IN THE NAME OF

ALLAH

THE MOST BENEFICENT THE MOST MERCIFUL

 

SALT RESISTANCE OF HALOPEPLIS PERFOLIATA: A COASTAL SALT MARSH STEM SUCCULENT HALOPHYTE

SARWAT GHULAM RASOOL

INSTITUTE OF SUSTAINABLE HALOPHYTE UTILIZATION

UNIVERSITY OF KARACHI

KARACHI, PAKISTAN

January, 2016

 

SALT RESISTANCE OF HALOPEPLIS PERFOLIATA: A COASTAL

SALT MARSH STEM SUCCULENT HALOPHYTE

THESIS

Submitted to the Faculty of Science, University of Karachi

in Fulfillment of the Requirement for the Degree of

DOCTOR OF PHILOSOPHY IN BOTANY

By

SARWAT GHULAM RASOOL

INSTITUTE OF SUSTAINABLE HALOPHYTE UTILIZATION

UNIVERSITY OF KARACHI

KARACHI, PAKISTAN

January, 2016

 

SALT RESISTANCE OF HALOPEPLIS PERFOLIATA: A COASTAL

SALT MARSH STEM SUCCULENT HALOPHYTE

THESIS APPROVED

RESEARCH SUPERVISOR: __________________________ (PROF. DR. BILQUEES GUL)

EXTERNAL EXAMINER: ____________________________

 

Acknowledgments

First of all, I am thankful to Almighty Allah for showering his blessings upon me in every step of my life. I would like to thank my advisor Dr. Bilquees Gul (Professor and Director, Institute of Sustainable Halophyte Utilization - ISHU) for urging me to join her lab to do my PhD work. I cannot forget her continuous support and encouragement in various ways over the years towards my struggle for higher education. Special thanks to Dr. Ajmal Khan (Professor and former Director, Institute of Sustainable Halophyte Utilization - ISHU) for collecting the seeds of Halopeplis perfoliata from Saudi Arabia for my research and also for providing a conducive work environment, constant support, and advice whenever needed for my research.

I am thankful to Dr. Abdul Hameed for his guidance, encouragement and help throughout my lab work, dissertation and manuscripts writing. Thanks to Dr. Salman Gulzar who helped in taking photosynthesis data with Licor and PAM, in data analyses and dissertation writing. I would also like to thank Dr. Irfan Aziz and Dr. Zaheer Ahmed for their thought provoking ideas, helpful discussions and critical comments during my work. I am also thankful to Dr. Raziuddin Ansari for reviewing my thesis. Many thanks to all of my dear friends and colleagues who contributed a lot during various stages of my research, particularly to Dr. Ayesha Rasheed for her generous friendship and continuous guidance, Ms. Hina Asrar for Rubisco quantification, Ms. Sadaf Tauqeer, Ms. Farah Nisar and Mr. Zaheer Shah for their moral support, Ms. Erum Shoukat and Ms. Shazia Anjum Qadri for their help during lab work, Mr. Irfanuddin Taimuri and Mr. Noman Masood for their assistance in computer work and of course to sweet Ms. Mishal Birgees Khan for her kind friendship.

I would like to extend my gratitude to the Hon. Prime Minister of Pakistan, Mr. Mian Muhammad Nawaz Sharif and the Higher Education Commission (HEC) Islamabad for providing me a laptop through the Prime Minister’s LapTop Scheme which was instrumental during dissertation writing. This thesis was partially supported with funds from Higher Education Commission, Islamabad through the Pak-US collaborative research grant to Dr. Bilquees Gul. Help of University of Karachi for providing facilities and logistic support is gratefully acknowledged. I would like to express my appreciation for Prof. Dr. Hans-Werner Koyro, Giessen University, Germany for his thought provoking lectures and discussions during his visits at ISHU in better understanding of halophyte eco-physiology. Thanks are also due to Drs. Gerald Edwards and Ray Lee at Washington State University for their kind help in analyzing carbon and nitrogen isotope ratios on plant samples.

Last but not least, thanks to my family. My heartfelt thanks to my beloved mother Mrs. Farooqa Ghulam Rasool who always supported me and made it possible to successfully complete this doctorate degree program. I am also thankful to my dear brothers and sisters for their kindness, generosity, encouragement and support. Finally, I pray for my father Mr. Ghulam Rasool (late) for all his love and support throughout my educational carrier.

VII 

 

Table of Contents

Page No.

List of Tables IX

List of Figures XI

List of Abbreviations XVI

Summary in Urdu 1

Summary in English 2

Chapter 1 General Introduction 4

References 13

Chapter 2 Effects of environmental factors, seed storage conditions, and exogenous chemical treatments in modulating seed germination of Halopeplis perfoliata

21

Abstract 22

Introduction 23

Materials and Methods 26

Results 29

Discussion 40

References 45

Chapter 3 Comparison of osmotic and ionic effects of various salts on germination inhibition and recovery responses of Halopeplis perfoliata seeds

52

Abstract 53

Introduction 54

Materials and Methods 55

Results 56

Discussion 63

References 66

VIII 

 

Chapter 4 Growth and physio-chemical adaptations of Halopeplis perfoliata under saline condition

72

Abstract 73

Introduction 74

Materials and Methods 77

Results 83

Discussion 99

References 105

Chapter 5 General Conclusions 116

IX 

 

List of Tables

Page No.

Table 2.1: Characteristic features of Halopeplis perfoliata seeds

collected from a coastal salt marsh in Jizan, Saudi Arabia.

29

Table 2.2: Three-way ANOVA indicating effects of photoperiod

(Phot.), temperature (Temp.), salinity (Salt) and their

interactions on mean final germination (MFG), rate of

germination (GRate), recovery from salinity (SR) and

recovery from dark (DR) of Halopeplis perfoliata seeds.

Numbers are F-values and asterisks in superscript are

significance levels at P < 0.05 (*** = P < 0.001).

30

Table 2.3: Two-way ANOVA indicating effects of storage time (Days)

temperature (Temp.), hyper-salinity (Salt), and their

interactions on mean final germination (MFG), recovery

from salinity (SR), viability (Viab.) and mortality (Mort.) of

Halopeplis perfoliata seeds. Numbers are F-values and

asterisks in superscript are significance levels at P < 0.05 (ns

= non-significant, * = P < 0.05, ** = P < 0.01 and *** = P <

0.001).

33

Table 2.4: Four-way ANOVA showing effects of dry storage (Stor.)

photoperiod (Phot.), temperature (Temp.), salinity (Salt) and

their interactions on mean final germination (MFG) of

Halopeplis perfoliata seeds. Numbers are F-values and

asterisks in superscript are significance levels at P < 0.05 (*

= P < 0.05 and *** = P < 0.001).

35

Table 2.5: Three-way ANOVA showing effects of dormancy regulating

chemicals (DRCs), photoperiod (Phot.), salinity (Salt) and

their interactions on mean final germination (MFG) of

Halopeplis perfoliata seeds. Numbers are F-values and

asterisks in superscript are significance levels at P < 0.05 (**

37

X 

 

= P < 0.01 and *** = P < 0.001).

Table 3.1: One-way ANOVA of the effects of salinity on growth, water

and ion relations, pigments, photosynthesis, isotope ratios,

stress markers, antioxidant enzymes and substrates of

Halopeplis perfoliata grown under salinity treatments (0,

1.50, 0.30 and 0.60 mol L-1 NaCl) for 30 days. Numbers are

F-values and asterisks in superscript are significance levels at

P < 0.05 (ns = non-significant, * = P < 0.05, ** = P < 0.01

and *** = P < 0.001).

58

Table 4.1: One-way ANOVA of the effect of salinity on different

parameters of shoot and root of Halopeplis perfoliata.

Numbers are the F-values with the level of significance (ns

= non-significant, * = P < 0.05, ** = P < 0.01 and *** = P <

0.001). Details of each parameter given below are

mentioned in Materials and Methods section.

84

Table 4.2: Photosynthetic pigments [chlorophyll a (Chl. a), chlorophyll

b (Chl. b), carotenoids (CAR.), total chlorophyll (Chl. a+b)

and chl. a/b ratio of Halopeplis perfoliata grown in different

salinity treatments (0, 1.5, 0.3 and 0.6 mol L-1 NaCl)

measured on (A) fresh weight (FW) (B) dry weight (DW) and

(C) leaf area basis. Values represent means ± S.E. Different

alphabets show significant difference among the salinity

treatments within each parameter; Bonferroni test; P < 0.05.

90

Table 4.3: Net photosynthetic rate (PN), respiration (RD), stomatal conductance (gS), transpiration (E), water use efficiency (WUE), and intercellular CO2 concentration (Ci) measured on photosynthetic shoots of Halopeplis perfoliata in response to NaCl treatments (0, 1.5, 0.3 and 0.6 mol L-1 NaCl). Values represent means ± S.E. Different alphabets show significant differences among salinity treatments. Bonferroni test; P < 0.05.

91

 

XI 

 

List of Figures

Page No.

Figure 1.1:

Halopeplis perfoliata A) plants and B) in the natural environmental

conditions.

11

Figure 2.1:

Morphology of a) un-germinated and b) germinated seeds of

Halopeplis perfoliata as seen under a light microscope.

29

Figure 2.2: Germination and recovery percentages of Halopeplis perfoliata seeds

after different salinity treatments (0, 0.1, 0.2 0.3, 0.4, 0.5 and 0.6 mol

L-1 NaCl); temperatures (10/20, 15/25, 20/30 and 25/35 oC) and light

(12-h photoperiod and complete darkness) regimes. Vertical bars are

means ± S.E. Different alphabets show significant differences among

salinity treatments; Bonferroni test; P < 0.05.

31

Figure 2.3: Rate of germination responses of Halopeplis perfoliata seeds after

different salinity treatments (0, 0.1, 0.2 0.3, 0.4, 0.5 and 0.6 mol L-1

NaCl); temperatures (10/20, 15/25, 20/30 and 25/35 oC) and light

(12-h photoperiod and 24- darkness) regimes. Vertical bars are means

± S.E. Different alphabets show significant differences among

salinity treatments; Bonferroni test; P < 0.05.

32

Figure 2.4: Germination, recovery, viability and mortality percentages of

Halopeplis perfoliata seeds after prolonged exposure (20, 40 and 60

days) in hyper-saline (0.5, 1.0, 1.5 and 2.0 mol L-1 NaCl) treatment,

temperatures (20/30 and 25/35 oC); and light (12-h photoperiod)

regimes. Values are means of 4 replicates.

34

Figure 2.5: Comparison germination percentages of fresh and 1-year stored

seeds of Halopeplis perfoliata in salinity treatments (0, 0.1, 0.2 0.3,

0.4, 0.5 and 0.6 mol L-1 NaCl); temperatures (10/20, 15/25, 20/30

and 25/35 oC) and light (12-h photoperiod and 24- darkness)

regimes. Different alphabets show significant differences among

salinity treatments; Bonferroni test; P < 0.05. Asterisks (*) shows

36

XII 

 

significant differences in fresh and stored seeds by Student’s t-test;

P < 0.05.

Figure 2.6: Germination percentages of Halopeplis perfoliata treated with

different dormancy regulating chemicals (DRCs) under different

salinity treatments (0.3 and 0.6 mol L-1 NaCl) at 20/30 oC temperature

and 12-h photoperiod. Vertical bars are means ± S.E. Asterisks (*)

show significant differences among salinity treatments within each

chemical treatment by Student’s t-test; P < 0.05.

38

Figure 2.7: Germination percentages of Halopeplis perfoliata treated with

different dormancy regulating chemicals (DRCs) under different

salinity treatments (0.3 and 0.6 mol L-1 NaCl) at 20/30 oC temperature

and complete darkness. Vertical bars are means ± S.E. Asterisks (*)

show significant differences among salinity treatments within each

chemical treatment by Student’s t-test; P < 0.05.

39

Figure 2.8: Regulation of seed germination in Halopeplis perfoliata by light,

temperature, salinity and chemicals (DRCs).

44

Figure 3.1: Effects of NaCl, Na2SO4, KCl, K2SO4, MgCl2, MgSO4, CaCl2 and

sea-salt on mean final germination (MFG) and germination recovery

in distilled water (SR) at 20/30 oC temperature and 12-h

photoperiod. Vertical bars are means ± S.E. Different alphabets

show significant differences among isotonic treatments within each

parameter; Bonferroni test; P < 0.05.

59

Figure 3.2: Effects of NaCl, Na2SO4, KCl, K2SO4, MgCl2, MgSO4, CaCl2 and

sea-salt on rate of germination at 20/30 oC temperature and 12-h

photoperiod. Vertical bars are means ± S.E. Different alphabets show

significant differences among isotonic treatments within indiviual

salt; Bonferroni test; P < 0.05.

60

Figure 3.3: Effects of isotonic treatments of NaCl, Na2SO4, KCl, K2SO4,

MgCl2, MgSO4, CaCl2 and sea-salt on mean final germination

(MFG), recovery from Salinity (SR) and recovery from Dark (DR)

61

XIII 

 

at 20/30 oC temperature and complete darkness. Vertical bars are

means ± S.E. Different alphabets show significant differences

among isotonic treatments within each paramerter of individual

salt; Bonferroni test; P < 0.05.

Figure 3.4: Comparative effects of NaCl and PEG on mean final germination

(MFG), Germination rate (GRate); recovery from salinity (SR) and

recovery from dark (DR) under 12-h photoperiod at 20/30 oC

temperature and complete darkness. Vertical bars are means ± S.E.

Different alphabets show significant differences among sainity

treatments within each parameter; Bonferroni test; P < 0.05. Asterisks

(*) show significant differences in germination within an isotonic

treatment of NaCl and PEG by student’s t-test; P < 0.05.

62

Figure 3.5: Differential inhibitory effects of iso-osmotic treatments with NaCl,

seasalt, and various chloride (Cl-) and sulphate (SO4 --) salts on seed

germination of Halopeplis perfoliata.

66

Figure 4.1: Scheme showing salinity treatment regimes administered for growth

experiment on Halopeplis perfoliata under greenhouse conditions.

78

Figure 4.2: Comparison of Halopeplis perfoliata grown under various salinity

treatments (0, 0.5, 0.3 and 0.6 mol L-1NaCl) for 30 days under

greenhouse conditions.

85

Figure 4.3: Growth analysis of Halopeplis perfoliata in salinity treatments (0,

0.15, 0.3 and 0.6 mol L-1 NaCl). Shoot FW (A), Root FW (B), Shoot

DW (C) Root DW (D), Shoot succulence (E) Root succulence (F).

Vertical bars are means ± S.E. Different alphabets are significantly

differences among salinity treatments within each parameter;

Bonferroni test; P < 0.05.

86

Figure 4.4: Shoot osmotic potential (A) and relative water content (B) in

response to salinity treatments (0, 0.15, 0.3 and 0.6 mol L-1 NaCl).

Vertical bars are means ± S.E. Different alphabets are significantly

differences among salinity treatments within each parameter;

87

XIV 

 

Bonferroni test; P < 0.05.

Figure 4.5: Na+ (A), K+ (B), Ca++ (C), Mg++ (D), Na+/K++ ratio (E), and K+ shoot

to root ratio in response to salinity treatment (0, 0.15, 0.3 and 0.6 mol

L-1 NaCl). Vertical bars are means ± standard error. Different

alphabets show significant differences among the salinity treatments

within each parameter; Bonferroni test; P < 0.05.

88

Figure 4.6: Relative levels of Rubisco quantified on total protein basis where 50

µg protein was applied based on SDS-PAGE gel electrophoresis on

photosynthetic shoot of Halopeplis perfoliata plants grown under

different salinity treatments (0, 0.15, 0.3 and 0.6 mol L-1 NaCl).

92

Figure 4.7: Carbon and Nitrogen isotopes discrimination in salinity treatments (0,

0.15, 0.3 and 0.6 mol L-1 NaCl). Vertical bars are means ± standard

error. Different alphabets show significant differences among salinity

treatments within each parameter; Bonferroni test; P < 0.05.

93

Figure 4.8: Maximum quantum efficiency (Fv/Fm), actual yield (PSII), electron

transport rate (ETR), photochemical (qL) and non-photochemical

quenching (NPQ) on photosynthetic shoot of H. perfoliata under a

range of irradiance and in salinity treatments (0, 0.15, 0.3 and 0.6 mol

L-1 NaCl). Values are mean of 3 replicates with means ± S.E.

94

Figure 4.9: Hydrogen peroxide in shoot (A) and root (B), Malondialdehyde in

shoot (C) and root (D) of Halopeplis perfoliata in salinity treatments

(0, 0.15, 0.3 and 0.6 mol L-1 NaCl). Vertical bars are means ± S.E.

Different alphabets show significant differences among the salinity

treatments within each parameter; Bonferroni test; P < 0.05.

95

Figure 4.10: Antioxidant enzyme activities in shoots and roots of Halopeplis

perfoliata in response to salinity treatments (0, 0.15, 0.3 and 0.6 mol

L-1 NaCl). Vertical bars are means ± S.E. Different alphabets show

significant differences salinity treatments within each enzyme

activity data; Bonferroni test; P < 0.05.

96

XV 

 

Figure 4.11: Reduced glutathione (GSH) in shoot (A), and root (B), ascorbic acid

(ASA) in shoot (C) and root (D) of Halpeplis perfoliata in response

to salinity treatments (0, 0.15, 0.3 and 0.6 mol L-1 NaCl). Vertical

bars are means ± S.E. Different alphabets show significant

differences among salinity treatments within each parameter;

Bonferroni test; P < 0.05.

97

Figure 4.12: Total soluble sugars (TSS) in response to salinity treatments (0, 0.15,

0.3 and 0.6 mol L-1 NaCl). Vertical bars are means ± S.E. Different

alphabets show significant differences among salinity treatments in

shoot and root of Halopeplis perfoliata; Bonferroni test; P < 0.05.

98

Figure 4.13:

Eco-physiological adaptations of water and ion relations,

photosynthesis and antioxidant defense system of Halopeplis

perfoliata under high salinity concentrations. (~ = similar response; ↑

= increased response).

104

Figure 5.1:

Summary of the stage-specific eco-physiological responses of

Halopeplis perfoliata in response to environmental factors.

118

XVI 

 

Abbreviations

ANOVA Analysis of variance

ASA Ascorbic acid

APX Ascorbate peroxidase

CAT Catalase

  δ13C 13 Carbon isotope discrmination

CE Carboxylation efficiency

CAR Carotenoids

Chl Chlorophyll

RD Dark respiration

DRCS Dormancy regulating chemicals

DW Dry weight

ETR Electron transport rate

FW Fresh weight

GRate Germination rate

GR Glutathione reductase

CE* Gross carboxylation efficiency

GPX Guaicol peroxidase

h Hour

H2O2 Hydrogen peroxide

Ci Intercellular carbon dioxide concentration

MDA Malondialdehyde

MFG Mean final germination

Mort. Mortality

δ15N 15 Nitrogen isotope discrmination

ΨS Osmotic potential

Photo. Photoperiod

PN Photosynthesis

RL Photorespiration

PEG Polyethyleneglycol-6000

PVPP Polyvinylpyrrolidone

XVII 

 

ROS Reactive oxygen species

DR Recovery of germination from dark

SR Recovery of germination from salinity

GSH Reduced glutathione

RWC Relative water content

SDS Sodiumdodecylsulfate

gs Stomatal conductance

SOD Superoxide dismutase

Temp. Temperature

T. Chl Total chlorophyll

TSS Total soluble sugars

E Transpiration

TCA Trichloroacetic acid

TW Turgid weight

Viab Viability

WUE Water use efficiency

1  

 

2 

 

Summary

Halopeplis perfoliata Forssk. is a C3 perennial halophyte found in coastal salt

marshes of Saharo-Sindian, Mediterranean and Western Irano-Turanian regions.

The plant has several ecological and economic usages but little is known about its

salt resistance strategies. Therefore, salt tolerance mechanism at seed germination

and growth stages of H. perfoliata were investigated. Increases in salinity

concentration decreased seed germination at all temperature regimes, while some

seeds could still germinate in 0.6 mol L-1 NaCl (equivalent to seawater salinity).

Hyper-saline conditions and complete darkness induced conditional dormancy.

Among salts tested, sea-salt inhibited seed germination more than isotonic NaCl

treatments. Among anions, chloride salts inhibited germination more than

isotonic sulfate salts. Dry storage of seeds for 1-year at room temperature

increased their tolerance to moderate salinities (0.2-0.3 mol L-1 NaCl) and

complete darkness, but decreased seed tolerance to higher temperature (25/35 oC)

as compared to freshly collected seeds. All dormancy regulating chemicals

(except for proline) alleviated dark-enforced dormancy under non-saline

conditions. Halopeplis perfoliata grew optimally at 0.15 mol L-1 NaCl treatment

and with increasing salinity (0.3 and 0.6 mol L-1 NaCl) treatments plant biomass

was comparable to non-saline controls. Lower (more negative) shoot osmotic

potentials and Na+ accumulation indicated capacity for osmotic adjustment with

increasing salinity. In general, photosynthetic CO2 fixation, light harvesting

ability of PSII and cation (K+, Ca++ and Mg++) concentrations were unchanged

with increasing salinity treatments. Higher CAT activity and H2O2 and the less

negative carbon isotope ratios in photosynthetic shoots indicated high rates of

photorespiration under saline conditions. Halopeplis perfoliata displayed unique

adaptations at seed germination and growth to survive under saline conditions.

  

3 

 

Halopeplis perfoliata

4 

 

Chapter 1

General Introduction

5 

 

Introduction

Salt marshes are highly productive yet threatened ecosystems

Coastal salt marshes are transition zones between marine and terrestrial ecosystems

(Teixeira et al., 2014). They inundated diurnally by seawater therefore marsh sediments

have high salinity (equivalent to > 0.6 mol L-1 NaCl) (Aziz et al., 2001; Khan et al.,

2005). Salt marshes in sub-tropical regions often lack sufficient rainfall which leads to

even higher soil salinity (Böer, 1996; Boorman, 2003). Most, salt marshes are

characterized by dense stands of few highly salt-tolerant species (Anjum et al., 2014). In

marshy habitats, halophytes perform key role in carbon cycling (Gribsholt et al., 2003;

Sousa et al., 2010) by serving as a net sinks of carbon from atmosphere and produce

between 100-1000 g carbon m−2 y-1 (McLusky and Elliot, 2004; Sousa et al., 2010)

which makes the marsh environment highly productive. However, these habitats face

various natural and anthropogenic threats (Yasseen and Al-Thani, 2007; Ramadan et al.,

2013). According to a recently published t of United Nations report, the global climate

changes are causing global warming and mean sea level which could have drastic effects

on these habitats (IPCC, 2014). In addition, intense construction activities for resorts,

hotels and picnic points in coastal areas are also a serious threat to these important

habitats. Hence, coastal marshes and their vegetation require special attention both in

research and conservation/protection activities.

Salt marsh vegetation of coastal areas is under serious threat

Common plant species of warm coastal salt marshes can be categorized into perennials,

succulents, shrubs, stolonifers and hemicrytophytes (Ghazanfar, 2006) and include many

highly salt tolerant succulent species such as Arthrocnemum macrostachyum,

Halocnemum strobilaceum, Halopeplis perfoliata, Haloxylon persicum, Salicornia

europea, Zygophyllum mandavielli, Salsola imbricata, Sesuvium verrucosum, Suaeda

maritima and Suaeda vermiculata. (Gairola et al., 2015). These salt marsh plants provide

habitat for the coastal fauna, beside many other ecological and economic benefits

(Barbier, 2013). However, anthropogenic activities such as installation of reverse

osmosis facilities, construction of excursion points and buildings are posing serious

threat to these salt marshes. Therefore, salt marshes have received special attention both

6 

 

from researchers and Government, which is evident from a growing body of research

works (Mahmoud et al., 1983, Böer, 1991, Al-Zaharani and Hajar, 1998; Ghazanfar,

2006; Anjum et al., 2015 and Meirland et al., 2015). Most of these research deals with

species composition, edaphology and threats to salt marshes. However, information on

eco-physiology and salt resistance mechanisms of the plants is missing.

Multiple factors regulate seed germination responses of marsh halophytes

Seed germination is the most sensitive stage in the life cycle of halophytes and is

influenced by a number of environmental factors, of which salinity is considered

foremost (Ungar 1995; Katembe et al., 1998; Gul et al., 2013). Insufficient rainfall in

sub-tropical regions results in high salinity in salt marsh sediments (Böer, 1996;

Boorman, 2003). Therefore, salinity of upper soil layers in such salt marshes may reach

44 ppt (equivalent 70 dS m-1) (Mandora et al., 1987). Seeds of most of the coastal plants

deposited in highly saline soils, hence they have to endure highly hostile conditions

(Ungar, 1978; Gul and Khan, 1998; Gul et al., 2013).

Seeds of most succulent halophytes of salt marshes are generally high salt

tolerant and can germinate in salinities higher than seawater salinity e.g. Salicornia

herbacea (1.7 mol L-1; Chapman 1960), Salicornia europaea (0.6 mmol L-1 NaCl;

Keiffer and Ungar, 1997), Arthrocnemum macrostachyum (1.0 mol L-1 NaCl; Khan and

Gul, 1998), Halosarica pergranulata (0.8 mol L-1 NaCl; Short and Colmer, 1999),

Halocnemum salicornicum (0.8-0.9 mmol L-1 NaCl; El-Keblawi and Al- Shamsi, 2008)

and Sarcocornia ambigua (45 g NaCl L-1 ~ 770 mmol L-1 NaCl; Freitas and Costa,

2014). In addition, seeds of these succulent halophytes can endure hypersaline conditions

by undergoing enforced dormancy (yet maintaining their viability) and show high

recovery of seed germination when salinity of the salt marshes is reduced following

rainfall (Ungar, 1995; Khan and Ungar, 1997; Gul et al., 2013). Seeds of most non-

succulent salt marsh halophytes such as the Atriplex, Crithmum and Limonium species on

the other hand could germinate at or below seawater salinity (Katembe et al., 1998; Zia

and Khan, 2008; Atia et al., 2009).

Seed germination of marsh halophytes is not only influenced by total salinity, but

also by different salts (Ryan et al., 1975; Khan and Gul, 2006). For example, germination

of stem succulent salt marsh halophytes Arthrocnemum macrostachyum, Halocnemum

strobilaceum, Sarcocornia fruticosa and Salicornia ramosissima varied under similar

7 

 

levels of Cl- and SO4-- salts, and was linked to differential osmotic rather than ionic

effects of these salts (Pujol et al., 2000). Furthermore, comparison of NaCl and sea-salt

on salt marsh halophyte Arthrocnemum indicum indicated that NaCl reduced germination

more than the sea-salt (Saeed et al., 2011). However, little information is available on the

effects of different salts and seawater on salt marsh halophytes in comparison to the

effects of NaCl.

Beside salinity, many other factors such as light, temperature, storage, and levels

of phyto-hormones also influence seed germination of halophytes (Baskin and Baskin,

1994; Khan and Gul, 2006). According to Baskin and Baskin (1998) out of 91

halophytes, seed of 23 species were germinated in the presence of light. Similar

responses were observed for various other stem succulent halophytes like Salicornia

pacifica (Khan and Weber, 1986), Allenrolfea occidentalis (Gul et al., 2000).

Arthrocnemum indicum (Saeed et al., 2011), Halocnemum strobilaceum and Halopeplis

perfoliata (El-Keblawy and Bhatt, 2015). In addition, temperature also affects seed

germination of halophytes (Bewley and Black 1994; Gul and Weber 1999; Khan et al.,

2001; Zheng et al., 2004, 2005). Seeds of salt marsh stem succulent halophytes generally

germinate best at the temperature 20/30 oC. For example Allenrolfea occidentalis (Gul

and Weber, 1999), Salicornia rubra (Khan et al., 2000) and Arthrocnemum indicum

(Saeed et al., 2011) germinated best at 20/30 oC. According to Mahmoud et al. (1983)

germination success in salt marshes of Arabian Peninsula depends on temperature.

However, this kind of information on the marsh halophytes of Arabian-Peninsula is

limited to just few studies (Gul et al., 2013).

Growth hormones are reported to enhance the seed germination under salinity

stress condition (Ahmed et al., 2014; Li et al., 2014). Alternation in endogenous

dormancy regulating compounds at the appropriate salinity, temperature and light could

facilitate seed germination (Ahmed and Khan, 2010; El-Kablawy et al., 2010). It has

been reported that ethephon, fusicoccin, GA3 and kinetin enhanced germination in stem

succulent halophytes such as Salicornia utahensis (Khan and Weber, 1986; Gul and

Khan, 2003), Allenrolfia occidentalis (Gul and Weber, 1998), Arthrocnemum indicum

(Khan et al., 1998) and Salicornia rubra (Khan et al., 2002). However, this information

on the salt marsh halophytes of Arabian-Peninsula is largely missing.

8 

 

Most salt marsh halophytes have “obligate” salt requirement for optimal growth

Succulent halophytes particularly those of Salicornoideae are reported to dominate

coastal salt marshes (McKee et al., 2012). Halophytes of Salicornoideae are supposed to

have higher salt tolerance than other halophytes. For example Arthrocnemum

macrostachyum (up to 1.0 mol L-1 NaCl, Khan et al., 2005), Salicornia europaea and S.

persica (up to 0.6 mmol L-1 NaCl, Aghaleh et al., 2009) and Tecticornia pergranulata

(syn. Halosarcia pergranulata) (up to 0.8 mol L-1 NaCl, Colmer et al., 2009). A number

of Salicornoideae members are in fact “obligate halophytes” (Flower and Colmer, 2008;

Rozema and Schat, 2013) which require some salt for their optimal growth (Flowers and

Colmer, 2008). For instance, Sarcocornia natalensis (300 mmol L-1 NaCl, Naidoo and

Rughunanan, 1990), Salicornia bigellovii (0.2 mol L-1 NaCl, Ayala and O’Leary, 1995),

Salicornia persica (Aghaleh et al., 2009) and Salicornia dolichostachya (0.3 mol L-1

NaCl, Katschnig et al., 2013) required salt for optimal growth. Hence, salt marsh

halophytes are well-adapted for surviving highly hostile marsh conditions. Owing to

their obligate halophyte nature, they could be interesting organisms for studying salt

tolerance mechanisms.

Salient features of salt tolerance mechanisms in salt marsh halophytes

Salt marsh halophytes are mostly “includers” which accumulate large amounts of salt in

their tissues and show tissue tolerance to increasing salinity (Aziz and Khan, 2001). Salt

tolerance is a highly complex phenomenon that incorporates a number of

cellular/molecular as well as whole plant level processes (Flowers and Colmer, 2008).

Generally, we can classify salt tolerance mechanisms of halophytes into four groups: 1)

osmotic adjustment, 2) ion homeostasis, 3) protection of metabolic activities such as

photosynthesis and 4) oxidative stress management (Jitesh et al., 2006; Flowers and

Colmer, 2015). Osmotic adjustment in halophytes helps to absorb and retain water in tissues

under hypersaline conditions (Shabala and Mackay, 2011; Flowers and Colmer, 2015).

This is achieved by compartmentalizing salts (mostly as Na+ and Cl-) in the apoplast and

cell vacuoles, with concomitant accumulation of organic osmolytes such as glycine

betaine, proline, free amino acids, sugars and sugar alcohols in cell cytosols (Flowers

and Colmer, 2008; Slama et al., 2015). Sequestration of salts in vacuoles is energetically

less costly than synthesis of organic osmolytes as vacuoles occupy the bulk of the cells’

9 

 

internal volume (Flowers and Colmer, 2008). Organic osmolytes in cytoplasm provide

additional advantage by protecting macromolecules under stress, also referred as

‘osmoprotection’ (Slama et al., 2015). Plant growth may be compromised for osmotic

adjustment with salinity increments due to the high energy cost of organic osmolytes

(Flowers and Colmer, 2008). Salt accumulation in halophytes for osmotic adjustment is a tightly regulated

multi-facted phenomenon (Shabala and Mackay, 2011). Salt exclusion at the level of

root, restrict its entry into the xylem stream (Flowers and Colmer, 2015) and most of the

woody halophytes of tidal marshes are reported to exclude 90 to 99% of the external Na+

(Reef and Lovelock 2015). Stomatal size and density as well as number reduces

transpiration rates which may also help in restricting Na+ entry into the shoots/leaves

(Shabala and Mackay, 2011). Some non-succulent salt marsh halophytes particularly

grasses get-rid of excess salts by secreting them via salt glands/hairs (Flowers et al.,

2010; Céccoli et al., 2015). At the cellular level, using a complex machinery of

membrane transporter proteins, halophytes keep Na+ and Cl- within tolerable ranges

along with efficient acquisition of K+ and Mg++ (Mansour, 2014; Flowers and Colmer,

2015). High external salinity may however results in ion imbalance which leads to

metabolic disruption causing tissue injury. Effective osmotic adjustment and ion-

homeostasis for plant survival under saline conditions would require the ability to

maintain significant photosynthetic CO2 gain at minimum water loss (Álvarez et al.,

2012; Naidoo et al., 2012).

Salinity is known to affect photosynthesis due to stomatal limitation (Lawlor and

Cornic, 2002; Flexas et al. 2004; Lambers et al., 2008). A decrease in stomatal

conductance reduces the influx of CO2 which ultimately reduces photosynthetic rate

(Maricle et al., 2007). However, many succulent halophytes of salt marshes are reported

to maintain significant photosynthetic rates under high salinities (Maricle et al., 2007).

Unlike photosynthetic CO2 assimilation, light-harvesting mechanisms of halophytes are

generally more resilient. For example, photochemistry was generally unaffected by

salinity in succulent halophytes Suaeda salsa (James et al., 2002) and Sarcocornia

fruticosa (Redondo-Gómez et al, 2006). Under decreased CO2 assimilation such as in

response to salinity, use of alternate electron sinks become essential for the survival of

plants (Taiz and Zeiger, 2010). Non-photochemical quenching (NPQ), which is the

dissipation of surplus energy mainly by heat production is considered an important route

10 

 

in plants (Taiz and Zeiger, 2010; Lambrev et al., 2012). For example, NPQ increased

with increases in salinity in Atriplex centralasiatica (Qiu et al., 2003) and Cakile

maritima (Megdiche et al., 2008). Rise in NPQ is a photo-protective mechanism that

minimizes the incidence of electron flow from photosystems to oxygen (O2) which

otherwise accelerates the formation of cytotoxic reactive oxygen species (ROS) (Jithesh

et al., 2006; Taiz and Zeiger, 2010; Demidchik, 2015). Accumulation of ROS can cause

oxidative damage to cell components such as membrane lipids, proteins, chlorophyll and

nucleic acids (Müller et al., 2001, Jithesh et al., 2006; Møller et al., 2007; Hameed and

Khan, 2011; Demidchik, 2015). Therefore, the antioxidant defense system, which

comprises of both enzymatic and non-enzymatic components, plays an important role in

defending cell components from oxidative damage (Jithesh et al., 2006). Superoxide

dismutase (SOD), catalase (CAT), and enzymes of Foyer-Halliwell-Asada pathway are

important antioxidant enzymes, while ascorbate (ASA) and glutathione (GSH) are key

antioxidant substances, which are implicated in protecting cells from salinity-induced

oxidative stress (Jithesh et al., 2006). However, efficacy of antioxidant system in

protecting plants from salinity effects may vary considerable with species and the

magnitude of salinity imposed, among other factors (Hameed and Khan, 2011).

Halopeplis perfoliata is a common halophyte of coastal salt marshes

Halopeplis perfoliata Forssk. is a succulent C3 perennial shrub found in coastal salt

marshes of Arabian peninsula (Böer, 1996; Zahran and Ansari, 1999; Al-Oudat and

Qadir, 2011), saline desert habitats along Arabian Gulf coast (El-Keblawy and Bhatt,

2015) and also occupies the first zone (near coast) of the many Red Sea salt marshes

(Migahid, 1978). There are many ecological and economic usages of this halophyte. For

example, H. perfoliata is used for soap manufacture by local communities (Al-Oudat and

Qadir, 2011). It can also be used for sand dune stabilization (Zreik, 1990). Halopeplis

perfoliata is an important primary producer of intertidal coastal zones and provides

habitat for wild life (Pilcher et al., 2003). However, little is known about the seed

germination (Mahmoud et al., 1983; El-Keblawy and Bhatt, 2015) and growth (Al-

Zaharani and Hajar, 1998) of this halophyte. Furthermore, underlying mechanisms for

salt tolerance of halophytes from the Arabian Peninsula have seldom been studied.

Halopeplis perfoliata is an interesting plant, owing to its naturally high salt tolerance and

economic potentials.

11 

 

A

B

Fig. 1.1. Halopeplis perfoliata A) plants and B) in the natural environmental conditions.

12 

 

Research questions The following question were addressed in the present study:

1. What is the salt tolerance limit of Halopeplis perfoliata during seed germination

stage under various photoperiod, temperature, and storage environments?

2. Can salinity-induced seed germination inhibition be mitigated by the exogenous

application of various dormancy regulating chemicals?

3. Are seeds of Halopeplis perfoliata equally tolerant to all types of salts?

4. What is the salt tolerance limit of Halopeplis perfoliata at growth stage?

5. What are different physiological and biochemical adaptations which enable

Halopeplis perfoliata to tolerate high salinity?

13 

 

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21 

 

Chapter 2

Effects of environmental factors, seed storage conditions, and exogenous chemical treatments in modulating seed germination of

Halopeplis perfoliata

22 

 

Abstract Halopeplis perfoliata is a coastal marsh halophyte with several ecological and economic

usages. Information about seed germination ecology of this plant is scanty. Therefore

this study was conducted to investigate the germination, recovery and viability responses

of H. perfoliata seeds to photoperiod (light/dark), temperature, salinity, hyper-salinity,

long-term storage and dormancy regulating chemicals (DRCs). Seeds were small and

non-dormant, and displayed highest germination in distilled water irrespective of

incubation temperatures. Seeds germinated better in light (12-h photoperiod) than in 24-h

dark. Increases in salinity decreased seed germination; however some seeds could

germinate in 0.6 mol L-1 NaCl (equivalent to seawater salinity) under 12-h photoperiod.

High salinity (> 0.3 mol L-1 NaCl) and darkness imposed conditional dormancy in seeds

and they showed high germination recovery when transferred to distilled water and light

respectively. Seeds of H. perfolaita could endure hyper salinity (> 1.0 mol L-1 NaCl) by

entering into a state of conditional dormancy. However, high incubation temperatures

resulted in higher seed mortality under hyper-saline condition. Dry storage of seeds for

one year at 25 oC increased their tolerance to moderate salinity and dark, but

substantially decreased tolerance to high temperature compared to fresh seeds. All DRCs

(except proline) alleviated dark-enforced dormancy but were generally ineffective in

reversing inhibitory effects of salinity. Hence it appears that individual effects of

photoperiod, salinity and temperature influenced seed germination more than their

interactive effects.

23 

 

Introduction

Arabian Peninsula salt marshes are facing various natural and anthropogenic threats

Coastal salt marshes are among the most productive ecosystems of the world and harbor

unique halophytic vegetation bearing specialized adaptations to produce enormous

biomass despite high seawater salinity (Teixeira et al., 2014). Their location at the

interface of sea and land makes them ecologically important. These habitats serve as an

important carbon and nitrogen sink, thereby help in CO2 sequestration and prevention of

eutrophication in coastal areas, beside many other ecosystem services (Seitzinger, 1988;

Valiela and Cole, 2002; Teixeira et al., 2014). However, these habitats are facing various

natural and anthropogenic threats (Yasseen and Al-Thani, 2007; Ramadan et al., 2013).

Sub-tropical salt marshes especially those along the coastline of Arabian Peninsula often

face lack of sufficient rainfall which leads to increased sediment salinity in salt marshes

(Böer, 1996; Boorman, 2003). Salinity of upper soil layer in such salt marshes could be

as high as 44 ppt (equivalent 70 dS m-1) (Mandora et al., 1987) which could be

detrimental for the prevailing vegetation (Ungar, 1991; Böer, 1996). Since seeds of most

coastal plants are deposited in top soil layers, which have substantially higher salinity

than lower layers, therefore conditions for seeds are harsher in comparison to mature

plants (Ungar, 1978; Gul and Khan, 1998; Gul et al., 2013).

Seed germination responses of salt marsh halophytes are highly variable

Salinity is one of the main factors influencing vegetation zonation in coastal salt marshes

mainly via its effects on seed germination (Bertness et al., 1992; Pujol et al., 2000;

Hameed et al., 2006; Elsey-Quirk et al., 2009; Gul et al., 2013). Seed germination of

halophytes generally decreases with increases in salinity (Khan and Gul, 2006).

However, seeds of most salt marsh halophytes could germinate in as high as seawater (>

0.6 mol L-1 NaCl) or even higher salinity (Gul et al., 2013). For instance, seeds of salt

marsh halophytes Arthrocnemum indicum (1.0 mol L-1 NaCl, Khan and Gul, 1998;

Ruppia tuberosa (90 g L-1 sea-salt or ~1.5 mol L-1 NaCl, Kim et al., 2013) and

Sarcocornia ambigua (0.77 mol L-1 NaCl, Freitas and Costa, 2014) showed high salinity

tolerance during germination. Seeds of most salt marsh halophytes can even endure

hyper-saline conditions (Gul et al., 2013) in which they although do not germinate but

maintain their viability and hence quickly germinate (germination recovery, sensu Khan

and Ungar, 1997) following sufficient rains which dilute soil/sediment salinity (Khan

24 

 

and Gul, 2006). Salinity tolerance of halophyte seeds also varies with changes in ambient

temperature and presence/absence of light (Gul et al., 2013). Sub/supra-optimal

temperatures and dark (mainly due to burial) decrease both germination as well as

salinity tolerance of halophyte seeds (Baskin and Baskin, 2001; Zia and Khan, 2004; El-

Keblawy and Al-Rawai, 2006; Hameed et al., 2013). However, large variations exist in

responses of the seeds of salt marsh halophytes to temperature and photoperiod changes.

For example, seeds of only four out of eight salt marsh halophytes responded to changes

in temperature and light/dark during germination (Noe and Zedler, 2000). Seeds of

another salt marsh halophyte Arthrocnemum indicum responded to changes in

temperature but not to light/dark (Saeed et al., 2011). Changes in temperature and

light/dark also influence seed viability and dormancy of halophytes (Gulzar et al., 2013;

Hameed et al., 2013). However, information about the seed germination of salt marsh

halophytes is limited (Saeed et al., 2011; Gul et al., 2013).

Salinity tolerance of halophyte seeds, as discussed above, is a variable trait and is

also influenced by storage conditions and duration (El-Kablawy, 2013a; Cao et al.,

2014). Recently El-Keblawy (2013a) studied effect of various storage conditions on

seed germination of two succulent halophytes Haloxylon salicornicum and Salsola

imbricata. He found that the seed storage for three months significantly increased

germination but longer than nine month storage at room and warm temperatures caused

significant reduction or complete inhibition in the germination of the aforementioned

halophytes. While seeds of some halophytes such as Allenrolfea occidentalis (Gul and

Weber, 1999), Salicornia rubra (Khan et al., 2000), Kochia scoparia (Khan et al., 2001)

and Salsola iberica (Khan et al., 2002) could maintain viability and germinablity after 20

days of high salinity (1.0 mol L-1 NaCl) exposure. In another study, Keiffer and Ungar

(1997) found variable viability and germination responses of the seeds of Atriplex

prostrata, Hordeum jubatum, Salicornia europaea, Spergularia marina, and Suaeda

calceoliformi to storage under high salinity for different time periods. Hence, these data

indicate that seed responses of halophytes to storage under high salinity could be variable

and species specific.

Dormancy regulating compounds can improve seed germination under stress conditions

Germination and salinity tolerance of the seeds of halophytes could be improved by

exogenous application of certain chemicals (Mehrun-Nisa et al., 2007; Atia et al., 2009;

25 

 

Ahmed et al., 2014). GA3, kinetin and thiourea are widely used chemicals, which can

mitigate salinity effects and improve seed germination of halophytes (Khan and Gul,

2006; Gul and Khan 2008; El-Keblawy et al., 2010). Recently, Ahmed et al. (2014)

reported that thiourea could significantly improve seed germination of three salt playa

halophytes under saline condition, while GA3 and kinetin had species-specific effects.

Hence, information about effects of chemicals on salinity tolerance and germination of

halophyte seeds appears inconclusive and could be species and/or chemical specific (Gul

and Khan 2008; El-Keblawy et al., 2010; Ahmed et al., 2014).

Seed germination eco-physiology of H. perfoliata is largely unstudied

Halopeplis perfoliata Forssk. is a succulent halophyte from family Amaranthaceae, that

occupies coastal salt marshes of Arabian peninsula (Al-Oudat and Qadir, 2011). This

species has many ecological and potential economic uses. For instance, it can be utilized

for sand dune stabilization in coastal deserts (Zreik, 1990) and also in soap industries

(Al-Oudat and Qadir, 2011). This plant is an important intertidal/terrestrial producer and

provides habitat structure for wild life (Pilcher et al., 2003). Seeds of this plant could

germinate up to 0.25 mol L-1 NaCl treatment (Mahmoud et al., 1983). However, little is

known about the seed germination eco-physiology or the two other members of genus

Halopeplis. Therefore detailed studies on salinity tolerance mechanisms of H. perfloiata

were performed to understand how its seeds withstand highly saline salt marsh

conditions. The objectives of this research were to find out: 1) salinity tolerance limit and

optimal germination conditions (temperature and light/dark regimes) for H. perfoliata

seeds, 2) viability and germination response of H. perfoliata seeds to storage under

hyper-saline conditions, 3) germination characteristics after long-term (1-year) dry-

storage in laboratory condition, and 4) salinity tolerance and germination responses to

exogenous application of various dormancy regulating chemicals (DRCs).

26 

 

Materials and Methods

Study site and Species description

Mature inflorescence of Halopeplis perfoliata were collected from the coast of Jizan,

Saudi Arabia in 2012. Study site was a salt marsh with frequent inundation, where mean

ambient temperature reportedly ranges between 36 and 21 oC (Alfarhan et al., 2005).

Mean annual rainfall of the area is 169 mm and humidity ranges between 39 and 86%

(Alfarhan et al., 2005). Seeds were collected from large number of plants randomly to

ensure adequate representation of the genetic diversity. Seeds were separated from their

inflorescence husk manually and brought to the laboratory in Pakistan. Seeds were

surface sterilized using 1% bleach solution for 1 min, followed by thorough rinsing with

distilled water and air-drying. Sterilized seeds were then used in experiments.

Seed characteristics

Fresh weight (FW) of 1000 seeds was measured using an analytical balance (0.0001 g).

Dry weight (DW) of the seeds was determined by placing 1000 seeds in an oven at 105 oC for 48-h. Moisture content was then calculated as difference between FW and DW. Ash

or inorganic content of the seeds was determined by igniting oven dried seeds in a

furnace at 550 oC for 3-h. Size of the seeds was measured using photographs of about

randomly chosen 100 seeds with the help of ImageJ software

(http://imagej.nih.gov/ij/images/). Seed texture was determined by observing seeds under

a mini-microscope (Dini-Lite digital Microscope, ANMO Electronics Corporation). Seed

color was matched against the catalog of Ralcolors (www.ralcolors.com).

Experiment. 1: salinity tolerance limit and optimal germination conditions

Germination was carried out in 50 x 9 mm (Gelman No. 7232) tight-fitting plastic petri-

plates with 5 ml of test solution. Seven concentrations of NaCl solution (0, 0.1, 0.2, 0.3,

0.4, 0.5 and 0.6 mol L-1) were used. Petri plates were placed in a germination chambers

set at temperature regimes of 10/20, 15/25, 20/30 and 25/35 oC, where low temperature

coincided with 12-h dark and high temperature with 12-h light (~25 µmol m-2 s-1, 400-

750 nm, Philips cool-white fluorescent lamps). Under similar temperature and salinity

treatments seeds were also incubated in complete darkness (24-h dark) by using dark

photographic envelops. Four replicates with twenty-five seeds per petri-plate were used

for each treatment. Petri-plates placed under 12-h photoperiod were monitored at 2 days

27 

 

intervals for 20 days and percent seed germination were noted. Seeds were considered to

be germinated with the emergence of the radical (Bewley and Black, 1994). After 20

days of incubation, rate of germination was estimated by using a modified Timson’s

index of germination velocity given below:

                                        

Where G is the percentage of seed germination at 2d intervals and t is the total

germination period (Khan and Ungar, 1985). The greater the value, the more rapid was

germination. While, germination of seeds incubated in dark was noted once after 20

days. After 20 days light exposed (12-h) un-germinated seeds were transferred to

distilled water, while un-germinated seeds from dark incubation were exposed to light in

order to study the recovery of germination. Seed germination during recovery

experiment was recorded at 2 days intervals for another 20 days period. The recovery

percentage was calculated by using following formula given below: 

                                            

Where, A is total number of seeds germinated after being transferred to distilled

water, B is the number of seeds germinated in saline solution and C is total number of

seeds. The seeds which did not germinate were further tested for their viability using 1%

(w/v) 2,3,5-triphenyle-tetrazolium chloride solution (MacKay, 1972; Bradbeer, 1998).

Experiment. 2: germination and viability responses of seeds to hyper-saline storage

To study the tolerance of H. perfoliata seeds to hyper-saline conditions, seeds were

exposed to four high salinity treatments (0.5, 1.0, 1.5 and 2.0 mol L-1 NaCl) at two

temperature regimes (20/30 and 25/35 oC) under 12-h light: 12-h dark photoperiod for

different time periods (20, 40 and 60 days). Seeds were also incubated for 24-h in dark.

Germination, recovery and viability of the seeds were examined according to the

methods described above.

28 

 

Experiment. 3: germination characteristics after long-term dry storage

Seeds of H. perfoliata were stored at room temperature (~25 oC) for 1-year and then

tested for germination, recovery and viability responses to salinity, temperature and

light/dark treatments, as described in experiment 1.

Experiment. 4: salinity tolerance and germination responses to dormancy regulating chemicals

To study if salinity tolerance of H. perfoliata seeds could be improved using different

dormancy regulating chemicals (DRCs), seeds were germinated in different salinity

treatments (0, 0.3 and 0.6 mol L-1 NaCl) in light (12-h photoperiod) as well as dark under

optimal temperature (20/30 oC) in presence and absence of different DRCs. Betaine (1

mmol L-1), ethaphon (10 mmol L-1), fusicoccin (5 µmol L-1), kinetin (0.05 mmol L-1),

proline (0.1 mmol L-1) and thiourea (10 mmol L-1) were used. Germination and recovery

were noted, as described above.

Statistical analyses

Germination data were arcsine transformed before statistical analysis. Analyses of

variance (ANOVAs) were used to determine if treatments (photoperiod, temperature,

salinity, hyper-saline storage, dry storage and DRCs) had significant effect on seed

parameters. While, a Post-hoc Bonferroni test was carried out to compare individual

mean values for significant (P < 0.05) differences. SPSS Version 11.0 (SPSS, 2011) was

used for data analysis. Student t-test (P < 0.05) was performed to compare mean values

for experiment 3 and 4.

29 

 

Results

Seed characteristics

Seeds of H. perfoliata were ellipsoid, small (0.28 mm Ф) and rough textured (Fig. 2.1).

Fresh weight of 1000 seeds was 92.86 mg and they had about 4.88% moisture. There

were no perianth or hairs attached to the fully mature seeds.

Fig. 2.1 Morphology of a) un-germinated and b) germinated seeds of Halopeplis perfoliata as seen under a light microscope. Table 2.1. Characteristic features of Halopeplis perfoliata seeds collected from a coastal

salt marsh in Jizan, Saudi Arabia.

Characteristic Description

Color Terra Brown*

Texture Rough

Shape Ellipsoid

Size (ф, mm) 0.28

FW (mg 1000-1 seeds) 92.86

DW (mg 1000-1 seeds) 88.33

Moisture (% of FW) 4.88

Ash (% of FW) 3.87

*RAL-8028, www.ralcolors.com

B A

30 

 

Experiment. 1: salinity tolerance limit and optimal germination conditions

Threee way Analysis of variance (ANOVAs) indicated significant (P < 0.001) effect of

temperature, photoperiod, salinity and their interaction on mean final germination

(MFG), rate of germination (GRate), recovery of germination from salinity (SR) and

recovery of germination from dark (DR) of H. perfoliata seeds (Table 2.2).

Table 2.2. Three-way ANOVA indicating effects of photoperiod (Phot.), temperature

(Temp.), salinity (Salt) and their interactions on mean final germination (MFG), rate of

germination (GRate), recovery from salinity (SR) and recovery from dark (DR) of

Halopeplis perfoliata seeds. Numbers are F-values and asterisks in superscript are

significance levels at P < 0.05 (*** = P < 0.001).

Seeds were non-dormant and germinated maximally (~90%) in distilled water

irrespective of incubation temperature (Fig. 2.2) however increase in salinity (NaCl

concentration) decreased MFG at all temperature regimes and there was > 10% MFG in

0.6 mol L-1 NaCl treatment. Comparatively higher MFG and GRate values were observed

in > 0.5 mol L-1 NaCl treatments at 20/30 oC (Fig. 2.2 and 2.3). There was substantially

low MFG in dark in comparison to 12-h photoperiod in all salinity and temperature

treatments (Fig. 2.2). Salinity and temperature treatments had a similar effect on GRate as

in case of MFG (Fig. 2.3).

When un-germinated seeds from salinity and 12-h photoperiod were transferred

to distilled water and incubated at the respective temperature for another 20 days, almost

Factor MFG GRate SR DR

Phot. 1746.58*** - - -

Temp. 80.37*** 87.85*** 24.80*** 24.70***

Salt 404.20*** 656.80*** 284.46*** 62.43***

Phot. * Temp. 41.94*** - - -

Phot. * Salt 215.50*** - - -

Temp. * Salt 14.28*** 19.34*** 7.790*** 5.08***

Phot. * Temp. * Salt 12.03*** - - -

31 

 

25/35oC

0.0 0.1 0.2 0.3 0.4 0.5 0.6

20/30oC

NaCl (mol L-1)

15/25oC

10/20oC

Recovery

Dark

aa a a

b

a

bc

a a

a

aa

a

b b

a a a a

bbab

b b b

b

cc

c cc

ccc cc

c c c c

b

c

a

b

a a

a

b

a

b

a

c

a

25/35oC

0.0 0.1 0.2 0.3 0.4 0.5 0.60

20406080

100

20/30oC

020406080

100

15/25oC

020406080

100

10/20oC

Ger

min

atio

n (%

)

020406080

100

a

b

cc

cc

d

a

b

d

a

d

c

c

e

d db

c

Germination

Light

a

a

e

f

c

a a

b b

cc

a b

bbb

a a

a

c

b b

c

b

dbb

a

bb

b

a

b b

d

c

Fig. 2.2. Germination and recovery percentages of Halopeplis perfoliata seeds after

different salinity treatments (0, 0.1, 0.2 0.3, 0.4, 0.5 and 0.6 mol L-1 NaCl); temperatures

(10/20, 15/25, 20/30 and 25/35 oC) and light (12-h photoperiod and complete darkness)

regimes. Vertical bars are means ± S.E. Different alphabets show significant differences

among salinity treatments; Bonferroni test; P < 0.05.

32 

 

10/20oC

0

10

20

30

40

50

25/35oC

NaCl (mol L-1)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

20/30oC

0.0 0.1 0.2 0.3 0.4 0.5 0.6

Ger

min

atio

n R

ate

0

10

20

30

40

50

15/25oCa

aa

aa

a a

ab

c

bb

b

c

b

cc

d

e

f

b

c c

d d d

c c c

all seeds were germinated (SR), hence total germination (MFG under saline condition +

SR) approach to the level of MFG in distilled water. Likewise, when un-germinated seeds

from dark incubation were exposed to 12-h photoperiod for 20 days, they germinated

(DR) and total germination (MFG in dark + DR) was comparable to MFG in respective

salinity treatments under 12-h photoperiod (Fig. 2.2).

Fig. 2.3. Rate of germination responses of Halopeplis perfoliata seeds after different

salinity treatments (0, 0.1, 0.2 0.3, 0.4, 0.5 and 0.6 mol L-1 NaCl); temperatures (10/20,

15/25, 20/30 and 25/35 oC) and light (12-h photoperiod and 24- darkness) regimes.

Vertical bars are means ± S.E. Different alphabets show significant differences among

salinity treatments; Bonferroni test; P < 0.05.

33 

 

Experiment. 2: germination and viability responses of seeds to hyper-saline storage

A three-way ANOVA indicated significant individual effects of duration, temperature,

hyper-salinity and their interaction on MFG, SR, viability and mortality of the seeds of H.

perfoliata (Table 2.3). Maximum (85%) germination occurred in distilled water within

20 days and no seed germinated after this period (Fig. 2.4). Some seeds were germinated

(> 20%) was noted in 0.5 mol L-1 NaCl treatment and no seed could germinate in hyper-

salinity treatments (1.0, 1.5 and 2.0 mol L-1 NaCl) even after 60 days (Fig. 2.4).

Comparatively higher MFG was found in distilled water and 0.5 mol L-1 NaCl treatments

at 20/30 oC than at 25/30 oC. About 70% of the un-germinated seeds from hyper-salinity

germinated when transferred to distilled water (SR) at 20/30 oC, even after 60 days of

exposure. Exposure of seeds to hyper-salinity for 60 days caused significant (P < 0.05)

reduction in SR value at warmer incubation temperature 25/35 oC (Fig. 2.4). About 10-

15% seeds were found dead in all salinity treatments at 20/30 oC after various exposure

times. High temperature (25/35 oC) treatments resulted in mortality of about 50% seeds

after 60 days under saline condition (Fig. 2.4).

Table 2.3. Two-way ANOVA indicating effects of storage time (Days) temperature

(Temp.), hyper-salinity (Salt), and their interactions on mean final germination (MFG),

recovery from salinity (SR), viability (Viab.) and mortality (Mort.) of Halopeplis

perfoliata seeds. Numbers are F-values and asterisks in superscript are significance

levels at P < 0.05 (ns = non-significant, * = P < 0.05, ** = P < 0.01 and *** = P <

0.001).

Factor MFG SR Viab. Mort.

Days 2.127ns 8.45*** 25.32*** 8.70*** Temp. 8.55** 58.41*** 35.99*** 26.07*** Salt 416.19*** 143.92*** 6.48*** 1.85ns Days * Temp. 2.13ns 1.493ns 9.01*** 2.50ns Temp. * Salt 3.30* 9.70*** 5.41** 3.04* Days * Salt 0.83ns 3.00* 1.52ns 1.04ns Temp. * Days *Salt 3.28** 1.70ns 2.27* 1.04ns

34 

 

NaCl (mM)

0.0 0.5 1.0 1.5 2.00

20

40

60

80

100

NaCl (mol L-1)0.0 0.5 1.0 1.5 2.0

0

20

40

60

80

100

20/30oC 25/35oC

60 Days

Germination % Recovery % Viability % Mortality%

20 Days

Ger

min

atio

n (%

)

0

20

40

60

80

10040 Days

0.0 0.5 1.0 1.5 2.0

60 Days

40 Days

20 Days

40 Days

Fig. 2.4. Germination, recovery, viability and mortality percentages of Halopeplis

perfoliata seeds after prolonged exposure (20, 40 and 60 days) in hyper-saline (0.5, 1.0,

1.5 and 2.0 mol L-1 NaCl) treatment, temperatures (20/30 and 25/35 oC); and light (12-h

photoperiod) regimes. Values are means of 4 replicates.

35 

 

Experiment. 3: germination characteristics after dry storage

A three- way ANOVA showed significant individual effect of storage, photoperiod,

temperature, salinity, and their interaction on seed germination of H. perfoliata (Table

2.4). Maximum germination of both fresh and stored seeds occurred in distilled water

and increase in salinity decreased germination of both fresh and stored seeds at all

temperature regimes under 12-h photoperiod (Fig. 2.5). Relatively higher germination

was observed in stored seeds as compared to fresh ones under moderately saline (0.30-

0.50 mol L-1 NaCl) conditions at 10/20, 15/25 and 20/30 oC. However, stored seeds did

not germinate at 25/35 oC under 12-h photoperiod). There was no difference in

germination of fresh and stored seeds under dark at low (10/20 and 15/25 oC) and warm

(25/35 oC), while stored seeds germinated better under dark at moderate (20/30 oC)

temperature (Fig. 2.5).

Table 2.4. Four-way ANOVA showing effects of dry storage (Stor.) photoperiod

(Phot.), temperature (Temp.), salinity (Salt) and their interactions on mean final

germination (MFG) of Halopeplis perfoliata seeds. Numbers are F-values and asterisks

in superscript are significance levels at P < 0.05 (* = P < 0.05 and *** = P < 0.001).

Factor MFG Stor. 21.07*** Phot. 2302.68*** Temp. 303.30*** Salt 240.28*** Stor. * Phot. 5.80* Stor. * Temp. 44.70*** Phot. * Temp. 125.28*** Stor. * Salt 6.86*** Phot. * Salt 100.60*** Temp. * Salt 19.40*** Stor. * Phot. * Temp. 20.91*** Stor. * Phot. * Salt 27.00*** Stor. * Temp. * Salt 4.98*** Phot. * Temp. * Salt 16.97*** Stor. * Phot. * Temp. * Salt 4.49***

36 

 

25/35oC

0.0 0.1 0.2 0.3 0.4 0.5 0.6

20/30oC

NaCl (mol L-1)

15/25oC

10/20oC

Stored

Dark

b

a

cc

d

ab

c

a a

a

aa

a

b b

a a ab bb

abb b b

a

c

c cc

bc c c c cc

d d d de e

b aa a b a b a b a b a

25/35oC

0.0 0.1 0.2 0.3 0.4 0.5 0.60

20

40

6080

100

20/30oC

0

20

4060

80

100

15/25oC

0

20

40

60

80

100

10/20oC

Ger

min

atio

n (%

)

0

20

40

60

80

100

a

b

cc

eed

a

b

b

a

b

c

c

cd d

a a

Fresh

Light

aa a a ab

d

c

a a

b b

cc

a a

aaaa a

a

c

b b

c

b

d

a

b

a a a a a aa

* *

** *

* *

*

*

*

* *

*

**

*

Fig. 2.5. Comparison germination percentages of fresh and 1-year stored seeds of

Halopeplis perfoliata in salinity treatments (0, 0.1, 0.2 0.3, 0.4, 0.5 and 0.6 mol L-1

NaCl); temperatures (10/20, 15/25, 20/30 and 25/35 oC) and light (12-h photoperiod

and 24- darkness) regimes. Different alphabets show significant differences among

salinity treatments; Bonferroni test; P < 0.05. Asterisks (*) shows significant

differences in fresh and stored seeds by Student’s t-test; P < 0.05.

37 

 

Experiment. 4: salinity tolerance and germination responses to dormancy regulating chemicals

There was a significant effect of dormancy regulating chemicals (DRCs), photoperiod,

salinity and their interaction on MFG of H. perfoliata (Table 2.5). Ethephon improved

MFG in high salinity (0.6 mol L-1 NaCl) only; kinetin was inhibitory, while betaine,

fusicoccin, proline and thiourea had no effect on MFG of H. perfoliata in distilled

water as well as in saline condition under 12-h photoperiod (Fig. 2.6). However, all

DRCs (except proline) improved MFG under complete darkness in distilled water as

well as under saline condition (Fig. 2.7). There was no effect of DRCs on recovery

(both SR and DR) responses of seeds (data not given).

Table 2.5. Three-way ANOVA showing effects of dormancy regulating chemicals

(DRCs), photoperiod (Phot.), salinity (Salt) and their interactions on mean final

germination (MFG) of Halopeplis perfoliata seeds. Numbers are F-values and asterisks

in superscript are significance levels at P < 0.05 (** = P < 0.01 and *** = P < 0.001).

Factor MFG

DRCS 4.07**

Phot. 1119.73***

Salt 470.31***

DRCS * Phot. 15.59***

DRCS * Salt 3.65 ***

Phot. * Salt 105.73***

DRCS * Phot. * Salt 3.78***

38 

 

NaCl (mol L-1)

0.0 0.3 0.60

20406080

1000

20406080

100

0.0 0.3 0.6

020406080

100

NaCl Thiourea

NaCl Fusicoccin

NaCl Proline NaCl Betaine

NaCl Ethephon

NaCl Kinetin

*

**

Ger

min

atio

n (%

)

Fig. 2.6. Germination percentages of Halopeplis perfoliata treated with different

dormancy regulating chemicals (DRCs) under different salinity treatments (0.3 and 0.6

mol L-1 NaCl) at 20/30 oC temperature and 12-h photoperiod. Vertical bars are means ±

S.E. Asterisks (*) show significant differences among salinity treatments within each

chemical treatment by Student’s t-test; P < 0.05.

39 

 

Fig. 2.7. Germination percentages of Halopeplis perfoliata treated with different

dormancy regulating chemicals (DRCs) under different salinity treatments (0.3 and 0.6

mol L-1 NaCl) at 20/30 oC temperature and complete darkness. Vertical bars are means ±

S.E. Asterisks (*) show significant differences among salinity treatments within each

chemical treatment by Student’s t-test; P < 0.05.

0.0 0.3 0.6

NaCl (mol L-1)0.0 0.3 0.6

020406080

100

()

020406080

1000

20406080

100

NaCl Betain

NaCl Fusicoccin

NaCl KinetinNaCl Thiourea

NaCl Ethephon

NaCl Proline

*

*

**

*

*

*

40 

 

Discussion

Seed size and moisture content of Halopeplis perfoliata Seeds of H. perfoliata were small (> 1 mm in diameter) like H. amplexicaulis and H.

pygmaea seeds (Shepherd et al., 2005; Anonymous, 2014) and also had low (> 5%)

moisture like many other halophytes such as Limonium stocksii and Suaeda fruticosa

(Hameed et al., 2014). In absence of hairs and perianth, small size could be an effective

adaptation for dispersal and also reduces their predation (Fenner, 1985; Leishman et al.,

2000; Shepherd et al., 2005). Stromberg and Boudell (2013) found that small seed size

was associated with adaption to disturbance and wet environments. Low moisture

content on the other hand is reportedly essential for prolonged longevity of the seeds

(Tompsett, 1986; Ellis, 1991; Rajjou and Debeaujon, 2008). Hence, small size and low

moisture content of the seeds are important adaptations of H. perfolaita for survival in

salt marsh, as reported for another subtropical halophyte Arthrocnemum macrostahyum

(Gul and Khan, 1998).

Seed germination responses of H. perfoliata are influenced by salinity, temperature and photoperiod Innate dormancy is generally absent in perennial halophytes (Gul et al., 2013), which

may be an adaptive strategy to take advantage of water availability after rain (Song et al.,

2005). Similarly, mature seeds of H. perfoliata were non-dormant and germinated

maximally (> 90%) in distilled water under 12-h photoperiod irrespective of incubation

temperature. Increases in salinity decreased both rate and final seed germination of H.

perfoliata, however a few seeds could also germinate in 0.6 mol L-1 NaCl (equivalent to

seawater salinity) under 12-h photoperiod. Some other Salicornioideae species such as

Arthrocnemum indicum (1.0 mol L-1 NaCl, Khan and Gul, 1998) and Sarcocornia

ambigua (0.77 mol L-1 NaCl, Freitas and Costa, 2014) also showed high salinity

tolerance during seed germination. When transferred to distilled water from saline

solutions, ungerminated seeds showed a high recovery of germination, as reported for

most other halophytes (Gul et al., 2013). This enforced or conditional dormancy

(inability to germinate yet maintaining their viability) due to salinity is an important

adaptive feature of halophyte seeds that distinguishes them from glycophytes and is a

key to constitute persistent soil seed bank so that chance for seedling recruitment only

after sufficient rain (Khan and Ungar, 1984; Khan and Gul, 2006; Cao et al., 2014).

41 

 

Changes in temperature affect a number of processes regulating the seed

germination such as membrane permeability, activity of membrane-bound proteins and

cytosol enzymes (Gul and Weber, 1999; Bewley et al., 2013). Change in temperature

changes may also act as a germination timing sensor (Ekstam et al., 1999). Hence,

changes in incubation temperature also influence germination and salinity tolerance of

halophyte seeds (Hameed et al., 2013; Gul et al., 2013). However, seeds of some

halophytes are more sensitive to temperature changes than others (Khan and Gul, 2006).

In this study, relatively higher germination and salinity tolerance of H. perfolaita seeds

was observed at 20/30 oC in comparison to other temperature regimes used. Recently Gul

et al. (2013) reviewed that seed of most subtropical halophytes germinate better at 20/30 oC, which resembles the post-rain ambient temperature in many subtropical areas. Higher

(25/35 oC) temperature was more inhibitory for the seed germination of H. perfoliata

than lower (10/20 and 15/25 oC) temperatures, as reported for another salt marsh

halophyte Arthrocnemum indicum (Saeed et al., 2011). Changes in temperature also

influence recovery of germination of halophyte seeds (Khan and Ungar, 1997). Recovery

of germination was also inhibited significantly at higher temperature regime, as in case

of A. indicum (Saeed et al., 2011). Hence, higher temperature and salinity appear to act

as regulatory factors by preventing germination of H. perfolaita in summer when there is

high salinity in marsh sediments owing to no/low precipitation and high

evapotranspiration due to high ambient temperatures.

Light has also been recognized as an important factor regulating seed germination

of many plant taxa including most halophytes (Pons, 2000; Baskin and Baskin, 2001;

Flores et al., 2006; Kettenring et al., 2006; Gul et al., 2013). Halophytes vary in their

light requirement for seed germination (Gul et al., 2013). Baskin and Baskin (1995)

reported that out of 41 halophytes, seed germination of 20 species was promoted by

light, 10 geminated in dark, and 11 species germinated equally well in light or dark.

However, most light requiring (positive photoblastic) seeds are small (Grime et al.,

1981). Seeds of H. perfolaita are also small and germinated better in presence of light

(12-h photoperiod) than in dark. In addition un-germinated (conditionally dormant) seeds

from dark treatment showed recovery of germination when exposed to 12-h photoperiod,

thus indicating their positive photoblastic nature. Milberg et al. (2000) suggested that

light requirement for seed germination is a co-evolved adaptation of small seeds to

ensure germination only when close to the soil surface. Light requirement is important

42 

 

for small seeds because they contain too little food reserve to support seedling

emergence from deep burial in soil (Winn, 1985; Bonfil, 1998; Seiwa, 2000). In our test

species, light requirement would favor germination of seeds at/near soil surface, to

ensure light access to nascent seedlings, thereby maximizing their survival in marsh

environment.

Seeds of Halopeplis perfoliata can endure hyper-salinity

Seeds of most halophytes can even withstand hyper salinity (equivalent to 1.0 mol L-1

NaCl or higher) by undergoing conditional dormancy, in which although they do not

germinate but remain viable and germinate quickly when salinity is diluted/alleviated

(Keiffer and Ungar, 1997; Gul et al., 2013). Seeds of the salt marsh halophyte H.

perfolaita also exhibited tolerance to hyper salinity by entering into a state of conditional

dormancy and showed high (80%) recovery when transferred to distilled water after 60

days of exposure to 2.0 mol L-1 NaCl at 20/30 oC. Similarly, seeds of another chenopod

halophyte Salsola affinis showed 37% recovery of germination, after 14 days of exposure

to 2.0 mol L-1 NaCl salinity (Wei et al., 2008). Seeds of some other halophytes Suaeda

depressa, Salicornia europaea, Suaeda linearis and Spergularia marina could also

withstand 0.85 mol L-1 NaCl treatment for 30 days in state of conditional dormancy and

showed substantial recovery in distilled water (Ungar, 1995). Hence, germination

inhibition in halophytes including our test species appears a consequence of osmotic

effect rather than ionic toxicity. However, high (25/35 oC) incubation temperature was

detrimental to seed viability and about 50% seeds of H. perfoliata seeds died after 60

days of exposure to 2.0 mol L-1 NaCl at 25/35 oC as compared to 20/30 oC, at which

there was only ~20% seed mortality. High storage temperatures reportedly reduce

longevity of seeds, especially under moist condition (Egley, 1990; Rajjou and

Debeaujon, 2008).

Storage influences seed germination of Halopeplis perfoliata

Long term storage of seeds is often reported to affect their germination and viability

(Kibinza et al., 2006; Rajjou and Debeaujon, 2008; Zhang et al., 2011). Studies on crop

seeds showed that germination requirements may become less specific after dry storage

(Probert, 2000). However, little is known about effects of long term storage on stress

tolerance of the seeds of non-crops such as halophytes (El-Keblawy, 2013a; Gul et al.,

2013). Dry storage of the seeds of two succulent halophytes Haloxylon salicornicum and

43 

 

Salsola imbricata for three months significantly increased germination but longer than

nine month storage at room temperature caused significant reduction or complete

inhibition of germination (El-Keblawy, 2013a). Similarly, another study showed that the

light requirement for germination reduced significantly in Prosopis juliflora seeds after 8

months of dry-storage at room temperature (El-Keblawy and Al-Rawai, 2006). In this

study, 1-year dry storage enhanced tolerance of H. perfoliata seeds to moderate salinity

and dark, but substantially decreased high temperature tolerance, in comparison to fresh

seeds. The ecological advantage of reduction in light requirement for seed germination

after storage although seems unknown, however increase in germination under moderate

salinity but not at high temperature could be to provide chance to seeds stored in distal

dry marsh sediments to germinate after winter rains but to prevent germination after light

summer rains which follow fast soil drying due to high temperatures.

Application of dormancy regulating chemicals can mitigate dark but not salinity effects

Various dormancy regulating chemicals (DRCs) such as betaine, ethephon, fusicoccin,

kinetin, proline and thiourea are often reported to increase germination and stress

tolerance of halophyte seeds (Khan et al., 1998; Gul et al., 2000; Khan et al., 2004;

Mehrun-Nisa et al., 2007; Atia et al., 2009; El-Keblawy et al., 2010; Zehra et al., 2013;

Ahmed et al., 2014). However, actions of these DRCs could be both species and stress

specific (Gul et al., 2000; Ahmed et al., 2014). In this study, different DRCs generally

had little/no effect on salinity tolerance of H. perfoliata but all (except proline) improved

MFG under complete darkness as compared un-treated seeds. Ethephon and thiourea

(Gul and Weber, 1998), betaine and kinetin (Gul et al., 2000) and fusicoccin (El-

Keblawy et al., 2010; El-Keblawy, 2013b; Zehra et al., 2013) and could also substitute

for light requirement for the seed germination of many other halophytes. As in this study,

proline did not improve salinity as well as dark enforced dormancy in two other

halophytes Halogeton glomeratus and Peganum harmala (Ahmed et al., 2014).

44 

 

Conclusions

Halopeplis perfoliata seeds were non-dormant and germinated quickly in distilled water

irrespective of incubation temperature but required light for germination. Seeds showed

high tolerance to salinity and some seeds could germinate in as high as seawater salinity

(0.6 mol L-1 NaCl) under 12-h photoperiod. High salinity (> 0.3 mol L-1 NaCl) and dark

imparted enforced/conditional dormancy in seeds, as they showed high recovery of

germination when transferred to distilled water and light. Seeds of our test species could

withstand hyper salinity (> 1.0 mol L-1 NaCl) by entering a state of enforced dormancy.

However, high incubation temperature enhanced seed mortality under hyper-saline

condition. Dry storage for 1-year increased tolerance of H. perfoliata seeds to moderate

salinity and dark, but substantially decreased high temperature tolerance, in comparison

to fresh seeds. Whereas, dormancy regulating chemicals could compensate for light

requirement but had no effect on salinity-enforced dormancy. Hence, it appears that

salinity enforced dormancy has osmotic, while dark enforced dormancy has biochemical

basis.

Fig. 2.8. Regulation of seed germination in Halopeplis perfoliata by light, temperature,

salinity and chemicals (DRCs).

45 

 

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Action of plant growth regulators in alleviating salinity and temperature effects

on the germination of Phragmites karka. Pakistan Journal of Botany, 45, 1919-

1924.

Zhang, Y., Wang, Y., and Li, S. (2011). Effects of different storage conditions and

durations on seed viability and germination of five desert plants in West

Erdos. Scientia Silvae Sinicae, 47, 36-41.

Zia, S., and Khan, M. A. (2004). Effect of light, salinity, and temperature on seed

germination of Limonium stocksii. Canadian Journal of Botany, 82, 151-157.

Zreik, R. (1990). The domestication and economic cultivation of halophytes. Developing

World Agriculture (pp. 74-79). London: Grosvenor Press.

52 

 

 

 

 

 

 

 

Chapter 3

Comparison of osmotic and ionic effects of various salts on germination inhibition and recovery responses of

Halopeplis perfoliata seeds

 

 

 

 

 

 

53 

 

Abstract

Salinity affects seed germination of halophytes by inducing ionic toxicity, osmotic

constraint or both. Information about the effects of salinity on seed germination of a

large number of halophytes exists, but little is known about the basis of this salinity-

induced germination inhibition. In order to partition salinity effects, we studied seed

germination and recovery responses of a coastal salt marsh halophyte Halopeplis

perfoliata to different isotonic treatments (ΨS: -0.5, -1.0, -1.5, -2.0 and -2.5, MPa) of

various salts and polythylene glycol (PEG) under two light regimes (12-h light

photoperiod and 24-h complete darkness). Highest seed germination was observed in

distilled water under 12-h light photoperiod and reduction in osmotic potential of the

solution decreased seed germination. However, some seeds of H. perfoliata could

germinate in as low as -2.5 MPa (~0.6 mol L-1 NaCl), which is equivalent to seawater

salinity. Sea-salt treatment was more inhibitory than isotonic NaCl at the lowest osmotic

potential (ΨS -2.5 MPa). Generally, chloride salts inhibited germination more than the

isotonic sulfate salts. Comparable germination responses of seeds in NaCl and isotonic

PEG treatments as well as high recovery of germination in un-germinated seeds after

alleviation of NaCl salinity indicated prevalence of osmotic constraint. Hence seed

germination inhibition in H. perfoliata under different salts is a combination of osmotic

constraint and their differential ionic effects.

54 

 

Introduction

Seeds of salt marsh halophytes are under direct influence of seawater

Coastal salt marshes are transition between marine and terrestrial ecosystems,

characterized by highly salt tolerant halophyte vegetation and offer a number of

ecological and economic services to mankind (Böer, 1996; Pennings and Bertness 2001;

Lee et al., 2006; Gedan et al., 2009; Teixeira et al., 2014). These habitats are however

under threat due to various natural and anthropogenic reasons (Yasseen and Al-Thani,

2007; Gedan et al., 2011; Ramadan et al., 2013). Therefore, salt marsh ecosystems

warrant special attention in research, which would eventually help in their protection and

conservation. Establishment of plants in salt marshes is reportedly dependent on the seed

germination responses which are influenced by a number of factors especially salt

concentrations in the marsh sediments (Pujol et al., 2000; Khan et al., 2001; Hameed et

al., 2006; Gul et al., 2013). However, data on seed germination responses of succulent

halophytes of subtropical salt marshes is scanty (Saeed et al., 2011; Gul et al., 2013).

Seeds of salt marsh halophytes are directly influenced by seawater owing to diurnal and

seasonal inundations (Gul and Khan, 1998; Gul et al., 2013; Teixeira et al., 2014). NaCl

is although dominant but other chloride and sulfate salts and their interactions may also

play a significant role in germination, radical emergence and seedling growth of coastal

halophytes (Gul and Khan, 1998; Khan, 2003; Zia and Khan, 2008). Most research on

the seed germination of halophytes has focused on responses to NaCl (reviewed by Gul

et al., 2013), with few studies on comparison of the effects of NaCl and sea-salt (Zia and

Khan, 2002; Atia et al., 2006; Hameed et al., 2006; Liu et al., 2006; Saeed et al., 2011;

Shaikh et al., 2013) and even fewer studies on effects of different salts (Ungar, 1991;

Egan et al., 1997; Panuccio et al., 2014). Likewise, investigations to partition osmotic

and ionic constraints of various salts (Munns et al., 1995) by comparing their effects on

seed germination with germination responses in non-ionic solutes such as polyethylene

glycol (PEG) are also limited and inconclusive (Tobe et al., 2000; Sosa et al., 2005;

Hameed et al., 2013).

Salt marsh halophyte Halopeplis perfoliata is largely unstudied

Halopeplis perfoliata Forssk. (Amaranthaceae) is a succulent halophyte of coastal salt

marshes along Arabian Peninsula (Al-Oudat and Qadir, 2011), where it acts as an

important primary producer and provides shelter to marine life (Pilcher et al., 2003). A

55 

 

previous report showed that the seeds of H. perfoliata can germinate in up to 0.25 mol L-

1 NaCl treatment (Mahmoud et al., 1983). This study was therefore designed to

comparatively investigate the effects of 1) sea-salt 2) various chloride and sulfate salts

and 3) polyethylene glycol 6000 (PEG-6000) on germination and recovery of the seeds

of H. perfoliata.

Materials and methods

Study site and seed collection

Seeds of Halopeplis perfoliata were collected from the coast of Jizan, Saudi Arabia in

2012. Seeds were separated from inflorescence husk manually; surface sterilized using

1% bleach solution for 1 min, followed by rinsing with distilled water and air-drying in

the laboratory.

Treatments and experimental conditions

Germination experiments were carried out in clear-lid plastic petri-plates (50 mm

diameter x 15 mm depth) with 5 ml of test solution. Seeds were immersed in distilled

water (0 MPa) and isotonic solutions of various salts (NaCl, Na2SO4, KCl, K2SO4,

MgCl2, MgSO4, CaCl2 and Sea-salt) and polyethylene glycol 6000 (PEG). Five isotonic

levels (ΨS: -0.5, -1.0, -1.5, -2.0 and -2.5, MPa) were used. The osmotic potential of the

above solutions was measured with a vapor pressure osmo-meter (5520 Wescor, Inc.).

There were four replicates of 25 seeds each per treatment. The petri-plates were placed in

programmed incubators maintained at 20/30 °C (optimum temperature for the seed

germination of sub-tropical halophytes, Gul et al., 2013), where low temperature

coincided with 12-h dark and high temperature with 12-h light (~25 µ mol m-2 s-1, 400-

750 nm, Philips cool -white fluorescent lamps). Seed germination percentage (radicle

emergance, Bewley and Black, 1994) was recorded at 2 days intervals for 20 days. Rate

of germination (GRate) was estimated by using a modified Timson index of germination

velocity given below:

                                                      

Where G is the percentage of seed germination at 2 days intervals and t is the

total germination period (Khan and Ungar, 1985). The greater the value, the more rapid

is germination. After 20 days un-germinated seeds were transferred to distilled water for

56 

 

another 20 days and recovery of germination (SR) from various osmotic treatments of

solutes (salts and PEG) was recorded. The recovery percentage was determined by

counting the recovered seeds from total number of seeds. The recovery percentage was

calculated by using given formula:

                                                  

                                                                           Where, A is total number of seeds germinated after being transferred to distilled

water, B is the number of seeds germinated in saline solution and C is total number of

seeds. A parallel set of experiment was conducted by placing petri-plates in dark-

envelops under aforementioned temperature and osmotic treatments for 20 days.

Germination of this experiment was noted once after 20 days. After 20 days un-

germinated seeds were exposed to light in order to study the recovery of germination

from dark (DR).

Statistical analyses

Data were statistically analyzed using SPSS version 11.0 (SPSS, 2011). Analysis of

variance (ANOVA) was performed to determine if various treatments affected the seed

germination parameters significantly. A Bonferroni post-hoc was conducted to indicate

significant (P < 0.05) differences among mean values.

Results

A four-way ANOVA indicated significant effects of osmotic potential (ΨS), photoperiod

anions and but not of cations on mean final germination (MFG) of H. perfoliata seeds

(Table 3.1).

Comparitive effects of NaCl and Sea-salt

Seeds were non-dormant and germinated maximally (~90%) in distilled water under 12-h

photoperiod (Fig. 3.1). Effects of NaCl and sea-salt were comparable in up to -1.0 MPa,

wherein there was maximal (~90%) MFG as in distilled water under 12-h photoperiod

(Fig. 3.1). While, in lowest (-2.5 MPa) ΨS treatment, sea-salt (MFG = ~10%) was found

more inhibitory than NaCl (MFG = ~25%). Rate of germination in -2.5 MPa was also

lowest in sea-salt compared to NaCl (Fig. 3.2). Un-germinated seeds from low ΨS

treatments of both NaCl and sea-salt, showed recovery (SR) when transferred to distilled

57 

 

water and almost all un-germinated seeds recovered (Fig. 3.1). Dark inhibited seed

germination of test species in both NaCl and sea-salt treatments (Fig. 3.3). When un-

germinated seeds from dark were transferred to 12-h photoperiod for another 20 days, a

significant recovery (DR) was seen in either salt type. Still un-germinated seeds from low

ΨS treatments of both NaCl and sea-salt showed complete recovery (SR) when transferred

to distilled water (Fig. 3.3).

Comparison of the effects of different salts

A decrease of up to -1.0 MPa (equivalent to 0. 215 mol L-1 NaCl) for chloride salts and -

1.5 MPa (equivalent to 0.4 mol L-1 NaCl) for sulfate salts had no inhibitory effect on

seed germination of test species (Fig. 3.1). K2SO4 was less inhibitory, in which over 70%

seeds germinated in lowest (-2.5 MPa) ΨS treatment. On the other hand, CaCl2 and NaCl

reduced MFG to ~50% in -1.5 MPa treatments (Fig. 3.1). Inhibitory effects of chloride

salts were in following order: CaCl2 > NaCl > KCl > MgCl2. While, MFG inhibition

among sulfate salts was as follows: Na2SO4 > MgSO4 > K2SO4. Rate of germination also

decreased with decreases in ΨS of salts (Fig. 3.2). However, there was a significant

decrease in rate of germination in -1.0 MPa for chloride salts and in -1.5 MPa for sulfate

salts. While similar to MFG, NaCl and CaCl2 had higher inhibition and K2SO4 caused

less inhibition to the rate of germination (Fig. 3.2). Almost all un-germinated seeds

showed recovery of germination (SR) when transferred to distilled water and generally

highest SR was observed from the lowest (-2.5 MPa) ΨS treatments of all salts (Fig. 3.1).

Dark caused substantial inhibition to the MFG of H. perfoliata, irrespective of the salt

and ΨS treatments (Fig. 3.3). When un-germinated seeds from dark were exposed to 12-h

photoperiod after 20 days, a significant recovery of germination (DR) was observed. The

DR was highest in distilled water and there was decrease in DR in solution of low ΨS (Fig.

3.3). When still un-germinated seeds from low ΨS treatments were transferred to distilled

water for another 20 days, most of them showed recovery of germination (SR),

irrespective of salt used (Fig. 3.3).

Comparison of the effects of NaCl and PEG

Effects of NaCl and PEG on MFG and germination rate were generally comparable at

higher osmotic levels and these germination parameters decreased with decreases in ΨS

under 12-h photoperiod (Fig. 3.4 A and 4 C). Un-germinated seeds from low ΨS

58 

 

treatments of both NaCl and PEG showed high recovery (SR; Fig. 3.4 B) complete

darkness caused substantial inhibition to MFG (Fig. 3.4. D). Un-germinated seeds from

the dark showed high recovery (DR) upon transfer to 12-h photoperiod condition (Fig.

3.4 E). However, relatively higher DR was observed in moderately low (-1.0 to -1.5 MPa)

PEG compared to isotonic NaCl treatments. Remaining un-germinated seeds from low

ΨS treatments of both NaCl and PEG germinated (SR) when transferred to distilled water

(Fig. 3.4 F). Table 3.1. One-way ANOVA of the effects of salinity on growth, water and ion

relations, pigments, photosynthesis, isotope ratios, stress markers, antioxidant enzymes

and substrates of Halopeplis perfoliata grown under salinity treatments (0, 1.50, 0.30

and 0.60 mol L-1 NaCl) for 30 days. Numbers are F-values and asterisks in superscript

are significance levels at P < 0.05 (ns = non-significant, * = P < 0.05, ** = P < 0.01 and

*** = P < 0.001).

Factors MFG GRate Rec.

Phot. 4341.52*** 372.55*** - ΨS 98.50*** 4.02** 500.63*** Anion 13.63*** 0.53ns 28.65*** Cat. 2.16ns 1.96ns 5.71** Phot. * ΨS 97.76*** 189.41*** - Phot.* Ani 14.39*** 45.74*** - Phot.* Cat. 2.35ns 9.08*** - ΨS*Ani 4.65*** 5.45*** 9.31*** ΨS *Cat. 0.78ns 1.58ns 4.29*** Ani* Cat. 2.46ns 9.20*** 10.58*** Phot.*Ani* Cat. 2.26ns 3.51* - Phot.* ΨS *Ani 4.50** 3.13** - Phot.* ΨS *Cat. 0.74ns 1.45ns - ΨS *Ani* Cat. 2.91** 2.91** 16.72*** Phot.* ΨS*Ani*Cat. 2.95** 1.99* -

59 

 

SeaSalt

0.0 0.5 1.0 1.5 2.0 2.50.0 0.5 1.0 1.5 2.0 2.5

20

40

60

80

100

MgSO4

20

40

60

80

100Ger

min

atio

n (%

)

20

40

60

80

100

20

40

60

80

100

NaCl

KCl

MgCl2

CaCl2

K2SO4

a

a

a

b

bb b

b

a

aaabab

a a

c

a

a

aa

a a

b

b

b

aa

b

b

a

a a

d

c

c

d

d

b

b

aa

b

b

aa

ab

a

aa

a

a

a

aa

bb

a

a

a

bc

b

c

c

c

a

a

aa

a

a a

a

a a a

b

Germination Recovery

ψs (-MPa)

b

b

aa

a aa

b b

Na2SO4

Fig. 3.1. Effects of NaCl, Na2SO4, KCl, K2SO4, MgCl2, MgSO4, CaCl2 and sea-salt on

mean final germination (MFG) and germination recovery in distilled water (SR) at 20/30 oC temperature and 12-h photoperiod. Vertical bars are means ± S.E. Different alphabets

show significant differences among isotonic treatments within each parameter;

Bonferroni test; P < 0.05.

60 

 

Sea-salt

0.0 0.5 1.0 1.5 2.0 2.5

MgSO4

K2SO4

CaCl2

0.0 0.5 1.0 1.5 2.0 2.50

1020304050

MgCl2

01020304050

KCl

01020304050

NaClG

erm

inat

ion

Rat

e

01020304050 Na2SO4

ψs (-MPa)

a a

aa

c

b

b

c

d

c

b

b

a a a

a

b

b

a aaab

c

a a a

b

c

d

a a a

b

d

c

d

a aab

b b

c

a a

bb

cc

Fig. 3.2. Effects of NaCl, Na2SO4, KCl, K2SO4, MgCl2, MgSO4, CaCl2 and sea-salt on

rate of germination at 20/30 oC temperature and 12-h photoperiod. Vertical bars are

means ± S.E. Different alphabets show significant differences among isotonic treatments

within indiviual salt; Bonferroni test; P < 0.05.

61 

 

Sea-salt

0.0 0.5 1.0 1.5 2.0 2.5

MgSO4

K2SO4

CaCl2

0.0 0.5 1.0 1.5 2.0 2.50

20406080

100

MgCl2

020406080

100

KCl

Ger

min

atio

n (%

)

020406080

100

NaCl

020406080

100Na2SO4

MFG SR DR

a

bb

cd

ea a

b

b

cbc

ca

a a

a a

a a

ab b

ca

b

c

a

a

aab

a a

bb

c

bb

c

b

aa

aba a

b

c

da a

b

cc

a

a

aa

ab

b

b ba

ab

b

c a

a

a

aa

a

bb

a

b

c

b

c

a a

aaa

a

ab

a

ab

bc

ca

a

a a

a

bab

b

c

bb

bb

ψs (-MPa)

Fig. 3.3. Effects of isotonic treatments of NaCl, Na2SO4, KCl, K2SO4, MgCl2,

MgSO4, CaCl2 and sea-salt on mean final germination (MFG), recovery from Salinity

(SR) and recovery from Dark (DR) at 20/30 oC temperature and complete darkness.

Vertical bars are means ± S.E. Different alphabets show significant differences among

isotonic treatments within each paramerter of individual salt; Bonferroni test; P <

0.05.

62 

 

DR (%

)

Ger

min

atio

n (%

)

Ger

min

atio

n (%

)S R

(%)

ψs (-MPa)

2D Graph 1

0.0 0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

0.0 0.5 1.0 1.5 2.0 2.5

Ger

min

atio

n R

ate

0

10

20

30

40

500

20

40

60

80

100

Dark

0

20

40

60

80

100

S R (%

)

0

20

40

60

80

100

0

20

40

60

80

100

aab

a

a

a bb

ccc

dd

aa

b

bb

a a a a

b

c

aa

bb

b

a a a

b

c a aa

a

b

bb

cd

ec c

a aa

a

b

c

c

b

b

c

bcc

*

*

**

*

* **

*

A

B

C

E

F

Light

NaCl

PEG

aa

D

Fig. 3.4. Comparative effects of NaCl and PEG on mean final germination (MFG),

Germination rate (GRate); recovery from salinity (SR) and recovery from dark (DR) under

12-h photoperiod at 20/30 oC temperature and complete darkness. Vertical bars are

means ± S.E. Different alphabets show significant differences among sainity treatments

within each parameter; Bonferroni test; P < 0.05. Asterisks (*) show significant

differences in germination within an isotonic treatment of NaCl and PEG by student’s t-

test; P < 0.05.

63 

 

Discussion

Sea-salt is more inhibitory for germination than NaCl

Seeds of H. perfoliata were non-dormant and showed maximum germination (~90%) in

distilled water under 12-h photoperiod, as reported for many other marsh halophytes

such as Aeluropus lagopoides (Khan and Gulzar, 2003), Limonium stocksii (Zia and

Khan, 2004) and Arthrocnemum indicum (Saeed et al., 2011). Recently, Gul et al. (2013)

reviewed that lack of innate dormancy in most subtropical perennial halophytes is

probably a common adaptation to take advantage of brief water availability after rains. In

addition, seeds of H. perfoliata displayed a high salt tolerance, as some seeds could

germinate in as high as seawater (-2.5 MPa NaCl) salinity. Seeds of many other

Salicornioideae halophytes such as Arthrocnemum indicum (Khan and Gul, 1998) and

Sarcocornia ambigua (Freitas and Costa, 2014) also showed tolerance to seawater or

higher salinity during seed germination. Furthermore, un-germinated seeds of H.

perfoliata showed a high recovery of germination when transferred to distilled water

from saline solutions like many other perennial halophytes, indicating that salinity

treatments resulted in enforced/conditional dormancy and were no detrimental to seed

viability (Khan and Gul, 2006; Cao et al., 2014). Hence, it appears that seeds of H.

perfoliata are well-adapted for saline environment. However, whether these seed

responses to NaCl based salinity are comparable to seawater salinity, have seldom been

studied. Most studies on effects of salinity on seed germination of halophytes are based

on NaCl treatments and generally little is known about effects of seawater on seed

germination of halophytes (Zia and Khan, 2002; Atia et al., 2006; Hameed et al., 2006;

Liu et al., 2006; Saeed et al., 2011; Shaikh et al., 2013). In this study, effects of NaCl and

sea-salt were comparable in up to -1.0 MPa but at lowest (-2.5 MPa) ΨS treatment sea-

salt was more inhibitory than isotonic NaCl. Many studies showed that the sea-salt

inhibits seed germination more in comparison to NaCl. For instance, higher

concentrations (or lower ΨS treatments) of sea-salt were more inhibitory for the seed

germination of Limonium stocksii (Zia and Khan, 2002), Aeluropus lagopoides,

Desmostachya bipinnata and Suaeda fruticosa in comparison to NaCl (Hameed et al.,

2006). However, NaCl inhibited seed germination of Hipophae rhamnoides (Tirmizi et

al., 1993) and Crithmum maritimum (Atia et al., 2006) more as compared to sea-salt.

Effects of NaCl and sea-salt on seed germination of Haloxylon stocksii comparable

64 

 

(Hameed et al., 2006) also indicated that the effects of NaCl and seawater on seed

germination of halophytes are species-specific.

Sulfate salts are less detrimental than chloride salts for seed germination of Halopeplis

perfoliata

Seawater is composed of many salts, of which NaCl is the major (> 85%) fraction

(Murray, 2004; Khan and Gul, 2006). Other notable cations of seawater in order of

magnitude are Mg++ > Ca++> K+ and SO4-- is second major anion after Cl- (Murray,

2004). Typically, six ions constitute > 99% of the seawater (http://oceanplasma.org

/documents/chemistry.html). Therefore, I examined the effects of these ions on seed

germination of coastal marsh halophyte H. perfoliata found significant effect of anions

but not of cations on seed germination of test species. A decrease of up to -1.0 MPa

(equivalent to ~0.2 mol L-1 NaCl) for chloride salts and -1.5 MPa (equivalent to 0.4 mol

L-1 NaCl) for sulfate salts had no inhibitory effect on seed germination of test species but

a further decrease in ΨS of solutions was inhibitory. Likewise, seed germination of three

perennial salt marsh halophytes Arthrocnemum macrostachyum, Juncus acutus and

Schoenus nigricans was inhibited more in chloride than sulfate salts of sodium and

magnesium (Vicente et al., 2007). Seed germination characteristics of Kalidium

capsicum (Tobe et al., 2002) Haloxylon ammodendron (Tobe et al., 2004) and

Halostachys caspica (Assareh et al., 2011) were also more sensitive to chloride than

sulfate salts. These data including the present study indicate that sulfate salts are

comparatively less detrimental to seed germination of most halophytes than isotonic

chloride salts. However eco-physiological significance of this differential effect is yet to

be determined by further research.

Halopeplis perfoliata seeds show differential response to isotonic NaCl and PEG

treatments

Salinity affects seed germination either by reducing soil water potential (i.e.

compromising imbibition an osmotic effect) and/or by excessive entry of Na+ which is

toxic to metabolism (ionic toxicity) (Khan and Gul, 2006; Kranner and Seal, 2013;

Hameed et al., 2014). Uptake of salt could lower seed’s water potential thereby

facilitating imbibition, however ionic toxicity may overshadow such beneficial osmotic

effects (Collis-George and Sands, 1962; Khan et al., 1987; Song et al., 2005; Gul et al.,

2013). In order to partition osmotic and ionic components of salinity, often seed

65 

 

germination response in a salt solution is compared with that in an isotonic solution of a

non-ionic solute such as polyethylene glycol (Hardegree and Emmerich, 1990; Bajji et

al., 2002; Song et al., 2005; Hameed et al., 2013). In this study, effects of NaCl and PEG

on seed germination of H. perfoliata were generally comparable, especially under high

osmotic levels. Similar effects of NaCl and non-ionic solutes (PEG or mannitol) were

observed on seed germination of Chrysothamnus nauseosus (Dodd and Donovan, 1999)

and Atriplex halimus (Bajji et al., 2002). Greater incidence of osmotic constraint of

salinity on seed germination than ionic toxicity was also observed in this study. High

recovery of un-germinated seeds in this study after alleviation of salinity also supports

this assumption. On the other hand, salinity caused ionic toxicity in some species like

Artemesia ordosica (Tobe et al., 1999), Aristida adscensionis and Prosopis strombulifera

(Sosa et al., 2005) and Suaeda heterophylla (Hameed et al., 2013).

Conclusions

Halopeplis perfoliata seeds appear highly tolerant to salinity, as some seeds could

germinate in NaCl treatments as low as -2.5 MPa (~0.6 mol L-1), which is equivalent to

seawater salinity. However, a sea-salt treatment of -2.5 MPa inhibited seed germination

more than isotonic NaCl treatment (Fig. 3.5). Furthermore, chloride salts were generally

more inhibitory to seed germination than isotonic sulfate salts (Fig. 3.5). Un-germinated

seeds from low osmotic potential treatments of all salts showed high recovery of

germination when transferred to distilled water, indicating osmotic rather than ionic

effect of salts (Fig. 3.5). Similar germination response of H. perfolaita seeds in isotonic

NaCl and PEG treatments at higher osmotic levels also indicate osmotic effect rather

than ionic effect of NaCl salinity.

66 

 

Fig. 3.5. Differential inhibitory effects of iso-osmotic treatments with NaCl, seasalt, and

various chloride (Cl-) and sulphate (SO4 --) salts on seed germination of Halopeplis

perfoliata.

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for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria, 8, 186.

Assareh, M. H., Rasouli, B., and Amiri, B. (2011). Effects of NaCl and Na2SO4 on

germination and initial growth phase of Halostachys caspica. Desert, 15, 119-

125.

Atia, A., Hamed, K. B., Debez, A., and Abdelly, C. (2006). Salt and seawater effects on

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B. eds. pp. 29-33). Switzerland.

Bajji, M., Kinet, J. M., and Lutts, S. (2002). Osmotic and ionic effects of NaCl on

germination, early seedling growth, and ion content of Atriplex halimus

(Chenopodiaceae). Canadian Journal of Botany, 80, 297-304.

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Bewley, J. D., and Black, M. (1994). Seeds: physiology of development and germination.

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Böer, B. (1996). Plants as soil indicators along the Saudi coast of the Arabian Gulf

Journal of Arid Environments, 33, 417-423.

Cao, D., Baskin, C. C., Baskin, J. M., Yang, F., and Huang, Z. (2014). Dormancy cycling

and persistence of seeds in soil of a cold desert halophyte shrub. Annals of

Botany, 113, 171-179.

Collis-George, N., and Sands, J. E. (1962). Comparison of the effect of physical and

chemical component of soil water energy on seed germination. Australian

Journal of Botany, 13, 575-584.

Dodd, G. L., and Donovan, L. A. (1999). Water potential and ionic effects on

germination and seedling growth of two cold desert shrubs. American Journal of

Botany, 86, 1146-1153.

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Chapter 4

Growth and physio-chemical adaptations of Halopeplis perfoliata under saline conditions

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Abstract

Halopeplis perfoliata, a C3 salt marsh halophyte with succulent stems, was grown in a

greenhouse to study its adaptive responses for ion regulation, antioxidant defense system,

photosynthesis and growth after one month exposure to saline conditions. Plants were

grown in sand culture with and without 0.15, 0.3 and 0.6 mol L-1 NaCl with Hoagland’s

nutrient solution and sampled for measurements of biomass, osmotic adjustment, cations,

photosynthetic pigments, RubisCo content, CO2 gas exchange, chlorophyll fluorescence

and antioxidant enzymes and substrates. After four weeks of exposure to salinity,

biomass was highest in the 0.15 mol L-1 NaCl treatment but similar to non-saline controls

at higher salinities. There was little difference in the effect of salinity on the water

relations, as indicated by the constitutively high shoot succulence, osmotic potential and

relative water content except for the highest salinity (0.6 mol L-1 NaCl) treatment where

decreased succulence and more negative values of ΨS, indicated signs of water stress.

Plants were able to maintain adequate K+ levels even at 0.6 mol L-1 NaCl where Na+

accumulation was highest. Photosynthetic rates were unaffected by salinity whereas,

transpiration (E) rates and stomatal conductance (gs) increased in low and moderate

salinity treatments. Decreased E and gs induced higher WUE in the 0.6 mol L-1 NaCl

treatment. The larger fall in the value of carbon isotope discrimination measured in

photosynthetic shoots at 0.6 mol L-1 NaCl also indicated greater transpiration efficiency

when exposed to high saline conditions. Chlorophyll fluorescence measurements failed

to indicate damage to photochemical pathways. However, decreased ETR/(PN+RL+RD)

ratios indicated a down regulation of photochemistry under saline conditions and little

need for alternative electron sinks for ROS detoxification. Increased H2O2 production

and CAT activity, higher CO2 compensation point and lower carbon isotope

discrimination indicated increased photorespiration rates at the highest salinity to avoid

photo-inhibition. These data indicate that H. perfoliata is an obligate halophyte that can

grow up to seawater salinities by down regulating its photochemistry and growth, high

osmotic adjustment, an efficient ion homeostasis and antioxidant defense system.

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Introduction

Growth

Halophytes distributed in salt marsh environments have developed specialized

adaptations for growing in high light, salinity and inundation regimes (Flowers et al.,

2015; Flower and Colmer, 2015). Extreme variations in these abiotic conditions results

in additional stress for plants. Characterization of the salt resistance traits could help in

predicting and improving plant performance under increasing salinity. Succulent eu-

halophytes such as Arthrocnemum macrostachyum and Suaeda fruticosa are reported to

endure up to 1.0 mol L-1 NaCl salinity during vegetative growth (Gul and Khan, 2006;

Hameed et al., 2012). Growth of these succulent halophytes appears to be promoted

between 0.2-0.4 mol L-1 NaCl in contrast to succulent xero-halophytes such as Haloxylon

recurvum syn. Haloxylon stocksii (Khan et al., 2000), Atriplex halimus (Khedr et al.,

2011) and Zygophyllum xanthoxylum (Yue et al., 2012). Little is known about the eco-

physiology of salt resistant Chenopods (Amaranthaceae) of the warm Saharo-Sindian

region which may vary considerably among species (Moinuddin et al., 2014).

Water and ion relations

Salinity affects the plant growth via osmotic or ionic stress. Osmotic stress affects the

plants growth by limiting the water availability. Halophytes adapt to seawater or higher

salinity through osmotic adjustment via ion compartmentalization in cell vacuoles, by

accumulating compatible organic solutes, increased succulence, and by means of salt-

secreting glands or bladders (reviewed by Shabala, 2013). Flowers and Colmer (2015)

also highlighted the role of ion exclusion besides excretion from photosynthetic tissues

which would otherwise result in the accumulation of salts to precipitate in the xylem

stream in a plant growing under seawater salinity. Ion exclusion from the xylem involves

membrane transporter proteins to maintain tissue levels of Na+ and Cl- within tolerable

range with efficient acquisition of K+ and Mg++ (Mansour, 2014; Flowers and Colmer,

2015). In addition, loading of excess salt in old senescing leaves also contribute to

management of excess ions (mainly Na+ and Cl-) at the whole-plant level (Flower and

Colmer, 2008; Shabala, 2013; Reef and Lovelock, 2015). Na+ and Cl- retained in

growing plant tissues are actively compartmentalized in cell vacuoles which minimize

metabolic toxicity and contribute towards osmoregulation to acquire and retain water.

Accumulation of organic solutes such as glycine betaine, proline and sugars in the

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cytoplasm may be involved in osmotic adjustment and osmoprotection (Munns and

Tester, 2008; Hameed and Khan, 2011). Higher amount of organic solutes were mostly

found in Chenopods such as Atriplex nummularia, Halocnemum strobilaceum and

Salicornia europaea, (reviewed by Lokhande and Suprasanna, 2012).

Photosynthesis and photorespiration

At the leaf level, growth is driven by CO2 fixation which is more sensitive to salinity

than light harvesting ability of photosynthetic machinery (Osmond 1994; Maricle et al.,

2007; Taiz and Zeiger, 2010). Increasing salinity generally decreases CO2 assimilation

by reducing mesophyll CO2 availability as a result of stomatal closure to minimize water

loss (Álvarez et al., 2012; Naidoo et al., 2012). Stomatal closure in salt-stressed leaves

has been shown to result in enhanced rates of photorespiration as reviewed by Wingler et

al. (2000). At the biochemical level, photosynthetic efficiency could be limited under

saline conditions due to reduced rubisCo activity, rubisCo activase, or chlorophyll

content (Rivelli et al., 2002; Koyro et al., 2013). At higher leaf Na+ accumulation under

saline conditions, C3 wheat responded to reduced internal CO2 concentration by

increasing water use efficiency (lower carbon isotope discrimination) (Rivelli et al.,

2002).

δ13C values can indicate whether C3 or C4 pathways are used for carbon fixation

(Zhu and Meinzer, 1999). The use of C-isotope discrimination in plant biomass is an

efficient tool for determining changes in carboxylation efficiency of photosynthetic

machinery under salinity stress (Michener and Lajtha, 2008). In C3 plants, δ13C

enrichment of biomass with increasing salinity treatments is attributed to either low Ci

values by stomatal closure and/or increased rates of photorespiration (higher

oxygenation/carboxylation) (Ivlev et al., 2013). Under both the above conditions, δ13C

values tend to become less negative (greater 13C enrichment in biomass) (Michener and

Lajtha, 2008; Ivlev et al., 2013). Atmospheric nitrogen has a δ15N value of zero. High

δ15N values are found in salt marsh halophytes such as Spartina sp. (6%) (Wigand et al.,

2007). A number of pot culture studies have reported significant discrimination between

plant tissues and the N in solution, there is general agreement that discrimination is only

observed when plant N demand is low compared with N supply. Proteins are generally

δ15N enriched relative to the bulk δ15N of the plant cell, while secondary products such

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as chlorophyll, lipids, amino sugars and alkaloids are depleted in δ15N (Michener and

Lajtha, 2008).

Chlorophyll Fluorescence

Although salinity stress causes damage to the light harvesting machinery of most plants,

many halophytes show no evidence of photo-inhibition (Qiu et al., 2003). Growth under

higher salinity could ultimately lead to over-reduction of photosynthetic electron

transport chains resulting in higher demands for alternative sinks to prevent photo-

inhibition of PSII (Demmig-Adams and Adams, 1992). Dissipation of excess energy in

form of heat via xanthophyll cycle at PSII acts as an efficient front-line defense in this

regard (Demmig-Adams and Adams, 1996; Müller et al., 2001; Terashima et al., 2009).

Whereas, water-water cycle contributes in protection of PSI by quenching reactive

oxygen species (ROS) produced by electron leakage to di-oxygen from over-reduced

PSII (Mehler, 1957; Asada, 1999). However, the magnitude and efficiency of these

mechanisms in protecting photosynthetic machinery may vary among species (Driever

and Baker, 2011; García-Plazaola et al., 2012).

Oxidative stress

Halophytes possess an efficient antioxidant system that keeps cellular levels of ROS

within a range, which is essential for their signaling and other roles (Jithesh et al., 2006;

Ozgur et al., 2013; Uzilday et al., 2015). Antioxidant system is composed of various

antioxidant enzymes such as superoxide dismutases (SODs), catalases (CATs) and

Foyer-Halliwell-Asada pathway enzymes and different low-molecular weight antioxidant

substances such as ascorbate (ASA) and glutathione (GSH) (Sharma et al., 2012; Ozgur

et al., 2013). These enzymatic and non-enzymatic antioxidants quench excess ROS;

thereby prevent oxidative damages to important cell components such as membrane

lipids, proteins, chlorophyll and nucleic acids (Sharma et al., 2012). However, under

highly saline conditions these protective activities become inadequate, thereby oxidative

damages results on species.

Halopeplis perfoliata Forssk. is a stem succulent C3 perennial halophyte from

family Amaranthaceae, which is commonly found in the coastal salt marshes and coastal

salt deserts of Arabian gulf coast (Boer and Saenger, 2006; Al-Oudat and Qadir, 2011;

El-Keblawy and Bhatt, 2015). An earlier study showed that this plant can tolerate as high

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as ~ 0.5 mol L-1 NaCl salinity by maintaining Na+ and Cl- contents to low levels (Al-

Zaharani and Hajar, 1998). Besides, nothing is known about the salt tolerance

mechanisms of this halophyte. The aim of this study was therefore to understand the

effects of increasing NaCl salinity on growth, water relations, ion relations,

photosynthesis and the antioxidant system of H. perfoliata seedlings.

It has been hypothesized that (1) Halopeplis perfoliata will tolerate near seawater

concentrations of NaCl treatments; (2) Halopeplis perfoliata will accumulate high shoot

Na+ concentrations with increasing salinity; (3) Shoot succulence will increase with

increasing shoot Na+ accumulation; (4) Plants will maintain K+ ion homeostasis across

all salinity treatments; (5) Light harvesting efficiency of PSII will be unaffected in

comparison with gas exchange processes; (6) Halopeplis perfoliata will maintain an

efficient constitutive antioxidant defense system under saline conditions.

Materials and Methods

Seed collection site

Halopeplis perfoliata inflorescence were collected from a salt marsh of Jizan Island,

Saudi Arabia (160 20` N to 170 40` N and 410 55` E to 430 20` E) in 2012. Study area is

frequently inundated by seawater and mean ambient temperature ranges between 36 and

21 oC (Alfarhan et al., 2005). Mean annual rainfall of the area is 169 mm, while humidity

ranges between 39 and 86% (Alfarhan et al., 2005). Seeds were collected from large

number of plants randomly to ensure adequate representation of the genetic diversity.

Seeds were separated from inflorescence husk manually and brought to laboratory in

Pakistan.

Growth experiment

Seedlings were raised in plastic pots (19 cm Ф x 25 cm depth) containing sandy soil

through sub-irrigated with half strength Hoagland’s nutrient solution (Epstein, 1972) in a

green net house (PAR ~600 umol m-2 s-1) under semi-ambient environmental conditions.

Temperature during the day-time ranged between 32 to 40 oC, while relative humidity

ranged from 40 to 60%.When seedlings attained a height of ~10 cm salinity (0, 0.15, 0.3

and 0.6 mol L-1 NaCl) was introduced gradually (at the rate of 0.05 mol L-1) NaCl after

every 12-h to avoid osmotic shock) in such a manner that all final salinity concentrations

were achieved on the same day. There were four replicate pots of one plant each per

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treatment. Plants were harvested 5 weeks after final salinity concentrations achieved.

Plant fresh weight (FW) were recorded immediately after harvest. Dry weight (DW) was

determined after drying vegetative parts in an oven at 60 oC for 48-h.

Fig. 4.1. Scheme showing salinity treatment regimes administered for growth experiment

on Halopeplis perfoliata under greenhouse conditions.

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Water and ion relations

Shoot succulence

Succulence was measured on a dry weight basis by using the following formula:

Succulence (H2O g g-1 DW) = (FW – DW)/DW

Relative water content (RWC)

Shoot RWC was calculated with the help of the following formula:

RWC (%) = (FW – DW)/(TW – DW)×100

Where, turgid weight (TW) of the shoot was determined after rehydration in distilled

water for 24-h at room temperature (25 oC).

Osmotic potential (ΨS)

Shoot osmolality was measured in pressed sap with the help of vapor pressure

osmometer (VAPRO-5520; Wescor Inc., Logan, UTAH, USA) (Gucci et al., 1991) and

osmotic potential (ΨS) was calculated by using van’t Hoff equation (Kramer and Boyer,

1995).

Cations regulation

Soluble Na+, K+, Ca++ and Mg++ in shoot and root sap were determined by using atomic absorption spectrometry (AA-700; Perkin Elmer, Santa Clara, CA, USA). Photosynthetic system

Shoot Photosynthetic pigments

Photosynthetic pigments (chlorophyll a, b and carotenoid) were extracted from freshly

harvested shoots in 80% (v/v) ice-cold acetone and estimated with the method of

Lichtenthaler (1987).

CO2 Gas-Exchange Measurements

Net photosynthesis rate (PN) on intact green shoots was estimated with the help of a Li-

Cor 6400XT portable gas exchange system (Li-Cor Biosciences Inc., Lincoln, Nebraska,

U.S.A.) by providing [CO2] of 400 µmol m-2 with a flow rate of 300 µmol m-2 s-1 and leaf

chamber relative humidity between 60-80%. A CO2 cartridge was used, and a 6400-01

CO2 Injector system controlled the CO2 partial pressure entering the cuvette. For all

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salinity treatments of H. perfoliata, measurements were made on one photosynthetic

shoot on three different plants (n = 3). In general, shoots in the same position as those

sampled for other biochemical tests in this experiment were chosen. The mid to distal

portion of individual branch was inserted in the conifer chamber for gas-exchange

measurements. All the readings were recorded between 10:00 am and 2:00 pm standard

time in a room with air temperature around 35 oC. Digital images of overlapping

projected area of photosynthetic shoots were used to calculate the complex leaf area

assuming that photosynthesis reading are predominantly dependent on surface area to

exposed incident light. The ImageJ 1.43 u software (NIH, U.S.A) was used to calculate

the projected shoot area. Carboxylation efficiency (CE) and gross Carboxylation

efficiency (CE*) were calculated from photosynthetic gas exchange data as:

CE = PN / PPFD

CE* = PN / PPFD * Leaf Absorbance

The risk of oxidative damage was indicated by the ratio of gross ETR (ETR*)

(from chlorophyll fluorescence data) and the sum of photosynthesis, photorespiration and

dark respiration (from gas exchange data)

= ETR*/(PN + RL +RD)

Where PN = rate of photosynthesis; E = rate of transpiration; F = flow rate; Cs =

sample CO2 concentration; Cr = reference CO2 concentration; Ca = ambient CO2

concentration; k = diffusion effect; S = leaf area.

Quantification of Rubisco

Rubisco (Larege Sub Unit) in photosynthetic shoots was extracted by grinding 0.05 g

plant material in liquid nitrogen and adding 0.05 g polyvinyl-polypyrrolidon (PVPP) in a

buffer (100 mmol L-1 Imidazol; 1.25 mmol L-1 EDTA; pH 7.8). Proteins were denatured

with sodiumdodecylsulfate (SDS) and protein content calculated on fresh weight basis

(Bradford, 1976). Proteins were separated by polyacrylamide gel electrophoresis

(agarose 6% (g/g) for stacking, 12.5% (g/g) for separation) according to Laemmli (1970).

Rubisco content to the total protein content was calculated using the integrals of the

signal strength on the gel using the image processing and analysis software ImageJ

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(National Institute of Health, Maryland, USA). Rubisco total amount was calculated with

the protein analysis mentioned above.

δ13C and δ15N values

Shoot δ13C values were determined relative to VPDB (Vienna Pee Dee Belemnite) and

δ15N values were determined relative to atmospheric N2 (Ehleringer and Osmond, 1989).

Three photosynthetic shoots were sampled each from a different potted plant 30 days

after salinity treatments were initiated. Shoot samples were oven dried at 60 oC for 48-h,

and ground to a fine powder in a mixer mill (Retch GmbH, Haan, Germany).

Chlorophyll Fluorescence Measurements

Chlorophyll fluorescence of intact green shoots was measured with a PAM-2500

flourometer (Heinz Walz GmbH, Effeltrich, Germany) under conditions similar to those

used for the gas exchange data. Maximum quantum efficiency of PSII (Fv/Fm) and

effective Quantum yield of photosystem II (PSII) was estimated by the method of Genty

et al. (1989) respectively. Non-photochemical fluorescence quenching (NPQ) was

estimated according to Bilger and Björkman (1990) and the coefficient of photochemical

fluorescence quenching (qL) as formulated by (Oxborough and Baker, 1997). ETR was

calculated according to the method of Krall and Edwards (1992) by using the shoot

absorbance data with a Licor Li-1800-12 integrating sphere (Li-Cor Biosciences Inc.,

Lincoln, Nebraska, U.S.A.).

Oxidative stress markers

Trichloroacetic acid (TCA) extracts were prepared by homogenizing finely ground plant

samples (0.5 g) in 5 ml of 3% (w/v) ice-cold TCA, followed by centrifugation at

12000×g for 15 min at 4 oC. Supernatant was stored at -80 oC in form of aliquots prior to

biochemical assays, which were conducted at 25 oC. Hydrogen peroxide (H2O2) was

determined by the method of Loreto and Velikova (2001) with minor modifications.

TCA extract (0.5 ml) was mixed with 0.5 ml of 0.5 mol L-1 potassium phosphate buffer

(pH 7.0) and 1.0 ml of 1 mol L-1 potassium iodide (KI). Mixture was incubated at room

temperature (25 oC) for 10 min and absorbance was recorded at 390 nm.

Malondialdehyde (MDA) contents were quantified with slightly modified method

of Heath and Packer (1968). TCA extract (0.5 ml) was mixed with 0.5 ml of 20% (w/v)

TCA containing 0.5% (w/v) 2-thiobarbituric acid and heated at 95 oC for 30 min in a

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shaking water bath. The reaction was terminated in an ice bath, followed by

centrifugation at 12000×g for 10 min at 4 oC. Absorbance of the supernatant was

measured at 532 nm and 600 nm (Heath and Packer, 1968).

Antioxidant enzyme activities

Antioxidant enzymes were extracted by the method of Polle et al. (1994). Finely ground

plant samples (0.5 g) were homogenized in 5 ml of ice-chilled 0.1 M potassium

phosphate buffer (pH 7.0) containing 2% (w/v) polyvinyl-pyrrolidone, 5 mmol L-1

disodium ethylene-diamine-tetra-acetic acid and 1 mmol L-1 L-ascorbic acid.

Homogenate was then centrifuged at 12000×g for 20 min at 4 oC. Aliquots of the

supernatants were stored at -80 oC prior to antioxidant enzyme assays, which were

carried out at 25 oC. Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed at

560 nm according to Beauchamp and Fridovich (1971), catalase (CAT, EC 1.11.1.6)

activity was measured at 240 nm by the method of Abey (1984), guaicol peroxidase

(GPX, EC1.11.17) activity was determined at 470 nm using the method Zaharieva et al.

(1999); ascorbate peroxidase (APX, EC 1.11.1.11) activity was estimated at 290 nm by

ascorbic acid oxidation method of Nakano and Asada (1981), and glutathione reductase

(GR, EC 1.6.4.2) activity was assayed at 340 nm by NADPH oxidation method of Foyer

and Halliwell (1976). Enzyme activities were defined as in original methods and

expressed as units of enzyme activity/mg protein which was determined according to the

method of Bradford (1976).

Antioxidant substances

Reduced glutathione (GSH) was determined in TCA extracts according to Guri (1983).

Briefly, TCA extracts (0.5 ml) were mixed with 0.5 ml of potassium phosphate buffer

(pH 7.0, 200 mmol L-1) and 0.1 ml of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and

incubated at room temperature (25 oC) for 30 min. Absorbance was measured at 412 nm.

Concentrations were estimated by using a standard curve prepared from analytical grade

GSH. Reduced ascorbate (ASA) was determined in TCA extracts by following the

method of Luwe et al. (1993). TCA extracts (0.2 ml) were mixed with potassium

phosphate buffer (pH 7.4, 150 mmol L-1) containing 10% TCA, 44% H3PO4, 4%

bipyridyl and 3% FeCl3 then incubated at 37 oC for 1-h and absorbance was measured at

525 nm. Concentrations were estimated by using a standard curve. Total soluble sugars

(TSS) in tissue sap were analyzed by using anthrone reagent according to Yemm and

83 

 

Willis (1954). Sap of different treatments were homogenized with anthrone reagent and

incubated in a water bath at 100 ºC for 11 min. The reaction was immediately stopped in

an ice bath and absorbance was noted at 630 nm (Yemm and Willis, 1954).

Statistical Analyses

Statistical analysis of the data was carried out by using SPSS Version 11.0 (SPSS, 2011).

One-way analysis of variance (ANOVA) was used to determine whether salinity had

significant effects on growth and different physio-chemical parameters. A Bonferroni

post-hoc test was carried out to determine if significant (P < 0.05) differences occurred

between individual treatments.

Results

Growth

One-way analysis of variance indicated significant effects of salinity on fresh weight

(FW) and dry weight (DW) of shoot (P < 0.05) and root (P < 0.01) and on succulence in

shoot (P < 0.001) and root (P < 0.01) tissues of H. perfoliata (Table 4.1). Shoot biomass

and succulence were highest at 0.15 mol L-1 NaCl compared to the other saline and non-

saline treatments (Fig. 4.3 A, B, C, D, E). However, root succulence increased

progressively with increasing salinity (Fig. 4.3 F).

84 

 

Table 4.1. One-way ANOVA of the effect of salinity on different parameters of shoot

and root of Halopeplis perfoliata. Numbers are the F-values with the level of

significance (ns = non-significant, * = P < 0.05, ** = P < 0.01 and *** = P < 0.001).

Details of each parameter given below are mentioned in Materials and Methods section.

Parameters Shoot Root Parameters Shoot Root

Growth CO2 Gas-Exchange FW 8.68* 20.90** PN 0.428ns - DW 9.12* 15.90** DR 12.12*** - Succulence 104.86*** 15.85** gs 18.24*** - Height 1.54ns 1.63ns Ci 1.38ns - Water and ion relations E 10.68*** - ΨS 32.60*** - WUE 131.44*** RWC 0.27ns - Isotopes Cations regulation δ13C 21.14*** - Na+ 1157.73*** 58.29*** δ15N 12.11*** - K+ 7.03* 0.86ns Stress markers Mg++ 8.94* 14.23*** H2O2 10.52*** 14.02** Ca++ 3.95ns 0.75ns MDA 6.57** 14.34** Na/K ratio 108.35*** 30.77*** Antioxidant enzymes N L/R 13.80*** - SOD 7.04* 4.43ns K L/R 0.51ns - APX 15.24** 4.90ns Shoot Photosynthetic Pigments CAT 7.13* 4.02ns Chl. a 24.13*** - GPX 10.19* 7.44* Chl. b 24.19*** - GR 0.48ns 2.52ns CAR. 22.27*** - Antioxidant Substances T. Chl. 24.34*** - GSH 25.58*** 51.88*** Chl. a/b ratio 3.63ns - ASA 96.26*** 37.63*** TSS 20.025*** 19.20***

85 

 

Fig. 4.2. Comparison of Halopeplis perfoliata plants grown under various salinity treatments (0, 0.15, 0.3 and 0.6 mol L-1

NaCl for 30 days uder greenhouse conditions.

86 

 

0 0.15 0.3 0.60

2

4

6

0

1

2

3

NaCl (mol L-1)

0 0.15 0.3 0.60

2

4

6

0

1

2

3

Shoot

0

8

16

24Root

0

8

16

24

F

W

(g p

lant

-1)

Succ

ulen

ce (H

2O g

g-1

dry

wt)

D

W

(g p

lant

-1)

a

a

b

a

ab

a ab

a

b

a

a

ab ab

a

a

ba

c

aab b

c

A B

C D

E F

F

W

(g p

lant

-1)

D

W

(g p

lant

-1)

Succ

ulen

ce (H

2O g

g-1

dry

wt)

Fig. 4.3. Growth analysis of Halopeplis perfoliata in salinity treatments (0, 0.15, 0.3 and

0.6 mol L-1 NaCl). Shoot FW (A), Root FW (B), Shoot DW (C) Root DW (D), Shoot

succulence (E) Root succulence (F). Vertical bars are means ± S.E. Different alphabets

show significant differences among salinity treatments within each parameter;

Bonferroni test; P < 0.05.

87 

 

NaCl (mol L-1)

0 0.15 0.3 0.6

RW

C (%

)

020406080

100120-10

-8

-6

-4

-2

0

aa a

b

A

B

ψs (

MPa

)

Water and ion relations

Osmotic potential and Relative Water Content

One-way ANOVA showed significant (P < 0.001) effect of salinity on shoot sap solute

potential (ΨS) (Table 4.1). There was a two-fold decrease in Ψs (P < 0.001) under 0.6

mol L-1 treatment compared to non-saline control (Fig. 4.4 A). RWC of shoots was

unaffected by increases in salinity (Fig. 4.4 B).

Fig. 4.4. Shoot osmotic potential (A) and relative water content (B) in response to

salinity treatments (0, 0.15, 0.3 and 0.6 mol L-1 NaCl). Vertical bars are means ± S.E.

Different alphabets show significant differences among salinity treatments within each

parameter; Bonferroni test; P < 0.05.

Cation regulation

Increase in salinity had significant effect on Na+, Mg++ and Na+/K+ ratio but not on K+

and Ca++ content in shoots and roots of H. perfoliata (ANOVA; Table 4.1). Shoot Na+

increased linearly with increases in salinity, whereas root Na+ increased only in the 0.60

mol L-1 NaCl treatment. Shoot Na+ was substantially higher than in root (Fig. 4.5 A). K+

in both shoots and roots remained largely unaffected by salinity increments; however in

shoots it was substantially higher than roots (Fig. 4.5 B). Ca++ in both shoots and roots

88 

 

0 0.15 0.3 0.6K

Sho

ot/R

oot

0

2

4

6

8

10

Mg++

(mm

ol /

L-1)

15

30

45

60

75

NaCl (mol L-1)

0 0.15 0.3 0.6

Na+ / K

+

0

4

8

12

16

Ca++

(mm

ol /

L-1)

0

15

30

45

60

75

N

a+

(mm

ol /

L-1)

200400600800

100012001400

Shoot Root

K

+ (m

mol

/ L-1

)

20406080100120

aa

aa a a

b

aa

a

b

ab

b b

aa

a

a

a aaa

a a a a

A

E

C D

b

c

a

a aa

a

b

a

a a

b aa

a

a

a

B

F

also remained unaltered by salinity, but roots had significantly (P < 0.05) higher Ca++

than shoots (Fig. 4.5 C). Mg++ in shoots increased at 0.6 mol L-1 NaCl compared to other

treatments, but was generally lower in roots under saline conditions compared to no

salinity control (Fig. 4.5 D) treatments (Fig. 4.5 E) while shoot to root K+ remained

unaffected by salinity (Fig. 4.5 F).

Fig. 4.5. Na+ (A), K+ (B), Ca++ (C), Mg++ (D), Na+/K++ ratio (E), and K+ shoot to root

ratio in response to salinity treatment (0, 0.15, 0.3 and 0.6 mol L-1 NaCl). Vertical bars

are means ± standard error. Different alphabets show significant differences among the

salinity treatments within each parameter; Bonferroni test; P < 0.05.

89 

 

Salinity effects on Photosynthesis

Shoot Photosynthetic Pigments

ANOVA indicated significant effects of salinity on contents of chlorophyll a (Chl. a),

chlorophyll b (Chl. b), carotenoids (CAR.) and total chlorophyll (T. Chl.) (Table 4.1). On

fresh weight and dry weight basis contents of photosynthetic pigments were decreased

under high salinity (Table 4.2 A and B). While on leaf area basis no significant

difference was found in photosynthetic pigments between salinity treatments (Table 4.2

C).

CO2 Gas-Exchange

Net photosynthesis (PN), measured on incident area basis, was unaffected by increases in

salinity (Table 4.1; Table 4.3). Dark respiration (RD) increased with increases in salinity

(Table 4.3). Stomatal conductance (gs) and transpiration rate (E) increased in up to 0.3

mol L-1 NaCl and declined afterwards (Table 4.3). A transient decrease in water use

efficiency (WUE) was observed under low (0.15 mol L-1 NaCl) and moderate (0.30 mol

L-1 NaCl) salinity treatments compared to no and high (0.6 mol L-1 NaCl) salinity

treatments. Salinity had no effect on intercellular carbon dioxide (Ci) concentration

(Table 4.3). However carboxylation efficiency (CE) and gross carboxylation efficiency

(CE*) increased at optimal salinity (0.15 mol L-1) as compared to other treatments.

However, oxidative risk indicated by the ratio of ETR and sum of photosynthesis (PN),

photorespiration (RL) and dark respiration (RD) was highest under non-saline control and

lowest at optimal salinity level (0.15 mol L-1 NaCl) (Fig. 4.3).

90 

 

Table 4.2 Photosynthetic pigments [chlorophyll a (Chl. a), chlorophyll b (Chl. b),

carotenoids (CAR.), total chlorophyll (Chl. a+b) and chl. a/b ratio of Halopeplis

perfoliata grown in different salinity treatments (0, 1.5, 0.3 and 0.6 mol L-1 NaCl)

measured on (A) fresh weight (FW) (B) dry weight (DW) and (C) leaf area basis. Values

represent means ± S.E. Different alphabets show significant difference among the

salinity treatments within each parameter; Bonferroni test; P < 0.05.

Parameters 0 0.15 0. 3 (mol L-1)

0.6

(A) Fresh weight basis (mg g-1 FW) Chl. a 0.23±0.03a 0.20±0.01ab 0.14±0.01b 0.14±0.03b Chl. b 0.09±0.01a 0.07±0.00b 0.04±0.00c 0.04±0.01c CAR. 0.06±0.01a 0.05±0.01ab 0.04±0.00b 0.04±0.00b T. Chl. (a + b) 0.32±0.07a 0.25±0.01b 0.2±0.01b 0.23±0.01b Chl. a/b ratio 2.64±0.11a 3.00±0.1ab 3.7±0.2b 3.08±0.19b (B) Dry weight basis (mg g-1 DW) Chl. a 7.41±0.30a 9.45±2.20a 3.58±0.02b 0.59±0.01c Chl. b 2.90±0.02a 3.12±0.04a 1.10±0.06b 0.20±0.04b CAR. 2.06±0.05a 2.72±0.64a 0.98±0.02b 0.22±0.01c T. Chl. (a + b) 10.316±0.50a 8.14±1.50a 4.70±0.04b 0.78±0.11c Chl. a/b ratio 2.645±0.11a 2.98±0.07a 3.45±0.25a 3.08±0.11a

(C) Shoot area basis (g m-2) Chl. a 0.32±0.01a 0.31±0.04a 0.23±0.03a 0.29±0.05a Chl. b 0.12±0.01a 0.08±0.01ab 0.07±0.07b 0.01±0.01ab CAR. 0.09±0.00a 0.09±0.01a 0.07±0.07a 0.01±0.01a T. Chl. (a + b) 0.44±0.02a 0.41±0.05a 0.30±0.04a 0.40±0.07a Chl. a/b ratio 2.75±0.04a 3.03±0.00ac 3.70±0.20b 3.30±0.10c

91 

 

Table 4.3. Net photosynthetic rate (PN), respiration (RD), stomatal conductance (gS),

transpiration (E), water use efficiency (WUE), and intercellular CO2 concentration (Ci)

measured on photosynthetic shoots of Halopeplis perfoliata in different salinity

treatments (0, 1.5, 0.3 and 0.6 mol L-1 NaCl). Values represent means ± S.E. Different

alphabets show significant differences among salinity treatments within each parameter;

Bonferroni test; P < 0.05.

Parametrs 0 0.15 0.3 0.6 (mol L-1)PN (µmol m-2 s-1) 5.99±1.71a 7.51±1.25a 6.71±1.08a 5.51±1.19a

RD (mol m-2 s-1) -1.49±0.01a -2.40±0.02b -2.71±0.07bc -3.21±0.04c

gS (mol m-2 s-1) 0.06±0.00a 0.11±0.00b 0.13±0.01b 0.08±0.01c

E (mol m-2 s-1) 1.43±0.05a 2.30±0.07b 2.78±0.44b 1.46±0.10a

Ci (µmol mol-1) 286.88±18.00a 301.18±17.90a 295.89±17.71a 274.85±26.08a

WUE 69.91±3.57a 41.31±1.51b 34.22±1.78b 89.35±1.17c

CE 0.01±0.00a 0.02±0.00a 0.02±0.00a 0.02±0.00a

CE* 0.01±0.00a 0.03±0.00a 0.02±0.00a 0.02±0.00a

ETR/(PN+RD+RL) 5.06±0.88a 3.46±0.64a 4.16±0.54a 3.51±0.83a

92 

 

NaCl (mol L-1)

0 0.15 0.3 0.60

500

1000

1500

2000

2500

3000R

ubis

co c

onte

nt (r

elat

ive

unit)

Quantification for Rubisco

Rubisco content peaked at 0.15 mol L-1 NaCl with substantially lower values in all other

treatments (0, 0.3 and 0.6 mol L-1NaCl) used (Fig. 4.6).

Fig. 4.6. Relative levels of Rubisco quantified on total protein basis where 50 µg protein

was applied based on SDS-PAGE gel electrophoresis on photosynthetic shoots of

Halopeplis pefoliata plants grown under different salinity treatments (0, 0.15, 0.3 and

0.6 mol L-1 NaCl).

δ13C and δ15N values

One-way ANOVA showed that significant (P < 0.001) effects of salinity on δ13C and

δ15N in shoot tissues (Table 4.1). H. perfoliata exhibit a similar pattern of δ13C values to

other C3 species. Lower values of δ13C were determined under salinity compared to

control (non-saline) (Fig. 4.7 A). However δ15N values increased at 0.15 mol L-1 and 0.3

mol L-1 NaCl treatments than in the non-saline control and 0.6 mol L-1 NaCl treatment

(Fig. 4.7 B).

93 

 

NaCl (mol L-1)

0 0.15 0.3 0.6

δ15N

(‰)

0

5

10

15

δ13C

(‰)

-36

-34

-32

-30

aa

a

b

b

bb

b

Fig. 4.7. Carbon and Nitrogen isotopes discrimination in salinity treatments (0, 0.15, 0.3

and 0.6 mol L-1 NaCl). Vertical bars are means ± standard error. Different alphabets

show significant differences among salinity treatments within each parameter;

Bonferroni test; P < 0.05.

Chlorophyll fluorescence

Maximum efficiency of PSII (Fv/Fm) was not affected by low or high irradiance and

salinity and it values ranged from 0.68 to 0.71 (Fig. 4.8 A). Actual yield of PSII was

largely unaffected by salinity but increases in light decreased it (Fig. 4.8 B). ETR*

values increased with increasing irradiance with little variation between treatments

however, high salinity (0.6 mol L-1) had lower electron transport rates than others (Fig.

4.8 C). Photochemical quenching (qL) gradually decreased with increasing irradiance

94 

 

YII

0.1

0.2

0.3

0.4

0.5

ETR

*

0

10

20

30

40

50

60

NPQ

PAR

60 107

185

300

632

900

1298

1852

0.0

0.5

1.0

1.5

2.0

qL

60 107

185

300

632

900

1298

1852

0.0

0.2

0.4

0.6

0.8

Fv/F

m

0.68

0.70

0.72

0.74

PAR

A B

C D

E

however qL values were higher in non-saline control and 0.15-0.3 mol L-1 but lower at

0.6 mol L-1 (Fig. 4.8 D). Non-photochemical quenching (NPQ) increased at high salinity

treatments by a factor of 0.4 - 2.0 in comparison to the control at high irradiance (~1850

µmol m-2 s-1) (Fig. 4.8 E).

Fig. 4.8. Maximum quantum efficiency (Fv/Fm), actual yield (PSII), electron transport

rate (ETR), photochemical (qL) and non-photochemical quenching (NPQ) on

photosynthetic shoot of Halopeplis perfoliata under a range of irradiance and in salinity

treatments [0 (ο), 0.15 (∆), 0.3 (◊) and 0.6 (□) mol L-1 NaCl]. Values are mean of 3

replicates with means ± S.E.

Oxidative stress markers

One-way ANOVA showed significant effects of salinity on endogenous H2O2 and MDA

content (Table 4.1). In the shoot, endogenous H2O2 and MDA were higher in 0.3 and 0.6

mol L-1 NaCl treatments in comparison with control (Fig. 4.9 A and C). Endogenous

95 

 

NaCl (mol L-1)0 0.15 0.3 0.6

0

10

20

30

40

50

0 0.15 0.3 0.6

MD

A

(nm

ol g

-1D

W)

0

10

20

30

40

Root

0

5

10

15

20

25Shoot

H2O

2

( µm

ol g

-1 D

W)

MD

A

(nm

ol g

-1D

W)

a ab

bcb

a

b

a

c

a

b

a

b

a

a

b b

A B

C D

a a

b bH2O

2

( µm

ol g

-1 D

W)

0

5

10

15

20

a a

b b

A

H2O2 and MDA contents of the roots decreased under high salinity compared to other

NaCl treatments (Fig. 4.9 B and D).

Fig. 4.9. Hydrogen peroxide in shoot (A) and root (B), Malondialdehyde in shoot (C)

and root (D) of Halopeplis perfoliata in salinity treatments (0, 0.15, 0.3 and 0.6 mol L-1

NaCl). Vertical bars are means ± S.E. Different alphabets show significant differences

among the salinity treatments within each parameter; Bonferroni test; P < 0.05.

Antioxidant enzymes

Salinity had significant effects on antioxidant enzyme activities (one-way ANOVA;

Table 4.1). In shoots, activities of SOD (Fig. 4.10 A) and APX (Fig. 4.10 G) decreased,

that of CAT (Fig. 4.10 C) increased, while those of GPX (Fig. 4.10 E) and GR (Fig. 4.10

I) remained unaffected under saline conditions as compared to non- saline controls.

While in roots, SOD activity showed a transient decrease under moderately saline

conditions (Fig. 4.10 B), CAT activity decreased in 0.6 mol L-1 NaCl (Fig. 4.10 D), GPX

activity increased with increases in salinity (Fig. 4.10 F), while activities of APX and GR

transiently increases in 0.15 mol L-1 NaCl (Fig. 4.10 H and J).

96 

 

Fig. 4.10. Antioxidant enzyme activities in shoots and roots of Halopeplis perfoliata in

response to salinity treatments (0, 0.15, 0.3 and 0.6 mol L-1 NaCl). Vertical bars are

means ± S.E. Different alphabets show significant differences among salinity treatments

within each enzyme activity data; Bonferroni test; P < 0.05.

0 0.15 0.3 0.6

SOD

(u

nits

/mg

prot

ein)

0.0

0.1

0.2

0.3

0.4

CA

T (u

nits

/mg

prot

ein)

0.000.010.020.030.040.05

GR

(uni

ts/m

g pr

otei

n)

0.0

0.5

1.0

1.5

2.0

GPX

(uni

ts/m

g pr

otei

n)

0.00.20.40.60.81.01.21.41.6

NaCl (mol L-1)0 0.15 0.3 0.6

SOD

(uni

ts/m

g pr

otie

n)

0.0

0.1

0.2

0.3

0.4

CA

T (u

nits

/mg

prot

ein)

0.000.010.020.030.040.05

GR

(u

nits

/mg

prot

ein)

0.0

0.5

1.0

1.5

2.0

GPX

(u

nits

/mg

prot

ein)

0.000.010.020.031.001.201.401.60

Shoot

APX

(uni

ts/m

g pr

otei

n)1

2

3

Root

APX

(u

nit/m

g pr

otei

n)

1

2

3

ab b b

a

a

a a

a a a a a

a

bab

aa

a

a

a

a

a a

a

b

cc

a

a a

a

ab

b b

a

b

c

abca

A B

D

E F

G H

I J

C

97 

 

NaCl (mol L-1)0 0.15 0.3 0.6

0

5

10

15

20

0 0.15 0.3 0.6

AsA

(µ

mol

g-1

DW

)

0

5

10

15

200.0

0.5

1.0

1.5

2.0

0.0

0.5

1.0

1.5

2.0

GSH

(µ

mol

g-1

DW

)

GSH

(µ

mol

g-1

DW

)

ba a

c

a

b

c

b

0.3 0.60.0000.0070.0140.0210.028

ab

c

d b

a

ccA B

C D

AsA

(µ

mol

g-1

DW

)

Shoot Root

Antioxidant substrates

Analysis of variance indicated significant effects of salinity on reduced glutathione

(GSH) and reduced ascorbate (ASA) contents of the shoots and roots of H. perfoliata

(Table 4.1). Shoot GSH content increased under saline conditions with highest values in

0.3 mol L-1 NaCl (4.11 A). While, root GSH content decreased with increasing salinity

treatments and levels were substantially lower in 0.3 and 0.6 mol L-1 NaCl (Fig. 4.11 A

and B). Shoot ASA content increased with increases in salinity (Fig. 4.11. C), while root

ASA increased in up to 0.3 mol L-1 NaCl followed by a substantial decline in 0.6 mol L-1

NaCl (Fig. 4.11 D).

Fig. 4.11. Reduced glutathione (GSH) in shoot (A), and root (B), ascorbic acid (ASA) in

shoot (C) and root (D) of Halopeplis perfoliata in response to salinity treatments (0,

0.15, 0.3 and 0.6 mol L-1 NaCl). Vertical bars are means ± S.E. Different alphabets show

significant differences among treatments within each parameter; Bonferroni test; P <

0.05.

98 

 

Root

NaCl (mol L-1)0 0.15 0.3 0.6

0

10

20

30

0

10

20

30

40Shoot

TSS

(mm

ol L

-1Pl

ant w

ater

) a

b b

b

a

b

a

b

Soluble Sugars

ANOVA table showed that salinity has significant effect on soluble sugars on shoots (P

< 0.001) and roots (P < 0.01) (Table 4.1). Sugars were significantly higher in 0.3 mol L-

1 and 0.6 mol L-1 NaCl treatments in shoots (Fig. 4.12. A). While lower in roots at the

same concentrations than control and 0.15 mol L-1 of NaCl treatments (Fig. 4.12 B).

Fig. 4.12. Total soluble sugars (TSS) in response to salinity treatments (0, 0.15, 0.3 and

0.6 mol L-1 NaCl). Vertical bars are means ± S.E. Different alphabets show significant

differences among salinity treatments in shoot and root of Halopeplis perfoliata;

Bonferroni test; P < 0.05.

99 

 

Discussion

Growth experiment indicated that Halopeplis perfoliata is an obligate halophyte

Succulent eu-halophytes in the subfamily Salicornioideae (Amaranthaceae) could

optimally grow optimally in the presence of salinity (Flower and Colmer, 2008; Rozema

and Schat, 2013). For example, Sarcocornia natalensis (0.3 mol L-1 NaCl, Naidoo and

Rughunanan, 1990); Salicornia bigellovii (0.2 mol L-1 NaCl, Ayala and O’Leary, 1995);

Halocnemum strobilaceum (0.34 mol L-1 NaCl, Pujol et al., 2001); Arthrocnemum

macrostachyum (0.4 mol L-1 NaCl, Khan et al., 2005); Salicornia utahensis 0.6 mol L-1

NaCl, Gul et al., 2009)and Salicornia dolichostachya (0.3 mol L-1 NaCl, Katschnig et al.,

2013) showed optimal growth under saline conditions. Inhibition of growth above

optimal salinity appears to compromise strategy to ensure survival by investing in

mechanisms of salt resistance and was referred to as a “life insurance” policy under salt

stress by Pujol et al. (2001). In this study, optimal growth of Halopeplis perfoliata at

0.15 mol L-1 NaCl coincided with increased shoot succulence. Succulence could help

plants to avoid physiological drought, reduce ion toxicity (Sánchez et al., 2004;

Touchette et al., 2009; Rozema and Schat, 2013) and buffer thermal changes (Medina,

1999).

Osmotic adjustment and succulence maintain sufficient tissue hydration for growth under saline conditions

Succulent halophytes such as Sueada fruticosa could constitutively maintain low tissue

osmotic potentials for water uptake under saline conditions (Hameed et al., 2012).

Halopeplis perfoliata also maintained low osmotic potentials up to 0.3 mol L-1 NaCl

which was not coupled with turgidity loss (high RWC) indicating high ability for

osmotic adjustment. Decreased succulence and a sudden decrease in ΨS at 0.6 mol L-1

NaCl treatment were suggestive of water stress. Vacuolar Na+ sequestration contributes

significantly in osmotic adjustment accounting for ~50% of the sap Op (Shabala and

Mackay, 2011) witha parallel accumulation of organic solutes (Hussin et al., 2013;

Jogaiah et al., 2014; Flowers and Colmer, 2015). Similar results were reported for

Halosarcia pergranulata (syn. Tecticornia pergranulata) (Short and Colmer, 1999)

Salicornia dolichostachya (Katschnig et al., 2013). Compromised K+ acquisition from

soil and replacement of K+ by Na+ in plant tissues under saline condition and is

deleterious for plant metabolism. However, highly tolerant halophytes of Salicornioideae

100 

 

such as Tecticornia pergranulata (Colmer et al., 2009) and Tecticornia indica (English

and Colmer, 2013) appeared to maintain K+ homeostasis under saline conditions which

has several important roles in plants metabolism (Munns and Tester, 2008; Benito et al.,

2013; Hussin et al., 2013). Several membrane-embedded transport proteins such as

K+/H+ symporters, outward rectifying K+ channels (KOR), members of high affinity K+

transporter family (KUP/HAK/KT) and inward rectifying K+ channels (KIR) are

responsible for K+ homeostasis in plants (Mansour, 2014). H. perfoliata also maintained

sufficient tissue K+ under saline conditions in all salinity treatments. Two to four fold

higher shoot to root K+ indicated that K+ transport to shoot was also not compromised by

salinity increments. Ca++ is also known to be involved in SOS pathway mediated Na+

efflux (Ji et al., 2013), inhibition of voltage independent cation channel (VIC) for Na+

influx (Maathius and Amtmann, 1999; White and Davenport, 2002). In addition, Ca++

regulates stress signaling (Spalding and Harper, 2011), enzyme activation (Plieth, 2005)

membrane and cell wall integrity (Marschner, 1995). Shoot and root Ca++ of H.

perfoliata remained unaffected by salinity increments, as reported for Ocimum Basilicum

(Ning et al., 2015). Magnesium (Mg++) is also required for activation of many enzymes,

ribosome aggregation and chlorophyll synthesis (Shaul, 2002). At 0.6 mol L-1 NaCl peak

shoot Mg++ levels compared to root in H. perfoliata compared to saline and non-saline

treatments could be linked to higher transport to shoots. Similar rise in Mg++ under saline

condition were observed in Salicornia europaea (McNulty, 1985) and Limonium stocksii

(Zia et al., 2008). In sunflower leaves this rise was due to dehydration stress imposed by

low water potential treatment (Rao et al., 1987). Peak shoot Mg++ levels at the highest

NaCl treatment in H. perfoliata could be a consequence of water stress and/or

chlorophyll degradation (Stocking and Ongun, 1962).

Down regulation of photosynthetic machinery helps to maintain the photosynthetic

efficiency

Chlorophyll content may not always be linked with growth reduction or inhibition with

increasing substrate salinity and may either increase, decrease or remain unchanged

(Moinuddin et al., 2014). NaCl-induced reduction in photosynthetic pigments could be

related to decrease in size and number of chloroplasts (Grigore et al., 2014).

Photosynthetic pigments decreased in response to increasing salinity in members of

Salicornioideae such as Salicornia persica, S. europaea (Aghaleh et al., 2009),

101 

 

Arthrocnemum macrostachyum (Redondo-Gómez et al., 2010) and Sarcocornia fruticosa

(Duarte et al., 2013). In H. perfoliata, changes in shoot photosynthetic pigments

appeared to be masked by on a leaf area basis by variations in shoot succulence with

increasing salinity. However, pigment levels decreased on fresh weight and particularly

on dry weight basis indicating either chlorophyll degradation or decreased allocation of

plant resources toward synthesis.

Members of Salicornioideae family utilize C3 mode of CO2 assimilation and have

the ability to maintain (Salicornia europaea, Kuramoto and Brest, 1979) or enhance

(Sarcocornia fruticosa; Redondo-Gómez et al., 2006 and Arthrocnemum

macrostachyum; Redondo-Gómez et al., 2010) photosynthetic rates under saline

conditions. This could be related to efficient storage of salt in the vacuole and dilution

effect which protects the chloroplast (Kuramoto and Brest, 1979). CO2 compensation

points of H. perfoliata indicated C3 function. Maintenance of CO2 assimilation rates (PN)

and internal carbon dioxide (Ci) concentrations of H. perfoliata were similar to

Tecticornia pergranulata with increasing salinity (Colmer et al., 2009). Decrease in

WUE under moderately low and moderate salinity (0.15 and 0.3 mol L-1 NaCl) could

indicate fixed response similar to natural salt marsh conditions with no water stress

whereas high salinity (0.6 mol L-1 NaCl) stimulated stomatal closure along with

decreased shoot succulence could be a preemptive protection against further dehydration.

Dark respiration (RD) in H. perfoliata increased with increasing salinity such as reported

by (Burchett et al., 1989; Koyro, 2006; Colmer et al., 2009), supporting the view that

salinity tolerance increases energy requirements such as for ion transport processes (Yeo,

1983). Reduction in biomass alongside sustained photosynthesis in H. perfolaita also

supports this assumption that most photosynthetic carbon gain was consumed in salt

tolerance processes. Decreased photosynthetic rates were attributed to inhibition

(synthesis or activity) of rubisco by salinity (Eisa et al., 2012; Koyro et al., 2013).

Decrease in rubisco levels (per protein basis) of H. perfoliata did not correlate well with

photosynthetic rates (leaf area basis) but could explain lack of plant growth at more than

optimal salinity (0.15 mol L-1 NaCl).

102 

 

Carbon and Nitrogen isotope discrimination values indicate variations in photosynthetic

efficiency and nutrient uptake

C3 plants generally show a linear correlation of carbon isotope composition vs. salinity

or close to it particularly under controlled laboratory studies for example Puccinellia

nutelliana and Salicornia europeae sub sp. rubra (Ivlev et al., 2013). Similar results

were obtained in the present study indicating a progressively higher δ13C enrichment of

biomass with increasing salinity treatments apparently due to increased rates of

photorespiration rather than due to stomatal closure. In H. perfoliata, a sharp increase

δ15N values up to 0.30 mol L-1 NaCl could be attributed to a higher demand for biomass

production at 0.15 mol L-1 NaCl and for nitrogen to produce osmotically active

molecules at 0.30 mol L-1 NaCl, as well as to a decrease in nitrate uptake at 0.60 mol L-1

NaCl (Song et al., 2009).

Chlorophyll fluorescence data (Fv/Fm) for H. perfoliata indicated little influence

of salinity on the integrity or function of light harvesting system (PSII) as reported in

Sarcocornia fruticosa (Redondo-Gómez et al., 2006); Salicornia veneta (Pagliano et al.,

2009) and Arthrocnemum macrostachyum (Redondo-Gómez et al., 2010). Decrease in

ETR* under saline conditions indicates dynamic photo-inhibition, a down-regulation of

the PSII activity to reduce the need for alternative electron sinks and to avoid oxidative

stress (Qiu et al., 2003).

In C3 plants, stress caused due to high light and temperature and low CO2 and

water results in increased photorespiration to avoid photo-inhibition of the

photosynthetic machinery (Taiz and Zeiger, 2010). Salt stress is also known to increase

photorespiration at initial stages due to stomatal reasons (similar to drought effects) i.e.,

by decreased stomatal conductance (lower Ci) or at later stages by non-stomatal factors,

such as high Cl- levels in the photosynthetic tissues (Wingler et al., 2002). In addition,

zeaxanthin may also be involved in dissipating additional photon energy to avoid photo-

inhibition at moderate light under saline conditions (Sharma and Hall, 1992; Duarte et

al., 2013). Elevated CO2 compensation point H. perfoliata indicated increased rates of

photorespiration at 0.6 mol L-1 NaCl possibly linked with high shoot accumulation of

salts (high osmotic potential). Higher rates of photorespiration under salt stress may

contribute in increased H2O2 production and lower CAT activity (Wingler et al., 2002).

103 

 

Antioxidative defense system protects Halopeplis perfoliata from salt stress

ROS accumulation generally leads to oxidative damages to important cell components

such as to membrane lipids (Jithesh et al., 2006). In this study, H2O2 production

gradually increased with increasing salinity in photosynthetic shoots similar to previous

studies such as in Arabidopsis thaliana (Ellouzi et al., 2011) and in some other plants

(Corpas et al., 1993; Noctor et al., 2002; Jithesh et al., 2006; Ozgur et al., 2013). Increase

in MDA content (an indicator of membrane lipids’ peroxidation) with increasing NaCl

was also observed in shoots of H. perfoliata. Similarly, MDA content increased under

salinity in many other halophytes (Jithesh et al., 2006; Ozgur et al., 2013; Hameed et al.,

2015). The relationship between MDA levels and plant performance under stress is

unresolved (Hameed et al., 2012; Alhdad et al., 2013), however, some stress signaling

roles of MDA have been reported (Weber et al., 2004). Higher MDA concentrations in

Suaeda fruticosa were associated with improved growth (Hameed et al., 2012). In this

study, MDA contents under high salinity were not too high and absence of visible

necrosis indicated that they might be involved in stress signaling.

Halophytes employ several enzymatic and non-enzymatic antioxidants to avoid

oxidative damage (Jithesh et al., 2006; Ozgur et al., 2013). In this study, activity of

antioxidant enzyme superoxide dismutase (SOD) declined and of ascorbate peroxidase

(APX) and glutathione reductase (GR) remained unchanged under saline conditions.

Unaltered photosynthesis rates under saline condition also indicate little incidence of

ROS production via over-reduction of photosynthetic electron transport chain, hence

enhanced activities of water-water cycle antioxidant enzyme (SOD and APX) were not

needed in H. perfolaita. Furthermore, unchanged photosynthesis alongside higher CAT

activity under saline condition, indicate that the rise in shoot H2O2 might be associated

with photorespiration rather than over-reduction of photosynthetic electron transport

chain. Shoot antioxidant substrates ascorbate (ASA) and glutathione (GSH) increased

with increasing salinity in H. perfoliata as in Sphaerophysa kotschyana (Yildiztugay et

al., 2013) and Limonium stocksii (Hameed et al., 2015). Accumulation of ASA and GSH

are suggestive of an efficient antioxidant system (Foyer and Halliwell, 1976; Jithesh et

al., 2006; Ozgur et al., 2013) besides their roles in photosynthesis (Foyer and Shigeoka,

2011). Increasing shoot soluble sugars could be linked with higher ASA biosynthesis

under saline conditions in H. perfoliata (Nishikawa et al., 2005).

104 

 

Conclusions

Halopeplis perfolaita could grow optimally at 0.15 mol L-1 NaCl treatment while with

increasing salinity (0.3 and 0.6 mol L-1 NaCl) treatments, it could still produce biomass

comparable to non-saline controls (Fig. 4. 13). Lower tissue osmotic potentials along

with Na+ accumulation indicated higher capacity for osmotic adjustment with increasing

salinity (Fig. 4. 13). In general, photosynthetic CO2 fixation and light harvesting ability

of PSII remained unaltered with increasing salinity treatments (Fig. 4. 13). Higher CAT

activity and H2O2 levels and also the less negative carbon isotope ratios in photosynthetic

shoot tissues indicate changes in rates of photorespiration under saline conditions.

Further investigations into the molecular arguments for these responses could reveal

opportunities for identification of these traits.

Fig. 4.13. Ecophysiological adaptations of water and ion relations, photosynthesis and

antioxidant defense system of Halopeplis perfoliata under high salinity concentrations.

(~ = similar response; ↑ = increased response across all salinity treatments).

105 

 

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Chapter 5

General conclusions

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Eco-physiological investigations into novel salt tolerance mechanisms among

halophytic plants growing under saline conditions are essential for sustainable

utilization of such species for conventional and non-conventional agriculture. The

results obtained in this study indicate that Halopeplis perfoliata is an obligate halophyte

with high salt tolerance at both the germination and mature vegetative stages of its life

cycle. Most freshly collected seeds germinated in distilled water in the presence of light

and in moderate temperature regimes indicating their non-dormant nature and their

preference for less stressful environmental conditions. Non-deep conditional dormancy

under hyper-saline conditions and in complete darkness was completely reversed upon

transfer to non-saline conditions in the presence of light. Isotonic treatments of NaCl,

PEG and sea-salt on seed germination revealed primarily an osmotic effect rather than

toxic effect of salts. Among various chemicals used in this study, chloride salts proved

to be more inhibitory for germination compared to isotonic sulfate salts. All seeds kept

in dry storage for one year at room temperature (25 oC) were viable and showed

enhanced tolerance to moderate salinities and darkness, but had lower tolerance to

higher temperatures in comparison to freshly collected seeds. All dormancy regulating

chemicals (except proline) alleviated dark-enforced dormancy under non-saline

conditions but were generally ineffective in reversing inhibitory effects of high salinity

(except for betaine, kinetin and thiourea at 0.3 mol L-1 NaCl). Optimum growth of H.

perfoliata in 0.15 mol L-1 NaCl treatment indicated its obligate salt requirement (Fig.

5.1) while at higher salinities (0.3 and 0.6 mol L-1 NaCl) biomass production was

comparable to non-saline controls. Plants appeared to maintain water uptake by

regulating succulence to achieve constant relative water content. Shoot ionic balance

(K+, Ca++ and Mg++) generally remained unaltered in response to increasing salinity.

Photosynthetic rates on leaf area basis were also unaffected by salinity which appear to

be more than compensated for by an increase in shoot area per plant and shoot

succulence per gram dry weight at low and moderate salinity treatments. This was

accompanied by higher transpiration rates (E) and stomatal conductance (gs) possibly

suggestive of adaptation for growth in a salt marsh environment. Decreased

transpiration rates in the 0.6 mol L-1 NaCl treatment resulted in higher WUE which was

also indicated by less negative carbon isotope discrimination values in photosynthetic

shoots. Chlorophyll fluorescence measurements ruled out any signs of damage to the

photochemical machinery while decreased ETR/(PN+RL+RD) ratios indicated a down

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regulation of photochemistry under saline conditions and therefore reduced requirement

for alternative electron sinks and ROS detoxification. In conclusion, H. perfoliata

appears to be a high salt tolerant plant which is well adapted for growth in salt marsh

habitats.

Fig. 5.1. Summary of eco-physiological adaptatons of Halopeplis perfoliata during seed germination and vegetative growth.