, stomatal conductance and transpiration were

significantly higher in Wyalkatchem than in Gutha after short-term treatment. However, there

was no significant of K and Na treatments or the interaction between them in terms of

stomatal conductance and transpiration (Table 6.1).

107

Table 6.1 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient)

and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2

and 2 mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for a further 48

hours (Experiment 1).


Parameters Solution K

Solution Na


Photosynthesis n.s n.s n.s * n.s n.s n.s


n.s n.s ** n.s n.s n.s n.s

Transpiration n.s n.s *** n.s n.s n.s n.s

108

0.2 mM 2 mM

Tra

nspi

ratio

n (m

mol

H2O

/m2 .s

)

0

2

4

6

K concentration (mM)

0.2 mM 2 mM

0 Na 10 Na 20 Na

Sto

mat

al c

ondu

ctan

ce (

mm

olH 2O

/m2 .s

)

0

100

200

300

400

Wyalkatchem

Leaf

ne

t ph

otos

ynth

esi

s (µ

mol

CO 2/m

2 .s)

0

5

10

15

20

25

Gutha

Fig. 6.1 Leaf net photosynthesis, stomatal conductance, and transpiration of cultivars

Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for

two weeks, followed by two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM)

(±SE, n=4) and measured 42 hours after the treatments (Experiment 1).

6.3.1.3 K, Na and Rb concentrations in shoot and root

Before the K and Na treatments and Rb addition, the shoots contained 46.6 mg K/g, 0.19 mg

Na/g in cv. Wyalkatchem and 48.2 mg K/g, 0.15 mg Na/g in cv. Gutha on a dry-weight basis.

Rubidium concentration was below the detectable limit (< 0.02 mg/g) in both cultivars. Forty

eight hours after K, Na treatments and Rb addition, shoot K concentration in both cultivars

was higher in 2 mM K than 0.2 mM K treatment without Na, but was similar between the two

K treatments with Na supply (Table 6.2). The interaction between K and Na was significant

109

for shoot K concentration (P ≤ 0.05), and three way interactions between K, Na and cultivars

was not significant (Table 6.3). Sodium addition did not have a significant effect on shoot K

concentrations 48 hours after treatment addition. Shoot Na concentration of both cultivars

increased with addition of Na but more strongly in low K treatments (Table 6.2). There was

nearly two fold decrease in shoot Rb concentrations with high K supply when compared with

low K supply.

Table 6.2 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-

efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K

levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a

further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with


K (mM) Na (mM) Wyalkatchem Gutha

Ion concentrations in shoot (mg/g, dry wt.) (n=4)

K Na Rb K Na Rb

0.2 0 43.8ef 0.26d 5.79ab 48.7b-e 0.19d 7.18a

0.2 10 44.4def 1.57bc 5.91ab 49.2b-e 1.12cd 7.62a

0.2 20 39.3f 3.48a 4.90abc 46.4def 2.26b 6.56a

2.0 0 52.9a-d 0.20d 2.88bc 58.2a 0.18d 3.09bc

2.0 10 50.7a-e 1.13cd 2.42c 55.7abc 1.46bc 2.45c

2.0 20 47.3c-f 1.76bc 2.00c 56.5ab 1.24bcd 3.11bc

Ion concentrations in root (mg/g, dry wt.) (n=4)

K Na Rb K Na Rb

0.2 0 23.1bcd 0.77d 12.4a 23.1bcd 0.77d 15.8a

0.2 10 27.0abc 4.48c 12.8a 20.8cd 4.92c 15.1a

0.2 20 20.4cd 8.49ab 10.2ab 17.8d 9.93a 14.7a

2.0 0 31.5a 0.86d 5.79bc 33.2a 0.54d 5.50bc

2.0 10 30.7ab 5.38c 4.96bc 32.3a 6.39bc 5.05bc

2.0 20 29.5ab 6.93bc 3.51c 30.4ab 7.00bc 4.96bc

110

Prior to the treatments, the roots contained 33 mg K/g, 0.43 mg Na/g in cv. Wyalkatchem,

and 33 mg K/g and 0.31 mg Na/g in Gutha. Rubidium concentrations were below the

detectable level. The response of post-treatment root K, Na and Rb concentrations showed

similar trends to those of shoot concentrations (Tables 6.3, 6.4). However, roots had lower K

concentration and higher Na and Rb concentrations than shoot (Table 6.2).

Table 6.3 Statistical summary of shoot and root K, Na, Rb concentrations and contents in

cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM

K) for 2 weeks and two K levels (0.2 and 2 mM K), three Na levels (0, 10 and 20 mM Na)

and Rb tracer (0.5 mM) for 48 hours (Experiment 1; n=4).


Parameters Solution K Solution Na Cultivar K×Na K×cv Na×cv K×Na×cv

Shoot K conc. * n.s *** * n.s n.s n.s

Shoot Na conc. n.s n.s *** n.s n.s n.s n.s

Shoot Rb conc. * n.s *** n.s n.s n.s n.s

Root K conc. n.s n.s n.s * n.s n.s n.s

Root Na conc. n.s n.s ** n.s n.s n.s n.s

Root Rb conc. n.s n.s *** n.s n.s n.s n.s

Shoot K content * n.s *** * n.s n.s n.s

Shoot Na content n.s n.s *** n.s n.s n.s n.s

Shoot Rb content * n.s *** n.s n.s n.s n.s

Root K content n.s n.s n.s * n.s n.s n.s

Root Na content n.s n.s ** n.s n.s n.s n.s

Root Rb content n.s n.s *** n.s n.s n.s n.s

6.3.1.4 K, Na and Rb contents in shoot and root

Pre-treatment shoot K and Na contents were 1.67 and 0.007 mg/plant in cv. Wyalkatchem,

5.13 and 0.016 mg/plant in cv. Gutha. The root K and Na contents were 0.64 and 0.008

mg/plant in cv. Wyalkatchem, 1.71 and 0.01 mg/plant in cv. Gutha.

Forty eight hours after treatment addition, shoot K content was not significantly different

among the treatments in both cultivars (Table 6.4). Gutha had considerably higher shoot K

111

content than Wyalkatchem because of greater biomass. Shoot Na content was dependent on

solution Na concentrations. Similar to shoot K content, shoot Rb did not show significant

difference among treatments. Root K, Na and Rb contents followed similar response as shoot

contents. Roots accumulated less K than in shoot, especially cv. Gutha which had a low root:

shoot ratio. Plant K uptake prior to treatment addition was 2.3 mg in Wyalkatchem and 6.84

mg in Gutha. Forty eight hours after Na addition, plant K uptake was increased by 39 to 53 %

in Wyalkatchem, and 23 to 46 % in Gutha (Table 6.5).

Table 6.4 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and

Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2

mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours

(harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters

differ at P≤0.05.


Ion content in shoot (mg/plant) (n=4)

K Na Rb K Na Rb

0.2 0 3.01c 0.02e 0.39bc 9.1ab 0.03e 1.34a

0.2 10 3.47c 0.12cde 0.45bc 9.18ab 0.21bcd 1.41a

0.2 20 2.95c 0.25b 0.36bc 8.24b 0.40a 1.16a

2.0 0 3.88c 0.01e 0.21bc 10.5a 0.03e 0.55b

2.0 10 3.57c 0.08e 0.17bc 9.17ab 0.23b 0.40bc

2.0 20 3.18c 0.11de 0.13c 10.2a 0.23bc 0.56b

Ion content in root (mg/plant) (n=4)

K Na Rb K Na Rb

0.2 0 0.84c 0.03de 0.45b 1.77ab 0.06de 1.22a

0.2 10 1.08bc 0.17cde 0.50b 1.54bc 0.37bc 1.13a

0.2 20 0.84c 0.34bc 0.41b 1.32bc 0.73a 1.09a

2.0 0 1.09bc 0.03e 0.19b 2.29a 0.03de 0.38b

2.0 10 1.11bc 0.19cde 0.18b 2.29a 0.45b 0.36b

2.0 20 1.01c 0.23cd 0.12b 2.30a 0.53ab 0.37b

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Table 6.5 The whole plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient)

and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2

and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours


differ at P≤0.05.


Plant content (mg/plant) (n=4)

K Na Rb K Na Rb

0.2 0 3.86d 0.05e 0.84b 10.9bc 0.09de 2.56a

0.2 10 4.55d 0.29de 0.95b 10.7bc 0.58bc 2.54a

0.2 20 3.79d 0.59bc 0.77b 9.56c 1.13a 2.25a

2.0 0 4.97d 0.04e 0.40b 12.8a 0.07e 0.93b

2.0 10 4.68d 0.27de 0.35b 11.5abc 0.68b 0.76b

2.0 20 4.20d 0.34cd 0.25b 12.5ab 0.75b 0.93b

6.3.2 Experiment- 2

6.3.2.1 Plant growth

Pre-treatment shoot dry weight and root dry weight were 0.2 and 0.08 g/plant, respectively in

cv. Wyalkatchem, and 0.27 and 0.10 g/plant, respectively in cv. Gutha. The growth response

to K and Na treatments for 48 hours was mostly consistent with Experiment 1. There was no

significant difference among the treatments (Appendix 4).

6.3.2.2 Leaf gas exchange

Photosynthesis measured 2 days after the K and Na treatments showed no significant

difference among the treatments and between two varieties (Fig. 6.2; Table 6.6).

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Table 6.6 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient)

and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels

(0.05 and 2 mM K), two Na levels (0, 2 and 10 mM Na) and Rb (0.5 mM) (n=4) added for 48

hours (Experiment 2).



Solution Na

cultivar K×Na K×cv Na×cv K×Na×cv

Photosynthesis n.s * n.s n.s ** n.s n.s

Stomatal conductance n.s n.s n.s n.s n.s n.s n.s

Transpiration n.s n.s n.s n.s n.s n.s n.s

114

0.05 mM 2 mM

Tran

spir

atio

n (m

mol

H2O

/m2 .s

)

0

2

4

6

0 Na 2 Na 10 Na


0.05 mM 2 mM

Sto

mat

al c

ondu

ctan

ce (

mm

olH 2O

/m2 .s

)

0

100

200

300

400

Wy alkatchem

Leaf

ne

t ph

otos

ynth

esi

s (µ

mol

CO 2/m

2 .s)

0

5

10

15

20

25

Gutha

Fig. 6.2 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars

Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for

two weeks, followed by two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM)

(±SE, n=4) and measured 42 hours after the treatments (Experiment 2).

6.3.2.3 K, Na and Rb concentrations in shoot and root

Before the K and Na treatments and Rb addition, the shoots contained 18 mg K/g, 0.11 mg

Na/g in cv. Wyalkatchem and 17 mg K/g, 0.12 mg Na/g in cv. Gutha on a dry-weight basis.

Rubidium concentration was below detectable limits (<0.02 mg/g) in both cultivars.

After treatment with high K (2 mM) for 2 days, shoot K concentrations increased

significantly compared with continuous low K treatment in both Wyalkatchem and Gutha,

and the interaction between K and Na was significant for shoot K concentration, and there

115

was a relatively weak interaction between K, Na and cultivar (P= 0.09) (Table 6.7). Addition

of 2 and 10 mM Na increased shoot Na concentrations in both cultivars compared with nil Na

supply, and the three way interaction was significant for shoot Na concentrations (P ≤0.05;

Table 6.7). Shoot Rb concentration in high K treatments was significantly lower than low K

treated plants. The addition of 2 mM Na to low K (0.2 mM K) pots increased shoot Rb

concentration of Wyalkatchem but not Gutha (Table 6.8), and the interaction between K and

Na was significant (Table 6.7). However, in high K treatment, Na addition had no effect on

shoot Rb concentrations in both cultivars.

Table 6.7 Statistical summary of shoot and root K, Na, Rb concentrations and contents in

cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM

K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb

tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2;

n=4). *P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant


Solution Na


Shoot K conc. *** *** n.s *** * n.s n.s

Shoot Na conc. *** *** n.s ** *** ** ***

Shoot Rb conc. *** *** n.s *** * n.s n.s

Root K conc. *** *** * *** n.s n.s n.s

Root Na conc. n.s *** ** n.s n.s ** n.s

Root Rb conc. *** ** * n.s n.s ** n.s

Shoot K content *** *** *** *** *** * n.s

Shoot Na content *** *** *** n.s *** ** ***

Shoot Rb content *** n.s *** * *** n.s n.s

Root K content *** ** *** ** ** * *

Root Na content n.s *** *** n.s n.s *** n.s

Root Rb content *** n.s *** n.s ** n.s n.s

116

Table 6.8 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-

efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K

levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a

further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with



Shoot ion concentration (mg/g, dry wt.) (n=4)

K Na Rb K Na Rb

0.05 0 16.9d 0.35f 29.2c 16.5d 0.44f 30.8bc

0.05 2 18.4d 1.02de 33.9a 17.7d 1.22cd 33.4ab

0.05 10 17.0d 2.17a 30.1c 17.5d 1.41bc 31.8abc

2 0 50.9ab 0.32f 4.95d 54.8a 0.29f 4.49d

2 2 47.8b 0.80e 5.40d 50.3b 1.12cde 4.08d

2 10 42.8c 1.08cde 4.29d 42.6c 1.62b 3.64d

Root ion concentration (mg/g, dry wt.) (n=4)

K Na Rb K Na Rb

0.05 0 4.68d 0.50d 21.0b 4.80d 0.42d 23.2ab

0.05 2 5.34d 3.94c 24.3a 5.02d 4.04c 25.4a

0.05 10 5.21d 5.31ab 23.2ab 4.11d 5.65ab 23.2ab

2 0 30.1a 0.38d 6.17c 30.5a 0.32d 6.66c

2 2 26.8b 3.97c 5.84c 25.5b 4.05c 6.48c

2 10 24.2bc 5.04b 4.65c 21.4c 5.81a 5.42c

Prior to the K and Na treatments, the roots had 9.12 mg K/g, 0.79 mg Na/g in cv.

Wyalkatchem, and 8.06 mg K/g and 0.76 mg Na/g in Gutha. Rubidium concentrations were

below detection level (<0.02 mg/g) for both cultivars.

Root K, Na and Rb concentrations showed similar response pattern as the shoot

concentrations (Table 6.8). While roots had much lower K concentration than shoots, root K

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concentrations in both Wyalkatchem and Gutha were significantly higher at high K than low

K supply. Root Na concentrations were related to the solution Na levels, and the roots had

higher Na concentration than shoots at 2 and 10 mM Na. Similar to shoot Rb concentrations,

root Rb concentrations were significantly higher in low K treatment than high K treatment

(Table 6.8). The addition of 2 mM Na to low K treatment (0.05 mM) increased root Rb

concentration of Wyalkatchem significantly (Table 6.8).

6.3.2.4 K, Na and Rb contents in shoot and root

Pre-treatment shoot K and Na contents were 3.62 and 0.02 mg per plant in cv. Wyalkatchem,

4.64 and 0.03 mg in cv. Gutha. The root K and Na contents were 0.71 and 0.06 mg in cv.

Wyalkatchem, 0.84 and 0.08 mg in cv. Gutha.

High K treatments had higher shoot K content than low K treatments in both cultivars. At

high K supply, the shoots of Gutha accumulated more K than Wyalkatchem because of

greater biomass (Table 6.9). Shoot Na content increased with increasing Na levels. At low K

supply, Rb content in shoots of Wyalkatchem increased significantly with 2 mM Na addition

when compared with nil Na treatment, however, the increase was not significant in Gutha.

Roots accumulated much lower K than shoots. Also at low K treatment, Rb content in roots

was nearly 3 fold less than shoot Rb content. Whole plant Rb content of Wyalkatchem was

significantly increased with addition of 2 mM Na to 0.05 mM K treatment (Table 6.10).

118


Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05

and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours


differ at P≤0.05.


Shoot ion content (mg/plant) (n=4)

K Na Rb K Na Rb

0.05 0 5.76e 0.12g 9.93c 8.14de 0.22efg 15.2a

0.05 2 6.25de 0.35ef 11.5b 8.70d 0.60bcd 16.4a

0.05 10 6.25de 0.80a 11.1bc 8.55de 0.69abc 15.6a

2 0 18.1c 0.11g 1.76d 27.9a 0.15fg 2.31d

2 2 16.8c 0.28efg 1.88d 25.2a 0.58cd 2.06d

2 10 16.3c 0.41de 1.62d 21.9b 0.84a 1.87d

Root ion content (mg/plant) (n=4)

K Na Rb K Na Rb

0.05 0 0.68c 0.07c 3.05c 0.91c 0.08c 4.42ab

0.05 2 0.82c 0.62b 3.82abc 0.90c 0.73b 4.60a

0.05 10 0.79c 0.80b 3.50bc 0.81c 1.10a 4.57a

2 0 4.22b 0.05c 0.87d 6.46a 0.07c 1.41d

2 2 4.38b 0.64b 0.94d 4.66b 0.74b 1.18d

2 10 3.82b 0.79b 0.73d 4.55b 1.24a 1.15d

119

Table 6.10 Plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha

(K-inefficient) in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with

low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and

10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting)

(Experiment 2). Means (n=4) with different letters differ at P≤0.05.


Plant ion content (mg/plant) (n=4)

K Na Rb K Na Rb

0.05 0 6.44d 0.19f 12.9c 9.05d 0.29f 19.6a

0.05 2 7.08d 0.97e 15.4b 9.61d 1.33cd 21.0a

0.05 10 7.04d 1.60bc 14.5bc 9.36d 1.79ab 20.2a

2 0 22.3c 0.17f 2.63d 34.4a 0.22f 3.71d

2 2 21.2c 0.93e 2.83d 29.9b 1.31cd 3.24d

2 10 20.1c 1.2de 2.35d 26.5b 2.07a 3.03d

6.4 Discussion

In both short-term experiments, the duration of K and Na treatments was 48 h, which was too

short to cause any significant growth changes in terms of shoot and root weight, and leaf gas

exchange. The big difference in biomass production of both cultivars between two

experiments was due to difference in growth conditions, especially temperature. However, 48

hours after the treatments were applied the difference in shoot growth rate between the two

harvests of Gutha was higher than Wyalkatchem, while, the root growth did not show much

difference among the cultivars.

In Experiment 1, with 0.2 mM K pre-treatment shoot K concentrations were 46.6 and 48.2

mg K/g dry weight in Wyalkatchem and Gutha, respectively. The K concentrations are in

sufficient range for wheat growth (Reuter et al., 1997). With sufficient K concentrations in

shoot already, there would be no need for Na substitution of K functions in plants, regardless

of the levels of Na concentrations applied in Experiment 1. However, previous study by

Rubio et al. (1995) in wheat showed an increased K+ uptake (measured as Rb+ uptake) due to

Na addition at the much lower external Na concentration of 1 mM Na. Therefore, in the

120

follow-up short-term experiment low K treatment was lowered from 0.2 mM K to 0.05 mM

K, and the Na concentrations also lowered from 10 and 20 mM Na to 2 and 10 mM Na,

respectively. Experiment 2 had pre-treatment shoot K concentrations of 18 and 17 mg K/g,

dry weight in Wyalkatchem and Gutha, respectively, which was very much lower than

Experiment 1 and with in the deficient range for wheat growth (Reuter et al., 1997).

Shoot K and Rb concentrations in both cultivars showed no change with Na addition in

Experiment 1. However, shoot and root Rb concentrations increased significantly in cv.

Wyalkatchem treated with 0.05 mM K and 2 mM Na in Experiment 2. Rubidium content in

Wyalkatchem also increased significantly with 2 mM Na addition in Experiment 2, while K

content did not increase significantly. Since Rb+ is often used as an analogue for K+ uptake

(Drobner & Tyler, 1998), the short-term Rb+ increase suggested the capacity for increased K+

uptake in Wyalkatchem with 2 mM Na+ addition to low K solution in this study. Similarly an

increased K+ uptake in wheat due to Na+ via high-affinity K+ uptake activated by added Na+

was observed by Rubio et al. (1995). However, the finding is in contrast with previous K+

uptake study using Rb+ as a tracer in wheat, which reported no stimulation in K+ uptake in

presence of low Na+ concentrations while Na+ stimulated growth at low external K (Box &

Schachtman, 2000).

Plant roots have low and high affinity K+ uptake mechanisms to take up K+ from the

extracellular medium (Britto & Kronzucker, 2008; Szczerba et al., 2009). The external K+

concentration directly influences the activities of K+ channels and transporters and also

regulates the two K+ uptake mechanisms to maintain a steady state flux of K+ (Wang & Wu,

2010). In this experiment, K+ acquisition from adequate K supply (2 mM) would be an

energetically passive process, while that from low external K concentrations (0.05 mM) is

usually considered to be an energy-demanding process (Britto & Kronzucker, 2008), and at

least two high-affinity K+ uptake transporters, KUP/HAK (K uptake transporter/ high affinity

K+ transporter) and HKT1 (High affinity K+ transporter) are shown to be induced by K+

starvation (Szczerba et al., 2009). One of the high affinity K+ uptake mechanisms is a Na+-

energized high-affinity K+ symport HKT1 (Schachtman & Liu, 1999), and it may play an

important role in K+ acquisition when external Na+ concentrations are low (Box &

Schachtman, 2000; Rubio et al., 1995).

The HKT transporter mediates high-affinity K+ uptake and high or low-affinity Na+ uptake

depending on external Na+ and K+ concentrations (Benito et al., 2014). At low external Na+

and K+ concentrations, some transporters function as Na+-K+ symporters, as demonstrated by

121

Na+- stimulated K+ uptake and K+- stimulated Na+ uptake, however, at high external Na+

concentrations, some of these transporters become Na+ uniporters, no longer transporting K+

(Benito et al., 2014; Rubio et al., 1995). Furthermore, recent studies suggest that HKT

transporters discriminate less between K+ and Na+ or even select for Na+ over K+ (Benito et

al., 2014).

Another possible effect of Na+ on transporters as a mechanism for increased K+ uptake could

be mediated by the low-affinity K+ uptake system (such as AKT). At high Na levels (80 mM

NaCl or above), Na+ crosses the plasma membrane causing a significant membrane

depolarization and increases K+ leakage through depolarization-activated outward-rectifying

channels (Shabala & Cuin, 2008). In sharp contrast to 80 mM NaCl treatment, K+ efflux in 20

mM NaCl treatment was found to be very short-lived and K+ uptake became dominant from

elusive ‘osmosensing mechanism’ (Chen et al., 2005). At moderate salinity (20 mM NaCl in

barley), Na+ hyperpolarized the plasma membrane and increased K+ uptake via inward-

rectifying hyperpolarized-activated K+ channels (Chen et al., 2005; Shabala & Cuin, 2008).

Therefore, Na-induced K+ flux was clearly dose dependent, and could possibly explain

increased K+ uptake at low and moderate Na levels in the present study.

In the present short-term experiments in wheat, there was increased Rb+ uptake (as the tracer

for K+ uptake) only at 2 mM Na but not at higher Na levels. The difference in external Na+

concentrations between barley (20 mM Na) and wheat (2 mM Na) where K+ uptake was

increased could be due to the difference between barley and wheat in Na+ uptake and use. For

example, wheat has 10 to 15 fold higher K+/Na+ ratio than barley, indicating that high soil Na

would have greater effect on wheat than in barley (Ma et al., 2011). Also, in comparison to

current results, Na+ stimulated Rb+ uptake via a K+/Na+ symport at low external Na+

concentration of 1 mM Na, however, but not at higher concentration of about 16 mM Na

(Rubio et al., 1995).

The increase in Rb+ or K+ uptake due to Na-stimulated root elongation in wheat is an another

possibility as root elongation can determine nutrient uptake by providing access to additional

nutrient supply (Barber & Silberbush, 1984). However, in this study there was no effect of Na

at deficient K supply in root growth parameters in both cultivars, and therefore it is unlikely

that increased K+ uptake was a direct effect of increased root elongation, but more likely due

to Na+-energised K+ uptake through mechanisms explained above.

122

The results of this study indicate that K-use efficiency of the cultivars had an impact on K+

uptake. We found that Na stimulated greater Rb+ uptake (as a tracer of K+ uptake) in the K-

efficient cultivar Wyalkatchem but not in the K-inefficient Gutha with low external Na

addition (2 mM Na+) for 48 hours. The main mechanism identified by Damon and Rengel

(2007) for K efficiency in wheat cultivars like Wyalkatchem was greater utilization efficiency

of shoot K. However, K-utilization efficiency in terms of growth or yield was not measured

due to short duration of experiment. This experiment was designed to measure short-term

response of K+ uptake with low external Na+. Long-term response in K+ uptake to low

external Na treatments will be measured in the following experiment (Chapter 7).

6.5 Conclusion

In this study, shoot or root growth parameters and leaf gas exchange of wheat cultivars were

not affected by K and Na treatments for 48 hours. Wheat K+ uptake varied with K-use

efficiency of cultivars with significantly increased Rb+ uptake (as a tracer of K+ uptake) in the

K-efficient Wyalkatchem but not in K-inefficient Gutha at deficient K supply (0.05 mM K)

and low external Na+ concentration (2 mM Na). The effect of Na+ on high-affinity and low-

affinity K+ transporters probably contributed to increased K+ uptake under low external Na+

conditions. A long-term solution culture experiment with similar treatments to study the

effect of low to moderate Na+ concentrations on K+ uptake and plant growth is warranted.

123

CHAPTER 7

SOLUTION CULTURE EXPERIMENT: LONG-TERM RESPONSES

Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium

(Na+) supply using rubidium (Rb+) tracer in a solution culture experiment: long-term

responses

7.1 Introduction

Sodium and potassium are structurally and chemically very similar elements and because of

this similarity, the presence of Na+ and its uptake by plants reduces the amount of K+ required

by many plants to meet basic osmotic functions (Benito et al., 2014). It is believed that under

limited K+ supply, Na+ can replace K+ in the vacuole as an alternative inorganic osmoticum,

and the released K+ is available for more K-specific processes (Kronzucker & Britto, 2011).

The presence of tissue Na+ is associated with reduced K+ content in leaves and reduced K+

requirement by plants (Benito et al., 2014). In contrast to this general understanding, my

earlier experiments showed that addition of low to moderate Na (25 and 50 mg Na/kg)

increased plant K uptake when soil K supply was deficient (Chapter 4) especially in K-

efficient wheat cultivars (Chapter 3). The Na-induced K uptake (Rb as a tracer) was evident

in a short-term solution culture experiment with a K-efficient but not in K-inefficient cultivar

after addition of K, Na treatments and Rb tracer for 48 hours (Chapter 6).

There are a number of K+ uptake mechanisms that operate in plants to ensure adequate K is

available for growth and metabolism. Depending on external K concentrations, at least two

transport systems are involved in K+ uptake: a low-affinity system that operates at high K

concentrations and a high-affinity system at lower concentrations (Nieves-Cordones et al.,

2014; Rubio et al., 1995). It has been suggested that one of the high-affinity K+ uptake

mechanisms, Na+- coupled K+ symport HKT1, may play an important role in K+ uptake when

external Na+ concentrations are low (Box & Schachtman, 2000). Solution culture

experiments were designed to investigate whether low external Na+ concentrations play a role

in energising K+ acquisition using Rb+ as a tracer for K+ uptake.

In previous soil culture experiments, there was increase in wheat K+ uptake, leaf

photosynthesis and stimulation of growth when low to moderate Na levels were added to K-

deficient soil (Chapter 3 and 4). Root growth at deficient K was better stimulated than shoot,

and K-efficient cultivars showed a stronger response (Chapter 3). In the solution culture

experiments, the effect of external Na concentration on root growth parameters was assessed

124

in detail by scanning roots as increase in root elongation could contribute to increased K

uptake by root. However, no root growth responses to Na were evident after the 2-day

treatment period (Chapter 6).

The main objective of the solution culture experiments was to examine the effect of Na on K

uptake, thereby avoiding any effect of Na on K availability that may occur in the soil

medium. The previous short-term uptake studies using Rb+ as a tracer suggested that Rb+

uptake in cv. Wyalkatchem was increased with 2 mM Na addition in low K solution (0.05

mM K) for 48 hours (Chapter 6). In this solution culture experiment, the long-term effects of

low external Na+ on K+ uptake and growth of K-efficient and K-inefficient cultivars were

examined.


7.2.1 Plant culture

Wheat (Triticum aestivum L.) cultivars Wyalkatchem (K-efficient) and Gutha (K- inefficient)

were grown in a naturally-lit glasshouse at Murdoch University, Perth, Western Australia

(32°04′S, 115°50′E) during late winter to early spring in a nutrient solution culture. Seeds

were germinated and transplanted to 4 L pots in the same procedure as described in Chapter

6. The seedlings were held in plastic lids of the pots supported by polystyrene foam, and the

nutrient solution was continuously bubbled with compressed air. The nutrient solution was

changed every 3 days throughout the experiment. The experiment had a factorial combination

of two wheat cultivars, two K levels, three Na levels and four replicates (see below).

7.2.2 Basal nutrient solution

A modified Hoagland’s solution was used in this experiment with the same composition of

nutrient solution described in previous chapter (Chapter 6).


Two K levels of 0.05 mM (low) and 2 mM K (adequate) and three Na levels of 0, 2 and 10

mM Na, the same as those in Experiment 2 of Chapter 6, were used in this experiment. The

plants were initially grown in 0.05 mM K plus basal nutrient solution without Na for two

weeks. A pre-treatment harvest was made by sampling two plants per pot 17 days after

transplanting to analyse initial ion concentrations. From day 17, half of the pots were

supplemented with extra K to raise the concentrations to 2 mM and the remaining pots were

continuously supplied with 0.05 mM, together with application of the three Na treatments.

125

Two weeks later a second harvest prior to Rb treatment was made with two plants per pot to

evaluate plant growth and ion concentrations. After the second harvest, RbCl was added as a

tracer (0.5 mM Rb) to the K and Na treatments to measure the uptake of Rb, K and Na. All

plants were harvested 48 hours after Rb addition. This experiment was long enough to

observe wheat growth response as plants received the K and Na treatments for 18 days before

final harvest, compared with the harvest was made 48 hours after the K and Na treatments

and Rb+ addition in the previous experiments (Chapter 6).

7.2.4 Measurements

Three harvests were taken: initial harvest of 2 plants/ pot before the K and Na treatments (17

days after transplanting), pre-Rb harvest of 2 plants/ pot after two weeks of K and Na

treatments (32 days after transplanting) and final harvest at 48 hours after Rb addition (35

days after transplanting) of 4 remaining plants/ pot. Plant phenology (leaf numbers, tiller

numbers) was recorded throughout the experiment. Leaf net photosynthesis, stomatal

conductance and transpiration rate were measured before and after treatment application

using LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The measurements

were made in fully expanded young leaves at ambient relative humidity of 50 %, leaf

temperature of 25˚C, reference CO2 of 380 µmol/mol, and photosynthetically active radiation

of 1500 µmol/m2·s.

At each harvest, shoot fresh and dry weights, and root dry weight were determined. At final

harvest, roots from one plant per pot were harvested for determining root length, surface area,

average diameter, root volume using a root scanner controlled by WinRhizo software

(WinRhizo Pro 2007, Regent Instruments Quebec, Canada). The shoots of four plants were

separated into old leaves (basal 3-5 leaves on main tillers including the leaves that showed K

deficiency/Na toxicity symptoms), young leaves (top 2- 3 leaves on each tiller) and the rest of

shoot for elemental analysis. Roots were collected after washing in DI water. The shoot and

root samples were dried in an oven at 60˚C for 48 hours and their dry weights were recorded.

The samples were then milled, digested as described in Chapter 3 and concentrations of K,

Na and Rb were measured using inductively-coupled plasma-atomic emission spectroscopy.


Statistical analyses were conducted using the statistical program SPSS 18.0. Three-way

analysis of variance was conducted to assess the effects of soil K and Na supply and their

126

interactions with cultivars. Tukey’s HSD was computed at P ≤ 0.05 for pair-wise comparison

of treatment means.

7.3 Results

7.3.1 Plant growth

Seventeen days after transplanting, the pre-treatment shoot dry weight and root dry weights

were 0.19 and 0.06 g/plant, respectively in cv. Wyalkatchem, and 0.33 and 0.09 g/plant,

respectively in cv. Gutha.

7.3.1.1 Pre- rubidium harvest

Plants with adequate K supply (2 mM) for two weeks had significantly higher shoot dry

weight than those with continuous low K (0.05 mM) in both Wyalkatchem and Gutha (Fig.

7.1). The addition of Na (2 and 10 mM) had no significant effect in shoot dry weight

compared with nil Na treatment at both K levels. The three way interactions between K, Na

and cultivars were not significant for shoot dry weight (P ≤0.05) (Table 7.1). Root dry weight

of adequate K plants was significantly higher than low K plants at all Na levels. The addition

of 2 mM Na to low K solution increased root dry weight significantly in Wyalkatchem (Fig.

7.1). However, at adequate K supply addition of 2 or 10 mM Na reduced root dry weight in

both Wyalkatchem and Gutha. The interaction between K and Na was significant (Table 7.1),

as was the three-way interaction with the cultivars (P ≤0.05).

127

Sho

ot d

ry w

eigh

t (g

/plt)

0.0

0.5

1.0

1.5

2.0

2.5

0.05 mM 2 mM

Roo

t dr

y w

eigh

t (g

/plt)

0.0

0.1

0.2

0.3

0.4


0.05 mM 2 mM

0 Na 2 Na 10 Na

Wy alkatchem Gutha

Fig. 7.1 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and

Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K

levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (harvested 32 days

after transplanting, pre-rubidium addition) (±SE, n=4).

128

Table 7.1 Statistical summary of plant growth in cultivars Wyalkatchem (K-efficient) and


levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium

harvest), and after Rb treatment for 48 hours (post-rubidium or final harvest).


Parameters K Na Cultivar K×Na K×cv Na×cv K×Na×cv

Pre- Rubidium harvest

Shoot dry wt *** n.s *** n.s *** n.s n.s

Root dry wt *** ** *** *** *** ** **

Post- Rubidium harvest

Shoot dry wt *** * *** *** *** * ***

Root dry wt *** n.s *** *** *** n.s *

Photosynthesis *** *** *** *** n.s n.s *


*** *** *** *** *** n.s n.s

Transpiration ** ** *** * n.s n.s n.s

Post-Rb harvest

Consistent with the pre-Rb harvest, shoot dry weights of adequate K plants at post-Rb harvest

were significantly higher than low K plants in both cultivars (Fig. 7.2). The addition of 2 mM

Na to low K nutrient solution significantly increased shoot dry weight of Gutha. However, at

adequate K, addition of 10 mM Na significantly reduced shoot dry weight (Fig. 7.2). The

interactions between K, Na and cultivars were significant for shoot dry weight (Table 7.1).

Root dry weight of adequate K plants was also higher than that of low K plants. Sodium

addition had no effect on root dry weight between treatments at post- Rb harvest, except in

Gutha at adequate K (2 mM K) there was significant reduction in root dry weight with 10

mM Na (Fig. 7.2). The three way interaction for root dry weight between K, Na, and cultivars

was significant (P ≤0.05; Table 7.1). Root growth was not affected with 2 mM and 10 mM

Na addition at low K supply, but 10 mM Na reduced root surface area, root volume, number

of tips and forks of Gutha at adequate K supply when compared with nil Na (Table 7.2).

129

Sho

ot d

ry w

eigh

t (g

/pla

nt)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.05 mM 2 mM

Roo

t dr

y w

eigh

t (g

/pla

nt)

0.0

0.1

0.2

0.3

0.4


0.05 mM 2 mM

0 Na 2 Na 10 Na

Wy alkatchem Gutha

Fig. 7.2 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and


levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium

harvest), and after Rb treatment for 48 hours (post-rubidium harvest, 35 days after

transplanting) (±SE, n=4).

130

Table 7.2 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3),

number of tips and forks in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient)

treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2

mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested after Rb treatment for

48 hours (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05.

Wyalkatchem Gutha

K (mM)

Na (mM)

Total length (cm)

Diameter (mm)

Surface area (cm2)

Total length (cm)

Diameter (mm)

Surface area (cm2)

0.05 0 3937cde 0.36abc 468d 2578e 0.41a 300e

0.05 2 4426cde 0.35bcd 508cd 3579de 0.38ab 431de

0.05 10 3791de 0.34bcd 418de 3616de 0.39ab 448d

2 0 7282a 0.32cd 744a 6535ab 0.35bcd 707a

2 2 6976a 0.30d 643abc 5561a-d 0.35bcd 614abc

2 10 5898abc 0.34bcd 689ab 4563b-e 0.36abc 548bcd

Wyalkatchem Gutha

K (mM)

Na (mM)

Root Volume (cm3) Tips Forks

Root volume (cm3) Tips Forks

0.05 0 4.07cde 6041d 16356de 3.23e 5199d 9167e

0.05 10 4.75bcd 7760cd 17815de 4.26cde 5110d 13357e

0.05 20 3.89de 5812d 13338e 4.22cde 6063d 13147e

2 0 6.07ab 17593a 47488a 6.33a 14832a 43124a

2 10 5.33a-d 16779a 41388ab 5.41abc 11460b 32353bc

2 20 5.18a-d 16825a 41294ab 4.69b-e 9217bc 25084cd


Leaf gas exchange parameters were measured 2 weeks after K and Na treatment and before

Rb addition. At low K, 2 mM Na addition significantly increased leaf net photosynthesis rate

of both cultivars compared with nil Na addition (Fig. 7.3). Gutha at low K with nil and 10

131

mM Na had the lowest photosynthesis rate among the treatments. The interactions between

K, Na and cultivars were significant for photosynthesis measurements (Table 7.1). Adequate

K treatment had significantly higher stomatal conductance (Gs) than low K, except Gutha at

10 mM Na. Similarly, addition of 2 mM Na to low K nutrient solution increased stomatal

conductance. Transpiration rate was the lowest in Gutha at low K without Na and the highest

in Wyalkatchem at high K with 2 mM Na. The interaction between K and Na was significant

for stomatal conductance and transpiration (P ≤0.05), but not with cultivars (Table 7.1).

Wy alkatchem

Leaf

ne

t ph

otos

ynth

esi

s (µ

mol

CO 2/m

2 .s)

0

5

10

15

20

25

30

Sto

mat

al c

ondu

ctan

ce (

mm

olH 2O

/m2 .s

)

0

200

400

600

800

0.05 mM 2 mM

Tran

spir

atio

n (m

mol

H2O

/m2 .s

)

0

2

4

6

8

Gutha


0.05 mM 2 mM

0 Na 2 Na 10 Na

Fig. 7.3 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars

Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks,

followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10

mM) for 2 weeks (±SE, n=4).

132

7.3.3 K, Na and Rb concentrations

Pre-treatment shoot K and Na concentrations (on a dry weight basis) were 19.7 mg K/g and

0.16 mg Na/g in Wyalkatchem, and 19.0 mg K/g and 0.18 mg Na/g in Gutha, respectively.

Root K and Na concentrations were 12.5 mg K/g and 0.77 mg Na/kg in Wyalkatchem, and

10.7 mg K/kg and 1.29 mg Na/kg in Gutha, respectively. Rubidium concentration was below

the detection level in shoots and roots of both cultivars (< 0.02 mg Rb/g).

Pre- Rb harvest

Seventeen days after K and Na treatments, shoots of both cultivars had nearly four- fold

higher K concentration at adequate K supply (53.3 mg K/kg) than at low K (14.9 mg K/kg)

(Table 7.3). At low K, addition of 2 mM Na did not increase shoot K concentration

significantly in either cultivar. Shoot Na concentrations were related to solution Na

concentrations, and increased with increasing Na concentrations in both cultivars from 0.15

mg Na/kg at nil Na to 1.54 mg Na/kg at 10 mM Na (Table 7.3). The three way interaction

between K, Na and cultivars was significant for shoot K concentrations but not for shoot Na

concentrations (P ≥0.05).

Shoot and root K concentration showed similar response to the K and Na treatments. Root K

concentration was 38 mg K/kg at adequate K supply compared with 6.8 mg K/kg at low K

supply. The addition of Na had no effect in root K concentrations at low K supply, whereas

root K concentration decreased with 20 mM Na in both cultivars at adequate K supply. Wheat

roots had lower K concentration than shoots in both cultivars, particularly at low K levels. In

contrast, roots had considerably higher Na concentration than shoots. Root Na concentration

increased with increase in solution Na concentrations. Similar to shoot, the interaction

between K and Na was significant for root K concentration but not for root Na concentration.

Also, the three way interaction between K, Na and cultivar was not significant (Table 7.4).

133

Table 7.3 Shoot and root K and Na concentrations in cultivars Wyalkatchem (K-efficient)

and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with

two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb

harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05.


Shoot ion concentration (mg/g, dry wt.) (n=4)

K Na K Na

0.05 0 15.5de 0.15g 12.6e 0.22fg

0.05 2 17.3d 0.77e 15.3de 0.89de

0.05 10 15.2de 1.22cd 13.6e 1.99a

2.0 0 53.4ab 0.12g 56.2a 0.11g

2.0 2 56.1a 0.77e 52.1bc 0.57ef

2.0 10 52.0bc 1.33bc 49.9c 1.62ab


K Na K Na

0.05 0 6.99d 0.52d 6.68d 0.48d

0.05 2 7.20d 3.01c 6.46d 3.86bc

0.05 10 6.98d 6.15a 6.63d 6.86a

2.0 0 36.2b 0.39d 44.1a 0.43d

2.0 2 37.5b 3.72bc 43.0a 4.50b

2.0 10 32.2c 5.89a 34.8bc 7.06a

134

Table 7.4 Statistical summary of shoot and root K, Na and Rb concentrations in cultivars

Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks

and then treated with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for

2 weeks (Pre-rubidium harvest), and harvested after Rb treatment for 48 hours (Post-

rubidium harvest). *P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant


Pre- rubidium harvest

Shoot K conc *** *** *** *** n.s ** ***

Shoot Na conc ** *** *** n.s ** *** n.s

Root K conc *** *** *** *** *** ** **

Root Na conc n.s *** *** n.s n.s * n.s

Post- rubidium harvest

Young leaves

K concentration *** *** *** *** n.s ** ***

Na concentration * *** * * n.s ** n.s

Rb concentration *** *** ** *** ** n.s n.s

Old leaves

K concentration *** *** n.s *** *** *** ***

Na concentration n.s *** n.s n.s n.s * n.s

Rb concentration *** *** *** *** *** n.s n.s

Rest of shoot

K concentration *** *** n.s n.s n.s n.s n.s

Na concentration ** *** ** n.s n.s n.s n.s

Rb concentration *** ** *** n.s *** n.s n.s

Root

K concentration *** *** n.s *** n.s n.s n.s

Na concentration *** *** *** * n.s * n.s

Rb concentration *** *** n.s *** n.s n.s n.s

135

Post-Rb harvest

Forty-eight hours after Rb addition, tissue K and Na concentrations remained similar to those

at pre-Rb harvest, i.e. young leaf K concentrations were higher at adequate K supply than low

K but did not vary with Na addition, except that 10 mM Na reduced young leaf K

concentration in Gutha with adequate K supply (Table 7.5). Young leaf Na concentrations

increased with addition of 2 and 10 mM Na. With Rb addition, young leaf Rb concentrations

in low K treatment were significantly higher than in adequate K treatment. At low K supply,

Rb concentrations in young leaves were significantly increased in both cultivars with addition

of 2 mM Na but decreased with 10 mM Na in Gutha, compared with nil Na (Table 7.5). At

adequate K, there was no change due to Na addition. The interaction between K and Na was

significant (P ≤0.05) for young leaf K, Na and Rb concentrations, while the three way

interaction among K, Na and cultivars was significant only for young leaf K concentrations

(Table 7.4).

Old leaf K concentration in low K treatment was lower than young leaf K concentrations.

Sodium concentration of old leaves was dependent on solution Na concentrations. Old leaf

Rb concentration increased significantly with 2 mM Na addition at low K treatment in

Wyalkatchem but not in Gutha. However, there was significant decrease in Rb concentration

with 10 mM Na addition at low K treatment in both cultivars. The interaction between K and

Na was significant for old leaf K and Rb concentrations while interaction between K, Na and

cultivar was significant only for K concentration (Table 7.4).

The rest of shoots also had similar treatment response as the leaves in terms of K and Na

concentrations. Adequate K treatment had considerably higher shoot K concentration than

low K treatment (Table 7.5). Low K treatment had significantly higher Rb concentration than

adequate K treatment, and the addition of 2 mM Na increased the Rb concentration

significantly in Wyalkatchem, but not in Gutha. The interaction between K, Na and cultivar

was not significant for rest of shoot K, Na and Rb concentrations (Table 7.4).

Roots had lower K concentrations compared with leaves and the rest of shoots. Adequate K

treatment resulted in nearly six-fold higher root K concentration than low K treatment, but Na

addition had no effect on root K concentration. Sodium addition increased root Na

concentration at both K levels. At low K, roots had considerably less Rb than the rest of

shoots, and Rb concentration varied significantly between low and adequate K treatments.

136

The addition of 2 mM Na to low K nutrient solution increased root Rb concentration in both

Wyalkatchem and Gutha.

Table 7.5 Young leaf, old leaf, and the rest of shoot and root K, Na, and Rb concentrations in

cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2

weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2

and 10 mM) for 2 weeks, and harvested 48 hours after Rb addition (35 days after

transplanting). Means (n=4) with different letters differ at P≤0.05.


Young leaf ion concentration (mg/g, dry wt.) (n=4)

K Na Rb K Na Rb

0.05 0 14.0cd 0.33d 20.4bc 12.3d 0.33d 21.3b

0.05 2 15.7c 1.67c 23.9a 13.8cd 1.66c 23.2a

0.05 10 13.8cd 2.70ab 19.5c 12.3d 2.95a 19.5c

2.0 0 45.5a 0.36d 3.39d 47.5a 0.36d 2.56de

2.0 2 47.9a 1.41c 3.27de 47.4a 1.48c 2.37de

2.0 10 45.3a 2.43b 2.54de 41.5b 2.85a 2.02e

Old leaf ion concentration (mg/g, dry wt.) (n=4)

K Na Rb K Na Rb

0.05 0 10.6de 0.65d 18.4b 7.88e 0.66d 15.2c

0.05 2 11.6d 2.63c 19.6a 8.77de 3.63abc 15.2c

0.05 10 10.3de 4.34a 15.2c 7.84e 4.52a 10.9d

2.0 0 45.0bc 0.65d 2.71ef 51.3a 0.60d 2.14ef

2.0 2 45.9b 2.61c 2.85e 44.6bc 3.04bc 2.03ef

2.0 10 42.6c 4.10ab 2.35ef 42.5c 3.95ab 1.68f

137

Rest of shoot ion concentration (mg/g, dry wt.) (n=4)

K Na Rb K Na Rb

0.05 0 15.1cde 0.37e 29.9b 12.8de 0.37e 26.4cd

0.05 2 17.8c 1.39cd 32.9a 16.5cd 1.23d 26.2cd

0.05 10 13.9cde 2.39ab 27.3bc 12.1e 2.76a 23.8d

2.0 0 59.9a 0.23e 5.05e 60.3a 0.22e 5.53e

2.0 2 59.5a 0.77de 4.65e 59.6a 1.27d 3.89e

2.0 10 54.2b 1.96bc 3.39e 54.0b 2.45ab 3.08e


K Na Rb K Na Rb

0.05 0 5.25c 0.90d 16.4bc 4.46c 0.84d 16.3bc

0.05 2 4.67c 4.18c 18.9a 4.07c 4.59c 18.0a

0.05 10 4.04c 6.36ab 15.8bc 3.82c 6.93a 15.0c

2.0 0 33.2a 0.72d 7.21de 33.4a 0.69a 7.52d

2.0 2 31.6a 3.93c 6.21de 32.4a 4.38c 6.52d

2.0 10 23.7a 5.72b 4.93ef 28.0ab 6.18b 3.41f

7.3.4 K, Na and Rb contents

At pre-Rb harvest shoot K content was significantly higher in adequate K treatment than low

K treatment (Table 7.6). There was little change in shoot K uptake due to Na addition or

between the cultivars Wyalkatchem and Gutha. Shoot Na content increased with increasing

Na levels (Table 7.6). Roots had lower K content but higher Na content than shoots (Table

7.6). The interaction between K and Na was significant for shoot and root K and Na content,

while three way interactions with cultivar were significant for shoot and root K content

(Table 7.7).

138

Table 7.6 Shoot and root K and Na contents in cultivars Wyalkatchem (K-efficient) and

Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, and harvested after treatment with

two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb

harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05.



K Na K Na

0.05 0 15.5de 0.15g 12.6e 0.22fg

0.05 2 17.3d 0.77e 15.3de 0.89de

0.05 10 15.2de 1.22cd 13.6e 1.99a

2.0 0 53.4ab 0.12g 56.2a 0.11g

2.0 2 56.1a 0.77e 52.1bc 0.57ef

2.0 10 52.0bc 1.33bc 49.9c 1.62ab


K Na K Na

0.05 0 6.99d 0.52d 6.68d 0.48d

0.05 2 7.20d 3.00c 6.46d 3.86bc

0.05 10 6.98d 6.15a 6.63d 6.86a

2.0 0 36.2b 0.39d 44.1a 0.43d

2.0 2 37.5b 3.72bc 43.0a 4.50b

2.0 10 32.2c 5.89a 34.8bc 7.06a

139

Table 7.7 Statistical summary of shoot and root K, Na and Rb contents in cultivars

Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks,

followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10

mM) for 2 weeks (Pre-Rb harvest) and harvested 48 hours after Rb treatment (post-rubidium

or final harvest).



Pre- rubidium harvest

Shoot K content *** *** *** *** *** *** ***

Shoot Na content *** *** *** *** n.s *** n.s

Root K content *** *** *** *** *** ** **

Root Na content *** *** * *** ** n.s n.s

Post- rubidium harvest

Shoot

K content *** *** *** *** *** *** ***

Na content *** *** *** * * *** n.s

Rb content *** *** *** *** *** n.s ***

Root

K content *** *** n.s *** ** n.s n.s

Na content n.s *** n.s n.s n.s n.s n.s

Rb content *** *** *** *** *** n.s ***

Post-Rb shoot K content at adequate K supply was higher than at low K supply in both

Wyalkatchem and Gutha. At low K supply, shoot K content was significantly higher with 2

mM Na addition than with 10 mM Na in both cultivars and with nil Na in Gutha. Shoot Na

content increased significantly with 2 and 10 mM Na addition (Table 7.8). Shoot Rb content

was lower at adequate K treatments than low K treatments. Shoot Rb increased significantly

with 2 mM Na addition to low K treatment. Root K and Rb contents were much lower than

the shoot contents (Table 7.8). The addition of 2 mM Na increased root Rb content of

140

Wyalkatchem and Gutha at low K. The interaction between K and Na was significant for

shoot K, Na and Rb contents, while three way interactions between K, Na and cultivar were

significant for shoot K and Rb contents. The interaction between K, Na and cultivars was also

significant for root Rb content.



levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested 48

hours after Rb addition (35 days after transplanting). Means (n=4) with different letters differ

at P≤0.05.



K Na Rb K Na Rb

0.05 0 17.2gh 0.49f 31.3c 18.6gh 0.61f 36.5b

0.05 2 19.8g 1.96e 34.7b 25.2f 2.86d 40.8a

0.05 10 15.5h 3.2cd 27.3d 19.8g 5.13b 36.2b

2.0 0 90.1d 0.51f 7.24fg 152a 0.78f 12.3e

2.0 2 92.2d 1.84e 6.9fg 134b 3.61cd 8.20f

2.0 10 83.5e 3.71c 5.05g 113c 6.07a 6.10fg


K Na Rb K Na Rb

0.05 0 1.95d 0.33c 6.12b 1.41d 0.26c 5.13d

0.05 2 1.84d 1.64b 7.46a 1.34d 1.52b 5.92bc

0.05 10 1.57d 2.48a 6.15b 1.35d 2.47a 5.35cd

2.0 0 14.6ab 0.32c 3.18ef 15.1a 0.32c 3.41e

2.0 2 14.0ab 1.73b 2.76fg 13.5b 1.82b 2.72fg

2.0 10 10.3c 2.49a 2.15g 11.2c 2.48a 1.35h

141

7.4 Discussion

This experiment showed no significant increase in shoot dry weight with Na addition for two

weeks to low K treatment in both cultivars at pre-Rb and post-Rb harvest. This is in contrast

to the previous split-root experiment (Chapter 4) where there was significant increase in

shoot dry weight in Wyalkatchem with Na addition of 50 mg Na/kg to one or both the

compartments at deficient soil K supply for 5 to 6 weeks. It is consistent however, with the

lack of shoot dry weight response to low Na in K-deficient wheat in Chapter 3. The root dry

weight of Wyalkatchem showed an increase with 2 mM Na addition to low K (0.05 mM K)

for 2 weeks. In previous pot experiment (Chapter 3), root dry weight increased only in K-

efficient cultivars (including cv. Wyalkatchem) with soil K deficiency and Na addition, and

also in split-root experiment at low K with 50 mg Na/kg Na addition, where the increase in

root dry weight was comparable to that with adequate K supply. The stimulation in shoot and

root dry weight due to Na in previous studies at 25 and 50 mg Na/kg was equivalent to 7.25

and 14.5 mM Na in soil solution at 15 % field capacity which was slightly higher than the

concentrations of 2 and 10 mM Na in the present study. Unlike the present study, there were

moderate to severe K deficiency symptoms observed in previous studies where positive

response to Na addition was observed in shoot and root growth. In an earlier solution culture

experiment with much lower Na concentrations, wheat biomass increased in one of the

experiments when 500 µM Na was added to nutrient solution with deficient external K (20

µM K) when there were low levels of light (Box & Schachtman, 2000). In the present study,

there was an increase in root dry weight of Wyalkatchem but no change in shoot dry weight.

Potassium has a vital role in regulating photosynthesis and stomatal aperture, and when plants

are K deficient, the rate of photosynthesis is depressed (Cakmak, 2005). In K deficient plants,

it was believed that Na+ substitutes K+ in maintaining ionic balance, osmotic pressure and

stomatal conductance (Marschner, 1995; Subbarao et al., 2003). Also, under K-deficient

conditions, low levels of Na can be beneficial to physiological processes, and this is mainly

attributed to the function of K+ as an osmoticum in vacuoles which can be replaced by Na+

(Gattward et al., 2012; Gierth & Mäser, 2007).

In this long-term solution culture experiment there was a significant increase in leaf net

photosynthesis rate and stomatal conductance with 2 mM Na addition to low K treatment

solution in both cultivars. The increase in leaf net photosynthesis rate as a result of 2 mM Na

was almost equal to the presence of adequate K in nutrient solution (2 mM K). Similarly, in a

study by Gattward et al. (2012) on cocoa a significant increase in photosynthesis, nearly

142

double than that of control (nil Na) was noticed with 40 % replacement of K+ by Na+ in the

soil medium. With increased photosynthesis in shoots of K-deficient wheat plants supplied

with low Na, more assimilates would be available for the need of root growth and nutrient

uptake. This was also the case in Chapter 3.

At the level of 2 mM K supply, there was enough K+ uptake to maintain adequate leaf

concentration and maximum growth. The K+ uptake mechanism which operates effectively at

2 mM K+ may, however, not operate efficiently to supply K+ required for plant growth at 0.05

mM K, as external K+ was too low to supply K requirements for growth. A recent evaluation

of mechanisms of K+ uptake at various external concentrations showed that non-selective

channels are the main pathway for K+ uptake at high concentrations (> 10 mM K). The

inward rectifying channel AKT1 dominates K+ uptake at intermediate concentrations (around

1 mM K), while at lower concentrations (0.1 mM K), AKT1 along with the high-affinity K+

uptake system (HAK) are the dominant K uptake systems. At extremely low concentrations

(< 0.01 mM), the only system capable of K+ acquisition is HAK5 or HAK1 (Nieves-

Cordones et al., 2014).

In this experiment, pre-Rb harvest showed no change in shoot or root K concentrations and K

uptake of both cultivars in the presence of low external Na+ at low K. Similarly the K

concentrations measured after Rb+ tracer addition for 48 hours showed no significant increase

in shoot parts of both Wyalkatchem and Gutha with Na addition to low K treatment solution.

However, 2 mM Na addition to 0.05 mM K treatment solution significantly increased Rb

concentrations (tracer for K+ uptake in this study) in young leaves, old leaves, rest of shoot,

i.e., in all parts of shoots and roots of Wyalkatchem, and in young leaves and roots of Gutha.

Shoot and root Rb+ uptake showed a significant increase in both cultivars with 2 mM Na

addition to low K treatments. Therefore, the findings showed evidence of Na+-coupled Rb+

influx in wheat roots and shoots with much stronger influx in K-efficient Wyalkatchem than

K-inefficient Gutha with external Na+ concentration of 2 mM Na but not at 10 mM Na.

In this experiment when Rb+ uptake was stimulated by low concentrations of Na+ addition,

K+ uptake increased but was not significant with Tukey’s HSD comparison. This may be due

to differences in Rb+ and K+ uptake behaviour. Negative feedback from K in plants may

decrease K+ uptake in long term studies but not Rb+ uptake since Rb do not have an essential

physiological role in plants. Moreover, the large amount of K in shoots already makes it

difficult to detect a small increase during the treatment period which can only be detected

using tracer such as Rb or radio-active K source.

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An earlier study in wheat using Rb+ as a tracer by Box and Schachtman (2000) showed that

low concentrations of Na+ do not increase K+ uptake to a large extent suggesting that Na+-

energised K+ is not a major mechanism for high-affinity K+ acquisition (Box & Schachtman,

2000). In this experiment, the increased K+ uptake with 2 mM Na addition to deficient supply

of K in nutrient solution observed could be explained by effect of Na+ on K transporters and

uptake mechanism. One possible mechanism is that in wheat roots, the high-affinity K+

uptake transporter HKT1 was shown to function as a K+-Na+ cotransporter with activation of

high-affinity K+ uptake by micromolar Na concentrations (Rubio et al., 1995). However,

more recent evidence emerged that HKT operates predominantly as a Na+ channel by

selecting Na+ over K+ (Benito et al., 2014). Another possible mechanism is mediated by low-

affinity K+ uptake involving Na+ hyper-polarization of the plasma membrane and increased

K+ uptake via inward-rectifying activated K+ transporters (Chen et al., 2005; Shabala & Cuin,

2008). The latter mechanism could also account for increased Rb+ uptake by wheat.

An increase in root Na concentration in K-deficient plants was shown to stimulate root

growth in cotton (Ali et al., 2009) and in previous soil culture experiments of wheat

(Chapters 3 and 4). Whether an increase in root elongation could contribute to increased plant

K uptake was examined by scanning of roots to assess length, surface area etc., as such an

effect could contribute to nutrient uptake by providing access to additional nutrient supply

(Barber & Silberbush, 1984). The results showed no significant increase in root dry weight,

or other parameters like root length, volume and surface area when Na+ was added to low K

treatments suggesting that increased root length and or surface area was not the reason for Na

stimulated K+ and Rb+ uptake..

The results from K+ uptake studies suggest that Na+-coupled Rb+ influx in wheat varied

between short-term and long-term experiments. In the short-term experiment, when Na

treatments and Rb were added for 48 hours, there was an increase in shoot and root Rb+

concentrations, and shoot Rb+ uptake in Wyalkatchem only, while in the long-term

experiment where plants were treated with Na treatments for 16 days before Rb+ addition

there was increased Rb+ influx due to Na+ in both cultivars. Hence the K-efficient cultivar

had quicker response to low to moderate Na supply relative to the K-inefficient cultivar as the

former increased Rb+ uptake with short-term exposure of Na treatment but the latter required

long-term Na exposure for showing Na+-energised Rb+ (K+) uptake. The solution culture

results showed that Na stimulated greater K uptake by K-efficient cultivars, consistent to the

finding from my pot experiment (Chapter 3).

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In previous study to rank wheat genotypes according to K-use efficiency there was no

examination of the role of Na (Damon & Rengel, 2007). In the present study, the relative K-

use efficiency of wheat cultivars was consistent across a range of Na levels from no added Na

up to toxic levels. Moreover in this study, K-efficient cultivar showed better response with

low to moderate Na supply relative to K-inefficient cultivar. In contrast to the suggestion that

increased capacity to substitute Na+ for K+ may be a mechanism underlying K-use efficiency

in wheat (Damon & Rengel, 2007), our results suggest that Na stimulated greater Rb+ uptake

(tracer for K+ uptake) by K-efficient cultivars. This raises the question of whether Na+

activation of K+ transporters should be re-examined in wheat cultivars with a range of K-use

efficiencies.

7.5 Conclusion

The addition of low Na (2 mM) to low K (0.05 mM) solution for 14 days increased root dry

weight of Wyalkatchem and increased leaf net photosynthesis and stomatal conductance in

both Wyalkatchem and Gutha. There was an 11 % increase in shoot Rb+ uptake in both

cultivars, and 22 % and 13 % increase in root Rb+ uptake of Wyalkatchem and Gutha,

respectively with addition of low Na. This study showed an increase in K+ uptake (as

measured with Rb+) suggesting the likely role of Na+ in energising K+ uptake by an effect of

Na+ on low-affinity K+ uptake involving Na+ hyper-polarization of the plasma membrane via

inward-rectifying activated K+ transporters or high-affinity transporter HKT1 which can

function as a K+-Na+ cotransporter.

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CHAPTER 8

GENERAL DISCUSSION AND CONCLUSIONS

8.1 Introduction

Under K deficiency, Na can substitute for K in maintaining ionic balance (Subbarao et al.,

2003), regulating osmotic pressure (Marschner, 1995), exerting vacuolar functions (Mäser et

al., 2002), and improving water balance via stomatal conductance (Gattward et al., 2012).

However, the degree of substitution varies among species and cultivars and so far there is a

paucity of information on effects of Na on K nutrition of wheat especially in soil-based

conditions (Ma et al., 2011). A good understanding of Na effects on K nutrition would

optimise soil K use and improve K fertilizer management and grain yield of wheat grown

under sodic and saline soils. Previously it has been suggested that cultivar differences in K-

use efficiency in wheat may be related to variation in Na substitution of K (Damon & Rengel,

2007) but there was no direct evidence available on whether the K-efficient genotypes would

be more or less responsive to soil Na supply than the K-inefficient genotypes. The main

objective of this thesis was to investigate the interaction between K and Na in wheat cultivars

at levels of NaCl that caused salinity and those that did not. A series of experiments were

designed to study the effect of NaCl on wheat K uptake, leaf gas exchange, cation ratios, and

plant growth in a range of K (deficient to adequate) and Na concentrations (low to toxic). The

experiments were conducted in both soil and solution culture systems and tested cultivars

differing in K-use efficiency and used a range of experimental approaches including split-

root, water deficit and tracer flux studies.

8.2 Growth stimulation by Na

Although Na is not an essential element for glycophytes such as wheat, the addition of Na

stimulated growth under limited K availability in three experiments of this study including

soils in standard pots, in split-root pots and in solution culture. Tiller number and shoot dry

weight were significantly increased in the split-root experiment when 50 mg Na/kg was

added to one or both compartments of 40 mg K/kg treatments which otherwise induced

severe K deficiency (Chapter 4). Low to moderate Na supply also alleviated K deficiency

symptoms in two pot experiments (Chapters 3 and 4). However, in the initial pot experiment

with cultivars differing in K-use efficiency, shoot dry weights were not significantly affected

by low to moderate Na (25 to 50 mg Na/kg equivalent to 7.25 and 14.5 mM Na in soil

solution at 15 % field capacity) in K deficient plants (Chapter 3). Similarly, shoot weight did

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not show significant stimulation with low or moderate Na additions to low K treatments in

the column experiment (Chapter 5) or solution culture experiments (Chapter 6 and 7).

Root growth was more responsive to Na at low K supply than shoot in experiments where

growth stimulation was observed. The root growth in K-efficient cultivars increased

significantly with low to moderate Na addition (25 to 50 mg Na/kg) under low soil K

(Chapter 3). Similarly, in the long-term solution culture experiment root growth stimulation

was seen at pre-Rb harvest only in K-efficient Wyalkatchem treated with 2 mM Na and 0.05

mM K (Chapter 7). In pot experiment and long-term solution culture experiment (Chapters 3

and 7), Na-induced growth stimulation was clearly evident in roots but not in shoots. The root

growth stimulation was much stronger in split-root experiment in Wyalkatchem with addition

of 50 mg Na/kg to one or both of the compartments at deficient soil K (40 mg K/kg). Indeed

the magnitude of root growth stimulation with 50 mg Na/kg was comparable to that with

adequate K supply (Chapter 4).

Even though the same levels of low K treatments (40 mg K/Kg) were used in all three soil-

based experiments, the plant to soil weight ratio varied considerably and this altered the

degree of K deficiency. Potassium deficiency was very severe in the split-root experiment

with a plant (number) to soil weight (kg) ratio of 1: 2.0 (Chapter 4), moderately severe in the

pot experiment with a ratio of 1: 2.07 (Chapter 3), and in the column experiment with plant to

soil weight ratio of 1: 5.0 (Chapter 5) wheat did not show any K deficiency symptoms or Na-

induced growth stimulation at low K supply. These experiments differed in the depth and

volume of soil that can be exploited by roots, and the pool of K available for K uptake in the

column experiment was 2.5 times greater with an extra 160 mg K/plant available than in the

pot and split-root experiments. As the available soil K can be locally depleted by root K

uptake in the pot, continued K uptake is dependent on root extension to access K available

elsewhere in the pot or deeper in the soil profile (Damon & Rengel, 2007). Therefore, in the

pot and split-root experiments as available K was depleted, added Na replaced some roles of

K by maintaining better plant growth than nil Na treatment. Hence in retrospect the research

questions posed for the column experiment (Chapter 5) would have been more effectively

tested if the ratio of plant number to soil weight was similar to that in the pot and split root

experiments (Chapters 3 and 4).

Wheat growth was stimulated when 0.5 mM Na was added to 0.02 mM K solution in an

earlier study, however, at much lower concentration of K and Na compared to this study, and

moreover, there was no differentiation between shoot and root growth in the study (Box &

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Schachtman, 2000). The present research in wheat showed contrasting effects of low to

moderate Na to those with barley where addition of 100 mg Na/kg stimulated significant

shoot growth increases but not root growth of barley (Ma et al., 2011). In salt-tolerant barley,

addition of 100 mg Na/kg stimulated significant shoot growth but not root growth, whereas

in wheat 100 mg Na/kg reduced the growth of K deficient plants (30 mg K/kg) (Ma et al.,

2011). Similar findings were obtained from the pot experiment in this thesis where 100 mg

Na/kg for 8 weeks had negative effects on plant growth (Chapter 3). The stimulative effect of

Na was at much lower Na levels in wheat (25- 50 mg Na/kg soil) than in barley (100 mg

Na/kg soil) and in wheat there was stronger response in roots than shoots.

8.3 Na effects on K deficient wheat

Typical K deficiency symptoms started to appear at low soil K (40 mg K/kg) with yellowing

of older leaves and brown spots in shoots at 6 weeks after germinated seeds were transferred

in the pot experiment in all wheat cultivars irrespective of K-use efficiency (Chapter 3). The

treatments of 25 and 50 mg Na/kg eliminated the deficiency symptoms and produced greener,

more erect leaves (Refer to earlier photos). Severe K deficiency symptoms developed and

persisted in the treatment of nil Na and low K supply. Under split-root condition, addition of

50 mg Na/kg in just one of the two root compartments was able to alleviate K deficiency

symptoms and produced healthy plants with increased tillering (Chapter 4). Previous study

also reported that rice plants grown without NaCl under K deficiency showed yellow

discoloration with droopy leaves and marginal necrosis, while in NaCl-treated plants the

leaves remained erect and greener (Yoshida & Castaneda, 1969).

Sodium supply had a beneficial role in leaf net photosynthesis, stomatal conductance and

transpiration at limited K supply in this study. At low K (40 mg K/kg), addition of low to

moderate Na (25 to 50 mg Na/kg) increased leaf photosynthesis and stomatal conductance to

measured values similar to those under adequate K and nil Na conditions (Chapter 3) and

there was three-fold increase in leaf gas exchange measurements in split-root experiment, at

low K, with addition of 50 mg Na/kg to just one of the compartments when compared with

low K without Na (Chapter 4). Similar increase in leaf gas exchange was seen in long-term

solution culture experiment with addition of 2 mM Na+ to 0.05 mM K solution (Chapter 7).

However, the lack of shoot growth response in the pot experiment even though gas exchange

increased with Na supply to low K plants is puzzling. It could be related to the late

appearance of K deficiency as symptoms first appeared after 6 weeks growth only 2 weeks

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before harvest in the pot experiment (Chapter 3). Hence it is possible that the shoot response

lagged behind that of the root and given time would have been measurable. This is consistent

with low root-shoot ratio being a pronounced response to low K (Ma et al., 2011). Hence the

stimulation of gas exchange by low to moderate Na in low K plants may increase

carbohydrate supply first to roots and later to shoots. Similarly, in long-term solution culture

experiment, an increase in root growth of Wyalkatchem and photosynthesis of both cultivars

were measured with 2 mM Na addition to low K plants, even though there was no significant

shoot response (Chapter 7). However, in split-root experiment K deficiency symptoms were

severe and early (3-4 weeks after transplanting) compared to other experiments and the

beneficial effects of Na was evident in both shoots and roots (Chapter 4).

The positive effect of Na on photosynthetic and respiratory CO2 exchange has been long

reported in sugar beet under K deficiency, suggesting that Na+ may have substituted for K+ in

stomatal opening either as an alternative cation to K or by conserving K supply (Terry &

Ulrich, 1973). However, the evidence gathered in the present study suggests that the low Na

supply that stimulated photosynthesis and stomatal conductance did not increase leaf or shoot

Na concentrations sufficiently to account for Na substitution of K. For example in pot

experiment in the low K plants, shoot K concentration was 13.9 mg K/g, dry weight

(equivalent to 89.5 mM K in tissue water) at 25 mg Na/kg of soil, whereas, depending on

cultivars there was only 0.18-0.33 mg Na/g, dry weight (mean tissue water Na concentration

of 2.8 mM) which was too low to replace K+ functions. Instead, the addition of Na increased

shoot K concentration/uptake and it is most likely that the photosynthesis and stomatal

conductance responses were due to increased leaf K concentration.

Sodium has the potential to replace K+ in certain non-specific metabolic functions in certain

plant species (Wakeel et al., 2011). High-affinity Na+ uptake in several plants take place

when K+ has been exhausted (Subbarao et al., 2003) suggesting that Na+ plays a

physiologically programmed role at insufficient K+ supply (Wakeel, 2013). In contrast to

previous suggestions, our study suggest that beneficial effect of Na+ in wheat can be

attributed to Na+-induced K+ uptake not to Na+ uptake.

8.4 Stimulation of K uptake by Na

The increase in K+ uptake due to addition of Na+ at deficient K supply was evident in wheat

in both soil-based and solution culture experiments. There was significant increase in shoot K

content of K-efficient cultivars, Wyalkatchem and Cranbrook with 25 and 50 mg Na/kg

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addition to low K soil (40 mg K/kg) (Chapter 3). Also, the addition of 50 mg Na/kg to one or

both of the compartments in split-root system with low K supply increased plant K content

significantly (Chapter 4). In tracer flux experiments, a short-term exposure to Rb+ (48 hours)

in low K solution (0.05 mM) containing 2 mM Na+ increased shoot Rb+ content of

Wyalkatchem (K-efficient cultivar) significantly (Chapter 6) but not Gutha (K-inefficient

cultivar). Long-term Na+ addition (14 days) showed Na+-induced K+ uptake (measured as

Rb+ uptake) in both cultivars. There was 11 % increase in shoot Rb+ uptake in both cultivars,

and 22 % and 13 % increase in root Rb+ uptake of Wyalkatchem and Gutha, respectively with

the treatment of 2 mM Na and 0.05 mM K (Chapter 7).

In this research, low to moderate Na induced beneficial effects in wheat at low K likely by

increasing K+ uptake. However, in an earlier research by Box and Schachtman (2000), there

was no evidence of enhanced K+ content in wheat due to Na supply, even though there was

an increase in wheat growth due to external Na+, indicating the positive effect of Na at low K

can be largely attributed to partial substitution of Na+ in wheat K functions and a direct effect

of Na+ on growth. In their experiment, tissue Na+ concentration increased significantly in the

presence of external Na+ when there was low solution K (20 µM K+), corresponding to

decrease in shoot K+ concentration. An increase in wheat K+ uptake (measured as Rb+

uptake) due to external low Na addition of 1 mM was seen in the uptake experiment by

Rubio et al. (1995) mediated by HKT1 transporter of wheat roots. Rubidium uptake was

increased significantly from 0.05 nmol/mg/min at nil Na to 0.40 nmol/mg/min at 1 mM Na in

solution containing 15 µM Rb+ as a tracer for K+ uptake (Rubio et al., 1995). Similarly in a

tomato growth experiment, there was significant increase in whole plant K content but not

root K+ content with 1 or 5 mM Na+ addition to low K (0.5 mM K+) treatments (Walker et al.,

2000). However, the increase in plant K uptake was not seen in their K+ uptake experiment of

tomato (Walker et al., 2000).

On the other hand, an increase in plant Na+ or shoot Na+ uptake and shoot Na+ concentrations

with addition of external Na at deficient K supply were attributed to Na+ substitution of K+

functions in few studies. There was an increase of up to 291 % in leaf Na+ content in cacao

tree with increasing replacement of K+ by Na+ at 2.5 mM soil K (Gattward et al., 2012), shoot

Na+ content of rice increased more than 10 fold with Na+ addition to K-deficient plants

(Yoshida & Castaneda, 1969), also there was significant increase in Na+ content of

eucalyptus (Almeida et al., 2010) and tomato (Tahal et al., 2000) where there was beneficial

effects seen with Na addition.

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8.5 Possible mechanisms of Na-induced K uptake

Plant roots have low and high-affinity K+ uptake mechanisms to absorb K+ from the

extracellular medium (Britto & Kronzucker, 2008; Szczerba et al., 2009). A possible

mechanism for increased K uptake is Na activation of K+ symporters in roots. Under K

deficiency, there is increased expression of the high-affinity K+ transporter (HKT) (Anschütz

et al., 2014). The HKT transporter mediates high-affinity K + uptake and high or low-affinity

Na+ uptake depending on external Na+ and K+ concentrations (Benito et al., 2014). At low

external Na+ and K+ concentrations, some transporters function as Na+-K+ symporters,

however, at high external Na+ concentrations, some of these transporters become Na+

uniporters, no longer transporting K+ (Benito et al., 2014; Rubio et al., 1995). Transporters of

the HKT-type discriminate less between K+ and Na+ or even select for Na+ over K+ (Benito et

al., 2014). However, Box and Schachtman (2000) reported the Na+ activation of K+

symporters increased K+ uptake only under low light conditions in wheat and concluded that

it was functionally a minor process for K+ uptake by wheat. Nevertheless, according to the

present study, there was increased K+ uptake due to Na+ in soil (Chapters 3 and 4), and

solution culture experiments (Chapter 7), suggesting there may be effects of Na+ on

transporters not identified by Box and Schachtman (2000).

An alternative mechanism for increased K+ uptake could be by a low-affinity K+ uptake

system (such as AKT). At high Na levels (80 mM NaCl or above in barley), Na+ causes a

significant membrane depolarization and increases K+ leakage, whereas, in sharp contrast to

80 mM NaCl treatment, K+ efflux in 20 mM NaCl treatment was found to be very short-lived

and K+ uptake became dominant from the elusive ‘osmosensing mechanism’ (Chen et al.,

2005). At moderate salinity (20 mM NaCl in barley), Na+ hyperpolarized the plasma

membrane and increased K+ uptake via inward-rectifying hyperpolarized-activated K+

channels (Chen et al., 2005; Shabala & Cuin, 2008). Therefore, Na-induced K+ flux was

clearly dose dependent, and could possibly explain increased K+ uptake at moderate Na

levels. In this study, estimated soil solution concentrations ranged from 0 to 60 mM Na (in

soil culture experiments, Chapter 3, 4 and 5) and the actual solution concentrations ranged

from 0 to 20 mM Na (solution culture experiments, Chapter 6 and 7). Since wheat is less salt-

tolerant than barley (Ma et al., 2011), it is possible for levels of 60 mM Na high enough to

cause membrane polarisation in wheat. However, the concentrations of Na+ that cause

membrane hyperpolarization and depolarization in wheat needs to be verified with

experiments.

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Other than Na+ effect on K+ transport across membranes that facilitate K+ uptake there may

be other possibilities that could contribute to increased K+ uptake. The Na-induced growth

stimulation and K+ uptake could be due to an increase in extractable soil K availability by soil

Na supply. In the incubation experiment, soil exchangeable and Colwell K showed no

significant increase with addition of Na (Chapter 3). However, in soil-based experiments for

a pot with 6 kg of soil, the increase in Colwell K was equivalent to around 18 mg in the 50

mg Na/kg treatment and provided 6 mg of extra K+ to each plant in the 3-plant pots, which

would account for part but not the entire increase of K content in Na-added plants. Clearly in

the solution culture experiment where there was also a Na stimulated uptake of K (Rb), the

effect on soil available K can be ruled out. Hence Na effects on soil available K are a possible

mechanism but not the only one or the most likely mechanism in these soils.

The Na+-induced increase in root growth due to Na+ could increase K+ uptake in wheat. The

increase in root Na concentrations may stimulate root elongation of K-deficient plants by

turgor effects on cell expansion in soil-based systems where root elongation has a major role

in determining nutrient uptake by providing access to additional nutrient supply (Barber &

Silberbush, 1984). An increase in root dry weight with low to moderate Na addition at low K

was seen in pot experiment particularly for K-efficient cultivars but there was no direct

evidence of increased root K uptake (Chapter 3). In split-root experiment the increase in root

dry weight of cv. Wyalkatchem with 50 mg Na/kg to one or both the compartments was

accompanied with significant increase in K uptake (Chapter 4). In long-term solution culture

experiment (Chapter 7), root dry weight of cv. Wyalkatchem increased only at Pre-Rb

harvest. Detailed root scanning results did not show any change in root length, root volume or

area with low external Na addition to low K solution in both short-term and long-term

experiments, despite an increase in root K+ uptake (as measured using Rb+ tracer) (Chapters 6

and 7). It is unlikely that Na supply under K deficiency would directly increase root dry

weight because root growth is impaired by limited assimilate translocation to roots in low K

and hence an increase in photosynthesis would have to be triggered first before root growth

would respond (Lemoine et al., 2013). In this study, low to moderate Na increased leaf

photosynthesis, which would lead to greater assimilate supply to roots and thus the

stimulation of root growth as appeared to occur in pot and split-root experiments and the

long-term solution culture experiment.

A possible increase in shoot K content could be due to increased K partitioning to shoots in

wheat. An increase in root Na concentrations at low to moderate Na may release vacuolar K+

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that is made available for cytoplasmic functions in the root cells or for translocation to the

shoot (Walker et al., 2000). In tomato roots, decreased K+ content and increased Na+ content

with 1 or 5 mM NaCl suggest release of K+ to maintain K+-dependent biochemical processes

in the cytoplasm or be available for translocation to shoots (Walker et al., 2000). In the

present experiments, Na+ content in roots of wheat greatly exceeded that in shoots, and

concentrations of Na+ in roots exceeded those of K+. The substitution of K by Na in roots

seems feasible and may release more K to the shoots which could account for increased shoot

K content of wheat with low to moderate Na addition to low K plants (Chapter 3). Shoot K

content increased with Na addition in K-efficient cultivars of wheat grown in pot-experiment

(Chapter 3) and short-term solution culture experiment (Chapter 6) without significant

increase in root K content.

8.6 Toxicity effects of Na

In this study, toxic effects of Na were evident at high soil Na concentrations (100 and 200 mg

Na/kg) and solution concentrations of 20 mM Na (Chapter 6) both at low and high soil K.

However, in this study less emphasis has been placed on Na toxicity effects on wheat as there

is a large body of research available on this topic already (Kronzucker et al., 2013) but much

less on effects of low to moderate Na levels. High Na levels reduced wheat growth (Chapters

3 to 5), and high Na levels of 200 mg Na/kg at both low (40 mg K/kg) and adequate K (100

mg K/kg) supply drastically reduced the photosynthesis and stomatal conductance of wheat

cultivars. It is suggested that high levels of NaCl can cause stomatal closure which limits CO2

diffusion to the chloroplast or inducing a stress-related decline in PSII photochemistry with

consequent PSII photoinhibition and/or photodamage (Degl'Innocenti et al., 2009).

Under NaCl stress conditions, reduction in K+ uptake occurs due to inhibition by Na+ ions of

K+ influx into the cell and stimulation of K+ efflux (Britto et al., 2010). The Na+-coupled K+

uptake mediated by both high-affinity and low-affinity transporters are affected by high Na

levels. According to Rubio et al. (1995), K+ uptake in wheat was stimulated at low external

Na+ (1 mM Na+) mediated by high-affinity K+ uptake transporter HKT1, but at

physiologically detrimental concentrations of Na+, K+ uptake mediated by HKT1 was

blocked and low-affinity Na+ uptake occurred (approximately 16 mM Na+), which correlated

to Na+ toxicity in plants. Moreover, at high Na levels (80 mM NaCl or above), Na+ crosses

the plasma membrane causing a significant membrane depolarization and increases K+

leakage through depolarization-activated outward-rectifying channels (Shabala & Cuin,

2008).

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8.7 Na effects on cultivars differing in K-use efficiency

The increased capacity to substitute Na+ for K+ was suggested as one possible mechanism

underlying K utilization efficiency (Rengel & Damon, 2008). However, in their previous

studies to rank wheat genotypes according to K-use efficiency (Damon & Rengel, 2007) there

was no examination of the role of Na in cultivar rankings. In this research, the ranking of

cultivars for K-use efficiency was consistent with those for the same cultivars in earlier report

in wheat by Damon and Rengel (2007). Moreover, the relative K-use efficiency of wheat

cultivars was consistent across a range of Na levels from no added Na up to toxic levels.

According to this study, K-efficient cultivars (Wyalkatchem and Cranbrook) had greater

response to low to moderate Na supply than K-inefficient cultivars (Gutha and Gamenya),

and the Na benefit was more restricted to K deficient plants. In contrast to the suggestion that

increased capacity to substitute Na+ for K+ may be a mechanism underlying K-use efficiency

in wheat, our results show that Na stimulated greater K uptake by K-efficient cultivars than

K-inefficient cultivars in experiments (Chapter 3 and 7). Sodium stimulation of

photosynthesis, stomatal conductance and root dry weight were greater in the K-efficient

cultivars (Chapter 3 and 7). These responses were consistent with greater utilization

efficiency of shoot K in the K-efficient cultivars which was the main mechanism identified

earlier by Damon and Rengel (2007) for K-use efficiency in wheat.

8.8 Implications of low to moderate Na for plant K nutrition

The present research on Na+ stimulation of growth by low-K wheat plants may be of

importance in fertiliser management strategies. There may be economic, nutritive and

environmental perspectives associated with K substitution by Na (Wakeel et al., 2011).

The application of expensive K+ fertilisers is hardly affordable by poor farmers especially in

developing countries, and partial substitution of K+ by Na+ in plant nutrition can decrease

cost of production. Particularly, soils dominant in clay minerals (vermiculite and smectite)

need a lot of K+ fertilisers, and Na+ can be a partial replacement for K+ fertilisers. Indeed,

smaller amounts of Na are required when compared to K as Na is not fixed by clay particles

(Wakeel et al., 2011). Research has shown that application of Na application at low rates

reduced K fertiliser requirement in sugar beet (Wakeel et al., 2010), and cotton was found to

grow under partial K substitution by Na (Ali et al., 2009; Ali et al., 2006). However, the

blending of Na in K fertilisers to decrease the K requirement and fertiliser cost, does not

appear to have been adopted in commercial agriculture.

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Potassium deficiency in wheat can depress root development more than shoot (Ma et al.,

2011). In this study, wheat root growth was better stimulated than shoot due to Na addition in

three instances (Chapter 3, 4 and 6). These results suggest that wheat grown under K-

deficiency and in presence of low to moderate Na may have greater benefit in stress than

well-watered conditions as Na can increase root biomass under drought. This could have an

implication under rainfed conditions where soil water is limited and where low K will further

hinder root elongation. However, under drought stress, root length or root: shoot ratio is

increased which help access to available water (Comas et al., 2013) but the effect on root

development on low K soils probably depends on which occurs first, drought or K-

deficiency.

The increase in wheat K uptake, photosynthesis, stomatal conductance and growth due to low

to moderate Na addition was evident in three experiments (Chapter 3, 4 and 7) when plants

suffered K deficiency symptoms. Moreover, the effect varied with K-use efficiency of

cultivars with K-efficient cultivars being more responsive to Na than K-inefficient cultivars.

In regards to practical implications under field conditions, based on this study, when K-

efficient wheat cultivars are grown under low to moderately saline conditions, K fertiliser

application can be reduced without risking yield declines, however, the substitution of K by

Na was not strong enough to recommend Na-based fertilisers in place of K in wheat.

Nevertheless, some encouraging results of alleviating K deficiency symptoms, stimulation in

growth and leaf gas exchange measurements by addition of moderate Na provide the

motivation for conducting further studies to improve our understanding and perspectives for

potassium fertilizer application in moderately saline and sodic soils. Further research

recommendations are discussed below.

8.9 Conclusions and recommendations

8.9.1 Conclusions

The main conclusions from this study can be summarised as follows:

• Wheat showed positive responses to low to moderate Na supply under K deficiency

with increased biomass production. Roots were more responsive to Na+ than shoot

when growth stimulation was observed.

• Wheat cultivars differing in K-use efficiency showed varied responses in growth

stimulation to added Na. There was a significant increase in root dry weight of K-

155

efficient cultivars in a standard pot soil culture experiment and long-term solution

culture experiment but not in K-inefficient cultivars.

• The growth response to NaCl varied with K and Na levels. High Na concentrations

(100 and 200 mg Na/kg in soil-based experiments) suppressed shoot and root dry

weight regardless of soil K levels.

• Genotypic differences in K-use efficiency influenced Na uptake and salt tolerance

with K-efficient cultivars being more salt tolerant than K-inefficient cultivars.

• When supplied with low to moderate Na under K deficiency, wheat cultivars showed

an increase in leaf net photosynthesis, transpiration and stomatal conductance in both

soil-based and long-term solution culture experiments with values comparable to

adequate K without Na.

• Split-root experiment showed that Na stimulation of growth in low-K plants occurred

regardless of the supply of K and Na in the same or different parts of the root system.

• Potassium uptake of wheat cultivars increased with low to moderate external Na+

supplied under K deficiency in this study in both soil-based and solution culture

experiments with Rb+ as a tracer. It was found that Na-induced increase in K+ uptake

was dose dependent.

• K-use efficiency of wheat cultivars influenced K+ uptake in presence of low to

moderate Na under K deficiency. There was a significant increase in shoot K content

of only K-efficient cultivars in a soil-based pot experiment and also in a short-term

solution culture experiment with Na addition for 48 hours using Rb+ as a tracer for

K+. However, in long-term solution culture experiment with Na addition for nearly 20

days there was significant Na-induced K+ uptake (measured as Rb+ uptake) in both K-

efficient and K-inefficient cultivars.

• The main mechanism for Na+-energized K+ uptake under limited K availability with

low external Na+ supply is attributed to the effect of Na+ on K+ transporters, both on

high-affinity and low-affinity K+ uptake transporters. However, under salinity stress

conditions, reduction in K+ uptake occurs due to inhibition of K+ influx into the cell

by Na+ ions and stimulation of K+ efflux by excess Na+.

• An increase in extractable soil K availability due to added Na may also increase root

elongation and contribute in part to the increase in K content of low K plants in soil-

based systems.

156

• This research facilitates novel understanding of the Na effects on K nutrition of wheat

cultivars differing in K-use efficiency, and will improve decision making and

management of K fertiliser in salt-affected, sodic and K-deficient soils.

8.9.2 Further research recommendations

The following recommendations are made based on the conclusions of this study to foster

new research:

• The present research based on glasshouse experiments needs to be evaluated under

field conditions with varying soil and agro-climatic conditions to define critical soil

levels of Na that stimulate wheat growth.

• More detailed research on Na+-activated low and high-affinity K+ uptake mechanisms

in wheat genotypes would improve our understanding on K uptake under moderately

saline conditions.

• It would be useful to enhance our understanding of K and Na interactions in relation

to varied levels of other major cations like Ca and Mg.

• Research on molecular expression underlying the effect of Na supply on low K wheat

genotypes. Molecular biology approach using mapped germplasm of wheat will help

to correlate the physiological responses of wheat to Na and K with the underlying

gene expression.

157

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166

APPENDIX 1

Chapter 3

1.1 K and Na concentrations in leaves, spikes and stem

Potassium concentration in young leaves, old leaves and stems of all four cultivars was

significantly higher with adequate K supply than K deficit conditions when soil Na levels

ranged from nil to moderate. In general, old leaves of K-efficient cultivars Wyalkatchem and

Cranbrook had considerably higher K concentrations at adequate K supply (100 mg K/kg). At

low K, high soil Na treatment (200 mg Na/kg) concentrated high K in leaves and stem mainly

due to concentration- effect. The interaction between soil K and Na levels was significant

(P≤0.05, Table A1.2) for leaf K concentrations; however, three way interactions between K,

Na and cultivars were not significant for K concentrations in leaves and stem. Ears/spikes had

almost similar K concentrations irrespective of K and Na treatments (Table A1.1).

Sodium concentration in leaves and stem were closely associated with soil Na levels in all

cultivars (Table A1.3). Old leaves and stem concentrated more Na than young leaves and

spikes, and concentration in young leaves was four times less than in old leaves and stem.

Sodium concentration in spikes was least influenced by soil Na irrespective of soil K and

genotypes, and there were only negligible concentrations of Na measured in spikes (Table

A1.3).

167

Table A1.1 Mean (n=3) K concentrations in young leaves, old leaves, stem and spikes (mg/g,

dry weight) of four wheat cultivars treated with two K levels (40 and 100 mg K/kg) and five

Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table A1.2 for significance of

treatment effects and interactions.

K concentrations

Na levels

Wyalkatchem Cranbrook Gutha Gamenya

K 40 K 100

K 40 K 100

K 40 K 100 K 40 K 100

Young leaves

mg/g

dry wt.

Nil Na 7.88 25.2 9.37 21.7 9.24 25.6 9.08 25.0

25 9.65 25.1 12.2 21.6 14.0 24.9 10.6 22.8

50 13.9 24.0 9.79 21.7 13.8 25.1 13.2 24.1

100 12.5 23.1 14.2 22.7 12.4 27.6 13.8 24.8

200 19.4 26.1 19.5 25.1 17.0 26.4 21.1 26.7

Old leaves

mg/g

dry wt.

Nil Na 6.64 29.8 10.8 29.7 8.7 25.7 8.15 25.9

25 11.1 28.8 19.1 28.8 14.5 23.0 11.6 25.6

50 11.0 28.5 18.4 29.4 14.6 25.5 11.6 27.9

100 8.88 30.1 13.4 31.8 13.6 26.5 10.8 27.7

200 17.9 32.1 19.4 32.6 19.4 31.9 17.1 28.2

Stem

mg/g

dry wt.

Nil Na 12.2 26.1 12.2 24.6 9.54 25.5 11.3 24.1

25 16.4 24.4 14.2 24.1 13.8 23.1 13.3 23.8

50 16.1 26.6 13.4 24.1 14.4 22.4 14.4 24.9

100 16.7 28.5 18.7 26.2 16.3 29.6 16.1 27.1

200 20.1 28.1 21.5 27.5 18.2 26.5 16.8 31.5

Spikes

mg/g

dry wt.

Nil Na 12.9 15.9 13.2 15.7 14.9 14.1 13.5 16.6

25 12.9 14.7 14.8 15.3 13.8 15.8 14.9 16.4

50 14.0 15.6 12.9 16.1 14.8 16.3 15.7 16.7

100 14.6 16.8 15.1 14.9 15.6 16.4 16.3 17.0

200 16.4 18.2 14.9 16.9 17.0 17.5 15.7 18.9

168

Table A1.2 Statistical summary of cation concentrations and contents in four wheat cultivars

(Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two levels of soil K (40 and

100 mg kg-1) and five levels of Na (0, 25, 50, 100 and 200 mg kg-1) for 8 weeks. *P≤0.05;

** P≤0.01; ***P≤0.001; n.s., not significant

Parameters Soil K Soil Na cultivar K×Na K×cv Na×cv K×Na×cv

K conc. young *** *** n.s *** n.s n.s n.s

K conc. old *** *** *** *** *** n.s n.s

K conc. stem *** *** n.s n.s n.s n.s n.s

K conc. spikes *** *** * n.s n.s n.s n.s

Na conc. young *** *** ** *** n.s * n.s

Na conc. old ** *** n.s n.s n.s n.s n.s

Na conc. stem ** *** n.s * * n.s n.s

Na conc. spikes n.s *** n.s n.s n.s n.s n.s

Ca conc. young *** *** *** ** *** n.s n.s

Ca conc. old *** *** n.s * ** * n.s

Mg conc. young *** n.s *** n.s *** n.s n.s

Mg conc. old *** *** * n.s *** n.s n.s

169

Table A1.3 Na concentrations in young leaves, old leaves, stem and spikes of four wheat

cultivars treated with two K levels (40 and 100 mg K/kg) and five Na levels (0, 25, 50, 100

and 200 mg Na/kg) for 8 weeks. Values are mean of three replicates. See Table A1.2 for

significance of treatment effects and their interactions.

Na concentrations

Na levels

Wyalkatchem Cranbrook Gutha Gamenya

K 40 K 100 K 40 K 100 K 40 K 100 K 40 K 100

Young leaves

mg/g

dry wt.

Nil Na 0.015 0.029 0.017 0.01 0.035 0.02 0.013 0.02

25 0.04 0.03 0.05 0.025 0.04 0.04 0.025 0.03

50 0.07 0.08 0.12 0.07 0.10 0.13 0.09 0.05

100 0.27 0.18 0.22 0.15 0.27 0.27 0.36 0.17

200 0.58 0.22 0.47 0.22 0.86 0.43 0.65 0.29

Old leaves

mg/g

dry wt.

Nil Na 0.07 0.08 0.08 0.05 0.065 0.06 0.08 0.06

25 0.22 0.17 0.39 0.12 0.29 0.27 0.30 0.34

50 0.57 0.56 0.89 0.51 0.81 0.77 0.80 0.75

100 1.36 1.20 1.55 1.38 1.86 1.16 2.07 1.07

200 3.90 2.98 4.00 4.25 4.97 3.89 4.14 3.08

Stem

mg/g

dry wt.

Nil Na 0.098 0.19 0.12 0.10 0.14 0.16 0.13 0.14

25 0.24 0.31 0.38 0.25 0.35 0.44 0.39 0.43

50 1.01 0.66 1.12 0.53 1.19 0.76 1.65 0.64

100 1.76 2.25 2.97 1.58 2.06 3.5 3.72 2.40

200 4.17 3.72 5.23 3.25 5.25 3.94 4.61 3.70

Spikes

mg/g

dry wt.

Nil Na 0.12 0.12 0.14 0.10 0.12 0.10 0.11 0.10

25 0.11 0.12 0.18 0.10 0.12 0.11 0.18 0.13

50 0.17 0.11 0.22 0.11 0.11 0.12 0.18 0.11

100 0.27 0.20 0.28 0.12 0.17 0.20 0.20 0.16

200 0.32 0.36 0.24 0.27 0.23 0.36 0.35 0.23

170

1.2 Ca and Mg concentrations in young and old leaves

Leaf Ca concentrations were lower in K adequate plants when compared with K deficient

plants (Fig. A1.1) at all Na levels which might be due to ‘dilution-effect’ as Ca content was

almost similar between the two K levels. The difference in Ca concentration between two soil

K levels was greater in Wyalkatchem and Cranbrook when compared with Gutha and

Gamenya. Leaf Ca concentrations in old leaves when compared with young leaves were

higher in genotypes Gutha and Gamenya, but was about the same in Wyalkatchem and

Cranbrook. Calcium concentrations in young leaves across various Na levels showed little

difference at adequate K supply. High Na treatments reduced Ca concentrations in both

young and old leaves at low soil K. Similarly, leaf Mg concentration was higher in the low K

soil than in the adequate K soil ranging from 0.8 to 2.1 mg Mg/g, dry weight (Fig. A1.2). The

interaction between K and Na levels among the genotypes was not significant for leaf Ca and

Mg concentrations (Table A1.2).

171

Gutha

0 50 100 150 2000

2

4

6

8 Gamenya

Soil Na levels (mg kg-1)

0 50 100 150 200

Wyalkatchem

Ca

conc

ent

ratio

n of

old

leav

es

(mg/

g, d

ry w

eig

ht)

0

2

4

6

8Cranbrook

Gutha

0

2

4

6

8 Gamenya

WyalkatchemC

a co

nce

ntra

tion

of y

oung

leav

es

(mg/

g, d

ry w

eig

ht)

0

2

4

6

8

10Cranbrook

Fig. A1.1 Ca concentration (mg/g, dry weight) in young (upper sub-figures) and old leaves

(lower sub-figures) (± S.E., n=3) of four wheat cultivars, treated with 40 mg K/kg (closed

circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg

Na/kg) for 8 weeks. See Table A1.2 for analysis of variance results.

172

Gutha

0 50 100 150 2000.0

0.5

1.0

1.5

2.0Gamenya

Soil Na levels (mg kg-1)

0 50 100 150 200

Wyalkatchem

Mg

conc

ent

ratio

n of

old

leav

es

(mg/

g, d

ry w

eig

ht)

0.0

0.5

1.0

1.5

2.0Cranbrook

Gutha

0.0

0.5

1.0

1.5

2.0 Gamenya

WyalkatchemM

g co

nce

ntra

tion

of y

oung

leav

es

(mg/

g dr

y w

eig

ht)

0.0

0.5

1.0

1.5

2.0

2.5Cranbrook

Fig. A1.2 Mg concentration (mg/g, dry weight) in young (upper sub-figures) and old leaves

(lower sub-figures) (± S.E., n=3) of four wheat cultivars, treated with 40 mg K/kg (closed

circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg

Na/kg) for 8 weeks. See Table A1.2 for analysis of variance results.

173

APPENDIX 2

Chapter 5

Root: Shoot ratios 5 weeks after sowing

Root: Shoot- harvest at 5 WAS

Soil K

40/40(W) 120/40(W)

root

: sho

ot ra

tio

0.0

0.1

0.2

0.3

0.4

0.5

0 Na 50 Na 200 Na

Fig. A2.1 Root: shoot ratios of wheat at 11 weeks after sowing (±SE, n=3). For treatment

descriptions refer to Table 5.1.

174

APPENDIX 3

Fig. A3.1 Experimental setup used in solution culture experiments

175

APPENDIX 4

Chapter 6

A4.1 Plant growth (Experiment 1)

Forty eight hours after K, Na treatments and Rb addition there was no significant treatment

effect on shoot or root biomass in both cvv Wyalkatchem and Gutha (Fig. A4.1). Gutha

produced nearly thrice the shoot weight and twice the root weight of Wyalkatchem (Table

A4.1). Root length measurements with the WinRhizo scanner showed no difference in

various parameters among the treatments (Table A4.2). Gutha had significantly higher root

length, projected root area, surface area, root volume and number of forks than Wyalkatchem.

0.2 mM 2 mM

Roo

t dry

wei

ght(

g/pl

ant)

0.00

0.02

0.04

0.06

0.08


0.2 mM 2 mM

0 Na 10 Na 20 Na

Sho

ot d

ry w

eigh

t(g/

plan

t)

0.00

0.05

0.10

0.15

0.20

0.25

Wyalkatchem Gutha

Fig. A4.1 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and


mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for 48 hours (harvested 17

days after transplanting) (Experiment 1) (±SE, n=4).

176

Table A4.1 Statistical summary of plant growth in cultivars Wyalkatchem (K-efficient) and


mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for a further 48 hours

(Experiment 1).



Solution Na


Shoot dry wt n.s n.s *** n.s n.s n.s n.s

Root dry wt * n.s *** n.s n.s n.s n.s

177

Table A4.2 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3),


(n=2 plants/replicate) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2

mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours

(harvested 17 days after transplanting) (Experiment 1). Means with different letters differ at

P≤0.05.

Wyalkatchem Gutha

K (mM)

Na (mM)

Total length (cm)

Diameter (mm)

Surface area (cm2)

Total length (cm) Diameter

(mm)

Surface area (cm2)

0.2 0 810b 0.39a 97.5b 1703a 0.37a 199a

0.2 10 908b 0.39a 113b 1861a 0.37a 214a

0.2 20 854b 0.37a 100b 1838a 0.37a 211a

2 0 993b 0.37a 116b 1843a 0.36a 210a

2 10 864b 0.38a 104b 1785a 0.37a 206a

2 20 881b 0.40a 110b 1884a 0.36a 215a

Wyalkatchem Gutha

K (mM)

Na (mM)

Root Volume (cm3) Tips Forks

Root Volume(cm3) Tips Forks

0.2 0 0.94b 1834b 1697b 1.86a 3962ab 4717a

0.2 10 1.11b 2882b 1960b 1.96a 4401ab 4388a

0.2 20 0.94b 3796ab 1766b 1.90a 3222b 4335a

2 0 1.08b 2311b 2057b 1.90a 3566b 4393a

2 10 1.01b 1952b 1604b 1.91a 4592ab 4302a

2 20 1.10b 2452b 1759b 1.96a 6801a 5300a

178

A4.2 Pre-treatment leaf gas exchange measurements (Experiment 1)

Table A4.3 Leaf net photosynthesis, stomatal conductance, and transpiration of wheat

cultivars Wyalkatchem and Gutha before treatment (±SE, n=24) at 14 days after transplanting

(Experiment 1).

Leaf gas exchange measurements Wyalkatchem Gutha

Photosynthesis (µmolCO2m-2 s-1) 14.4±0.17 15.6±0.71

Stomatal conductance (mmolH2Om-2s-1) 382±6.43 373±9.40

Transpiration (mmolH2O m-2 s-1) 6.39±0.16 5.95±0.29

A4.3 Plant growth (Experiment 2)

The growth response to K and Na treatments for 48 hours was mostly consistent with

Experiment 1. There was no significant difference among the treatments (Table A4.4),

despite higher biomass in Gutha than Wyalkatchem (Fig. A4.2).The growth difference

between the two cultivars was smaller when compared to Experiment 1. Total root length of

cv. Gutha treated with high K (2 mM) for 2 days were significantly higher than the

continuous low K treatment at all three Na levels (Table A4.5).

179

Table A4.4 Statistical summary of plant growth and leaf gas exchange in cultivars

Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2

weeks and two K levels (0.05 and 2 mM K), two Na levels (0, 2 and 10 mM Na) and Rb (0.5

mM) (n=4) added for 48 hours (Experiment 2).



Solution Na

cultivar K×Na K×cv Na×cv K×Na×cv

Shoot dry wt n.s n.s *** n.s n.s n.s n.s

Root dry wt n.s n.s *** n.s n.s * n.s

Photosynthesis n.s * n.s n.s ** n.s n.s

Stomatal conductance n.s n.s n.s n.s n.s n.s n.s

Transpiration n.s n.s n.s n.s n.s n.s n.s

180


0.05 mM 2 mM

Roo

t dr

y w

eigh

t (g

/pla

nt)

0.00

0.05

0.10

0.15

0.20

0.05 mM 2 mM

0 Na 2 Na 10 Na

Sho

ot d

ry w

eigh

t (g

/pla

nt)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Wy alkatchem Gutha

Fig. A4.2 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and

Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05

and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for 48 hours (harvested

19 days after transplanting) (Experiment 2) (±SE, n=4).

181

Table A4.5 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3),


treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na

levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days

after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05.

Wyalkatchem Gutha

K (mM)

Na (mM)

Total length (cm) Diameter (mm)

Surface area (cm2)

Total length (cm) Diameter

(mm)

Surface area (cm2)

0.05 0 1106d 0.41ab 146de 1462bc 0.41ab 183bcd

0.05 2 1173cd 0.40ab 147de 1520b 0.40ab 190abc

0.05 10 1083d 0.42a 143e 1443bc 0.41ab 184a-d

2 0 1198bcd 0.39ab 154cde 1906a 0.38ab 219ab

2 2 1377bcd 0.37ab 163cde 1880a 0.37ab 218ab

2 10 1256bcd 0.39ab 158cde 1945a 0.36b 223a

Wyalkatchem Gutha

K (mM)

Na (mM)

Root volume(cm3) Tips Forks

Root volume (cm3) Tips Forks

0.05 0 1.57cd 2558a 3799b 1.87a-d 2796a 3940b

0.05 2 1.55cd 2815a 3832b 1.93a-d 2930a 4166b

0.05 10 1.53d 2653a 3724b 1.93a-d 2463a 4211b

2 0 1.63bcd 2936a 3970b 2.13a 3212a 5446a

2 2 1.79a-d 3172a 4229b 2.03abc 3244a 5459a

2 10 1.65a-d 3078a 4189b 2.12ab 3268a 5780a

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