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EFFECT OF SALICYLIC ACID AND GIBBERELLIC ACID ON
MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES IN
DROUGHT-STRESSED WHEAT (TRITICUM AESTIVUM L.)
CROP
ANEELA ULFAT
Regd. No. 2004-Gkig-630
Session (2012-2015)
Department of Botany
Faculty of Sciences
University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan
ii
EFFECT OF SALICYLIC ACID AND GIBBERELLIC ACID ON
MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES IN
DROUGHT-STRESSED WHEAT (TRITICUM AESTIVUM L.)
CROP
By
ANEELA ULFAT
(Regd. No: 2004-Gkig-630)
A Thesis
submitted in partial fulfillment of the requirement for the degree of
Doctor of Philosophy
in
Botany
(Session 2012-2015)
Department of Botany
Faculty of Sciences
University of Azad Jammu and Kashmir Muzaffarabad, Pakistan
iii
iv
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TO MY LOVING PARENTS
Whose devotion and inspiration towards knowledge served me as ray of light, who
always pray for my success and prosperity. Whose encouragement, sacrifices and
generous support both morally and financially enabled me to achieve this goal.
viii
CONTENTS
Page No.
LIST OF CONTENTS vii-xii
LIST OF ABBREVIATIONS xiii
ACKNOWLEDGEMENT xv
ABSTRACT xx
1. INTRODUCTION 01
1.1CLIMATE CHANGE AND ITS EFFECT ON AGRICULTURE 02
1.2 PAKISTAN STATUS IN TERM OF CLIMATE CHANGE 03
1.3 ABIOTIC STRESSES 03
1.4 STRATEGIES TO OVERCOME STRESSES 04
1.5 JUSTIFICATION OF THE STUDY 05
2. REVIEW OF LITERATURE 07
2.1 WORLD WHEAT MARKET 07
2.2 WHEAT STATUS IN PAKISTAN AND AZAD JAMMU
AND KASHMIR 07
2.3 CLIMATE CHANGE AND ITS IMPACT ON AGRICULTURAL
CROPS 08
2.4 DEMAND AND AVAILABILITY OF WATER 08
2.5 DROUGHT STRESS 10
2.5.1 Problems during Drought 11
2.5.2 Strategies to Cope Drought Stress 13
2.5.2.1 Priming 14
2.5.2.2 Hormonal seed priming 15
2.5.2.2.1 Role of salicylic acid 15
ix
2.5.2.2.2 Role of gibberellic acid 16
2.5.2.3 Priming effect on biochemical, morphological and
physiological processes 17
2.6 TRANSGENERATIONAL EFFECT OF EFFECT OF ELEVATED
CO2 19
2.7 CONSIDERATIONS FOR THE FUTURE 19
3. MATERIAL AND METHOD 21
3.1 STUDY 21
3.2 PLANT MATERIAL 22
3.3 SEED PRIMING TREATMENTS 22
3.4 EXPERIMENTAL SET-UP 22
3.5 SEED BIOCHEMISTRY ATTRIBUTES 22
3.5.1 Oxidative Enzyme Assays 22
3.5.1.1 Protease activity 22
3.5.1.2 Estrase activity 23
3.5.1.3 Amylase activity 23
3.5.1.4 Superoxide dismutase activity 23
3.5.1.5 Peroxidase activity 23
3.5.1.6 Catalase activity 24
3.6 MORPHOLOGICAL ATTRIBUTES 25
3.7 BIOCHEMICAL ATTRIBUTES 25
3.7.1 Hydrolytic antioxidant 25
3.7.2 Enzymetic antioxidant 26
x
3.8 PHYSIOLOGICAL ATTRIBUTES 26
3.8.1 Malondialdehyde Contents 26
3.8.2 Total Oxidant Status 25
3.8.3 Relative Water Contents 26
3.8.4 Cell membrane thermostability 27
3.8.5 Pigments Analysis 27
3.8.5.1 Chlorophyll a,b, Carotenoid and Anthocyanin 27
3.9 METABOLITIES ACCUMULATION 28
3.9.1 Total soluble Sugars 28
3.9.2 Total Proteins 28
3.9.3 Proline accumulation 28
3.10 Mineral Elements 28
3.10.1 Potassium and calcium ratio 28
3.11 Seed Quality attributes 29
3.11.1 Wet Gluten (%) 29
3.11.2 Gluten Index (%) 29
3.11.3 Falling Number (Sec) 29
3.11.4 Proteins, Moisture and Starch (%) 30
3.12 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE
GELS 30
3.13 TRANS GENERATIONAL EFFECT OF ELEVATED CO2
ON WHEAT AT ANTITHESIS DROUGHT STRESS 30
3.13.1 Gaseous Exchange and Water Relations 30
3.13.2 Yield and Yield Components 31
xi
3.13.3 Key Enzyme of Carbohydrate Metabolism (C Enzymes) Assays 32
3.13.4 Key Enzyme of Carbohydrate Metabolism (A Enzymes) Assays 34
3.14 Statistical Analysis of Data 35
4. RESULTS AND DISCUSSION 36
4.1 SEED BIOCHEMISTRY 36
4.1.1 Oxidative enzymes assay 36
4.1.1.1 Protease activity 36
4.1.1.2 Amylase activity 37
4.1.1.3 Esterase activity 39
4.1.1.4 Superoxide dismutase activity 39
4.1.1.5 Peroxidase activity 40
4.1.1.6 Catalase activity 41
4.1.1.7 Cluster analysis based on seed biochemistry attributes 42
4.1.2 Conclusion 43
4.2 MORPHOLOGICAL ATTRIBUTES 44
4.2.1 Cluster analysis based on Morphological attributes 66
4.2.2 Conclusion 67
4.3 BIOCHEMICAL AND PHYSIOLOGICAL ATTRIBUTES 67
4.3.1 Oxidative Enzymes 67
4.3.1.1 Estrases activity 67
4.3.1.2 Amylase activity 68
4.3.1.3 Protease Activity 70
4.3.1.4 Superoxide dismutase activity 72
4.3.1.5 Peroxidase Activity 72
xii
4.3.1.6 Catalase activity 74
4.3.1.7 Ascorbate peroxidase activity 75
4.3.2 Cluster Analysis based on oxidative enzymes attributes in
flag leaf of wheat 76
4.3.3 Conclusion 77
4.4 PHYSIOLOGICAL ATTRIBUTES 79
4.4.1 Malondialdehyde Contents 79
4.4.2 Total oxidative status 79
4.4.3 Relative water contents 80
4.4.4 Cell membrane thermostability 81
4.4.5 Photosynthetic pigments 82
4.4.6 Conclusion 85
4.5 METABOLITE ACCUMULATION AND MINERAL ELEMENTS 85
4.5.1 Sugar contents 85
4.5.2 Protein contents 87
4.5.3 Proline accumulation 87
4.5.4 Potassium ratio 88
4.5.5 Calcium ratio 89
4.5.6 Cluster Analysis Based on Biochemical and Physiological
Attributes 91
4.5.7 Conclusion 92
4.6 WHEAT GRAIN QUALITY 93
4.6.1 Wet gluten contents 93
4.6.2 Gluten index 94
xiii
4.6.3 Falling number 96
4.6.4 Seed Storage Protein 98
4.6.5 Moisture Contents of seed 100
4.6.6 Starch content of seed 101
4.6.7 Conclusion 104
4.7 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE
GELS 104
4.8 TRANSGENERATIONAL EFFECT OF ELEVATED
CARBONDIOXIDE ON METABOLISM OF WINTER WHEAT
EXPOSED TO ANTHESIS DOUGHT 107
4.8.1 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on Yield
Attributes 107
4.8.2 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on physiological
attributes 109
4.8.3 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on Invertases
and Susy activity 111
4.8.4 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on the key
enzymes for Carbohydrate (C) Metabolism 116
4.8.5 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on (a) key
enzymes activities for Carbohydrate Metabolism 124
4.8.6 Conclusion 132
SUMMARY 136
LITERATURE CITED 138
APPENDIX 168
xiv
List of Abbreviation
SA Salicylic acid
GA Gibberellic acid
SOD Superoxide dismutase
POD Peroxidase
CAT Catalase
APX Ascorbate Peroxidase
GR Glutathione reductase
RWC Relative Water Contents
TOS Total Oxidant Status
MDA Malondialdehyde
CMT Cell Membrane Thermostability
K+ Potassium
Ca+ Calcium
CO2 Carbon dioxide
(a[CO2]) Ambient Carbon dioxide
(e[CO2]) Elevated Carbon dioxide
Tr Transpiration rate
An Photosynthesis
Gs Stomatal exchange
PVP Polyvinylpolypyrrolidone
CWInv Cell wall invertase
VACInv Vacuolar invertase
xv
CYTInv Cytoplasmic invertase
SUSY Sucrose synthase
AGPase ADP-glucose pyrophosphorylase
UGPase UDP-glucose pyrophosphorylase
PGM Phosphoglucomutase
PGI Phosphoglucoisomerase
G6PDH Glucose-6-phosphate dehydrogenase
Ald Aldolase
HXK Hexokinase
FK Fructokinase
PFK Phosphofructokinase
TAP Total antioxidant potential
xvi
ACKNOWLEDGEMENTS
Words are bound and knowledge is limited to praise ALMEIGHTY ALLAH,
the Lord of the world, the Omnipotent, the Beneficent, who gave me the requisite
potential and diligence for the successful accomplishment of this task. Special
praise to HAZRAT MUHAMMAD (PBUH), who is forever the source of
knowledge for whole mankind.
I am grateful for the financial assistance by Punjab Government (fee
reimbursement scheme) and Higher Education Commission (HEC) Islamabad
Pakistan, under the IRSIP PhD Fellowship Scheme.
I’m appreciative to my parents for all their love, prayers, sacrifices,
sympathies, guidance and encouragement which served as “beacon of hope” all
along my work. I offer my richest and heartiest gratitude to them.
I confess here that thesis would not be completed without the principal
contribution, affection, untiring help, invigorating encouragement and moral
support of my affectionate supervisor Prof. Dr. Syed Abdul Majid. I have never
seen such a polite and humble person. It is a great pleasure for me to be his student.
I owe special thanks and pray for his long life.
Here, I would like to express my gratitude to all my respected teachers of the
Botany Department specially, Dr. Ghulam Murtaza, Prof. Dr. M. Qayyuam Khan,
Dr. Rehana kausar, Dr. Hamayun shaheen, Dr. Sidiqa Firdous, Mis. Sidra Qayyum,
Dr. Rizwan Taj, Dr. Tariq Habib and Dr. Ejaz Dar for their direction, meaningful
suggestion and helpful attitude during the course of this degree. I extend my cordial
and profound thanks to my M.sc supervisor Dr. Altaf Hussain for his support and
guidance.
xvii
I am extremely grateful to Dr. Hamayun Shaheen, my teacher and member of
my supervisory committe for his continues guidance and support throughout my
Ph.D research till the completion mission impossible. I feel extremely privileged
for his cooperation, valuable suggestion, sympathetic attitude, words of
encouragement and advice over the course of my study. I’m also obliged to my co-
supervisor, Dr. Amjad Hameed, Principal Scientist (NIAB) for his help, guidance
and cooperation to achieve this task. I am thankful the services he provide me. He
is a supervisor whom I learned that “you cannot go wrong when research activities
are planned’.
I offer my cordial and profound thanks to Dr. Javeed Ahmad, Dr. Abrar, Dr
Ghulam Subhani, Miss Sadaf Afzal, Miss Hira Shair, Awais and Amir Hameed in
wheat research (AARI) Faislabad. They have been providing me the good facilities
and time during exhaustive moments of my work. I do not have words at my
command to express my heartiest thanks and gratitude to my external foreign
supervisors, Dr. Fulai Liu (Principal supervisor), Prof. Dr. Thomas Georg Roitsch
(Co-supervisor) and Xiagnan Li (Post doc fellow) at University of Copenhagen,
Denmark. Their visionary research activities made my investigations more fruitful
and I hope it will reshape my upcoming research directions.
I particularly wish to acknowledge the help rendered by my research fellows
in University of Copenhagen especially, Sajid Shokat Senior Scientist (NIAB) for
his input in my research works. The effectiveness of research work in Denmark
could not have been achieved without his help and cooperation.
xviii
I really have no words to express my cordial and sincere indebtness to my
brothers, Naveed, Nafees and Awais and beloved sisters, Nabila and Tayyeba for
their cooperation and deep love who supported me during the odd hours of life. I
would like to record my sincerest thanks to my uncles (Zahid and Shahid), all other
relatives and cousins for their cooperation during my studies. I wish to express my
profound gratitude and pray to my loving legends my grandmothers, grandfather
and uncle (late), though they are no more with me to see that their dream have
comes true. I love you and I will forever remember you.
I am highly indebted to my beloved rommates, Anila, Maria, Shamsa, Zara,
Safina, Nosheen, Hafsa, Amnah, Saba and Shaista. I wish to acknowledge my
everlasting friends and fellows, Khadija, Asia, Saima, Ghazala, Safina, Ammara,
Shazia, Sidra, Sidra and Anum. I can’t forget the love, cooperation and colorful
moments with my friends during my Ph.D. Special thanks to my friends from
NIAB, Fozia, Mehak, Sidra, Anum and Misbha. I extend my thanks to my fellows
at University of Copenhagen Rabecca, Milan, Lian, Sichen, Lamis, Shehnaz,
Rumana, Shumaila, Rizwan and especially Rehina for their love and they make my
time very special in Denmark
I cannot forget all those peoples who provided me spiritual and moral support
and they always make a silent prayer for me. I have only one sentence for all of
you, I love you all and your love led me every step to fruition. I would like to
acknowledge all the clerical staff and lab staff at all research institute and
universities for their help and good behavior. May the Almighty Allah shower his
blessing to all those who assisted me at different stages during my academic career.
ANEELA ULFAT
xix
ABSTRACT
Global warming and uneven climatic change have augmented the drought
prevalence. These dilemmas are enforcing the agri- scientist to develop some long
term future policies. The aim of present study was to examine the method for
improved growth and development of wheat under the premises of drought. Five
wheat cultivars were used to investigate the consequence of drought on plants and
it was also investigated that how hormonal priming can be helpful to cope drought.
Seed of these wheat cultivars were primed in 10-4 M Gibberellic acid and Salicylic
acid concentration. The response of antioxidant enzymes was variable among the
non primed and primed seeds for all studied genotypes. Shahkar had the highest
protease activity in primed seeds while AARI-11 had the highest amylase activity.
Similarly, AARI-11, Shahkar and Chakwal-50 had the highest Superoxide
dismutase activity while Shahkar and Pakistan-13 had the highest peroxidase
activity. Cell membrane thermo stability, proline and relative water contents were
decreased under drought stress. Hormonal priming with Gibberellic acid and
Salicylic acid improved the physiological response and antioxidant enzymes
activities in some genotypes under both conditions.
Yield and its contributing traits yield components were lessened under the
effect of drought. FSD-08 and Pakistan-13 showed maximum grain yield during
control and drought condition. Priming increased the grain yield in all varieties.
Grain quality characters were noticeably affected under drought stress. Hormonal
seed priming was able to maintain the grain quality by minimizing the adverse
effects of drought. FSD-08 was able to maintain the grain quality under normal and
stress conditions.
xx
Drought is unavoidable under changing climatic scenario however famine
can be avoided. The second experiment was conducted in order to know the
mechanism of trans-generational effect of elevated carbon dioxide on winter wheat.
Seeds obtained from the previous generations of ambient and elevated carbon
dioxide were regrown under ambient (400ppm) and elevated (800ppm) within the
green house. Drought stress was imposed for 4 days during anthesis stage and then
plants were re-watered. Flag leaves were used to analyze the activities of enzymes
involved in carbohydrate metabolism and antioxidant enzymes. This
transgenerational effect and its enzymatic basis were not investigated previously.
Results showed that glycolytic intermediates and antioxidants were enhanced under
elevated carbon which ultimately increased yield. Trans-generational effect
indicated that seeds had stress memory and thus maintained the effect of previous
exposure. We found within source (leaf) cytoplasmic invertases, sucrose synthase,
catalase and ascorbate peroxidase activities were decreased while the activity of
cell wall invertases was increased under drought and elevated carbon. Similarly,
the activities of glucose-6-phosphate dehydrogenase, superoxide dismutase,
ascorbate peroxidase and dialyzed peroxidase were decreased under drought and
elevated carbon within the sink (spike). Likewise, the activities of sucrose synthase
in the source and aldolase in the sink were increased upon re-watering indicating
that water is playing an important role to activate these enzymes. Similarly, lower
yield was recorded under ambient carbon dioxide. These results indicate that high
metabolism of sucrose synthase within the source; aldolase and glucose-6-
phosphate dehydrogenase within the sink can be helpful to mitigate the drought
stress under elevated carbon dioxide.
1
Chapter 1
INTRODUCTION
Wheat (Triticum aestivum L.) is most widely grown food crop across the globe
belonging to family poaceae, sub family pooidceae and tribe triticeae. This crop has been
improved extensively throughout the world and more than 5,000 cultivars of this species are
being used. It is estimated that more than 35,000 cultivars were developed in the past but
most of them were not able to get commercial fame among the farming community
(Feldman and Levy, 2015) and consequently disappeared quickly. Wheat is grown under
diverse climatic conditions from higher elevation to equator and this crop is well
acclimatized to from 30° and 60°N and 27° and 40°S latitudes (Walter and Breckle, 2013).
Across the world, wheat is being harvested anywhere during whole year due to its versatile
nature. Wheat is not only the oldest cultivated crop but also the staple food of European,
West Asian and North African civilizations for the last 8000 years. This king of cereals has
the highest consumers demand and it has biggest cultivation area among all crops, including
rice, maize and potatoes. Globally, wheat trade is higher than any other cereal (FAO, 2016).
Wheat is considered as world most imperative crop due to reason that it contains
many calories, vitamins, proteins and minerals. Its significance is consequent from the
properties of its gluten; a cohesive network of tough endosperm, proteins that stretch with
the expansion of fermenting dough. Wheat is used for making bread, unleavened bread,
used in pastry products, and for semolina products. Most of these uses, pooled with its
nutritive value and storage quality, have made wheat a staple food for more than one-third
of the world’s population. Among cereal, most of the food stuff were made with wheat
(Council, 2010). Being the staple diet of most of dominates all crops in acreage and
2
production. Global cropped area of wheat is around 240 million ha while world wheat
production during 2016 was 733.8, utilization 715.7, supply 945.0 and trade 164.9 (FAO,
2016).
In Pakistan, wheat is prime food crop in terms of area and consumption. Pakistan is
categorized among top ten wheat producers and consumers. Wheat is a major diet of whole
population and 3/5th of the daily dietary requirements are fulfilled by wheat and per capita
consumption is 125 kg. It also has key importance in country’s policy about food security.
In Pakistan wheat shares 2% in total GDP and around 10% in the value added products of
agriculture. It is estimated that around 0.6% increase in wheat cropped was observed during
2016 and area was increased from 9.204 to 9.260 million. Likewise, overall yield was
increased from 25.086 to 25.482 million tons and 1.6% yield increase was calculated (GoP,
2016).
1.1 CLIMATE CHANGE AND ITS EFFECT ON AGRICULTURE
Global warming and climate change influenced the socio-economic sector on one
hand and agriculture sector on another hand globally. Different plants have diverse
requirements for germination and for better growth. One important variable is plant habitat
and its ecology (Raven, 2008). Environmental change likewise conveys vulnerabilities to
the possibilities of development of wheat production.
As per the (International Maize and Wheat Improvement Center) (CIMMYT, 2016),
environmental change may influence wheat production through the immediate impacts on
yield by means of physiological process, through changes in sowing dates or expanded
precipitation, and through changes in the zones under production, as areas turn out to that
is not much reasonable for wheat. Increased carbon dioxide (CO2) focuses can possibly
build plant development and yield, fundamentally through extended photosynthesis. Before
the industrial revolution the global atmospheric CO2 was 35% less than today’s
3
amount. Although, current amount of atmospheric CO2 is less than 400 part per million yet,
it is expected to reach at 970 part per million at the end of this century (IPCC 4th report).
It is expected that poor people (almost 1.2 billion) have dependency on wheat but it is also
found that this crop is affected by environmental factors more. It is estimated (in South
Asia) that may be in near future about 2050, there is decline in wheat production. In
developing countries wheat demand increased 60% by 2050. In parallel, global change
increases temperature which is more effective in developing world to decline wheat
production by 20–30% (Wheat CRP).
1.2 PAKISTAN STATUS IN TERM OF CLIMATE CHANGE
Since 1947, Pakistan is prone to water shortage and drought conditions during
wheat grown cycle while on the other hand scarce rains coupled with high temperature
resulted in lower wheat production in both irrigated and rainfed regions. The most
economical solution to cop this problem is the development of drought and heat tolerant
varieties. Developing countries similar to Pakistan are also in front of troubles such as
glacier melting, flash floods, drought and heat index. May be we face melting of Pakistan’s
glaciers in 2035. It will bring a major terrible effect on fresh water flows (Stolton et al.,
2006). These climatic changes affect the economy of Pakistan because Pakistan is an
agriculture country and contributes 21% to GDP. Pakistan is 3rd among those countries that
are affected by climate change and stand in 135th number in terms of Co2 emissions (De
Vries, 2010). Among various climatic factors low water availability for irrigation is a
threatening issue and going to be increase as the time flows in Pakistan.
1.3 ABIOTIC STRESSES
All type of stresses weather biotic or abiotic affect the wheat growth and yield.
Environmental stresses appear in several forms, plant water status is badly affected by all
these stresses. It may be understood that all plants have the encoded capacity to response
4
to stress by signaling. Amid the diverse abiotic stresses, salinity, chilling, heat, and drought
stress affects the yield and growth of wheat crop (Shinozaki and Yamaguchi, 2000). Water
shortage reduces and affects the production of food crops up to seventy percent all over the
world (Akram and Ashraf, 2013). Plants adapt to drought stress in versatile way like
adaptations in morphology, physiology and metabolism (Moghadam et al., 2011). Drought
stress results in stunted growth of plants (Sairam and Saxena, 2000). Khan et al, (2010)
reported that growth and yield of wheat crops is affected by shortage of water (Khan et al.,
2010). When it occurs it acts as a limiting factor for the final produced crop. Serious water
stress in wheat amid the vegetative stages brings about diminished leaf region and this thus
influences tillering and spike measure (Denčić et al., 2000).
Grain yield has been found to be correlated with drought stress at critical growth
stages of wheat (Malik et al., 2010). Water scarcity results in changes in physiology of
wheat crop as well changes also occur at biochemical level. Many defense mechanisms i.e.,
ion homeostasis, osmoregulation, hormonal systems and antioxidant enzymes production
were occur in tolerant species which enable to survive them and develop properly before
reproductive stages (Ashraf, 2010).
1.4 STRATEGIES TO OVERCOME ABIOTIC STRESS
One of the most important steps is seed priming to overcome water shortage. By
this water is absorbed by seeds and metabolic processes start but radical does not emerge
from seed (Farooq et al., 2006b). Primed seeds frequently showed better results regarding
sprouting uniformity, germination rate and germination percentage (Kaya et al., 2006).
This technique has been applied to overcome the water shortage effects in many crop
species. Primed seeds during germination pass from different phases like imbibitions and
lag phase and are ready to grow under every condition (Eisvand et al., 2010). Certain
5
efforts have made to know the increase of yield under drought stress conditions when
grown under elevated CO2. Photosynthetic rate going to be increase with increase in CO2
level and it results in more photosynthate production along with enhancement of
antioxidants enzymes with increasing reducing power. This may improve the resistance
against environmental stresses, like drought (Hassanein et al., 2009). However, crop
improvement in context of mitigating the climate change would be an ideal strategy to
move forward for certain crop improvement schemes.
In Himalayan region of Pakistan like Azad Kashmir wheat cultivation and
production is almost neglected, although area has very good potential for the production of
spring as well as winter wheat varieties. Wheat is the staple food of the people of this
region.
1.5 JUSTIFICATION OF THE STUDY
Keeping in view the importance of climate change and to know about the plant’s
positive approach during this change especially in relation to the elevated CO2 and priming
the project was designed. It will probably help us to know the real mechanism of plants
stress tolerance. The rise in CO2 concentration has direct as well as indirect consequences
on agricultural production. Among many parameters growth of plant, physiology and
productivity are directly influenced by global climate change due to increase in
concentrations of CO2. Proper regulation of plant machinery under these environmental
changes is extremely important. Consequently, the plants have to create a balance from
source to sink along with stress tolerance (Godt and Roitsch, 2006). Plant source tissues
yieldsurplus of assimilates and these are either elated to the growing tissues or stored in the
form of different sugars. However, partitioning of these sugars can be estimated by the sink
strength (relatively) and by different abiotic along with biotic stress factors (Keunen et al.,
2013).
6
This study was designed to investigate the following myths;
To use the hormonal seed priming as shot gun approach to manage with drought
stress
To improve the wheat yield under drought stress grown under elevated CO2
To study the metabolic changes associated with drought stress, priming and
elevated CO2
To investigate the stress memory of trans-generational seed re-grown under
elevated CO2
To determine the effective ways to promote sustainable agriculture and to promote
the wheat production in Himalayan region of Pakistan like Azad Kashmir
7
Chapter 2
REVIEW OF LITERATURE
Wheat (Triticum aestivum L.) is a cereal grain, originated from Levant region of the
near East but now cultivated globally. Wheat is grown in most parts of the world, from
near-arctic to near-equator.
2.1 WORLD WHEAT MARKET
During the year 2011-12 wheat production was 697.4 million tons, supply 896.6
million tons, utilization 694.3 million tons, trade 148.5 million tons and Stock-to-
disappearance ratio was 18.2. In the year 2012-13 production of wheat was decreased as
compared to previous year. Wheat production increased upto 711.5 million tons during
2013-14, while during 2014-15 wheat production was 730.5 million tons, supply 913.7
million tons, utilization 703.6 million tons, trade 156.6 million tons and Stock-to-
disappearance ratio was 16.7. There was a significant increase during 2015-16 in
production 733.8 million tons, supply 945.0 million tons, utilization 715.7 million tons,
trade 164.9 million tons and Stock-to-disappearance ratio was 16.6. During 2016-17
production 742.4 million tons, supply 968.2 million tons, trade 730.5 million tons and
Stock-to-disappearance ratio 17.4 estimated (FAO, 2016).
2.2 WHEAT STATUS IN PAKISTAN AND AZAD JAMMU AND KASHMIR
Pakistan is ranked at sixth position among top wheat producing countries. Pakistan
is producing 25 million tons of wheat yearly though Punjab contributed its share of 19
million tons wheat to total production. Pakistan is self-sufficient nation in wheat production
as wheat is sown on more than 20 to 25 million acres of land in the country every year
consistently. In Azad Jammu and Kashmir (AJK) wheat is grown on around 92
8
thousand hectares with a yearly production of around 113 thousand tons. Azad Jammu and
Kashmir with a normal yield of 1226 kg for every hectare is for behind the normal yield of
Pakistan. The nearby wheat production can't satisfy household require and about 350
thousand tons of wheat were imported to AJK from Pakistan (PARC, 2016). The
atmosphere of Azad Kashmir is temperate to sub-tropical with a normal annual
precipitation of 1300mm. There is variation of height (sea level) ranges from south (360
meters) to north (6325 meters). The snow line (from ocean) varies from 1200 meters
(winter) to 3300 meters (summer) as reported in AJK Bureau of statistics (2015).
2.3 CLIMATE CHANGE AND ITS IMPACT ON AGRICULTURAL CROPS
Energy constraints, water availability, climate change and ecological degradation
are the largest threats that were facing agriculture (Kirschenmann, 2011). With every
passing year, the horticultural framework is affected by additional climate change
(IPCC).The ascent in CO2 fixation has immediate and circuitous impact on agriculture.
Plant development, profitability and physiology are straight forward affected by worldwide
environmental change by expanding in concentration of CO2. Subsequently, the best
possible direction of sugar production and appropriation is fundamental for plant
advancement and stress reaction. Critical variations in mRNA articulation levels and
actions of compounds associated with sugar digestion happen amid plant improvement,
separating starch leaf and spike tissues, that are essential in deciding the last bio-yield and
henceforth edit final yield and quality of grain (Godt and Roitsch, 2006).
2.4 DEMAND AND AVAILABILITY OF WATER
The expansion rate of human population of Pakistan requires an elevated food
progress while less water resources are provided for agriculture. This alarming condition
9
can only be resolved proficiently if water is managed more, so that crop produce per unit
of water utilization boosts. Agriculture is the major consumer of water generally in most
countries. On top of that, agriculture sector encounters the enormous obstacle of
development of crop, as almost 50% more food will be needed by 2030 and development
must be doubled by 2050. These goals should be achieved with less water, due to the fact
of growing stresses from urbanization, industrialization and environmental change (OECD,
2010).
Water is a generally critical constituent of the metabolism of every single alive
being, encouraging numerous imperative natural processes because water is good solvents,
medium of transportation and retain the property to impart cooling property by evaporation
(Mundree, et al.,2002). In all photoautotrophs including plants, water assumes the extra
part of giving the vitality important to initiate the process of photosynthesis. Water atoms
are split, in a procedure called autolysis, to yield the electrons that are utilized to drive the
vitality yielding photosystem II reaction center. It goes about as medium dissolvable in
which numerous biochemical procedures takes place. The proteins in the Calvin cycle and
Kreb's cycle are all skimming around in the stroma, which is only a fluid arrangement of
stuff. Numerous different pathways additionally include catalysts, substrates and item that
take part in whole processes. Water goes about as dissolvable bearer for mineral
nourishment. Water help to move the supplements in plants and those supplements move
upward in the xylem. Water creates turgor weight, which give strength to leaves and stems.
At the point when plants lose water, they lose turgor and shrink (Taiz and Zeiger, 2002).
Water constitute a fundamental prerequisite for germination. Growing seeds are
frequently dry and need to retain,a process of imbibition, a critical amount of water to avoid
desiccation through frequently drying according to seeds dry weight. Plants seeds require
in general require almost 35% to 45% of water contents for germination. Wheat plant needs
10
water at two basic stages, first at tillering that begins a week after rise so water system
ought to be connected not later than 20-25 later subsequent to seeding. The second water
system is essential amongst anthesis and grain arrangement if irrigation water is accessible.
For various developmental stages water supply is necessary in plants. For the most part 4
to 6 irrigations are required amid the entire yield cycle (Acevedo-Opazo et al., 2010).
2.5 DROUGHT STRESS
Drought is very important amongst the most widely recognized natural stresses that
influence growth and metabolism of plants. Drought stress keeps on being a critical
challenge to plant breeders and agricultural researchers. It is expected that by the year 2025,
around 1.8 billion individuals will confront supreme water lack and 65% of the total
population will live under drought stress situations (Xiong et al., 2006). It is estimated that
up to 2050 most of the arable land will face much problems due to drought (Vinocur and
Altman, 2005). Water shortage, restrict the development and profitability of crops and
damage more than other stresses. Drought is an overall issue, obliging worldwide less yield
production and environmental change has made this circumstance more genuine (Pan et
al., 2006).
Drought is multidimentional push influencing plants at different levels of their
growth. Dry season influences morphological, physiological and biochemical processes in
plants bringing about development hindrance, closure of stomata with back to back
lessening of transpiration, diminishing in chlorophyll substance and restraint of
photosynthesis (Demirevska et al., 2008) making it the major single component for yield
decrease all around the world (Narusaka et al., 2003). The reaction of plants to water
11
relies on a few components, for example, developmental stage, severity and length of stress
and cultivar hereditary qualities (Beltrano and Marta, 2008, Din et al., 2011).
2.5.1 Problems During Drought
Wheat is one of the essential grain crops on the planet. It can be developed in an
extensive variety of agrarian situations. Water accessibility is the most constraining
component for wheat production, drought stress antagonistically influences plant
development and advancement, seed germination, (Dash et al., 2010; Almaghrabi, 2012),
seedling development, enzyme action (Seckin et al., 2009), DNA, RNA as well as synthesis
of protein (Anuradha and Rao, 2001) and mitosis (Tabur and Demir, 2010). In wheat, most
delicate to drought stress stages are tillering, reproductive and germination (Passioura,
2007). Heat stress is the significant limitation to wheat in dry, semiarid, tropical and
subtropical areas of the world (Ashraf and Foolad, 2005). It influences the accessibility and
translocation of photosynthates to creating seed and starch combination, along these lines
antagonistically influencing the grain weight and quality (Mohammadi et al., 2004).
Better implementation of crops relies on accessibility of water. Among different
abiotic stresses, water deficiency is most important because it effect up to 70% of yield and
production of crops (Akram and Ashraf, 2013). Diverse abiotic components influence the
development and yield of the crop plants. Among these components, water condition is
most important because it decreases the yield and also effects its development (Kusvuran,
2012; Souza et al., 2004; Saensee et al., 2012).
12
Figure 2.1: Drought Stress effect on cell physiological processes
A few agents have portrayed the impact of water shortage on different physiological
traits of development in wheat. All in all, dirt water shortage brings about decline in relative
water contents (Tas and Tas, 2007), leaf succulence (Qi et al., 2009), chlorophyll content,
cell membrane stability index (Farooq and Azam, 2006; Tas and Tas, 2007), number of
grains per spike and weight of grains and grain yield (Sanjari Pireivatlou and Yazdansepas,
2010). Relative water contents identified with water take-up by the roots and water
misfortune by transpiration (Anjum et al., 2011). Cell membrane stability measured as rate
damage of leaf tissues of wheat cultivars, can be utilized for screening for drought stress.
The water stress diminishes in membrane stability index of all the wheat assortments.
Under water stress, tolerant cultivar showed higher membrane stability index support of
high RWC under dry season because roots develop more than the shoots and abscisic acid
actuated diminishment in stomatal opening has a tendency to keep up cell turgidity and
chlorophyll content (Keyvan, 2010).
Constrained water supply generally causes a decrease in chlorophyll content being
decidedly associated with yield (Zaharieva et al., 2001). Generally high chlorophyll
contents may add to the plant efficiency under stress conditions. Photosynthesis is
13
amongst the most delicate process to overcome the stress caused by drought (Chaves et al.,
2009). The inhibitory impacts of drought on photosynthesis might be connected with low
CO2 accessibility because of low stomata and mesophyll conductance (Flexas et al., 2008),
and/or impedances in carbon cycle (Peeva and Cornic, 2009). Stomata closure is an early
reaction to drought stress and an effective approach to lessen water availability in water-
restricting situations. Biochemical confinement of photosynthesis additionally assumes a
critical part under delayed times of drought stress (Flexas et al., 2008).
Water stress and high temperature are the major natural components influencing
wheat grain quality. It has been accounted for that times of heat stress with temperatures
higher than 35 °C may change flour quality. These impacts have been identified with an
expanded gliadins/glutenins proportion (Daniel and Triboi, 2000) and decreased the extent
of the bigger sub-atomic size glutenins (Wardlaw et al., 2002). It is realized that yield
diminishment that for the most part happens under drought stress is for the most part
connected due to protein content expansion (Rharrabti et al., 2003; Guttieri et al., 2005 ;
Garrido-Lestache et al., 2005; Pompa et al., 2009).
2.5.2 Strategies to Cope Drought Stress
These days different strategies are utilized to create plants that can withstand these
stresses. As of late, seed priming has been created as a basic strategy to produce tolerant
plants against different stresses (Ashraf et al., 2008). Drought stress impacts on seed
germination and seedling development of numerous plants. Seed priming could be utilized
to overcome the depressive impacts of drought. The enhancing impacts are affected by
numerous elements including priming strategies, plant species and drought stress intensity
(Farooq et al., 2009).
14
2.5.2.1 Priming
Diverse sorts of priming medications were recorded to upgrade drought resistance
in numerous plants. Scientists characterized priming as a technique utilized by
agriculturists in an extensive variety of plants, including wheat and chickpea. Seeds are
soaked with solution for around 6 h, 12 h, 24 h, then dried till to attain original weight that
was before soaking. Seed priming techniques includes hydro-priming (Farooq et al., 2013),
osmo-priming (Ghiyasi and Tajbakhsh, 2013), hardening with plant growth inducers
(Eivazi, 2012), and hormonal priming (Khan et al., 2009). Various priming agents
including ascorbic acid, salicylic acid, kinetin, CaCl2, abscisic acid are frequently reported
in literature for chemical priming of seeds (Jafar et al., 2012; Farooq et al., 2013).
Germination process can be mediated by soaking the seeds into water that helps in
imbibitions to break the dormancy as enactment of definite catalyst and so forth (Ajouri et
al., 2004). Various process animating germination are initiated by seed priming and hold
on taking after the redesiccation of the seed (Asgedom and Becker, 2001). The germination
procedure can be partitioned into stages: (i) quick imbibition (ii) beginning seed metabolic
process and (iii) consequent radical rise and carrying on the process growth (Fig 2.2).
Figure 2.2: Schematic diagram showed the effect of seed priming viz normal on seed germination process
(Source Rajjou et al.,2012)
15
2.5.2.2 Hormonal Seed Priming
During the most recent 20 years, phytohormones, drew the consideration of
researchers because of their capacity to initiate systemic acquired resistance (SAR) to
plants to various kinds of stresses (Tuna et al., 2007). Phytohormones can be used to
overcome the stress by priming of seeds and growth rate can be enhanced by foliar
application. During water deficiency, plants adapt the changes by different growth
hormones like salicylic acid (SA), Gibberellic acid (GA), cytokinase (CKS), absisic acid
(ABA) and Indole acetic acid (IAA) (Farooq et al., 2009).
2.5.2.2.1 Role of salicylic acid
Salicylic acid (SA) is regarded to persuade exclusive physiological and
biochemical activities of plants.
Salicylic acid take part in enhancing their activities and performance (Hayat et al.,
2010). SA helps in regulation of different physiological processes because it act as a
endogenous regulator due to phenolic nature (Hayat et al., 2010) furthermore gives security
against biotic as well as abiotic stresses, for example, stress induced by salt (Kaya et al.,
2006). SA prompted increment of the resistance in seedlings of wheat against stress induced
by salt (Shakirova et al., 2003).
Salicylic acid required in the growth regulation, development and advancement of
plants also their interaction to biotic as well as abiotic stress (Khan, 2013; Miura and Tada,
2014). SA is included in the regulation of different essential physiological processes for
example photosynthesis, nitrogen metabolism , proline, production of glycinebetaine
16
Antioxidant defense system and plants water relations during the condition of stress along
these lines gives insurance in plants against abiotic stresses (Khan, 2013).
It has been demonstrated that salicylic acid mitigate low-temperature stress in maize
and wheat plants during winter (Taşgín et al., 2003), or modulate different responses in
plants due to the stresses induced by salt (Borsani et al., 2001), ozone or ultraviolet light,
drought and herbicides (Ananieva et al., 2002). SA induces the defense process in plants to
mitigate salt stress (Afzal et al., 2011). SA helps a large number of crops against salt stress
for example, tomato (Tari et al., 2002) bean (Azooz, 2009), and maize (Gunes et al., 2007).
2.5.2.2.2 Role of Gibberellic Acid
Gibberellins (GA) are diterpenoids, regulating plant growth and development.
They are ordinarily utilized in present day in agriculture and were initially obtained from
pathogens especially on rice during 1938 known as Gibberella fujikuroi (Santner et al.,
2009; Yamaguchi, 2008).
They act all through the plant during cell division, help in cell during multiplication;
promote transitions mediated by developmental stages especially during breaking the seed
dormancy and germination during the adolescent and after that during developmental stages
also helps in reproduction improvement. In spite of the fact that GA activity is vital for
typical development and improvement, seedlings without the ability to incorporate or see
GAs will experience constrained advancement, even during the light conditions mediate
flowering (Griffiths et al., 2006; Ueguchi-Tanaka et al., 2005).
Gibberellins (GAs) are for the most part mandatory in progress and improvement.
They control germination of seed, leaf expansion, stretching of stem and flowering
(Magome et al., 2004). Gibberellic acid (GA) amasses quickly under all type of stresses.
17
Some researchers give more importance to Gibberellins (Hisamatsu et al., 2000).
Hydrolases synthesis and production can be enhanced by GA especially alpha amylase
helps in seeds germination. Gibberellins activates amylases especially proteases, Beta-
glucanases and alpha amylase (Ueguchi-Tanaka et al., 2005). During the production of
hormones especially gibberellins in plants, seed aleurone determine various transduction
pathways (Penfield et al., 2005). Drought stress alone delayed growth and elongation of
the hypocotyl, while use of gibberellic acid switched this impact. For this situation,
gibberellic acid somewhat expanded the water status of the seedlings and in part managed
protein synthesis (Taiz and Zeiger, 2006).
2.5.2.3 Priming effect on biochemical, morphological and physiological processes
To adapt stress plants prompts assorted biochemical and physiological mechanisms
for survival (Tas and Tas, 2007). Salicylic acid regulate the antioxidant enzyme activities
for example in tomato plants were sprayed with catalase and super oxide dismutase under
stress condition during drought (Hayat et al., 2010) or under the response of stress due to
presence of excess salt (Szepesi et al., 2009) and (Yusuf et al., 2008).
To lessen the unfavorable impact of drought stress plants have advanced some
protective systems, for example, a rise in the ROS (reactive oxygen species) (Miller et al.,
2010; Huang et al., 2012).
Reactive oxygen species (ROS), most of time produced in chloroplast also to found
to be in mitochondria, bringing on oxidative stress. Real ROS particals are leads Production
of singlet oxygen, anion radical, hydrogen peroxide and radicals of hydroxyl results in
ROS. Plants develop some defense mechanisms to protect themselves from harmful results
of oxidation especially during drought. The ROS scavenging mechanism is among the
18
common defense response against abiotic stresses (Vranová et al., 2000). To protect
themselves from ROS, plants can inherently create distinctive sorts of antioxidant that help
the plants to overcome the drought stress and face less harms due to oxidation. Peroxidase,
catalase and superoxide dismutase are important scavengers of free redicals (Khan et al.,
2008). The catalase (CAT) has potential to detoxify ROS in peroxisomes by dismutasing
the hydrogen peroxide into H2O and O2 (Prochazkova et al., 2001). Peroxidase and catalase
help in disposing of the H2O2 that is formed by SOD by detoxifying superoxide anion (O2-
) (Hasheminasab et al., 2012). Drought stress influences the development and yields of
wheat genotypes which brings about harmed development of the plants (Raza et al., 2014).
Under drought stress, different biochemical, physiological and molecular changes happen
in plants during drought stress condition (Arora et al., 2002). Consequently, amplified
superoxide dismutase transform in plants is the confirmation of pressure tolerance (Pan et
al., 2006; Hameed et al., 2011). Salicylic acid and its related compounds upon priming
cause initiation and inhibition in plants (Gill and Tuteja, 2010).
Salicylic acid advanced morphological trends that make contributions closer
to yield enhancement but, it depends on plant species, development and usage technique
(Arfan et al., 2007). Gibberellins (GAs) were for the most part required in development
and improvement. Wheat grain yield was increased by GA3-priming, it initiated regulation
of ion uptake and hormones homeostasis under salinity (Iqbal and Ashraf, 2013).
The enhancement in total protein in rice plants under drought stress by extrinsic use
of plant hormones might be because of their conceivable addition in water stress adjustment
(Tuna et al., 2008). The plants build proteins which are included in purification of free
radicals and along these lines assume very important for adjustment during the condition
of stress (Witzel et al., 2009) (Bandehagh et al., 2011;). Gluten
19
proteins are among the most complex protein arranges in nature because of ivarious
distinctive parts and diverse size brought about due to genotype, technological processes
and developing conditions (Wieser, 2007). They assume a key part in deciding the
interesting properties in relation to rheological dough and also used in baked products.
Bread wheat quality of assessment is utilized a considerable measure of quality analysis.
Some investigation strategies require a lot of test and result is acquired in the long time.
Quality properties in this study impact measuring the quality performance of genotypes.
These quality criteria can be utilized for quality evaluation as a part of early era breeding
programs.
2.6 TRANSGENERATIONALEFFECT OF ELEVATED CARBONDIOXIDE
Pre-exposure of plant to mild stress may activate the ‘stress memory’ that facilitate
safest protective response to the consequent stress events and happening (Boyko and
Kovalchuk, 2011). Stress memory can be defined as physiological changes at genetic level
and even epigenetic during stress conditions and to overcome this stress adjusts reactions
from generations to generations (Boyko and Kovalchuk, 2011). Stress memory in the
following generation supposed to be linked with improved tolerance in numerous species
regarding biotic as well as abiotic stresses of plants. Based on above literature survey, the
current study came up with the following major goals. There is need to develop drought
resistant cultivars and CO2 responsive genotypes to cope with coming environmental
issues.
2.8 CONSIDERATIONS FOR THE FUTURE
Due to the lack of good cultivars besides the inadequacy of related research, we are
not able to achieve mutual variation among wheat varieties and environments. Outcome
technical innovations to progress quality of wheat are the main challenge for whole world.
Additional studies must be directed towards the physiological, biochemical, and molecular
20
levels to reach a suitable conclusions about high yield and good wheat quality. There is
need to select special verities to achieve goals with respect to growth in special locations
and this in turn relate to increasing the probability of recognizing and predicting species
with maximum quality of grains in various environments. Similarly, genotypes that have
best response to elevated CO2 should be promoted. Water management and soil
conservation and irrigation techniques need to be improved. Promote agriculture in Azad
Jammu and Kashmir by using agricultural land technologies and strengthening of research
and technical services.
21
Chapter 3
MATERIAL AND METHOD
3.1 STUDY
This study was carried out in the field of University of Azad Jammu and
Kashmir Muzaffarabad. Azad Jammu and Kashmir consists of 13,297 Square Kilometer
area with latitude 33o–36o and longitude 73o–75o. It is a mountainous region and climate of
this state ranges from sub-tropical to alpine. Normal highest temperature is 45.2°C while
minimum temperature may go down to -2.6 °C (Anon, 2007). Study area is pointed out in
the map as shown below.
Fig 3.1 Map of the study area
3.2 PLANT MATERIAL
Seeds of wheat genotype AARI-11, Chakwal-50, Shahkar, Pakistan-13 and
Faisalabad-2008 were obtained from National Agricultural Research Center (NARC)
Islamabad Pakistan.
22
3.3 SEED PRIMING TREATMENTS
Priming was carried out by presoaking the seeds of each genotype in10-4M solution
of salicylic acid (SA) and gibberellic acid (GA) for eight hours. In addition, continuous
aeration was supplied by aquarium pumps, kept for drying and retried to original weight.
Half of the seeds were not primed which served as control.
3.4 EXPERIMENTAL SET-UP
The experiment was performed by split plot design by dividing the main plot into
two sub-plots i.e., well watered and drought followed via similarly cut up of sub-plots
into three sub-sub-plots appeared as replicates. Sub-sub-plots were supplied with
priming remedies and cultivars were completely randomized. Sub-sub-plots were provided
with priming treatments and cultivars were completely randomized. About fifteen sub-plots
were considered as control by supplying enough water while other fifteen sub-plots were
marked stress group without well watered condition and roofed with water proof sheet.
Sowing was done by hand drill, keeping seed to seed 1.5 cm and row x row distance of 20
cm.
3.5 SEED BIOCHEMISTRY ATTRIBUTES
3.5.1 Oxidative Enzyme Assays
3.5.1.1 Protease activity
Protease activity was estimated by the casein digestion assay established by
(Drapeau, 1974). Casein solution was prepared by mixing 6.5 mg/ml of casein in the 50mM
potassium phosphate buffer. For protease activity, seeds were homogenized in 50mM
potassium phosphate buffer (pH 7.8). One unit is that amount of enzyme, which releases
acid soluble fragments equivalent to 0.001 A280 per minute at 370C and pH 7.8. The
absorbance was recorded at wavelength of 660nm. Enzyme activity was expressed on fresh
weight basis.
23
3.5.1.2 Esterase activity
To measure the non–specific esterase namely α-esterase (alpha esterase) activity
(Van Asperen, 1962) method was used. The assay solution was prepared by taking 0.03 M
α or β–naphthyl acetate (substrate solution), 0.04 M phosphate buffer with pH 6.8 and
sample extract in each test tube separately. The absorption was recorded at 590nm by
spectrophotometer (HITACHI U-2800) when color developed for alpha esterase.
3.5.1.3 Amylase activity
To measure the α-amylase inhibition activity (Giancarlo § et al., 2006) method were
used. Starch solution (1%, w/v) was prepared by taking 1g of soluble starch, dissolved in
0.02M sodium phosphate buffer and sodium chloride (0.006 M) with pH 6.9. Dinitro
salicylic acid (DNS) color reagent was prepared by mixing 96 mM DNS solution with
sodium potassium tartrate solution (30.0g of SOD. K.) and absorbance was recorded at
540nm. Potassium phosphate buffer (pH 6.8) was used as blank.
3.5.1.4 Superoxide dismutase activity
The method of (Dixit et al., 2001) was followed for the estimation of Superoxide
dismutase (SOD) activity. Leaves were homogenized in a medium composed of 50mM
potassium phosphate buffer (pH 7.0), 0.1 mM EDTA and 1 mM dithiothreitol (DTT). The
SOD activity was evaluated by measuring its potential to inhibit the photochemical
reduction of nitroblue tetrazolium (NBT) as described by (Giannopolitis and Ries, 1977).
One unit of SOD activity was defined as the enzyme concentration that caused 50%
inhibition of NBT photochemical reduction.
3.5.1.5 Peroxidase activity
Peroxidase (POD) activity was determined by following the method of (Chance and
Maehly, 1955). The reaction was initiated by adding 0.1 ml enzyme extract in 50 mM
24
phosphate buffer (pH 7.0), 40mM H2O2 and 20mM guaiacol followed by recording in
absorbance at 470 nm after every 20s. One unit POD activity was defined as an absorbance
change of 0.01 units min_1.
3.5.1.6 Catalase activity
Catalase activity was determined by following the method of (Beers and Sizer,
1952). CAT activity was measured in assay solution (3mL) containing 50 mM phosphate
buffer (pH 7.0), 5.9mM H2O2 and 0.1 ml enzyme extract. After every 20 sec, decrease in
absorbance was recorded at 240 nm and absorbance change of 0.01 units min_1 was defined
as one unit CAT activity.
3.6 MORPHOLOGICAL ATTRIBUTES
Morphological attributes such as plant height (cm), spike length (cm), spikelets,
number of tillers, peduncle, extrusion length (cm), grains in pikes, total yield (kg/ha),
biomass yield (kg/ha), thousand grain weight (g) and harvest index (%) were recorded from
ten randomly selected plants at maturity. Yield and harvest index were calculated by.
Grain yield (kg /ha) = Grain Yiled
Sampled Area× 1000m−2
Harvest index = Grain Yield
Biological Yield× 100
While;
Stress tolerance index = values uder stress
Values under control×100
3.7 BIOCHEMICAL ATTRIBUTES
Fully emerged flag leaves were contribute grain yield directly up to 75% so fully
emerged flag leaves were collected for biochemical assay.
25
3.7.1 Hydrolytic antioxidant
Protease, esterase and amylase activities were done by using similar method as
already described for seeds.
3.7.2 Enzymatic antioxidant
Similarly, Superoxide dismutase (SOD), Peroxidase activity (POD), Catalase
(CAT) and Ascorbate Peroxidase (APX) activity was determined in leaf by using the same
method as used by seeds.
3.8 PHYSIOLOGICAL ATTRIBUTES
3.8.1 Malondialdehyde Contents
Malondialdehyde (MDA, a product of lipid peroxidation) contents from leaf tissues
were determined by method the method of (Heath and Packer, 1968) with some changes as
suggested by (Dhindsa et al., 1981). Samples were homogenized in 5 mL of 0.1% TCA and
centrifuged for 5 mints at 10,000rpm. In 1 mL aliquot of the supernatant, 4 mL of 20%
TCA containing 0.5% TBA were added. The mixture was heated at 950C for 30 min and
then quickly cooled in an ice-bath. The absorbance was recorded at 532 nm and the non-
specific absorption at 600 nm was subtracted. Extinction coefficient of 155 mM-1 cm-1 was
used to calculate MDA contents.
3.8.2 Total Oxidant Status
Total Oxidant Status (TOS) was evaluated by a novel automated method developed
by (Erel, 2005). Two types of reagent R1 and R2 were used and the results were expressed
in μmol H2O2 equivalents/L. Reagent R1, assay mixture contained (stock xylenol orange
solution (0.38g in 500μL of 25mM H2SO4), 0.49g NaCl, 500μL glycerol and volume up to
50mL with 25 mM H2SO4), sample extract and reagent R2
26
(0.0317g θ-anisidine, 0.0196g ferrous ammonium sulphate (II)). The absorption of each
assay mixture was measured at wavelength 560 nm after 5 minutes nm with the
spectrophotometer. The term μmoL H2O2 equivalents/L was used to express the results.
3.8.3 Relative Water Contents
According to (Weatherly, 1950), relative water contents (RWC) of the flag leaf
sample were estimated by measuring fresh weight, turgid weight and dry weight.
Relative water content = [Fresh weight–dry weight/turgid weight–dry weight] ×
100.
3.8.4 Cell Membrane Thermo stability
Plant material were placed into two sets of test tubes alongwith de-ionized water
and then put in a refrigerator at 10°C for 18 h. After that with de-ionized water plant sample
were washed and 15mL deionized water was added in the same test tube. Thereafter, one
set of the test tubes was kept at 45°C and the other half at 25°C for 1h. Now, to get
stabilization both samples sets were placed in a refrigerator for 18hr at 10°C. To take the
readings, conductivity meter was used for both sets, heat treated (T1) and control (C1). Now
samples were boiled for 1 hour samples. After cooling the samples second conductivity
reading (C2 and T2) was taken at 25°C. Cell membrane thermo stability was calculated by
the equation of (Blum et al., 2001).
MTS (%) = [1-(T1/T2)] x100
27
3.8.5 Pigments Analysis
3.8.5.1 Chlorophyll a, b, Carotenoid and Anthocyanin
Leaf samples were homogenized into 80% acetone for chl a, chl b and carotenoids,
while for anthocyanin methanol/HCl/water in a ratio of 90:1:1 instead of 80% acetone was
used. Centrifuged and measured optical density at the wavelength of 537, 647 and 663nm
(Sims and Gamon, 2002).
3.9 METABOLITIES ACCUMULATION
3.9.1 Total soluble Sugars
According to (Dubois et al., 1956) sugar content was measured. Leaf samples were
homogenized using a clean mortar in distilled water and centrifuged at 3000 rpm for 5 min.
Then in 0.1ml supernatant, 1ml phenol (5 % v/v) was added and left for 1 hr. incubation
was done by the addition of concentrated H2SO4. Concentration of unknown sample was
calculated by using standard curve of glucose. The absorbance was recorded at 420nm.
3.9.2 Total Proteins
The method of (Bradford, 1976) was used for the estimation of protein contents.
The Bradford’s reagent was made by mixing (25ml of 95% ethanol 50 with 50mg
Coomassie Blue G250 dye), after that mixture was added in 50mL of 85 % o-phosphoric
acid to make total volume of 500mL with distilled water. By using the solution (1 mg ml-
1) of BSA (Bovine Serum Albumin) a standard curve was made. Absorbance was observed
at 595nm on spectrophotometer (Boyer, 1993).
28
3.9.3 Proline Accumulation
Proline was determined from the sample by following the method of (Bates et al.,
1973). 0.15g, leaf sample was homogenoized in 10mL sulphosucilic acid solution. Solution
was prepared by taking 3g of sulphosucilic acid in 100mL of water. Then 2mL glacial acetic
acid and 2mL acidic ninhydrine prepared. 2.5 ninhydrine, 60mL glacial acetic acid, 30 mL
distilled water and 10mL orthophosphoric acid were added in the reaction mixture. After
boiling, cools the mixture and added 6mL toluen by shaking it thoroughly and then poured
it into separating funnel for proline extraction. Then proline was assayed at 520nm by using
spectrophotometer.
3.10 MINERAL ELEMENTS
3.10.1 Potassium and Calcium Ratio
Potassium (K+) and calcium (Ca+) were estimated by (Szabo-Nagy et al., 1992)
method. Suspension was prepared by boiling one gram of flag leaves in 10 mL of perchloric
acid for 30 minutes and then de ionized water was added to make the total volume one liter
in volumetric flask. Potassium and calcium contents were assayed with the help of
JENWAY PFP 7 Flame photometer and a standard curve was also made.
3.11 SEED QUALITY ATTRIBUTES
3.11.1 Wet Gluten (%)
Glutamate instrument ICC standard no 155 and 158 and AACC method 38-12 were
used for Glutametic test. About 10g sample was placed into glutametic washing chamber
on the top of polyester screen. Mixed and then washed the sample with a 2% NaCl salt
solution for 5 min. After washing, wet gluten was subjected for centrifugation and then
weight by using weighing balance.
29
3.11.2 Gluten Index (%)
The percentage of gluten that remained on the sieve during centrifugation is defined
as gluten index, which indicates gluten strength. Gluten index was calculated by separating
and weighting gluten on sieve and gluten that was passed from sieve.
3.11.3 Falling Number (Sec)
Alpha amylase activity was measured by using falling number instrument 1310ICC
standard no. 107/1 (1995) and AACC method 56-81B (1992). Flour sample of 7g with
25mL distilled water was added in the viscometer tube, shaking well with the help of
shaker, and then tube was placed in the water bath. After 5 sec automatic stirring started.
The total time in sec from the start of instrument until the stir has fallen. Time was
registered by instrument.
3.11.4 Proteins, Moisture and Starch (%)
Omega kernel analyzer was used to determine protein, moisture and starch contents.
500g sample placed in sample holder and set software accordingly and find all readings in
percentage.
3.12 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE GELS
For extraction of soluble proteins, leaves (0.5g) were ground in 50 mM phosphate
buffer (pH 7.8) and centrifuged in a micro-centrifuge machine for 10min at 14,000 rpm.
Protein concentration of extracts was measured by a dye binding assay as described by
(Bradford, 1976).The supernatant was decanted and used for protein profiling. Protein
profiling of samples was performed using SDS-polyacrylamide gels as described by
(Laemmli, 1970). The process of SDS-PAGE was repeated thrice. Gels were photographed
using UVIpro-platinum gel documentation system (UVItec UK). Computerized gel
analysis was performed using UVI pro Platinum 1.1 Version 12.9).
30
3.13 TRANSGENERATIONAL EFFECT OF ELEVATED CARBONDIOXIDE (CO)2 ON
WHEAT AT ANTHESIS DROUGHT STRESS
Another experiment was carried at crop science section of Plant and Environmental
Department (PLEN) at University of Copenhagen, Denmark. The experiment was carried
out on winter wheat (Triticum aestivum L. var. Lianmai 6). Grains harvested from three
successive previous generations were further exposed to two different levels of CO2 i.e.
ambient CO2 concentration (a[CO2], 400 mmol L _1) and elevated CO2 concentration
(e[CO2], 800 mmol L _1). These seed were sown in 4 L pots; pot size was 17 cm in diameter
and 16.5 cm in height with 4 drainage holes along with four replicates. Pots were filled
with peat material (Sphagnum, 32% organic matter, pH = 5.6–6.4 and EC = 0.45 ms cm
_1). The CO2 enrichment was achieved by emission of pure CO2 from a bottled tank,
released in one point and distributed in the greenhouse cells through internal ventilation.
The CO2 concentration in the greenhouse cells was monitored every six seconds by a CO2
Transmitter Series GMT220 (Vaisala Group, Helsinki, Finland). The climate conditions in
the greenhouse were set at: day/night temperature 20/16 0C, photoperiod 16h, relative
humidity 70%, supplemental light 400 mmolm _2s _1 was maintained by sunlight plus meta-
halide lamps. A and E was seeds from previous generation while a and e was current level
of (CO2). Experiment was carried in three series. Three sets were made before drought,
after drought and after recovery.
3.13.1 Gaseous Exchange and Water Relations
Photosynthesis (An), stomatal conductance (gs) transpiration rate (Tr) and leaf
water potential (Yl) was measured with LI6400 apparatus and pressure chambers.
31
3.13.2 Yield and Yield Components
Harvesting was done at the physiological maturity of the crop and data of different
parameters like, number of spike number, grain per spike, thousand kernels weight, grain
yield, biological yield and harvest index were measured by using similar method as already
described for first experiment.
3.13.3 Key Enzyme of Carbohydrate Metabolism (C Enzymes) Assays
A series of c-enzyme and a-enzyme involved in carbohydrate metabolism were
measured from flag leaf and spikes of wheat. Frozen plant material was used and grounded
in liquid nitrogen, also add 0.1% polyvinyl polypyrrolidone (PVPP). Homogenized
material centrifuged, pallet and supernatant was dialysed overnight against 20mM
potassium phosphate buffer (pH 7.4) at 4°C. Dialysed and cell wall both extract were shock
freeze in liquid nitrogen and stored at –20 °C.
For all kinetic enzyme activity assays UV-transmissive single-use microcuvettes
(Plastibrand®;Brand, Wertheim, Germany) was used in a total reaction volume of 200μl in
a spectrophotometer (U-3000; Hitachi, Tokyo, Japan). Assays were consequently made to
a 96-well micro titre plate format. For all measurement, aliquots (up to 25 μl) of the
different protein extracts were incubated in a plate reader (Ascent Multiskan; Thermo
Fisher Scientific) at 30°C for 40 min in UV-transmissive flat bottom 96-well plates (UV-
Star;Greiner Bio One, Kremsmünster, Austria) in a total reaction volume of 160μl with a
mixture of buffer components, substrate (s), auxiliary substance (s), and auxiliary enzymes
and absorbance at 340 nm was monitored throughout the entire period of incubation. All
assays were carried out in triplicate. For control reactions, substrate was neglected. The
change in absorbance per second during the linear phase of substrate conversion was used
as the basis for the calculation of specific enzyme activity in nkat g FW–1.
32
The activity of three types of invertases i.e cytoplasmic (CytInv), cell wall (CWInv)
and vacuolar (VacInv) invertases were examined based on the method of (Sung et al.,
1989). A flat bottom 96 well plate was selected (Sarstedt, Nümbrecht, Germany) and
extract up to 20μl were used. To calibrate curve, glucose standards (0–50 nmol) were used.
Sucrose was eliminated for the control reactions and all the measurements were carried out
in triplicate. The value of liberated glucose was determined by measuring the absorbance
at 405 nm in a plate reader (Ascent Multiskan; Thermo Fisher Scientific, Waltham, MA,
USA). Specific activities were expressed as nkat g FW–1.
Sucrose synthase (Susy) activity was determined by (Pelleschi et al., 1997) method.
Two reactions were performed; one was carried out by using 1mM UDP that detect the
cytInv and susy background activity. Second reaction was performed without 1mM UDP
to detect the cytInv background activity only. Susy activity was calculated by subtracting
cytInv background activity (2) from total activity (1).
For determination of UDP-glucose pyrophosphorylase (UGPase) and ADP-glucose
pyrophosphorylase (AGPase) activity, method of (Pelleschi et al., 1997) and (Appeldoorn
et al., 1999) were used respectively. For UDP-glucose, dialysed extract were used along
with 100 mM TRIS-HCl at pH 8.0, 5 mM MgCl2, 0.44 mM EDTA, , 0.1% BSA, 1.5 mMPPi,
2 mMUDPGlc, 1 mM NADP, 2 mM 3-PG, 0.432 U of PGM, and 1.28 U of G6PDH. For
AGPase activity dialysed extract and 100 mM TRIS-HCl at pH 8.0, 0.44 mM EDTA, 1.5
mMPPi, 5 mM MgCl2, 0.1% BSA, 2 mMADPGlc, 1 mM NADP, 2 mM 3-PG,0.432 U of
PGM, and 1.28 U of G6PDH.
Aldolase (Ald) activity was determined by method (Schwab et al., 2001). Dialysed
extract were used with 1 mMF1,6bisP, 1 mM EDTA, 5 mM MgCl2, 0.15 mM NADH, 0.48
U of TPI, and 0.8 U of GPDH in 50 mM TRIS-HCl at pH 8.0. For control reactions, F1,6
bisP was not used. For determination of fructokinase (FK) and hexokinase (HXK) standard
33
methods were used (Appeldoorn et al., 1999; Petreikov et al., 2001). For FK dialysed
extract with 5 mM fructose, 5 mM MgCl2, 2.5 mMATP, 1 mM NAD, 0.8 U of PGI, and
0.8 U of G6PDH in 50mM BisTris at pH 8.0. For HXK activity, similar method was used
as used for Fk. Only 5 mM glucose was used instead of 5 mM fructose.
For determination of phosphofructokinase (PFK) activity, method of (Klotz et al.,
2006) was used. Dialysed extract were taken in 50mM TRIS-HCl , pH 8.0, 5 mM MgCl2,
1 mM F6P, 1 mM EDTA, 0.2 mM ATP, 0.16 U of aldolase, 0.15 mM NADH, 0.48 U of
TPI, and 0.8U of G6PDH. For phosphogluco isomerase (PGI) activity, dialysed extract with
4 mM MgCl2, 4 mM DTT, 2 mM F6P, 0.25 mM NAD, and 0.32 mM G6PDH and 100 mM
TRIS-HCl with pH 8.0 (Zhou and Cheng, 2008). Phosphogluco mutase (PGM) activity
was measured by using (Manjunath et al., 1998) method. For it, ,4 mM DTT, 0.1 mM
G1,6bisP, 10 mM MgCl2, 1 mM G1P, 0.25 mM NAD, and 0.64 U of G6PDH with plant
extract in 20 mM TRIS-HCl at pH 8.0.
For determination of glucose-6-phosphate dehydrogenase (G6PDH) activity,
(Deschepper, 1982) method were used. Dialysed extract were used with, 1 mM G6P, 0.4
mM NADP in 100 mM TRIS-HCl at pH 7.6 and 5 mM MgCl2. The increase in absorbance
at 340 nm in all kinetics except phosphofructokinase (PFK) and Aldolase (Ald) due to
conversion of NADP to NADPH was monitored.
34
3.13.4 Key Enzyme of Carbohydrate Metabolism (A Enzymes) Assays
Activity of SOD was measured by method (Beauchamp and Fridovich, 1971).
Dialysed extract were incubated with buffer (50mMKPO4 PH 7.8 and 0.1mM EDTA)
along with 0.05 Cytochrome c and 10mM Xanthine. For control Xanthine were omitted.
Activity of CAT was determined by (Aebi, 1984). Dialysed extract were incubated with
(50mM buffer, AF 2040.11% and 9.8mM H2O2). For control H2O2 were not used. Activity
of APX were determined by (Nakano and Asada, 1981). Dialysed extract were incubated
with (50mM buffer, 50 mM ascorbate and 10mMH2O2). For control reaction H2O2 were
eliminated. Glutathione reductase (GR) activity was measured by using method (Edwards
et al., 1990). Dialysed extract were incubated (100Mm Thris Hcl pH 7.8, 0.2mM NADPH
and 0.6mM Glutathione oxidized (GSSG). For control GSSG were omitted.
The grounded material was put into a 10 ml centrifuge tube, where 5 ml of 80%
ethanol was added. The pellets were extracted two more times with 80% ethanol.
Supernatants were retained, combined and stored at − 20 °C and further soluble sugar were
determined. For starch determination ethanol-insoluble pellet was used. Glucose was used
as a standard. Concentration of soluble sugars and starch was expressed on a dry matter
basis. Total soluble sugar and starch concentration were sum up and then concentration of
non-structural carbohydrates was obtained.The analysis was made on HPLC with aminex
87H column at 37oC and 600 ml/min.
3.14 STATISTICAL ANALYSIS OF DATA
Microsoft Excel 2002 (Microsoft Corp., Redmond, WA, USA) was used for
statistical calculations and descriptive statistics were applied to organize and analyze the
data. Triplicate data were used; Factorial analysis were used to analyze data significance of
data was tested y Tucky’s test (Tukey, 1949). Values presented in table, graphs are mean
35
±SE; bars with different alphabets differ significantly from each other. For second
experiment four replications were used and microsoft excel was used for analysis. Three
ways ANOVA was used to see the differences.
36
Chapter 4
RESULTS AND DISCUSSION
Five wheat cultivars viz. AARI-11, Chakwal-50, Shahkar, Pakistan-13 and FSD-08
have been used to have a look at the impact of drought and to manage drought with
hormonal seed priming. Priming was done by exposing seeds of five genotypes in 10-4 M
aerated solution of SA and GA for 8h, non-primed seeds was also used. Following findings
were observed about seed biochemistry, yield attributes, leaf biochemical and
Physiological attributes and seed quality attributes.
4.1 SEED BIOCHEMISTRY
In this section of study, pre sowing treatments with plant growth hormones induced
biochemical changes in wheat seeds were investigated with main emphasis on different
oxidative enzyme modulations i.e. protease, amylase, esterase, superoxide dismutase,
peroxidase, catalase. Further we can use these biochemical markers for screening against
different stresses.
4.1.1 Oxidative Enzymes Assay
4.1.1.1 Protease activity
All genotypes depicted significant variation under normal and primed condition
regarding protease activity (Fig. 4.1). The highest Protease activity was found in Shahkar
(5965±285µM/min/g f.wt.) and the lowest Protease activity (3395±115µM/min/g f.wt.)
was found in AARI-11 genotypes in absence of any priming treatment. Shahkar also
expressed the highest Protease activity when primed with SA while AARI-11, Pakistan-13
and FSD-08 genotypes were lowest protease activity. Significant increase in protease
activity (7275±75µM/min/g f.wt.) was observed in Chakwal-50 and Shahkar
(9110±20µM/min/g f.wt.) on SA priming. FSD-08 and Pakistan-13 had the highest
37
protease activity on GA priming while other genotypes remain unaffected. Seed priming
with GA showed that Shahkar (10325±345µM/min/g f.wt.) performed better results than
AARI-11 (4015±25µM/min/g f. wt.) in ranking of enzyme activity.
Figure 4.1: Effect of priming on protease activity in wheat seeds; Np-non primed, SA-Salicylic acid and
GA-Gibberellic acid
Under water stress many antioxidant enzymes were induced among them proteases
were also induced (Bray, 2002; Carvalho et al., 2001). When plants were exposed to stress
intracellular proteases have an ability to degrade injure or unwanted proteins, metabolism
reorganization and also help in remobilization of nutrient (Feller et al., 2008). It was crucial
for the researchers to recognize the linking mechanism among proteolysis and plant concert
in water stress and remedies from stress. It was still not understandable that under stress
high proteolytic activity is beneficial for the plant to help in restructuring of protein model
or it leads to cell breakdown. Some investigational facts suggest that proteolytic activity
was maximum in drought sensitive species and varieties compared to resistant ones (Hieng
et al., 2004).
4.1.1.2 Amylase activity
All tested genotypes depicting the significant variations under normal and primed
condition (Fig. 4.2). In control seeds was present in FSD-08 had (11.69±1.50mg/g. f.wt.)
fgef
de
gfg
g
c
b
g gg
cd
a
cdde
0
2000
4000
6000
8000
10000
12000
AARI-11 Chakwal-50 Shahkar Pakistan-13 FSD-08
Pro
teas
e (µ
M/m
in/g
f.
wt) NP SA GA
38
the highest amylase activity followed by Pakistan-13 (9.24±0.943) and AARI-
11(9.24±0.189mg/g. f.wt.) genotypes. When priming with SA applied it enhanced amylase
activity in AARI-11, Shahkar and Pakistan-13 along with unaffected biochemical changes
in FSD-08 and Chakwal-50 genotypes was observed. Priming with GA also increased
amylase activity in all genotypes and double increased of enzymatic activity in Shahkar,
Pakistan-13 and AARI-11.
Figure 4.2: Effect of priming on amylase activity in wheat seeds
Literature in support of this study exhibited that when α-amylase activity increased
in the seeds accountable to analogous increase in non-reducing sugar level in the seeds by
chitosan priming (Farooq et al., 2006a). Previous findings showing that amylase and sugar
contents directly increased in primed rice kernels confirmed that seed priming either
induced the de novo synthesis or increases the activities of existing enzymes (Lee et al.,
2007). Seed germination is result of enzyme generation or enzyme activation essential for
the mobilization and degradation of seeds reserves (Subramani et al., 2011). During this
process α-amylase and proteases control starch digestion and protein digestion respectively
while the hydrolysis of different types of esters was done by esterase enzyme (Subramani
et al., 2011).
cdd d cd
cd
a
cdcd
ab
cd
bc
cd
abab
cd
0
10
20
30
40
50
AARI-11 Chakwal-50 Shahkar Pakistan-13 FSD-08
Am
yla
se (
mw
g/g
. f.
wt)
NP SA GA
39
4.1.1.3 Esterase activity
An increased esterase activity in priming with GA hormone was observed. Esterase
might be take part in metabolic process during seed germination and maturation of plants.
For growing embryos, esterase is responsible to release and provide stored food material
(Subramani et al., 2011). In recent study, among all genotypes under control and primed
seeds treatment esterase activity was slightly increased (Fig. 4.3). FSD-08 showed
significantly maximum esterase activity (1001± 7.86µM/min/g f.wt.) when primed with
GA. Seed germination is result of enzyme generation or enzyme activation essential for the
mobilization and degradation of seeds reserves (Subramani et al., 2011).
Figure 4.3: Effect of priming on esterase activity in wheat seeds
4.1.1.4 Superoxide dismutase activity
Superoxide dismutase (SOD) activity showed significant variability in tested
genotypes depicted the Shahkar at higher rank regarding SOD activity (18.41
±0.16units/buf used) while other genotypes had the same magnitude when non primed
seeds were observed (Fig. 4.4).Under seed priming with SA the highest SOD activity
(24.81±0.30units/buf used) showing genotype was Chakwal-50. Increasing trend in SOD
activity under treatment with SA was reported in Chakwal-50, AARI-11and Pakistan-13
while a decreased SOD activity was observed in FSD-08 and Shahkar genotypes. Under
b b bb bb b b b bb
b b ba
0
500
1000
1500
AARI-11 Chakwal-50 Shahkar Pakistan-13 FSD-08
Est
rase
(µ
M/m
in/g
f.
wt) NP SA GA
40
GA mediated seed priming AARI-11 genotype showed leading trend related to SOD
activity among all tested genotypes showing enhanced SOD activity.
Figure 4.4: Effect of priming on SOD activity in wheat seeds
To protect cell against oxidative and environmental stress SOD is consider as an
important enzyme. These findings support the previous research on seed priming and
increased superoxide dismutase activity in rice seeds (Xiaohuan et al., 2009). Due to seed
priming in Victoria and Victor seedlings, antioxidant enzymes i.e. Catalase, Superoxide
dismutase and Peroxidase showed increased activities (Zhang et al., 2007a).
4.1.1.5 Peroxidase activity
Significant variation was present in all tested genotypes; the highest peroxidase
(POD) activity (33533± 699 units/g f.wt.) was observed in FSD-08while the lowest POD
activity (9823± 1032 units/g f.wt.) was present inAARI-11 in non primed seeds(fig. 4.5).
The SA priming significantly leading position related to POD activity was enhanced in all
genotypes except FSD-08 which remain unaffected. In case of GA priming, POD activity
increased in all tested genotypes except FSD-08.
f ef
de
eff
abc ab
ef
abc
g
a
cd
abc
cd bcd
0
5
10
15
20
25
30
AARI-11 Chakwal-50 Shahkar Pakistan-13 FSD-08
SO
D (
unit
s/b
uf
use
d)
NP SA GA
41
Figure 4.5: Effect of priming on POD activity in wheat seeds
Increasing POD could significantly enhance seed tolerance to abiotic conditions
(Ansari and Sharif-Zadeh, 2012). The same results were reported on Berseem (Trifolium
alexandrinum L.) that depicted the improved activities of antioxidant enzyme like
superoxide dismutase, peroxidase and catalase in primed seeds as compare to those
untreated seeds (Rouhi et al., 2012).
4.1.1.6 Catalase activity
Catalase resists the cell towards the oxidative harm by using elimination of
unfastened radicals or ROS and consequently also quality of seed was improved (Yeh and
Sung, 2008). Under control condition, AARI-11 (3340±300units/g. f.wt.) and Shahkar
(3320±180units/g. f.wt.) had the same magnitude of CAT activity. Salicylic acid enhanced
the CAT activity in all genotypes (Fig. 4.6). In study of CAT activity AARI-11 genotype
had the leading position under both SA and GA mediated seed priming analysis while FSD-
08 had the lowest rank on CAT activity in SA seed priming analysis. In case of GA priming,
CAT activity significantly increased in all tested genotypes. (Ahmed et al., 2012)
investigated that catalase (CAT), protease, amylase and superoxide dismutase (SOD)
activates were improved when different priming agents were used. The ascorbate
peroxidase improvement in wheat is also associated with the seeds primed with salicylic
f fef
f
ab
abc bcd
ab a
bcd
defcde cde
cdef
cd
0
10000
20000
30000
40000
AARI-11 Chakwal-50 Shahkar Pakistan-13 FSD-08
PO
D (
Unit
s/g f
. w
t)
NP SA GA
42
acid and gibberellins in comparison to the non treated seeds (Tabatabaei, 2013).The
worldwide demand of wheat is increasing rapidly and may be exceed till 2050 up to 750
million tons (Mujeeb-Kazi, 2006).
Figure 4.6: Effect of priming on CAT activity in wheat seeds
Seed germination enhancement through priming is linked with incentive of
antioxidant enzymes activities (Afzal et al., 2011). The numerous valuable sound effects
of priming have been reported in various field crops including, sunflower, maize, soybean,
sugar beet and wheat (Kusvuran et al., 2014). Physiological feature of plants were enhanced
by seed priming through growth hormones under both drought and balanced conditions
with the increased activity of antioxidants i.e. CAT, POD, SOD and that protect the cell
adjacent to creation of free radical and prevent from oxidative stress (Eisvand et al., 2010).
4.1.1.7 Cluster analysis based on seed biochemistry attributes
Clusters analysis (CA) showed that genotypes possess similarity to each other form
main two groups (Fig 4.7). Group one and two further forms a similar cluster within group.
It was observed that the genotypes within group may be similar to each other while those
out of groups were not similar.
bcd
ef
ef
bcd
ef
f
ef
a bcd
e
ab bcd
e def
bc
bcd
e
bcd
e
ab
cd cdef
0
2000
4000
6000
8000
AARI-11 Chakwal-50 Shahkar Pakistan-13 FSD-08
CA
T (
Unit
s/g.
f. w
t)
NP SA GA
43
Figure 4.7: Dendogram derived from hierarchical cluster analysis of combined seed biochemical attributes
4.1.2 Conclusion
Priming with SA and GA increased the activity of oxidative and antioxidant
enzymes such as amylase, protease, catalase, superoxide dismutase and peroxidase.
However, genotypes response varied in term of different antioxidant enzymes. Shahkar had
the highest protease activity in primed seeds while AARI-11 had the highest amylase
activity. Similarly AARI-11, Shahkar and Chakwal-50 had the highest Superoxide
dismutase activity while Shahkar and Pakistan-13 had the highest peroxidase activity. By
using SA and GA as a priming agent abiotic stress of wheat plant can be overcome by
modulating the activities of antioxidants.
44
4.2 MORPHOLOGICAL ATTRIBUTES
This study was designed in order to discover the phenomenon for better wheat
growth under water deficit conditions. Significant variability was noticed for plant height
(cm) under optimum and water deficit growing conditions (Table 4.1). More height was
observed for AARI-11 (102.7±1.27cm) while Chakwal-50 was short stature (91.1±1.15cm)
under normal conditions in absence of priming. AARI-11, Chakwal-50 and Pakistan-13
respond more to plant height by SA priming while slight in case of GA priming, more
height was observed for Pakistan-13, Chakwal-50 and FSD-08.
Similar response was noticed for plant height under drought. Highest value of plant
height was recorded for AARI-11 (95.0±5.21cm) and lowest for Chakwal-50 (78.8±1.42
cm) in the absence of priming. Both SA and GA were helpful to maintain the plant height
under irrigated conditions however, under drought stress height was significantly reduced.
As lower plant height is suitable to obtain desirable yield.
Under both growing conditions a reasonable variability was observed for the
number of tillers (
Table 4. 2). In the absence of priming, tillers was increased in Shahkar under normal
and for Pakistan-13 under drought conditions (6.4±0.78 and 3.8±0.33) respectively.
Similarly, the lowest number of tillers was observed in drought conditions. By the
application of SA and GA slight increase in tillers number was observed for Pakistan-
13under normal with their individual effect (6.4±0.65) and (5.8±0.99) respectively under
while their combined effect was (5.9±0.4). Likewise, more number of tillers were recorded
for Pakistan-13 under drought conditions after their treatment with SA and GA (4.8±0.60
and 4.2±0.26) respectively and their combined effect was (4.2±0.26). The combined
analysis revealed that more effect of drought was observed during 2014.
45
Substantial variation for spike length (cm) was measured. Pakistan-13 expressed
more spike length under non-primed normal conditions (13.0±0.21cm) followed by AARI-
11, Chakwal-50 and FSD-08 (12.6±0.25cm), 11.2±0.15cm) and (11.0±0.27cm)
respectively while small spike length (10.3±0.22cm) was observed in Shahkar. SA caused
improvement in spike length in Shahkar, Pakistan-13 and AARI-11. Similarly, GA also
caused improvement in spike length but non-significant differences were observed in
Pakistan-13.Interestingly, Pakistan-13 showed the highest spike length (10.8±0.15cm)
under drought conditions without any priming (
Table 4. 3). However, increased spike length was observed for FSD-08
(10.8±0.21cm) and AARI-11 (11.2±0.19cm) by treatment with SA and GA hormones.
Conversely, spike length was significantly decreased under Drought however this effect
was overcome by their treatment with SA and GA. AARI-11 (11.950cm) was observed to
be tolerant line based upon its mean values under both conditions. The mean analysis of
both years a decreasing trend of spike length. Conversely, spike length was significantly
decreased under Drought however this effect was overcome by their treatment with SA and
GA. AARI-11 (11.950cm) was observed to be tolerant line based upon its mean values
under both conditions. The mean analysis of both years a decreasing trend of spike length.
46
Table 4. 1: Effect of seed priming and drought on plant height (cm) of wheat during 2014 and 2015; Np-non primed, SA-Salicylic acid and GA-
Gibberellic acid
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11 102.7±1.27g-I
103.9±0.81i
101.1±0.83g-i
102.6±0.60e
84.9±1.27a-c
85.4±2.04a-c
90.7±1.52cd
87.0±1.09c
94.92
Chakwal-50 91.1±1.15cd
94.6±0.92d
95.4±0.86
93.7±0.70d
78.8±1.42a
82.03±0.64a
82.3±1.32a
81.0±0.74a
87.40
Shahkar 95.6±1.44d-g
96.4±1.11d-h
96.0±1.44d-g
96.0±0.73d
79.0±1.07a
84.4±1.12a-c
80.6±0.44a
81.3±0.75a
88.70
Pakistan-13 101.0±0.72f-I
103.5±0.79hi
103.5±0.77i
102.6±0.50e
82.9±1.12ab
90.1±1.79b-d
85.8±1.24bc
86.2±1.05bc
94.47
FSD-08 96.5±0.56d-h
97.3±0.78ef-i
98.7±1.17fg-i
97.5±0.52d
80.7±2.07a
82.6±1.04a
84.6±1.08a-c
82.6±0.88ab
90.10
Years
2014 97.64 100.08 100.18 94.69 80.56
85.40 86.41 84.12 91.53a
2015 96.14 98.28 98.46 97.96 82.02
83.47
84.28
83.59 90.67a
Mean 96.89
99.18
99.27
98.61
81.29
84.94
85.32
83.69
91.15
47
Table 4. 2: Effect of seed priming and drought on number of tillars of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11 3.0±0.68a 4.3±0.84a 3.8±1.01a 4.7±0.48ab 3.1±0.48ab 3.3±0.22a 3.5±0.66a 3.3± 0.26a 4.067
Chakwal-50 4.6±0.36a 4.9±0.85a 4.7±0.51a 3.7±0.35a 2.9±0.22a 3.2±0.11a 3.5±0.71a 3.2±0.24a 3.519
Shahkar
6.4±0.78a 5.9±0.82 5.4±0.89a 4.7±0.45ab 3.5±0.15a 3.4±0.38a 2.9±0.28a 3.3±0.16a 4.039
Pakistan-13
5.7±0.62a 6.4±0.65a 5.8±0.99a 5.9±0.43b 3.8±0.33a 4.8±0.60a 4.0±0.37a 4.2±0.26ab 5.089
FSD-08 5.3±0.76a 4.9±0.76a 3.1±0.93a 6.02±0.45b 3.3±0.22a 3.9±0.65a 3.8±0.78a 3.7±0.33a 4.875
Years
2014 5.59 5.81 5.64 5.68 3.45 4.31 3.96 3.90 4.588a
2015 4.48 4.52 4.58 4.52
3.31 3.56 3.28 3.38 4.048a
Mean 5.04 5.15 5.11 5.10 3.38 3.94 3.62 3.64 4.37
48
The highest number of spikelets was recorded for Pakistan-13 under non-primed
(22.0±0.64), SA (22.3±0.47) and GA (22.8±0.41) hormones (Table 4.4). However, lowest
number spikelets were counted for Chakwal-50 under primed and non-primed conditions.
In contrast, FSD-08 showed the highest number of spikelet under drought non-
primed (18.8±0.27) and SA (19.6±0.20) treatment while Shahkar respond more to GA
under drought conditions (19.8±0.32). An increasing trend in number of spikelet was
observed by the application of priming except in FSD-08. Surprisingly, GA treatment
decreased the number of spikelets in comparison to control. Mean avalue of both growing
situations manifested that highest spikelet (20.52) was found in Pakistan-13 while the
lowest mean was recorded for Chakwal-50 (18.70). Like most of the studied traits have
same trend for both consecutive years (2014 and 2015).
Extrusion length (cm) also showed significant variation for all the genotypes under
normal and drought conditions. FSD-08 showed highest extrusion length (13.8±0.81cm)
under normal non-primed condition. For this trait Pakistan-13 exhibited higher extrusion
length (16.0±0.68cm and 16.5±0.40cm) for SA and GA treatments respectively. FSD-08
performed well under drought conditions and highest extrusion length (12.1±0.66cm and
2.6±0.68cm) were recorded for non-primed and GA treatment respectively. However,
Shahkar was more responsive to SA application and highest extrusion length was observed
in this genotype.
49
Table 4. 3: Effect of seed priming and drought on spikelength of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11
12.6±0.25e-h 13.7±0.35h 13.2±0.23h 13.1±0.19d 10.6±0.18bcd 10.3±0.25a-d 11.2±0.19de 10.7±0.14b 11.950
Chakwal-50
11.2±0.15de 11.0±0.22cd 11.7±0.37defg 11.3±0.16bc 8.8±0.23a 9.3±0.15ab 9.6±0.15abc 9.2±0.12a 10.300
Shahkar
10.3±0.22bcd 11.7±0.08d-g 11.6±0.26def 11.2±0.19bc 9.3±0.18ab 9.7±0.29abc 9.3±0.15ab 9.4±0.12a 10.350
Pakistan-13
13.0±0.21gh 13.3±0.20h 12.8±0.35fgh 13.0±0.15d 10.8±0.15cd 10.6±0.29bcd 10.71±0.14bcd 10.7±0.11b 11.900
FSD-08
11.0±0.27cd 11.5±0.24def 12.8±0.27fgh 11.8±0.23c 10.3±0.24bcd 10.8±0.21cd 10.8±0.30cd 10.6±0.14b 11.236
Years
2014 11.66 12.40 12.41 12.16 9.89 10.36 10.46 10.23 12.19a
2015 11.68 12.12 12.46 12.08 10.12 9.96 10.23 10.10 11.09a
Mean 11.67
12.26 12.44 12.12 10.00 10.16 10.34 10.17 11.14
50
Table 4.4: Effect of seed priming and drought on spikelets of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Over
all
mean NP SA GA NP SA GA
AARI-11 20.5±0.42d-
i 20.9±0.32e-i 21.5±0.32ghi 21.0±0.21b 18.5±0.46a-e 18.8±0.45a-f
18.9±0.38a-
f
18.7±0.23
a 19.88
Chakwal-50
17.9±0.41ab 19.8±0.73b-h 20.5±0.14c-i 19.4±0.37a 16.4±0.41a 18.6±0.52a-e
18.9±0.21a-
f
18.0±0.34
a 18.72
Shahkar 21.0±0.69e-
i 21.5±0.40ghi 21.4±0.42ghi 21.3±0.29b 18.0±0.47abc 19.1±0.26b-g
19.9±0.16b-
h
19.0±0.26
a 20.20
Pakistan-13 22.0±0.64e-
i 22.3±0.47f-i 22.8±0.41hi 21.4±0.302b 18.2±0.29a-d 18.7±0.18a-e
19.8±0.36b-
h
19.0±0.17
a 20.52
FSD-08 21.5±0.40gh
i 21.8±0.55hi 22.6±0.32i 21.9±0.26b 18.8±0.27a-f
19.6±0.20b
—h
18.6±0.32a-
e
18.9±0.22
a 20.20
Years
2014 20.92 21.74 21.88 21.51 18.74 19.96 20.11 19.94 20.05
a
2015 19.78 19.78 21.12 20.22 17.94 19.02 20.40 19.78 19.76
a
Mean 20.35
20.76 21.5 20.87 18.34 18.99 19.25 18.86 19.86
51
It was also observed that water deficiency significantly reduced the extrusion length
in all varieties. Mean value of drought and irrigated conditions exhibited that FSD-08 has
highest (13.61cm) extrusion length while Chakwal-50 had only 11.88cm. All the studied
genotypes showed non-significant variation for peduncle length under drought and normal
conditions. Pakistan-13 showed highest (34.5±1.16cm) while Chakwal-50 exhibited lowest
(30.5±0.80cm) peduncle length under normal and non-primed conditions. SA priming
slightly increased the peduncle length in all genotypes except in Shahkar. However, GA
Priming increased the peduncle length in all studied genotypes. However, FSD-08 and
Pakistan-13 performed well for peduncle length (31.7±0.49cm) under drought while lowest
length was recorded in Chakwal-50 (28.5±0.40cm). Even with the application of SA and
GA this trait was unaffected for all the genotypes. A small increase for peduncle length was
observed for AARI-11 and Chakwal-50 in non-primed genotypes. A significant reduction
in length of peduncle was found in all genotypes without water condition. Based upon the
mean data analysis for both
52
Table 4.5:Effect of seed priming and drought on extrusion length of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11 13.0±0.84a-e 15.1±1.03cde 14.9±0.77cde 14.3±0.53bc 10.1±0.56ab 11.5±0.58a-d 12.0±0.66a-e 11.2±0.38a 12.82
Chakwal-50
11.7±1.05a-d 13.1±0.72a-e 13.8±0.57a-e 12.9±0.48abc 9.7±0.35a 11.0±0.40abc 11.7±1.13a-d 10.81±0.43a 11.88
Shahkar
12.8±0.90a-e 14.6±0.75b-e 15.4±0.69c-e 14.32±0.50bc 11.5±0.56a-d 12.0±0.44a-e 12.4±0.71a-e 11.9±0.32ab 13.16
Pakistan-13 13.0±0.98a-e 16.0±0.68de 16.5±0.40e 15.20±0.54c 11.6±0.71a-d 11.9±0.83a-d 12.1±0.76a-e 11.9±0.42a 13.55
FSD-08 13.8±0.81a-e 15.3±1.14cde 15.9±0.63de 15.03±0.52c 12.1±0.66a-e 11.8±0.39a-d 12.6±0.61a-e 12.18±0.32ab 13.61
Years
2014 12.973 14.24 15.06 14.09 10.83 11.360 12.06 11.42 13.16a
2015 13.20 14.39 15.21 14.27 11.22 11.360 13.25 12.33 12.84a
Mean 13.09
14.31 15.13 14.18 11.03 11.36 12.30 11.75 12.96
53
Table 4.6: Effect of seed priming and drought on peduncle length (cm)of wheat during 2014 and 2015
Genotypes Control
Mean Drought
Mean Overall
mean NP SA GA NP SA GA
AARI-11 57.2±1.16f-j 59.3±0.71h-k 59.3±1.21g-k 58.6±0.62de 49.5±1.23 52.1±1.07 56.0±1.01 52.5±0.88bc 55.59
Chakwal-50 50.7±0.61b-f 55.3±0.82d-h 55.4±0.85d-h 53.839±0.67bc 43.6±0.68 48.3±0.99 49.4±0.92 47.1±0.77a 50.49
Shahkar 57.2±1.31f-j 56.5±0.62f-j 62.8±1.21jkl 58.8±0.90def 47.9±0.78 52.0±0.62 55.7±0.83 51.8±0.87b 55.37
Pakistan-13 62.3±1.24h-l 60.3±1.02h-l 64.9±1.12kl 62.5±0.76ef 55.6±1.07 54.6±1.52 59.4±1.71 56.5±0.93cd 59.82
FSD-08 60.7±1.99h-l 62.4±1.17i-l 66.0±1.09l 63.0±0.96f 53.1±0.59 57.2±0.87 59.3±0.86 56.5±0.75cd 59.55
Years
2014 59.47 59.36 62.84 60.56 50.38 53.98 57.10 53.16 57.86a
2015 55.82 58.24 60.58 58.21 49.58 53.74 54.89 52.73 55.47a
Mean 57.65 58.80 61.71 59.39 49.98 52.86 56.00 52.94 56.16
54
drought and irrigated Pakistan-13 was with highest mean value (33.39cm) and Chakwal-50
had lowest (31.90cm).
Also, significant variation was observed for grain numbers under normal and
drought conditions. Highest number of grains (62.3±1.24) was recorded for Pakistan-13
whiles the lowest for Chakwal-50 (50.7±0.61) under non-primed normal condition. SA and
GA improved this number in all studied genotypes, Pakistan-13 (55.6±1.07) showed
highest number of grains while Chakwal-50 (43.6±0.68) exhibited lowest value under
drought non-primed conditions. SA priming increased the grain number in all genotypes
except Pakistan-13. However, GA priming increased this number in all genotypes.
Significant reduction in grain number was observed under drought. However, mean data
analysis for drought and irrigated condition suggested that Pakistan-13 and FSD-08 are
superior varieties for this trait (59.0) while lowest value was recorded for Chakwal-50
(50.0).
55
Table 4.7: Effect of seed priming and drought on number of grains of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11 37.3± 0.95b-i 40.0±0.66f-i 39.8±1.22e-i 39.0±0.60cd 32.9±0.67abc 34.5±0.57a 34.3±0.74a-e 33.9±0.39a 36.52
Chakwal-50
35.3±0.78a-g 38.1±1.00b-i 38.3±1.32c-i 37.2±0.66bc 31.7±0.75a-f 32.7±1.04ab 33.5±0.76a-d 32.6±0.505a 34.98
Shahkar
40.6±0.82ghi 39.0±0.70d-i 39.3±1.44e-i 39.7±0.58cd 31.5±0.71a 34.9±0.67a-g 35.0±1.01a-f 33.8±0.59a 36.78
Pakistan-13
39.6±1.29e-i 41.0±0.82hi 41.6±0.60i 40.7±0.55d 34.3±1.04a-e 35.0±0.70a-h 35.6±0.82a-h 35.0±0.48ab 37.90
FSD-08
39.3±1.05e-i 39.7±1.10ghi 40.9±0.76hi 39.9±0.56cd 34.6±0.33a-f 36.9±0.91a-e 35.0±1.26a-g 35.2±0.51ab 37.62
Years
2014 40.01 40.99 42.44 41.55 34.65 36.45 37.43 36.51 37.25a
2015 35.50 37.35 38.95 37.27 31.17 33.86 34.64 33.89 36.27a
Mean 37.76
38.271 39.20 38.41 34.41 35.16 36.04 35.20 36.80
56
57
Significant variation was found for 1000-grain weight under both drought and normal
conditions (Table 4.). Shahkar and Chakwal-50 showed highest (40.6±0.82g) and lowest
(35.3±0.78g) 1000-grain weight respectively under normal and non-primed condition.
Priming with SA caused slight increase for this trait in AARI-11, Chakwal-50 and Pakistan-
13 while GA triggered more 1000 grain weight for Shahkar. In non primed samples under
drought 1000-grain weight was higher in Pakistan-13 (34.6±0.33g) and lowest in Shahkar
(31.5±0.71g). Treatment with both primers increased the thousand grain weight of all the
studied germplasm.
Like many agronomic traits Pakistan-13 exhibited highest (1910.1±30.79kg/ha)
grain yield under normal non-primed conditions while FSD-08 showed high output
(1583.5± 76.21kg/ha) under drought and non-primed conditions (Table 4.9). Lower grain
yield was recorded for Chakwal-50 under irrigated (1289.3±40.98kg/ha) and drought
(936.1± 26.70kg/ha) in non-primed conditions. However, SA priming increased the grain
yield in all genotypes under normal conditions. Likewise, priming with this growth
regulator was also effective for all genotypes under drought
Also GA priming was effective to increase the grain yield under irrigated conditions
but under drought it was effective, however FSD-08 showed non-significant differences.
Mean values of normal and drought conditions suggested that Pakistan-13 performed well
(1764.77kg/ha) while Chakwal-50 was more severely affected (1189.66kg/ha).
58
Table 4.8: Effect of seed priming and drought on thousand grain weight of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11 37.3± 0.95b-i 40.0±0.66f-i 39.8±1.22e-i 39.0±0.60cd 32.9±0.67abc 34.5±0.57a 34.3±0.74a-e 33.9±0.39a 36.52
Chakwal-50 35.3±0.78a-g 38.1±1.00b-i 38.3±1.32c-i 37.2±0.66bc 31.7±0.75a-f 32.7±1.04ab 33.5±0.76a-d 32.6±0.505a 34.98
Shahkar 40.6±0.82ghi 39.0±0.70d-i 39.3±1.44e-i 39.7±0.58cd 31.5±0.71a 34.9±0.67a-g 35.0±1.01a-f 33.8±0.59a 36.78
Pakistan-13 39.6±1.29e-i 41.0±0.82hi 41.6±0.60i 40.7±0.55d 34.3±1.04a-e 35.0±0.70a-h 35.6±0.82a-h 35.0±0.48ab 37.90
FSD-08 39.3±1.05e-i 39.7±1.10ghi 40.9±0.76hi 39.9±0.56cd 34.6±0.33a-f 36.9±0.91a-e 35.0±1.26a-g 35.2±0.51ab 37.62
Years
2014 40.01 40.99 42.44 41.55 34.65 36.45 37.43 36.51 37.25a
2015 35.50 37.35 38.95 37.27 31.17 33.86 34.64 33.89 36.27a
Mean 37.76
38.27 39.20 38.41 34.41 35.16 36.04 35.20 36.80
59
Table 4.9.: Effect of seed priming and drought on grain yield(kg/ha) of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11 1696±42.2i-l 1636±48.31-l 1632±59.7h-l 1654±28.3d 1220±30.13a-e 1227±31.6 a-f 1388±18.7e-i 1278±23.9b 1466.8
Chakwal-
50 1289±40.9b-h 1368±42.2c-l 1458±68.3d-i 1372±32.8bc 936±26.7a 987±27.3 ab 1097±21.1abc 1007±21.3a 1189.6
Shahkar 1533±56.7e-j 1641±46.6i-l 1659±63.1i-l 1611±33.1d 1146±42.7 a-d 1174±75.1a-d 1278±48.6a-g 1199±34.0b 1405.5
Pakistan-13
1910±30.7kl 1917±87.2kl 1911±60.5kl 1912±34.6e 1463±144.8d-i 1579±35.5 g-k 1648±35.1i-l 1616±29.8d 1764.7
FSD-08 1849±26.3g-l 1947±36.7l 1935.1±37.2l 1910±21.1e 1583±76.2 g-k 1618±40.0g-l 1571±108.2f-k 1537±59.1cd 1724.1
Years
2014 1719.7 1754.2 1742.8 1738.9 1384.1 1374.0 1475.8
1411.3 1566.2
2015 1531.6
1563.6
1595.5
1563.5
1108.9
1227.2
1311.1
1215.7 1454.1
60
The highest biological yield was recorded in FSD-08 under non-primed normal
(5873.3±119.44kg/ha) and drought (5120.1±95.15kg/ha) conditions however, all the
genotype varied significantly. Lowest biological yield was recorded for Chakwal-50 under
irrigated and drought conditions (4983.0±64.11kg/ha and 4579.8±75.74kg/ha respectively)
without any treatment. Priming with SA and GA revealed the increase in biological yield
in most of genotypes. All the genotypes were responsive to SA priming under drought
except FSD-08 however; GA priming was effective for all genotypes. Combined results for
stress and without stress exhibited that FSD-08 was with more biological yield
(5498.11kg/ha) and Chakwal-50 was with less yield (4859.83kg/ha).
Non-primed seed of Pakistan-13 performed well under well watered (31.8±1.18%)
and drought (27.0±2.32%) situations while Chakwal-50 was again at bottom for irrigated
drought conditions (24.4±0.74%) and (18.8±0.41%) respectively. Priming with SA
increased in AARI-11and Chakwal-50 under both conditions. Mean analysis of drought
and irrigated condition suggested that FSD-08 (29.350%) is a superior variety while
Chakwal-50 could not perform well (23.24%). Based upon all yield contributing traits 2014
was better year as compared to 2015though the results were non-significant. Likewise,
drought affected these entire traits more severely during 2015.
Amongst testing cultivars in non primed samples, AARI-11 had the highest
plant height under stress and without stress conditions. The consequences suggested on
this experiment also matched with the findings in literature that when GA3 was applied on
fenugreek as a foliar spray it significantly
61
Table 4.10: Effect of seed priming and drought on biological yield (kg/ha) of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overa
ll
mean NP SA GA NP SA GA
AARI-11 5827±87.3h-i 5709±128.5ghi 5949±125.5i 5829±66.9f 4709±66.8a-c 4762±97.2a-d 4896±59.8a-f 4789±45.6ab 5309.1
Chakwal-50 4983±64.1a-f 5166±46.5e-g 5487±186.3f-i 5212±81.0cd 4579±75.7a-c 4610±58.0a 4631±48.5ab 4507±37.7a 4859.8
Shahkar
5325±81.3d-h 5456±57.0f-i 5345±80.0d-h 5375±42.3de 4846±77.2a-e 5031±78.5b-f 5068±76.1b-f 4981±48.1bc 5178.8
Pakistan-13
5789±71.1hi 5813±132.5hi 5426±143.4e-i 5676±77.9ef 5076±103.1b-f 5134±89.3c-g 5225±62.6b-g 5111±47.4cd 5394.1
FSD-08 5873±119.4hi 5948±189.2i 5864±109.1hi 5895±78.4f 5120±95.1b-g 5078±59.9b-f 5103±69.0b-f 5100±41.5cd 5498.1
Years
2014 5670.80 5753.13 5706.00 5709.9 5094.80 5180.00 5272.53 5115.77 5303.9
2015 5288.93 5184.80 5363.26 5279.0 4810.93 4853.66 4930.93 4831.84 5192.0
Mean
5479.86
5468.96 5534.63 5494.4 4831.84 5215.77 4951.73 4954.86 5224.6
62
Table 4.11: Effect of seed priming and drought on harvest index (%)of wheat during 2014 and 2015
Genotypes Control Mean Drought Mean Overall
mean NP SA GA NP SA GA
AARI-11 27.0±0.57d-h 28.6±0.69fh 27.7±0.29d-h 27.8±0.33c-e 22.5±0.52 24.2±0.43b-f 20.8±0.27a-c 22.5±b0.40 25.20
Chakwal-50 24.4±0.74c-h 25.2±1.20d-h 25.0±0.34c-g 24.9±0.46bc 18.8±0.41a 20.5±0.24a-c 21.3±0.06ab 19.5±0.23a 23.24
Shahkar
28.5±0.29e-f 28.8±0.10f-h 28.7±0.62f-h 28.6±0.22d-f 20.8±0.27b-f 22.0±0.17a-c 24.4±0.50a-c 22.1±0.43ab 25.40
Pakistan-13 31.8±1.18h 28.9±0.70f-h 29.2±1.06 29.9±0.62ef 27.0±2.32d-h 28.3±0.85a-e 30.1±0.54gh 26.9±0.79cd 29.35
FSD-08
30.9±1.10h 31.2±1.22g-h 31.5±0.99 30.9±0.61f 23.3±0.85d-h 27.3±1.78d-h 28.9±1.10fgh 27.7±1.0cde 28.48
Years
2014 27.87 29.24 30.31 29.14 21.34 24.27 25.6 23.7 26.61a
2015 26.27 28.52 29.61 28.13 20.47 23.28 23.79 22.52 25.66a
Mean 27.07 28.88 29.96 28.64 20.91 23.783 24.70 23.13
25.88
63
increased plant height, branches and grain yield. The present work revealed a lessening in
the number of tillers in one plant, length of the spike, spikelets and a reduced thousand
kernel weight due to drought stress in comparison to the control, illustrating the effect of
water deficiency on wheat cultivars.
Preceding researchers discovered enormous decreased in spike length (m-2),
grain/spike and thousand kernel weight in a study on twelve wheat cultivars under drought
strain.Phloem translocation relies upon the turgor pressure and the water potential. It has
been observed that water deficiency reduce the capability of phloem which leads to reduced
grain yields. Furthermore, water pressure at the anthesis affected the grain filling rate that
induced reduction of grains per spike in comparison to different treatments. Literature
supported our findings that Plant regulators, specifically GA
have crucial function in improvement of wheat yield (Bari and Jones, 2009),
photosynthesis capacity, delay in leaf senescence and elevated seed quantity in wheat under
stress (Zheng et al., 2011).Drought stress remarkably affected the grain weight like plant’s
other morphological characteristics. Present study found that the wheat variety Shahkar
had the highest thousand kernels weights among all the tested varieties of wheat, which
shows it’s less sensitivity to drought strain (Table 4.8). But AARI-11, Chakwal-50 and
Pakistan-13 showed improvement of the trait under priming with SA and GA.
Decreased grain weight is caused by the moisture shortage at grain filling stage, as it
leads to unusual grain formation. Decline in Photosynthesis, reduced rate and duration of
grain development under drought is a leading cause of yield reduction (Pandey et al., 2001).
64
Keeping in picture the depth and duration of drought stress, a
pronounced drop in 1000-grain weight in wheat was recorded owning to the shortening of
grain filling period. The period of grain filling was shortened by water insufficiency after
anthesis, leading to endosperm desiccation and constraining embryo quantity (Gooding et
al., 2003). Similar behavior was observed by other researchers, reporting the reduced grain
weight due to drought at grain filling stage (Sharafizad et al., 2013).
Priming with SA and GA magnified the wheat potential in producing a better TKW.
Results were accordance to (Dawood et al., 2012) who informed that in sunflower and
mung-bean1000-kernel weight was increased through utilizing salicylic
acid. Drought strain disorder the absorption and translocation of photosynthetic assimilates
and consequently alters and reduce the yield components (Moghadam et al.,
2011). Salicylic acid priming and extended yield are related to the early flower
development, maximum flowers and greater number of pods (Kulshrestha et al., 2013).
Primed plants are capable of producing better yields than untreated seeds (Harris et al.,
2005). Salicylic acid is capable of boosting up the growth and yield of crop in either case,
stress or without stress(Moghaddam et al., 2011). Wheat yields are boosted up by Salicylic
acid (Shakirova et al., 2003). SA influences the plants responses, increase the translocation
of assimilates from leaf to grain that boost in yield and parameters of yield (Dawood et
al., 2012). FSD-08 had a better straw yield representing its better tolerance to drought
than the other examined cultivars. The reduced biological yield of the other genotypes was
compensated by priming with SA or GA. It was well documented in literature that in wheat
more biomass reduction occur in stress (Shamsi and Kobraee, 2013).
The extended biological yield might be due to the promising effects of
hormonal priming on biomass, number of seedlings and on plant nutrition (Zhang et al.,
65
2007b). Primed seeds have greater biomass and dry weights as compared to non-primed
(Rashid et al., 2002). Biological yield seems increased under normal conditions is due to
the precise vegetative growth. The broad leaf surface is the cause of higher photosynthetic
activity and therefore better organic yield. The genotype (Pakistan-13 and FSD-08) with
better grain yield showed highest harvest index. This is in accordance to the study of
(Reynolds et al., 2009)who reported that grain yield and harvest index is highly affiliated.
It was found that harvest index is markedly affected by the water shortage (Galavi and
Moghaddam, 2012) at anthesis stage.Distribution of
photosynthetic assimilatesamongst plant elementsis determined with the aid of harvest
index, drought stress reduced assimilates translocation into the grains and results in
reduced harvest index. Many researchers confirmed the lowered photosynthetic activity
due to decreased soil moisture, and in turn decreased translocation of assimilates to the
grain. In water deficit soil photosynthesis reduction cause assimilate to remobilize from
source to sink (Asseng and Van Herwaarden, 2003;Plaut et al., 2004). Drought at
specific growth stages in wheat plant contributes to reduce grain yield, organic yield,
harvest index and other yield contributing parameters (Harris et al., 2005).
66
Table 4.12: STI % of wheat genotypes based on morphological parameters across both years and priming
Genotypes Seedpr
iming
Plant
height
Tiller
s
Spikeleng
th
Spikelet
s
Extrusi
on
Peduncle Grains Grain
weight
Grain
Yield
Bio
Yield
Harv
est index
AARI-11 NP
82.69
62.7
1 84.26 90.04 78.03 93.88 86.49
88.2
5 71.95 80.80
83.2
7
SA
86.57
55.7
9 79.20 80.52 83.38 93.56 80.12
87.0
1 67.38 81.88
77.0
1
GA
82.61
61.8
8 90.18 85.88 89.77 90.33 83.67
77.6
5 74.74 91.00
85.5
9
Chakwal-
50
NP
82.08
74.2
9 82.68 87.06 88.66 91.85 89.36
86.8
0 76.59 87.67
86.1
1
SA
83.66
57.5
1 93.83 87.76 87.27 94.07 87.59
88.2
8 85.64 87.18
87.2
6
GA
82.17
63.2
1 75.33 89.59 75.96 94.54 87.84
86.3
1 74.99 83.40
84.6
6
Shahkar NP
86.65
74.9
0 84.29 94.12 84.12 95.53 87.38
83.2
2 72.13 85.36
81.6
5
SA
87.62
69.7
3 82.70 88.79 82.23 93.69 92.07
89.4
7 71.52 92.21
73.0
2
GA
87.05
81.2
3 80.20 88.03 74.61 90.00 90.47
85.4
4 82.37 88.33
80.6
8
Pakistan-
13
NP
84.90
62.0
8 93.78 89.92 77.12 86.23 91.57
90.5
7 83.11 85.38
90.2
1
SA
89.73
71.8
1 84.87 87.91 80.89 89.38 94.44
86.0
5 85.06 82.30
75.1
9
GA
86.34
72.6
0 82.10 92.20 84.96 90.53 89.18
87.5
0 75.27 82.59
77.2
1
FSD-08 NP
83.97
62.4
6 80.34 92.94 80.09 88.26 88.65
89.0
0 77.08 94.81
72.6
9
SA
82.93
62.0
2 83.62 90.02 73.66 86.64 91.55
85.7
9 82.21 94.45
96.1
0
GA
85.69
70.9
4 84.53 82.47 79.48 92.88 89.90
85.6
3 85.16 87.02
91.7
0
67
Table 4.13: Combine STI of wheat genotypes based on morphological parameters across both years
4.2.1 Cluster analysis based on Morphological attributes
The CA sequestrates genotypes into clusters which exhibit high homogeneity within
a same cluster. Shahkar-50 and AAR1-11 form same cluster while Pakistan-13 and FSD-
08 form same cluster and Chakwal-50 varied for other form different cluster (Fig 4.8).
Figure 4.8: Dendogram derived from hierarchical cluster analysis of all combined morphological attributes
of five wheat genotypes
AARI-11 84.83 70.43 81.37 89.16 78.29 92.54 89.63 86.84 77.26 82.16 81.06
Chakwal-50 86.52 86.32 81.85 82.39 84.19 93.18 87.58 85.28 73.39 86.48 74.63
Shahkar 84.74 69.07 84.18 89.23 83.71 90.72 88.13 87.68 74.45 92.67 77.08
Pakistan-13 84.04 70.74 82.14 88.40 78.30 89.42 90.48 86.00 80.39 90.06 89.88
FSD-08 84.76 61.90 90.44 86.66 81.08 91.01 89.71 88.13 84.62 86.52 89.74
68
4.2.2 Conclusion
Among genotypes it was concluded that FSD-08 and Pakistan-13 were top ranking.
Based on above results and discussion it is cleared that some genotypes have good tolerance
under stress. On the basis of this experiment we conclude and can recommended that
priming along with growth regulator overcome the coming environmental issues such as
drought. Temperature, humidity and environmental fluctuations were also responsible for
reduction in yield.
4.3 BIOCHEMICAL AND PHYSIOLOGICAL ATTRIBUTES
The rationale of this section of study was to explore the physiological and
biochemical mechanism of wheat genotypes along with growth regulators and drought
stress, as a result we are able to screen the suitable genotype to drought stress. Completely
emerged leaves (flag) were selected for biochemical and physiological analysis. This phase
is mainly considerable for the reason that flag leaf contribute grain yield directly make up
around 75% of effective leaf area.
4.3.1 Oxidative Enzymes
4.3.1.1 Estrases activity
Under normal condition, Shahkar (535 ±24.72µM/min/g f.wt.) had the maximum
esterase activity while Chakwal-50 had the lowest esterase activity (416±11.23µM/min/g
f. wt.) in non primed samples (Fig. 4.9). Esterase activity was increased notably upon
priming with SA in FSD-08 and Pakistan-13. When samples with GA priming were
studied, esterase activity was amplified significantly in Pakistan-13and Chakwal-50.
Under drought stress, FSD-08 had the highest esterase activity (778 ± 22.47µM/min/g f.wt.)
while AARI-11 had the lowest esterase activity (501± 11.23 µM/min/g f. wt.) in non
primed samples. Esterase activity becomes low on SA priming in Pakistan-13. When
69
stress was imposed esterase activity significantly increased in all genotypes. Priming with
SA increased the esterase activity in stress condition than well watered in AARI-11 and
Chakwal-50 while lower activity in Pakistan-13. When priming with GA was observed,
esterase activity amplified under drought inPakistan-13 while decreased in Chakwal-50.
Figure 4.9: Estrase activity in flag leaves of wheat genotypes grown under control and drought stress
4.3.1.2 Amylase activity
Under normal condition, Shahkar had the highest amylase activity
(17.73±2.26mg/g. f.wt.) while FSD-08 had the lowest amylase activity (2.45±0.18 mg/g.
f.wt.) in non primed samples (Fig. 4.10). Amylase activity was decrease considerably upon
priming with SA in AARI-11 and Chakwal-50 while increased in Pakistan-13, Shahkar and
FSD-08 under normal condition. When GA primed samples were analyzed, amylase
activity was increased appreciably in FSD-08 and Chakwal-50 while reduced in Shahkar.
During drought stress, FSD-08 had the maximum amylase activity (37.17±1.69 mg/g. f.wt.)
while Shahkar had the lowest amylase activity (4.52±0.75 mg/g. f.wt.) without any
priming. Amylase activity was significantly raised on SA priming only
efgh
ij
defg
hij
gh
ij
gh
ij
b
efg
bc bfg
hi
defg
bcd
a
bc efg
b
j
gh
ij
fgh
i
cde
b cdef
defg h
ij ij
efgh
fgh
i
b defg
0200400600800
10001200
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
Control Drought
Est
rase
act
ivit
y (
unit
/µM
/min
/g f
. w
t) NP SA GA
70
AARI-11and Shahkar decrease in Pakistan-13. Regarding GA priming samples,
increased amylase activity was present in all experienced cultivars except AARI-11.
Amylase activity was significantly increased under drought stress in Pakistan-13 while
lowered in Shahkar. Amylase activity was increases in SA treated samples under drought
in Pakistan-13, Chakwal-50 and AARI-11. Amylase activity also increased in term of GA
priming under drought except AARI-11.
Figure 4.10:Amylase activity in flag leaves of wheat genotypes grown under control and drought
stress
In cereal crops amylases take part in the breakdown of starch. It is clearly depicted
from literature that in primed wheat seeds amylase activity was uplifted as compared to
none primed. Previously, enhanced amylase activity has been reported in rice seeds that are
subjected to priming; however amylase activity varied among different priming treatments
(Farooq and Azam, 2006). Our findings were supported by previous literature. In many
areas of the world due to striking climate-change scenarios like rise in aridity main focus
of the researcher is on plant reaction to water stress. Several physiological and biochemical
changes have been extensively studied previously while, vegetation are expose to water
defg
hijk
fgh
ijk
cdefg
hi
hijk
k
defg
hijk
ijfgh
ijk
ijk
efgh
ijk
b
jk jk
bcd
e
bc
def
bcd
bcd
ef
a
cdefg
cdefg
hi
defg
hijk
bcd
efg gh
ijk
jk
cdefg
defg
hijk
a
a a
a
0
10
20
30
40
50
60
70
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
Control Drought
Am
yla
se a
ctiv
ity (
unit
/mg/g
. f.
wt)
NP SA GA
71
deficit (Passioura, 2007). The retort of plants to water stress is multifaceted and concerned
with alteration in their metabolism, physiology and morphology.
4.3.1.3 Protease Activity
Without stress condition, FSD-08 had the maximum protease activity
(6945±65.00g.f.wt.) while AARI-11 had the lowest protease activity (5385±75.00g. f. wt.)
in non primed samples (Fig. 4.11). Upon SA priming Protease activity significantly
decreased in Pakistan-13 while increased in Shahkar. When we see GA priming samples,
protease activity was improved notably in Shahkar while lessened in FSD-08. When stress
was imposed, Pakistan-13 had the maximum protease activity (6265±45.00g. f. wt.) while
other genotypes have same dimension in non primed samples. When SA priming was
applied protease activity was significantly higher only in Shahkar. Drought stress
significantly increased the protease activity in Pakistan-13 while other genotypes remain
unaffected. SA priming decreased the protease activity under drought in AARI-11 and
FSD-08. Priming with GA decrease protease activity in normal as well as when stress was
imposed in all tested genotype.
Figure 4.11: protease activity in flag leaves of wheat genotypes grown under control and drought stress
de
efgh
i
cde
cde
ab efg
h
ef
efgh
ij
ab
cdef
efgh
efg
ab
c de
ab
efg
hij hij
ab fg
hij
efg
bcd
cd
e
ab
bcd cd
e
fgh
ij ij j
gh
ij
hij
02000400060008000
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
Control DroughtPro
teas
e ac
tivit
y (
Unit
s/g.
f.
wt)
NP SA GA
72
Under water stress many antioxidants are induced among them proteases was also
induced (Cruz De Carvalho et al., 2001; Bray, 2002). When plants are exposed to stress
intracellular proteases take part in the degradation of injured or unwanted proteins,
metabolism reorganization and also help in remobilization of nutrient (Feller et al., 2008).
It is crucial for the researchers to recognize the mechanism and relation between proteolysis
and plant concert in water stress and remedies from stress. It is still not understandable that
under stress high proteolytic activity is beneficial for the plant to help in reorganization of
protein pattern or it leads to cell breakdown. Some experimental facts suggest that
proteolytic activity was maximum in drought sensitive species and varieties compared to
resistant ones (Hieng et al., 2004). Therefore, it is important to study the mechanism of
drought resistance of plant species in order to improve their biochemical and physiological
characters to facilitate developing cultivar with increased resistance. SA and GA played a
significant role in the protective response to stresses in different plant species (Shakirova
et al., 2003; Srivastava and Srivastava, 2007).
4.3.1.4 Superoxide dismutase activity
Without stress, Shahkar and AARI-11 has same superoxide dismutase (SOD)
activity while FSD-08 and Pakistan-13 has same activity in non primed samples (Fig. 4.12).
SOD activity was non significantly affected by when priming agent were used. Under the
condition of drought stress, Chakwal-50 had the highest SOD activity
(20.51±3.40unit/gf.wt.) while FSD-08 had the lowest SOD activity (16.96± 1.349
unit/gf.wt.) in non primed samples. SOD activity was notably increased AARI-11 and FSD-
08 when SA priming was applied while there was no effect in other genotypes. SOD activity
was increased in AARI-11 and FSD-08 when GA priming was applied. When stress was
imposed SOD activity increased in all genotypes as compared to without stress. SOD
activity was increased upon priming with GA and SA under drought as compared to well
73
watered in all tested genotypes. The SOD activity is conscientious for scavenging free (O2–
) radical to produce H2O2, greater than before within three drought days at tillering stage in
both drought susceptible and drought tolerant cultivars (Bano et al., 2012).
Figure 4.12: SOD activity in flag leaves of wheat genotypes grown under control and drought stress
It is accepted that H2O2play an chief role in signal transmission networks. Lot of
stress reactions involve H2O2, due to catalase regulating functionsand scavenging,the
homeostasis of H2O2 is maintained. Therefore, SOD activation may be triggred by H2O2
accumulation. So, to tolerate stress for adaptation and survival the balance between ROS
and the antioxidative system is necessary. Under water stress, some cultivar showed higher
membrane stability index and SOD activity. Drought tolerant genotype has higher
membrane stability and SOD activity (Sairam and Saxena, 2000). It also demonstrates that
under drought stress physiological parameters like proline, RWC and SOD activity could
be used as criteria for ranking of varieties for drought tolerance.
4.3.1.5 Peroxidase Activity
Under normal condition, AARI-11 has the highest peroxidase activity
(30003±323unit/g f.wt.) while Pakistan-13 had the lowest POD activity (20146 ± 209
unit/g f.wt.) in non primed samples (Fig. 4.13). Peroxidase activity was decreased upon
priming with SA in AARI-11 though improved in Shahkar and Pakistan-13. In AARI-11
and Pakistan-13 with GA priming POD activity was increased. Under drought stress, FSD-
bcd
e
bcd
e
bcd
e
de
de
ab
c a
ab
cd
ab
ab
cd
bcd
e
bcd
e
ab
cd
bcd
e de
a a
ab
cd
ab
cd a
cde
cde
bcd
e
e
bcd
e
a
ab
cde
ab
cde
ab
cd
a
05
1015202530
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
Control Drought
SO
D a
ctiv
ity (
unit
/gfr
esh
wei
ght)
NP SA GA
74
08 had the highest POD activity (37529±123unit/g f.wt.) while Pakistan-13 has the lowest
POD activity (21078±183unit/g f.wt.) in non primed samples. Priming with SA increased
POD activity in Pakistan-13, and Chakwal-50 while lowered in FSD-08. POD activity
increased in Pakistan-13 and Chakwal-50 with GA priming.
Peroxidase (POD) takes part in decomposition of H2O2. Researchers proved that
under drought stress condition scavenging system of hydrogen peroxide was more
aggressively induced in different wheat genotypes (Hameed et al., 2011). Generally,
hormones take part in defense mechanism to overcome stress. Effect of salicylic acid under
salinity stress on peroxidase and catalase activity was studied previously. It is demonstrated
that peroxidase activity was significantly increased when treatments was made with 0.05
mM SA solution. It is also confirmed that the exposure to exogenous SA accelerated the
drought and salt stress resistance of plants (Deef, 2007). During severe water stress excess
levels of H2O2 have the ability to overcome or prevent the synthesis of antioxidant enzymes
(Selote and Chopra, 2006).
Figure 4.13: POD activity in flag leaves of wheat genotypes grown under control and drought
stress
cdef efg
efg
fg
cdfg
bcd
bcd
bcd
fg
ab
cdefg efg
defg
defg
defg
bcd
ab
ab
c
a
defg
bcd
g
efg fg
cdefg
ab
c
ab ab
c
ab
c
cdefg
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
Control Drought
PO
D a
ctiv
ity
(unit
/g f
resh
wei
ght)
NP SA GA
75
4.3.1.6 Catalase activity
Without stress condition, the highest catalase activity was found in FSD-08
(2609±0.039units/g.f.wt.) while Chkwal-50 has the lowest catalase activity
(2171±0.008units/g. f.wt.) in non primed samples (Fig. 4.14). Catalase activity was
decreased extensively in AARI-11and FSD-08 while improved in Chakwal-50 and Shahkar
under normal condition alongwith SA priming. GA priming increased the catalase activity
in Chakwal-50 Pakistan-13 and FSD-08 while decrease in Shahkar and FSD-08.
In stressed samples, FSD-08 had the maximum catalase activity (3638±0.002
units/g.f.wt.) while Chakwal-50 had the lowest catalase activity (3292±0.001units/g.f.wt.)
in non primed samples. When SA priming were applied catalase activity was increased in
Pakistan-13 and Shahkar while decrease in other genotypes. Catalase activity was increased
on GA priming only in Shahkar while decreased in Chakwal-50. When stress was applied
catalase activity was increased in FSD-08 and Shahkar while minimum in Chakwal-50.
Catalase activity was increased on SA priming in stress condition in comparison without
stress in Pakistan-13while decreased in Chakwal-50 and AARI-11. Priming with GA
increased the catalase activity in stress in Shahkar while decreased in Chakwal-50 and
AARI-11.
Figure 4.14: CAT activity in flag leaves of wheat genotypes grown under control and drought
stress
fgh
i ij
hi
def
hij
bcd
e
bcd
e
bcd
e
bcd
ef
bcd
egh
i
hij
hij
a defg
bcd
bcd
bcd
ef
bcd
bcd
j
hij
fgh
i j
fgh
i
gh
i
bcd
e
bcd
bcd
e
bcd
0123456
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
Control Drought
CA
T (
Unit
s/g.
f. w
t)
NP SA GA
76
Our results are in accordance with Zhang et al., 2007 who said that accelerated
activities of most important antioxidant enzymes i.e. CAT, SOD and POD in Victoria and
Victor seedlings after priming treatments were found. Catalase is among the most important
enzyme for elimination or detoxification of too much hydrogen peroxide in the seeds.
Indeed, high level of antioxidative enzymes saves the cell alongside the oxidative damage
by free radicals removal of ROS. During stress as well as normal condition seed priming
with growth regulators improved physiological activities of wheat seedling. Meanwhile,
enhanced the activities of several antioxidants i.e. CAT, POD and SOD as a result it protect
the cell against free radical production and cellular damage due to oxidation (Eisvand et
al., 2010).
4.3.1.7 Ascorbate peroxidase activity
Under normal condition, AARI-11 has the highest ascorbate peroxidase (APX)
activity (1288±0.034units/g.f.wt.) while Chakwal-50 had the lowest ascorbate peroxidase
activity (0216±0.001units/g.f.wt.) in non primed samples (Fig. 4.15). When SA priming
was applied ascorbate peroxidase activity was lower in Pakistan-13 and AARI-11 however,
increased in Shahkar and Chakwal-50. During GA priming, Ascorbate peroxidase activity
was found to be accelerated significantly in Shahkar, Chakwal-50 and FSD-08 while
reduced in Pakistan-13.
Under drought stress, Pakistan-13 had the highest Ascorbate peroxidase (APX)
activity (1710±0.018units/g.f.wt.) while Shahkar had the lowest APX activity
(0808±0.071units/g.f.wt.) in non primed samples. Upon priming with SA ascorbate
peroxidase activity was appreciably increased in AARI-11while there was no effect in other
genotypes. Ascorbate peroxidase activity was increased in AARI-11 and Chakwal-50while
decreased in Shahkar alongwith GA priming.
77
Drought stress notably amplified the ascorbate peroxidase activity in Pakistan-13,
Chakwal-50, and FSD-08. Ascorbate peroxidase activity along with SA priming increased
in stressed samples than those that are controlled in FSD-08, Pakistan-13 and Chkwal-50.
Ascorbate peroxidase activity improved under stress same as in devoid of stress in
Chakwal, Pakistan-13, and FSD-08 on GA priming. Our findings were similar to previous
researchers who have found that anti-oxidative peroxidases or glutathione reductase
activity is increased when subject to the drought stress (Miller etal., 2010). Therefore,
these enzymes are excellent biochemical stress markers and their improved activity may
demonstrate the prospective for remediation. Under control and under drought stress
genotypes respond differently.
Figure 4.15:APX activity in flag leaves of wheat genotypes grown under control and drought stress
4.3.2 Cluster Analysis based on oxidative enzymes attributes in flag leaf of wheat
Major two cluster were formed which further form three sub cluster. Our data
represents the affinity of every grouped variable in one bunch to relate strongly to each
other (Fig. 4.16).
bc
h
h
de
bcd
efgh
bc
bcd
e
efgh
a ab
ccde
fgh
fgh
h
bcd
ef
bcd
e
bc
gh
ab
c
abcd
e
fg
fg
h
cdefg
h
a a
efgh
def
ab
c
0
1
1
2
2
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
AA
RI-
11
Chak
wal
-50
Sh
ahkar
Pak
ista
n-1
3
FS
D-0
8
Control Drought
AP
X (
unit
s/g.
f. w
t)
NP SA GA
78
Figure 4.16: Dendogram derived from hierarchical cluster analysis of all combined antioxidant activities of
five wheat genotype
4.3.3 Conclusion
Our experiment signifies that hormones play critical roles in plant responses to
drought. GA and SA induced drought tolerance by enhancing the antioxidant enzymes
activities i.e. superoxide dismutase, peroxidase, amylase and ascorbate peroxidase.
79
Table 4. 6: STI % of wheat genotypes based on oxidative enzymesacross genotypes and priming treatments
4.4 PHYSIOLOGICAL ATTRIBUTES
Priming induced physiological attributes against stress (Makhmudova et al., 2011).
4.4.1 Malondialdehyde Contents
There was non significant effect on Malondialdehyde (MDA) contents in all tested
genotypes under well water and drought. Priming with SA and GA slightly increased the
MDA (Fig 4.17). Generally, in literature under low water condition lipid peroxidation
significantly improved in wheat cultivars. However, if water level was changed from low
to medium or high level of lipids peroxidation could also change and varies genotype to
genotype (Hameed et al., 2011).
Genotypes Seed
priming
Estrase Amylase Protease SOD POD CAT APX
AARI-11 NP 104.48 105.26 87.28 142.12 142.12 143.93 56.86
Chakwal-50 NP 103.41 129.51 84.46 169.76 169.76 151.65 35.39
Shahkar NP 109.85 25.53 85.46 132.34 132.34 149.72 20.43
Pakistan-13 NP 91.12 160.00 91.33 225.14 225.14 114.58 47.03
FSD-08 NP 100.66 1515.38 77.97 209.55 209.55 139.44 39.09
AARI-11 SA 101.49 841.18 64.46 177.11 177.11 177.49 114.70
Chakwal-50 SA 92.19 393.94 85.50 164.73 164.73 152.55 284.14
Shahkar SA 119.01 233.57 100.16 174.21 174.21 131.39 81.18
Pakistan-13 SA 99.60 60.00 83.55 180.71 180.71 84.04 413.69
FSD-08 SA 110.67 131.51 78.77 231.74 231.74 127.49 146.00
AARI-11 GA 104.91 92.31 91.70 226.33 226.33 103.69 135.57
Chakwal-50 GA 97.12 243.44 77.54 179.15 179.15 146.13 303.35
Shahkar GA 102.33 520.75 59.29 121.92 121.92 154.58 116.37
Pakistan-13 GA 95.51 1652.94 67.10 325.70 325.70 154.82 325.37
FSD-08 GA 97.77 251.67 72.31 183.31 183.31 143.48 185.88
80
Figure 4.17: MDA contents in flag leaves of wheat genotypes grown under control and drought stress
4.4.2 Total oxidative status
Under normal condition, all genotypes have the same dimension in total oxidative
status (TOS) except Chakwal-50 which had the lowest TOS in non primed samples (Fig.
4.18).TOS was increased in Chakwal-50 and Pakistan-13 on SA priming while non
significant effect on other genotypes. Alongwith GA priming, TOS was improved much in
Chakwal-50. Under drought stress, all genotypes have same dimension in non primed
samples. TOS increased in Shahkar and Pakistan-13 on SA priming. Priming with GA has
non significant effect. Drought stress not significantly decreased TOS under normal
condition as compared to control. Priming with SA and GA raised maintained TOS to a
normal level under drought as in normal condition.
Figure 4.18: TOS in flag leaves of wheat genotypes grown under control and drought stress
a a
a a
a
a
a
a
a
a
a
a a a a
a
a
a
a
aa
a
a
a a a
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0100200300400500600700800
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A c
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/uM
/g f
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TO
S (
unit
/µM
/g.
f. w
t )
NP SA GA
81
4.4.3 Relative water contents
Under normal condition, AARI-11 had the highest RWC (82.690±0.887%) while
Chakwal-50 had the lowest RWC (57.72±0.103%) in non primed samples (Fig. 4.19). RWC
increased significantly on SA priming in Chakwal-50 while other genotypes were not
affected. In case of GA priming, relative water content was increased significantly in
Chakwal-50 and Pakistan-13. Under drought stress, Pakistan-13 had highest relative water
content (80.19±0.01%) while Chakwal-50 has lowest relative water content (61.58±1.77%)
in non primed samples. Relative water content was increased alongwith SA priming in
AARI-11 and Shahkar while lower in Chakwal-50. GA priming had no effect on all
experienced genotypes. Relative water content was decreased under drought stress in all
genotypes except Pakistan-13. SA priming raised the relative water content in stress
condition in comparison with non stress condition in AARI-11 and Shahkar. Relative water
content increased along GA priming under drought condition as well as normal condition
in AARI-11.To investigate the physiological responses of wheat against water stress
researchers reported that as a consequence of water stress, relative water content (RWC),
chlorophyll and carotenoid and membrane stability was decreased while proline
accumulation was increased (Chandrasekar et al., 2000) . Similar conclusions were
revealed by other authors (Azooz, 2009) they also proved that remarkable increase in RWC
when SA was applied under water deficient condition ultimately increased yield. Constant
reduction in RWC in retort to PEG-mediated water stress have been explored and reported
in wheat (Bajji et al., 2001) in Brassica (Swati and Ahmad, 2000) and in rice (Hsu et al.,
2003) Many scientists suggested that RWC and proline could be used as a tool to determine
stress induced in plants due to low amount of water (Strauss and Agenbag, 2000).
82
Figure 4.19: RWC contents in flag leaves of wheat genotypes grown under control and drought stress.
4.4.4 Cell membrane thermostability
Without stress, FSD-08 had the maximum cell membrane thermo stability (CMT)
(6.011±0.33%) while Chakwal-50 had the lowest CMT (3.604±0.236%) in non primed
samples. CMT was increased significantly on SA priming in Chakwal-50 and FSD-08
without stress (Fig. 4.20). CMT was increased notably along GA priming in AARI-11 and
FSD-08 while reduced in Pakistan-13. Under drought stress, Pakistan-13 had the highest
CMT (4.547±0.33%) while AARI-11had the lowest CMT (3.203± 0.33%) in absence of
any priming treatment. CMT was appreciably increased only in AARI-11 and Pakistan-13
while decreased in Chakwal-50 along SA priming. When GA Priming was applied CMT
increased in AARI-11 and FSD-08 while decreased in Chakwal-50. It is accepted that
plants facing drought showed a variety of changes in different processes to flourish under
water stress situations (Arora et al., 2002).
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RW
C (
%)
NP SA GA
83
Figure 4.20: CMT contents in flag leaves of wheat genotypes grown under control and drought stress
In preceding literature, numerous reports described a work on both the tolerant
genotypes and susceptible genotypes and concluded that under drought stress genotype
having low electrolyte leakage and H2O2 are more tolerant than sensitive genotypes.
Tolerant varieties showed more membrane stability than susceptible ones (Sairam and
Saxena, 2000). A series of adaptive responses of wheat plants to face drought like
membrane stability, oxidative damages to plant cells and antioxidant protection were
reported by many researchers (Chandrasekar et al., 2000; Hameed et al., 2011). Water
deficiency adversely affects the cell membrane stability by leakage of membrane in all
crops. However, stability of membrane can be prevented under water deficient condition
by using SNP treatment to wheat seedlings (Hao et al., 2008).
4.4.5 Photosynthetic pigments
Without stress condition, had the highest chlorophyll a contents (8.620±0.49) was
found in FSD-08 while lowest chl a contents was found in Chakwal-50 (6.677±0.120) in
non primed samples. Chlorophyll a contents was slightly increased in all genotypes under
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84
normal condition along SA priming. Chlorophyll a increased in all studied genotypes when
samples were primed with GA.
Under drought stress, Chl a contents decreased in all studied genotypes. SA and GA
Priming slightly increase the chl a in all tested genotypes. Chl a was decreasd in all
genotypes under drought in comparison with nomal condition. Many authors (Shakirova et
al., 2003) and (Iqbal and Ashraf, 2013) on wheat plants and (Amin et al., 2008) on maize
plants found that SA and GA results in significant increase in chlorophyll content. That
accretion of photosynthetic pigments may be due to exogenous application of SA that
results in photosynthetic efficiency increment as represented, by increasing in both chl a,
chlb and carotenoids content in the leaves of wheat plants grown under stressed condition.
Figure 4.21: Chl a contents in flag leaves of wheat genotypes grown under control and drought stress
In case of Chl b slight variation was present in all studied genotypes (Fig. 4.22).
Under drought stress, chl b decreased in all tested genotypes. Priming was able to increased
chl b under drought stress condition. Under balanced environmental condition, FSD-08 had
the uppermost carotenoid contents (3.233) while lowest in AARI-11 (2.092) in non primed
samples. Carotenoid contents were somewhat increased on SA and GA priming in all under
experiment genotypes (Fig. 4.23).
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Chla
unit
/g.
f. w
t
NP SA GA
85
.
Figure 4.22: Chlb contents in flag leaves of wheat genotypes grown under control and drought stress
Under drought stress, carotenoid contents were slightly decreased in tested
genotypes while SA and GA have slightly increased carotenoid contents in all tested
genotypes. Overall drought stress had significant effect on carotenoid contents in all tested
genotypes. Findings of (Hassanein, 2009) reported that in stressed wheat showed stunted
growth, accumulation of hydrogen peroxide and lipid peroxidation under high salinity and
found decline in Chl a, b, carotenoid and total pigments.
Figure 4.23: Carotenoids contents in flag leaves of wheat genotypes grown under control and drought stress
ab
b
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Chlb
unit
/g.
f. w
tNP SA GA
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Car
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/g.
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NP SA GA
86
Non significant difference in all tested genotypes in case of anthocyanine under
normal and stress due to drought (Fig. 4.24). SA and GA priming showed non significant
effect on anthocyanine contents under normal and drought stress. Some researchers Khan
et al., (2003) showed that photosynthetic rate was increased in SA treated corn and soybean.
The pretreated seeds with SA solution (10-2
mol/L) revealed higher chlorophyll and
anthocyanine content. Similar findings were reported by many authors and reported that
SA significantly enhances the pigment content under salt stress conditions (El-Tayeb,
2006).
Fig: 4.24 Anthocyanin contents in flag leaves of wheat genotypes grown under control and drought stress
4.4.6. Conclusion
Results revealed that priming with Gibberellic and Salicylic acid improved the
physiological response in studied genotypes under both drought and control conditions.
Relative water contents and cell membrane stability maintained under stress in primed
seeds. Similarly photosynthetic pigments increased with priming. Ultimately all these
altered physiological factors improved plant growth and enhanced yield under stress
condition.
4.5 METABOLITE ACCUMULATION AND MINERAL ELEMENTS
4.5.1 Sugar contents
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Without stress, maximum sugar contents were found in FSD-08 (2.76±0.050g.f.wt.)
while Chakwal-50 had the lowest sugar contents (2.09±0.029g.f.wt.) in non primed
samples. SA priming depicted that there was significant reduction Sugar contents in AARI-
11, whereas improved in Chakwal-50 and Shahkar without stress (Fig. 4.25). Priming with
GA increased the sugar contents in Pakistan-13 and Chakwal-50 while other remains
unaffected. Under drought stress, FSD-08 had the highest sugar contents
(2.84±0.081g.f.wt.) while Ckakwal-50 had the lowest sugar contents (1.743±0.033g.f.wt.)
in absence of any priming treatment. Sugar accretion was notably increased in Shahkar and
FSD-08 along SA priming while reduction was seen in other genotypes. When GA priming
was applied sugar contents only increased in Shahkar at the same time as decreased in
AARI-11, Chakwal-50 and Pakistan-13. In Shahkar under stress sugar contents increased
whereas decreased in AARI-11 and Chakwal-50. Under drought sugar contents was more
than control in Shahkar due to SA priming. Priming with GA increases the sugar contents
under drought in Shahkar while decreased in Pakistan-13 and AARI-11. Some researchers
worked on flag leaf of different wheat cultivars and concluded that amount of soluble
carbohydrate varied among different genotypes. Different wheat cultivars perform
differently depends on their tolerance and susceptibility. Hereditary composition justifies
the patience of wheat crop against stress. It was observed that more soluble sugars were
accumulated in tolerant species as compared to susceptible species(Nayyar and Walia,
2003).
88
Figure 4.25: Sugar contents in flag leaves of wheat genotypes grown under control and drought stress
4.5.2 Protein contents
There was non significant effect on protein contents in non primed samples.
Similarly under drought stress non significant effect was present. Only Chakwal-50 has the
highest protein under drought. All other genotype has the non significant effect.
Figure 4.26: Protein contents in flag leaves of wheat genotypes grown under control and drought stress
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tal
solu
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sugar
s (u
nit
/g f
. w
t)
NP SA GA
ab
ab ab
b
ab
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tein
co
nte
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(Unut/
mg/g
f.
wt.
)
NP SA GA
89
4.5.3 Proline accumulation
Without stress, FSD-08 had the maximum Proline accumulation
(2.945±0.025µmole/g f.wt.) while Chakwal-50 had the lowest Proline accumulation
(1.733±0.08 µmole/g f.wt.) in non primed samples (Fig. 4.27). Reduction in proline occurs
in FSD-08 and Shahkar whereas increased in Chakwal-50 along SA priming. More ever,
GA priming improved Proline accumulation in AARI-11 while decreased in Chakwal-50
and Pakistan-13. Under drought stress, Shahkar had the highest Proline accumulation
(3.540±0.055 µmole/g f.wt.) while Chakwal-50 had the lowest Proline accumulation
(2.220±0.036 µmole/g f.wt.) in lack of any priming treatment. Proline accumulation was
increased on SA priming in Chakwal-50 while decrease in AARI-11. There is significant
increased the Proline accumulation in all studied genotypes under drought conditions.
Priming with GA and SA significantly increase the proline accumulation under drought in
contrast to normal condition. Previously, a substantial enhance was found in proline levels
in the seedlings faced to saline stress and treated with SA in comparison with control salt
stressed seedlings. SA exogenously applied under salt and drought stress enhanced the salt
and drought tolerance of plants (Deef, 2007). We conclude from above results that these
cultivars attempt to put up with drought situation by the accumulation of relatively high
osmolyte accumulation. Researcher reported that proline significantly increased (p<0.01)
under drought stress (Keyvan, 2010).
90
Figure 4.27: Proline contents in flag leaves of wheat genotypes grown under control and drought stress
4.5.4 Potassium ratio
Without stress, Pakistan-13 had the maximum Potassium (K+) ratio
(28.123±0.248mg/g f.wt.) while Shahkar had the lowest K+ ratio (17.721±0.196 mg/g f.wt.)
in non primed samples (Fig. 4.28). SA priming reduced the K+ in Shahkar and FSD-08
while in Pakistan-13 ratio was increased. Priming with GA increased K+ ratio in AARI-11
and Pakistan-13 whereas reduction occur in FSD-08 and also in Shahkar. In drought
condition, maximum K+ ratio was observed in Chakwal-50 (23.403 ± 1.634 mg/g f.wt.)
while minimum K+ ratio was present in FSD-08 (13.549 ± 0.187 mg/g f.wt.) in nonprime
samples. Similarly, Potassium ratio decreased in FSD-08 and increased in Pakistan-13 and
Chakwal-50 along SA priming. GA priming was effective to improve the K+ ratio in AARI-
11 and Chakwal-50 although decreased in FSD-08. In many previous studies, under salinity
stress importance of K+ was widely studied, it was reported that significant diminution in
K+ was reported for wheat under saline condition. GA3 priming was effective to increase
the shoot K+ concentration in normal condition. It has been reported that sensitive cultivars
had more K+ than tolerant. Since, by reviewing the literature to increase K+ in wheat
cultivars under salinity stress (Nayyar and Walia, 2004). Normally under drought stress
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Pro
line
(unit
/µm
ole
/g f
.wt)
NP SA GA
91
nutrients uptake was reduced. It was observed that exogenous application of Si was at
anthesis stage was responsible for accumulating maximum plant nutrients. Under drought
stress control plants has highest nutrient then those treated with Si. A controlled plant has
high nutrients like K, Mg and Zn contents than those treated with Si (Bukhari et al., 2015).
Figure 4.28: K+ratio in flag leaves of wheat genotypes grown under control and drought stress
4.5.5 Calcium ratio
High Ca+ ratio was present in Pakistan-13 (28.12±0.248 mg/g f.wt.) while Ca+ ratio
was lower in FSD-08 (15.11±0.059 mg/g f.wt.) in nonprime samples without drought (Fig.
4.29). Low ratio of Ca+ was recorded in AARI-11 and Pakistan-13 and high ratio was
recorded in FSD-08 without stress condition along SA priming. Priming with GA revealed
the increase in Ca+ ratio in Pakistan-13 although decreased in Shahkar. Results showed that
Chakwal-50 had the maximum Ca+ ratio (23.40±1.63 mg/g f.wt.) whereas FSD-08 had the
less Ca+ ratio (13.54±0.187 mg/g f.wt.) under drought in non primed condition. Priming
with GA was effective in improving ratio of Ca+ in FSD-08, AARI-11and Pakistan-13.
Drought stress significant reduction was observed under drought in Ca+ ratio
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K+
rat
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unit
/mg/g
f.w
t.)
NP SA GA
92
in AARI-11 and Pakistan-13. Results revealed that SA priming increased the Ca+Pakistan-
13 under situation of drought in comparison with normal while in FSD-08 ratio was
declined. Priming with GA increased the Ca+ ratio under drought in Pakistan-13and AARI-
11 in comparison with control.
Figure 4.29: Ca+ ratio in flag leaves of wheat genotypes grown under control and drought stress
For predicting the tolerant plants against drought (Asghari et al., 2001)
premeditated in two wheat varieties mannitol content, ABA, K+ and Ca+possibally due to
the ratio of K+ / Ca2+. It is mainly depending on the interferingamong the aspects and
connection such as membrane permeability, membrane of Chloroplast and stomatal
membrane because it take part in stomatal aperture and regulation of guard-cell turgor.
However, on the position of stomata the real fact about the ratio of K+ / Ca2+ are still under
investigation (Asghari et al., 2001). Due to hyperosmotic stress Cytosolic Ca+ was
increased, this increased in cytosolic Ca+, under stress maintained the osmosensing
(Matsumoto et al., 2002).
It confirms from previous studies that seed priming induce many metabolic changes
in plants,as a result enhances stress tolerance (Jisha et al., 2013; Kubala et al., 2015).
Another study confirmed that, β-amino butyric acid seed priming in green gram enhanced
proline accumulation, amount of carbohydrate and total protein contents (Jisha and Puthur,
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Ca+
rati
o (
unit
/mg/g
f.w
t.)
NP SA GA
93
2016). Under stress, all parameters of plant’s growth along with development was increased
significantly by the application of salicylic acid exogenously. Increased chlorophyll
contents and significant increase in net CO2 assimilation has been reported (Martel and
Qaderi, 2016). Under stress conditions, applied SA regulates the plant physiological
processes like photosynthesis and proline metabolism thereby providing protection to plant
(Miura and Tada, 2014). The function of SA in thermo tolerance is well reported.
Among growth regulators, SA in particular has been well documented in improving
plant’s stress-tolerance (abiotic) through controlling plant’s major metabolic processes
(Khan et al., 2009). According to (Hasanuzzaman et al., 2014), damaging effect of abiotic
stress in plants can be reduced by the exogenous application of SA. Electrolyte leakage is
correlated with chlorophyll content, sugar, proline and protein.Increased levels of these
compatible solutes resulted decreased electrolyte leakage (Zhang et al., 2013).
4.5.6 Cluster Analysis Based on Biochemical and Physiological Attributes
Genotypes respond differently in term of physiological attributes. Two main
clusters were further divided into three sub cluster (Fig. 4.30). Our data reflected the
tendency of each grouped variables in one cluster to relate closely to each other.
94
Figure 4.30: Dendogram derived from hierarchical cluster analysis of all combined physiological and
biochemical attributes of five wheat genotypes
4.5.7 Conclusion
It was concluded from this section of study that, genotypes respond differently
under water stress due to adoptive changes in antioxidant and in other metabolic processes.
Cell membrane thermo stability, relative water contents are decreased while antioxidants
increased. However, growth regulator increased the physiological response and antioxidant
activities in some genotypes under stress and without stress conditions. Presoaking of seed
enhanced some enzymes that scavenge free radical like superoxide dismutase, catalase,
peroxidase, amylase, soluble sugars and proteins and trigger synthesis of proline that is an
osmo-protectant. Accumulation of proline increased under stress condition while ion
accumulation response was varied among genotypes.
95
Table 4.7: STI% of wheat genotypes based on physiological and biochemical attributes across
genotypes
4.6 WHEAT GRAIN QUALITY
Adding together with yield, wheat grain quality is essential to the welfare of beings.
The purpose of this section of study was to appraise the consequence of drought stress and
hormonal priming on physio-chemical properties of wheat grains.
4.6.1 Wet Gluten Contents
Significant variations were found among tested genotypes in wet gluten contents.
Under normal condition, Shahkar had the highest wet gluten contents (32.2 ± 0.28%) while
Chakwal-50 had the lowest wet gluten (24.6 ± 0.29 %) without any prior application of
growth regulators (Fig 4.31). There was non-significant under drought stress, FSD-08 has
the highest gluten strength (29.5 ± 0.73 %) while Pakistan-13 had the lowest wet gluten
(27.0 ± 0.34 %) in absence of any priming treatment. The SA priming had non-significant
Seed
primin
g
MDA TOS RW
C
CM
T
Chl
a
Chl b Caro
tenoi
d
Anthoc
yanin
Sugar Protein Proli
ne
K+ Ca+
AARI-11 NP 139 100 83 65 62 60 106 95 86 107 112 111 73
Chakwal-
50
NP
110 133 106
10
1 56 58 103 93 83 110 128 111 86
Shahkar NP
106 93 90
10
5 88 80 100 95 104 108 141 92 91
Pakistan-
13
NP
113 92 94 75 65 98 90 94 94 99 110 127 63
FSD-08 NP 66 89 86 62 68 51 84 92 103 102 107 92 89
AARI-11 SA 67 105 99 83 69 72 99 99 85 103 107 107 99
Chakwal-
50
SA
99 83 76 82 58 60 102 93 66 93 130 96 93
Shahkar SA 75 120 111 80 63 73 94 96 105 113 147 126 102
Pakistan-
13
SA
94 121 96 81 74 63 78 93 107 101 110 103 131
FSD-08 SA 98 94 83 63 70 63 78 94 104 99 114 119 62
AARI-11 GA 87 94 77 77 64 99 75 94 64 99 102 107 126
Chakwal-
50
GA
132 109 78 89 75 86 100 94 62 98 148 107 92
Shahkar GA 86 107 86 85 71 101 100 95 114 101 144 113 128
Pakistan-
13
GA
126 95 84 87 77 107 90 101 93 97 122 109 115
FSD-08 GA 110 116 84 65 89 95 86 101 107 99 119 103 102
96
effect on wet gluten. With GA increased the wet gluten in FSD-08 under drought however;
other genotypes are not affected. Wet gluten was increased under drought stress in
Ckakwal-50 (28.5±0.46%), while decreased in Shahkar (28.4±0.32%). The SA and GA
priming had non-significant difference in tested genotype on wet gluten content under
drought stress. The effect of both year was non significant.
Figure 4.31: Wet Gluten contents in seeds of wheat genotypes
Similar findings have been reported by many authors in which water stress apply at
the post anthesis period considerably increased the grain gliadin ratio (Fan et al., 2004).
Wet and dry gluten are also increased under drought stress in wheat grains. For end-use
quality, proteins are the major and vital components of grain of wheat. To maintain the
quality of dough the gluten proteins play an important role, dough quality is mainly
maintained by glutenins and gliadins as compared to (Balla et al., 2010) albumins and
globulins which are important in term of nutritional point of view and have little effect on
the dough quality. Albumins and globulins contain all essential amino acids that are
important for human health. (Balla and Veisz, 2007).
4.6.2 Gluten Index
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Control Drought
Wet
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97
Gluten strength can be measured by the by knowing about the gluten index. In our
finding gluten index increased in most of genotypes. Most of the quality analyses were
related to grain and grain flour including starch, hardness index , moisture percentage, wet
gluten protein percent, , falling number, , dry gluten, gluten index and sedimentation of
zeleny. These quality characters were varied from one genotype to other. Mostly quality
traits are affected by water stress during the grain filling period. In previous studies, it is
well studied gluten index and SDS sedimentation volume indirectly correlate with a
decrease in that when a terminal water stress applied an increase in protein percent, wet
and dry gluten, falling number, moisture content, bread volume, grain yield and 1000 grains
weight (Aslani et al., 2013).
In recent study significant difference was evaluated in all genotypes under both
treatments (Fig 4.32). Gluten index remained unaffected both the years. Without stress
condition, FSD-08 had the maximum gluten strength (90.5±0.27%) while Pakistan-13 had
the lowest gluten strength (72.2±0.05%) in non primed seeds. Gluten index decreased
significantly on SA priming in Chakwal-50(75.4±0.18%), and Shahkar (77.1±0.12%) while
increased in Pakistan-13(78.2±0.05%) without stress. When GA priming applied, gluten
index was improved drastically in AARI-11(88.6±0.10), Pakistan-13(83.4±4.22) and FSD-
08(94.0±0.02) while decreased in Chakwal-50(76.3±0.21).Under drought stress; FSD-08
had highest gluten strength (95.0±5.21%) while Shahkar had the lowest gluten strength
(80.4±0.17%) in non primed samples. Gluten index was significantly raised on SA priming
only AARI-11(84.3±0.05%) while other genotypes remained unaffected. GA Priming was
able to boost the gluten index only in Shahkar (83.4±0.11%) under drought.
Gluten index significantly increased under drought stress in AARI-11
(84.3±0.05%), Pakistan-13(89.0±0.02%) and FSD-08 (95.0±5.21%) while decreased in
Shahkar (80.4 ± 0.17). Priming with SA increased the gluten index in FSD-08
98
(98.0±0.02%), Pakistan-13(89.0±0.02%) and Shahkar (83.4±0.11%) under drought
stressed condition. GA priming increased gluten index under drought same as in without
stress in Chakwal-50 (84.2±0.07%), Pakistan-13(85.4±0.07%) while decreased in Shahkar
(83.4±0.11%).
Figure 4.32: Gluten index contents in seeds of wheat genotypes
4.6.3 Falling number (Sec)
In literature it is reported that under drought and salt stresses the amount of water
absorption by the flour causes a decline in the value of gluten, glutenin and increased
various attributes like falling number, grain protein, gliadin and grain hardness index
(Eyvazi et al., 2005). Some other researcher has worked on diverse cultivars of spring
wheat (red and white seeded) and found that germination index, falling number and
sprouting are mostly affected by genotypic variation then the environmental factors and it
is possible to develop genetically variant varieties (PHS resistant cultivars) than
environmental factors (Rasul et al., 2012).
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4 C
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4 D
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5 C
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5 D
Control Drought
Glu
ten i
nd
ex %
NP SA GA
99
Under normal condition (Fig. 4.33). FSD-08 had the highest falling number
(362.6±5.03 sec) while Shahkar had the lowest falling number (294.6±3.05 sec) in non
primed samples. Falling number was decreased significantly inChakwal-50 (280.3±7.76
sec) while increased in Pakistan-13(340.6±10.20sec) with SA priming under normal
condition. There is non-significant effect on all other genotypes. Priming with GA showed
that falling number was decreased appreciably in FSD-08 while higher in Pakistan-
13(355.6±3.51 sec). Under drought stress, FSD-08 had the highest falling number
(369.3±10.28 sec) while Chakwal-50 had the lowest falling number (298.6±3.05sec) in non
primed samples. Falling number was significantly decreased on SA priming in Chakwal-
50(266.3±12.0 sec) and Shahkar (306.3±5.13 sec). GA priming decreased the falling
number only in Chakwal-50(275.3±13.01sec) while other genotypes remain unaffected
under drought.
Drought stress raised the falling number only in one genotype Shahkar
(322.61±0.26sec) other genotypes have intermediate effect. In case of GA priming
droughtincreased the falling number in Shahkar(318.0±8.0sec) while decreased in
Pakistan-13(309.3±15.53sec), while on Falling number effect of year was non-significant.
100
Figure 4.33: F/No in seeds of wheat genotypes
Quality performance of wheat genotypes during grain filling is strongly affected by
weather conditions. The quality criterion for wheat in bread making is Hagberg falling
number (HFN) but it has a negative association with a-amylase activity. During pre-harvest
sprouting (PHS) or on other case late-maturity excessive levels of a-amylase are produced
(Major et al., 2001). During the time of grain filling in the absence of visible sprouting high
a-amylase activity or low HFN has been linked with high soil moisture and low
temperatures (Gooding et al., 2010; Gooding et al., 2013). Hagberg falling number (HFN)
is strongly affected by genotype-environment (Gooding et al., 2010), and agronomy
interactions in genotype- (Kindred et al., 2005).
4.6.4 Seed Storage Protein
Under normal condition, no difference was observed in tested genotypes in non
primed condition (Fig 4.34). During both years’ proteins were non significantly varied.
Protein percentage was increased significantly in Chakwal-50 while decreased in Pakistan-
13 (12.7±0.15%) when primed with SA. In term of GA priming protein percentage
increased in FSD-08 while other genotypes remained unaffected. Under drought stress,
Chakwal-50 (15.6± 0.49%) and Shahkar have the highest protein (15.7± 0.89%) while
ijkl
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ab
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mn
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ab
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AA
RI-
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Chak
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-50
Sh
ahkar
Pak
ista
n-1
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D-0
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AA
RI-
11
Chak
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Sh
ahkar
Pak
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n-1
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D-0
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201
4 C
201
4 D
201
5 C
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5 D
Control Drought
F/N
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NP SA GA
101
other three genotypes have same percentage without any priming. Protein percentage was
significantly raised on SA priming Chakwal-50 (16.7±0.20%) and Pakistan-
13(15.2±0.40%) while decrease in AARI-11(13.2±0.10).When GA priming was used it
help to increase the protein percentage in Chakwal- 50 (16.7±0.20) while significant
decrease in Pakistan-13(12.9±0.68%) under drought.
Drought stress significantly increased the protein percentage in Chakwal- 50
(15.6±0.49%) while decreased in FSD-08(13.4±0.36%).The SA priming raised the protein
percentage in Pakistan-13(15.2±0.40%) while decreased in AARI-11(13.2±0.10%) in
drought stressed in comparison with normal. Priming with GA increase protein in Shahkar
(15.7±0.15%) while decreased in AARI-11(15.7±0.15%) and FSD-08(14.2±0.15%) as
compared to normal.
Figure 4.34: Protein percent in seeds of wheat genotypes
It is notable that yield production fall that heppens under dry spell stretch situation
is for the most part in connection to an ascent in protein content (Pompa et al., 2009). Water
stress completely climbed protein content (Rharrabti et al., 2003).
Protein percentage was increased significantly under drought in the Chakwal- 50
while decreased in FSD-08. The SA priming raised the protein percentage in Pakistan-13
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Sh
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Pak
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D-0
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201
4 C
201
4 D
201
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Control Drought
Pro
tein
%
NP SA GA
102
while decreased in AARI-11under drought. Proteincontents increased under SA priming in
Shahkar while decreased in AARI-11 and FSD-08 as compared to normal.
Proteins are the major constituent of grain since wheat is widely used in many
industries and foods due to its high concentration of grain protein. Now a day in crop
sciences, quality of grain is becoming a challenging and broadly discussed topic.
Environmental factors affecting wheat quality directly. Due to food security and market
demand the main concern has now progressively shifted to the upgrading of processing
quality. Therefore, main focus of breeding is on giving out quality and improvement of
grain contents of protein (He et al., 2004; Yong et al., 2004; Wang et al., 2005).
In relative to drought stress effect on grain quality, we examined that protein
contents almost increases, similar findings have been earlier reported in literature in
different environment (Guttieri et al., 2005). Under irrigated and non-irrigated treatments
studied gliadin/glutenin ratio has studied and accomplished that there is no considerable
distinction between two treatments they also concluded that fraction of polymeric proteins
was higher under non irrigated treatment (Panozzo et al., 2001). Similar findings were
found previously bread wheat, researchers observed that when water availability becomes
limited non-significant change was seen in rate of accumulation of soluble and insoluble
proteins per degree day in bread wheat even though the polymer in solubilization in
progress earlier (Daniel and Triboi, 2000).
4.6.5 Moisture Contents of seed
Under normal condition, non-significant difference was found in all tested
genotypes under non priming treatment (Fig 4.35). Under SA priming, moisture contents
were decreased notably in Pakistan-13 (8.5±0.01%), AARI-11(8.7±0.15%) and Chakwal-
50 (8.2±0.10%), while increased in Shahkar (9.6±0.10%) and FSD-08 (9.3±0.11%) under
103
normal condition. priming with GA increased moisture contents appreciably in AARI-
11(9.5±0.08%), while decreased in Shahkar (9.1±0.10%) and FSD-08 (8.6±0.05%). Under
drought stress, AARI-11 had the highest moisture contents (9.2±0.10%) while Shahkar had
the lowest moisture contents (8.2±0.10%) in non primed samples. Moisture contents were
significantly raised on SA priming only AARI-11(9.5±0.05%) while decreased in
Chakwal-50 (7.7±0.10%). When GA Priming samples were examined it seems that GA
increased the protein in Shahkar under control, while significant decrease in Chakwal-50
(7.7±0.01%). Drought stress significantly decreased the moisture in Chakwal-50 (8.50.10),
Shahkar (8.2±0.10%) and in Pakistan-13(8.0±0.10%). Priming with SA raised the moisture
contents in AARI-11(9.5±0.05%) while reduced in Chakwal- 50 (7.7±0.10%) under
drought form as compared to control condition. Under observation genotypes have less
moisture contents under drought as compared to normal. During both years moisture
contents remained same and non significantly varied.
Figure 4.35: Moisture percent in seeds of wheat genotypes
The estimation of moisture content in seeds is an important factor as it influences
the storage life and seed quality. For optimum milling yield moisture content is important
for starch development as well as ensuring the filling out of the endosperm. Low moisture
content is expected during post-harvest which is essential for the storage life of the grain
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Control Drought
Mo
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104
prior to being milled for its future use. If moisture contents are in excess it can lead to
sprouting in storage, mold growth, toxin formation and insect infestation. In our study all
tested genotypes has good moisture range afterharvesting which is suitable criteria for seed
storage. Drought stress reduced the moisture contents in three genotypes while SA priming
enhanced moisture in some genotype.
4.6.6 Starch content of seed
Under normal condition, AARI-11 and Shahkar had the highest starch
(52.4±0.05%) while other genotypes have non-significant difference in non primed samples
(Fig. 4.36). Starch was increased significantly on SA priming in Shahkar (52.4±0.10%),
Chakwal-50 (52.2±0.10%), and FSD-08(51.6±0.05%) while decreased in AARI-11.
Protein was increased significantly in GA priming in Shahkar (52.1±0.15%) and FSD-
08(51.7±0.05%) while decreased in Chakwal-50 (50.0±0.10%) and AARI-
11(50.2±0.10%).Under drought stress, Chakwal-50(49.1±0.10%) had the lowest starch
contents in non primed samples. Starch was significantly decreased in maximum genotypes
on SA priming. Starch only increased in Pakistan-13(51.2±0.20%) while decreased in other
genotypes when GA priming was applied. Under the effect of drought stress starch was
decreased in all other genotypes except Pakistan -13 which remain unaffected. In case of
GA and SA priming the starch contents are declined in all studied genotypes under the
condition of drought in comparison to control condition. During both the years non
significant effect occurred on starch contents.
105
Figure 4.36: Starch percent in seeds of wheat genotypes
Due to reduction in starch content the yield losses are caused because starch occupy
65% of cereal grain (Barnabás et al., 2008). Sucrose of grains are related to the
accumulation of starch which is correlated with sucrose synthesis and rest enzymes which
are responsible for starch synthesis (Yan et al., 2008). Under stress it is suggests that
enzymatic activityreduced that are involved in synthesis of starch and accumulation sucrose
contents become low. According to (Labuschagne et al., 2009) high temperature is
responsible for starch synthase inactivation which is the main enzyme in the biosynthesis
of starch causingdecline the starch ratio in endosperm. Some researcher reported that when
heat stress applied on bread wheat flour the protein content in flour increasesed(Bencze et
al., 2004); (Balla and Veisz, 2007). Similar results are also noted by other authors (Daniel
and Triboi, 2000); (Hrušková and Švec, 2009). After flowering, heat and drought stress
coupled with yield losses because of decline in the starch synthesis however grain proteins
are increased (Fowler, 2003). The basic useful properties of starch, particularly starch or
flour has the high water absorption ability and the drought flexibility, are reliant on the ratio
of amylose-to-amylopectin and the starch granules size distribution (Labuschagne et
a
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4 D
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Control Drought
Sta
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NP SA GA
106
al., 2009). Most of the time wheat grain quality mainly affected by water stress and high
temperature.
Figure 4.37: Dendogram derived from hierarchical cluster analysis of all combined quality attributes of five
wheat genotypes
4.6.7 Conclusion
The magnitudes in which drought stresses affect quality depend on the sensitivity
and tolerance of genotype. Wheat grain quality characters were noticeably affected under
drought stress. The highest wet gluten was observed in Shahkar under normal condition
while under drought wet gluten decreased in Shahkar that showed the sensitivity of cultivar.
FSD-08 had the same values for wet gluten under normal and drought stress. GA improved
wet gluten in FSD-08. Gluten index and falling number was highest in FSD-08 followed
by Pakistan-13 and AARI-11. In Shahkar, under drought; gluten index and falling number
decreased while SA priming improved it in shahkar genotype. Grain protein content were
increased under drought stress in the Chakwal-50. Starch and moisture percentage were
highest in the Shahkar and AARI-11. Under drought, moisture and starch contents
107
decreased in all genotypes except Pakistan -13 in which these attributes remained
unaffected under drought.
4.7 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE GELS
In the present study five genotypes of wheat alongwith priming treatment were
evaluated by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) for the estimation of protein contents stored in seeds. Standard protocol was
followed to separate protein subunits on polyacrylamide gel (10%). SDS-PAGE technique
resulted in fifty eight bands of total scorable bands. Based on these bands, difference of
primed and non primed seeds of wheat remained screened. Among fifty eight polypeptide
bands, 37% were generally existing in all genotypes and called as monomorphic, whereas
58% revealed differences and reflected as polymorphic. The size of these bands ranged
from 10 to 98 kDa. A protein, marker (pre stained) with weight ranges from 14.4 to 97kDa
was used to calculate the weight of these obtained polupeptide bands.
Band size vary between non primed and primed samples and bands that were not
prominent in some samples that were not counted, some bands were missing, while some
new bands were recorded.
The bands of the wheat samples were in sequence of band number 1, 2, 5, and 6.
Band number 3 was present only in primed samples. In FSD-08, SA treated samples have
two band among 66-45 while GA has four bands that showed variation to other samples.
In SA and GA treated samples in FSD-08 band size ranged to almost 10. In Punjab and
AARI-11 genotypes many new bands were detected between 45-20. The most prominent
change that occur in seed protein profile due to priming was the induction of a new band
25, 30,33 kDa protein induced by SA priming. GA priming also induced a 28 and 42 kDa
protein which was changed from non priming treatments. Priming treatment also caused
108
disappearance of two peptides with approximate molecular weights of 25 and 27and 35
kDa from Pakistan-13and Shahkar. However these two peptides did not disappear in
nonprime seeds. Priming also induced a change in the expression of a 33, 38 and 25 kDa
peptide. This priming induced protein was expressed by all priming treatments with more
prominent expression in FSD-08 and AARI-11 and Pakistan-13. A 26 kDa protein appeared
after seedpriming with GA. Another 25 and 30 kDa protein was expressed in case of SA
priming treatments (Fig 4.38a-b).
The reported findings are in line with those of Din and Flower (2002) who reported
that under non-stress conditions 15, 18 and 23 kDa proteins in ABA treated seeds.
Likewise, Reddy et al., (1993) worked on rice callus treated with ABA and found 15 and
23 kDa proteins under conditions of water stress. The plants showed adaptations by making
few proteins to overcome the stresses induced by of biotic as well as abiotic factors, among
them some of these produced proteins deduct by phytohormones, for example, salicylic
acid that was reported earlier (Hussein et al., 2007). Plants forms proteins to with stand
abiotic stresses and a considerable lot of these produced proteins are prompted by action of
phytohormones, for example, ABA and SA showed same affect earlier (Jin et al., 2001).
Seed priming promotes the synthesis of some new Proteins while loss of some
protein in some samples. A few new proteins appeared in the seed protein profiles after
priming treatment in the present study. It can be hypothesized that this peptide may be
related to a defense mechanism or osmotic adjustment under stress conditions.
109
Figure 4.38a: Electrophorogram showing control and seed priming induced variations in five wheat seed
protein profiles.
Figure 4.38b: Electrophorogram showing control and seed priming induced variations in five wheat seed
protein profiles
4.8TRANSGENERATIONAL EFFECT OF ELEVATED CARBONDIOXIDE ON
METABOLISM OF WINTER WHEAT EXPOSED TO ANTHESIS DOUGHT
Our study supports the idea that during the 20th century that over a number of
regions, drought index has increased and climate changes have altered the breeding targets.
110
4.8.1 Effect of elevated e[CO2], ambient a[CO2] and Drought on Yield Attributes
Grain number was also significantly affected by CO2 and irrigation treatment.
Symbols denoted E and A was seeds from previous generation while a and e was current
levels of CO2.In drought stressed plants grain number was reduced to a significant level
and severe reduction was observed under Ea[CO2] and Ee[CO2] as compared to Aa[CO2]
and Ae[CO2]. Carbon dioxidetreatments significantly affected the thousand kernel weight
however, it was unaffected by irrigation treatment. Plants grown-up under Ae[CO2] and
Ee[CO2] had higher yield in well watered plants than drought stressed plants. Harvest Index
was significantly exaggerated by both CO2 and irrigation management. Plants grown-up
under Aa[CO2], Ae[CO2] and Ee[CO2] had higher HI in both controlled and drought
stressed plants (Fig. 4.39).
Drought stress effect different phonological stages of the crop. When drought stress
was imposed near the beginning of reproductive stage it cause spikelet and pollen abortion
which leads to decrease the number of grains in wheat and rice (Kato et al., 2008; Dolferus
et al., 2011). Pollen and ovary abortion occurred when plant face drought stress at anthesis
stage due to induction in poor dehiscence of anther and inhibits panicle exertion owing to
reduced peduncle length, which cause in superior spikelet sterility in crops especially rice,
maize and wheat (Powell et al., 2012; Rang et al., 2011; Aslam et al., 2013). Our results
were also in agreement with the previous reports and grain number was reduced under
drought condition even under e[CO2]. The fatal drought at grain filling stage in wheat and
barley results during early senescence by means of shorter grain-filling period and low
persistence of flag-leaf green area (Samarah, 2005). In previous studies, number of grains
per plant and HI was increased under e[CO2] (Dong and Liu, 2005). Similar findings were
observed in our study that drought had significant effect on yield and yield contributing
factors. It was observed that e[CO2] enhanced grain yield, in addition to biomass.
111
Under elevated carbon it was observed that grain number in wheat was increased.
These results were confirmed by (Pleijel et al., 2000). Our results were supported by some
previous studies that e[CO2] enhanced yield even when seeds of ambient generation was
put under e[CO2]. (Wu and Wang, 2000)shown that under elevated [CO2] bean yield was
higher as a result of increase in bean number.The idea behind it was that high carbon
assimilation fix more carbon that result in the full development of flowers and grains.
Regarding individual grain weight the effect of high [CO2] was not always significant with
increase or decrease (Heagle et al., 2000; Pleijel et al., 2000). Ther is need of introduction
of such genotypes that is responsive to CO2- (Ainsworth et al., 2008) especially under
drought and salinity with the intention of providing starting lines for the process of
breeding.
112
4.8.2 Effect of elevated [e(CO2)], ambient a[CO2] and drought on
physiologicalattributes
Symbols denoted E and A was seeds from previous generation while a and e was
levels of CO2. Plants grown under Ae[CO2] and Ee[CO2] had higher photosynthesis while
during drought seeds grown under Ae[CO2] had high photosynthesis. After recovery
photosynthesis was higher under Ee[CO2]. Statistical analysis indicated that slight variation
was observed in term of stomatal conductance. The effect of CO2 was statistically
significant on transpiration rate while, plants grown under Ea [CO2] and Ee[CO2] had more
transpiration rate than plants under Aa[CO2] and Ae[CO2]. However, in all drought
stressed plants transpiration rate was significantly reduced. Results indicate that plants
grown under Aa[CO2] and Ee[CO2] had more leaf water potential(LWP) as compared to
Ae[CO2] and Aa[CO2] . Interactive effect of CO2 with LWP was also significant (Fig. 4.40-
43).
113
Plants intellect and react to e[CO2] during improved photosynthesis and stomatal
conductance going to be decreased in large number of species under different stress
conditions (Ainsworth et al., 2008). The consequences from this learning specify that
photosynthetic rate; stomatal conductance and transpiration rate was higher under elevated
CO2 in all three series of experiment. After drought transpiration rate was dramatically
reduced under both level of CO2. High [CO2] compensate the effect of drought stress
(Bencze et al., 2014)by increasing the levels of carbohydrates for the new tissues
development or filling grain (Wall et al., 2006). Though, effect of elevated [CO2] is not
always positively correlated for the stress tolerance in some studies (Pleijel et al., 2000).
Usually, transpiration is reduced by high concentration of [CO2] (Morison, 1987)
however; larger leaf area is responsible to induce reduction in transpiration by doubled
[CO2] (Wu and Wang, 2000). It was examined in our work that leaf water potential
dramatically decreased under ambient and elevated level of CO2. It is well documented
114
in previous studies that the main cause for this lower potential is due to effect of
complimentary masking factors. The masking factors are may be the difference in term of
temperature gradient flanked by the ambient air and the leaf that will escort to a deviation
of internal vapor pressures of leaf. CO2 assimilation and WUE adversely affected by low
water potential of leaf because of variation in different reactions during the process of
photosynthesis (Chaves et al., 2003) and decreased conductance in mesophyll (Warren,
2004).
4.8.3 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on Invertases and
Sucrose synthase activity
Symbols denoted E and A was seeds from previous generation while a and e was
levels of CO2. In case of (VacInv) plants under Ee[CO2] had high vacInv activity followed
by plants grown under Ae[CO2]. In spike vacInv activity was increased under Aa[CO2] and
Ae[CO2] . This activity uplifted in both spike and leaf in drought stressed plants under
Ae[CO2] and Ea[CO2] after recovery. Cytoplasmic invertase (CytoInv) activity was
significantly affected by CO2. Higher activity was observed under Ae[CO2] while under
Aa[CO2] activity was reduced. In spike, dramatic increased activity was observed under
Aa[CO2], while it was reduced in all other [CO2] treatments. Cell wall invertase (CWInv)
activity was significantly increased under Aa[CO2] and Ae[CO2]. However, in case of spike
non-significant variation was observed. It is reported in previous literature that under
drought conditions, in mature maize leaves, cell wall invertase activity was not affected but
an increase in vacuolar invertase activity and accumulation of high hexoses was recorded
in the leaves. Cell wall invertases are considered the key enzymes in sucrose unloading and
in the source/sink balance within the plants (Tang et al., 1999), by supplying carbohydrates
to tissues through apoplastic pathway. Sucrose synthase (Susy) activity in leaf was showed
similar trend under all level of CO2 before stress were applied, in spikes CO2 had significant
Fig-40 Effect of optimum and elevated [CO
2] on the photosynthesis (An) before drought (a), after drought (b) and after recovery
115
effect on Susy activity. Susy activity was highest under Ea [CO2] and Ee[CO2] treatment.
In drought stressed plants Susy activity was reduced in leaf while again uplifted after
recovery. In spikes, different results were observed for Susy activity and it was high for
both well wtered and drought stressed plants under Aa[CO2] (Fig. 4.44-47).
116
117
A and E= seeds from previous generation, a and e= current level of CO2. W= irrigation, D= drought,
L=Leaf. S=Spike
Figure: 4. 48 Key enzymes (C) activities of primary carbohydrate metabolism under drought and
well watered in both source (L) and sink (S).
118
4.8.4 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on the key enzymes for
Carbohydrate Metabolism
Symbols denoted E and A was seeds from previous generation while a and e was
levels of CO2. Significant variations were observed regarding UDP-glucose
pyrophosphorylase (UGPASE) activity in flag leaf. UGPASE activity was significantly
increased under Aa[CO2],while in all other treatments similar trend was observed.
However, within spike UGPASE activity dramatically increased under Aa[CO2] and
Ee[CO2]. Phosphoglucomutase (PGM) activity was increased manifold under the exposure
of e[CO2] in the leaf. In contrast within spikes PGM activity was significantly affected and
seems higher under e[CO2] as well as a[CO2]. In drought stressed plants PGM activity
remained unchanged in leaf while activity reduced after recovery in all except Aa[CO2]
(Fig. 4.49-50).
119
120
High Phosphoglucoisomerase (PGI) activity was observed under Aa[CO2] and
Ae[CO2] in leaf. In spikes, PGI activity increased many folds under Aa[CO2]. Glucose-6-
phosphate dehydogenase (G6PDH) activity was higher under Aa[CO2] and Ae[CO2]. In
spikes G6PDH activity was increased under Ae[CO2], Ea[CO2] and Ee[CO2] treatments.
Plants grown under drought stress had high G6PDH activity under Ae[CO2] and Ea[CO2]
in leaf . While in spike high activity was observed under Ae[CO2] and Ee[CO2] treatment
(Fig. 4.51-52).
121
Frutokinase (FK) activity was significantly affected by [CO2] in flag leaf. The
activity of this enzyme was increased in the plants grown under Ea[CO2] and Ee[CO2] as
compared to Aa[CO2] and Ae [CO2]. However, in spikes this activity remained unaffected.
Within drought stressed plants FK activity increased under all levels of CO2. In flag leaf,
Hexokinase (HXK) activity was slightly higher under Ea [CO2] and Ee[CO2] than Ae[CO2]
and Aa[CO2]. In contrast, within spikes this HXK activity was increased under Ae[CO2]
and Ee[CO2], and it was decreased under Aa[CO2] and Ea[CO2]. In drought stressed plants
HXK activity enhanced manifolds under e[CO2] (Fig. 4.53-54).
122
In leaf the highest Phosphofrutokinase (PFK) activity was found under Ae[CO2]
and Ea[CO2] treatment as compared to Aa[CO2] and Ee[CO2]. In spikes, the highest activity
of PFK was recorded under Ae[CO2]. In drought stress plants PFK activity was
123
less than control plants. The activity of Aldolase (Ald) showed increasing pattern under
e[CO2] treatments. In spikes, enzyme activity was similar for different [CO2] treatments.
In Spikes this activity showed dramatic changes, Aldolase activity showed distinct pattern.
Significant variations were observed regarding AGPASE activity in flag leaf. Similarly, the
activity of ADP- glucose pyrophosphorylase (AGPASE) was increased under Aa[CO2] and
Ae[CO2] treatments. However, within spike AGPASE activity dramatically increased
under Aa[CO2] and Ee[CO2] (Fig. 4.55-57).
124
To reveal the accomplishment of enzyme activity within physiological phenotyping
approach, no previous studies were focused on trans-generational effect of winter wheat
and it is even more difficult to extrapolate from one generation to the next.
125
Our analysis was built upon the largest scenarios of climate change because the
understanding of climate direction is of outmost importance. Some authors consider
climatic change as positive one, combined with development in cultivation technologies,
may increase potential wheat yield by 37-101% by 2050(Pérez-Carrillo and Serna-Saldívar,
2007). In present experiment, the metabolism of enzymes was determined at different
stages from source and sink organs. We found that plants of even a[CO2] background had
the highest activity under e(CO2). However, we also observed some of the enzymes have
higher activities under a[CO2] in both the organs (leaf and spike).
The present work suggested that UDP- glucose pyrophosphorylase (UGPase)
showed highest activity under Ae(CO2), while ADP- glucose pyrophosphorylase (AGPase)
activity increased under both level of CO2. It may be due to the adaptive response to with
stand the drought stress by carbon portioning, it results in stunted growth due to glycolytic
enzymes decrease expression. Elevated [CO2] can increase the amount of carbohydrates
within the leaf, mainly in the form of starch.
In one of previous studies, in crops ADPG pyrophosphorylase resultant activity
under elevated condition [CO2] was 1.6-fold more than that of the ambient conditions.
Similarly manifold increased AGPase activity has been shown in our study. Probable
mechanisms could be that sucrose synthesis due to feed-back inhibition of or recycling of
sucrose due to the activity of invertase and stimulation of AGPase by an improved 3PGA:
phosphate ratio, and increased expression of AGPase result of sugar-mediated signaling
(Stitt and Krapp, 1999). The results of present experiment indicate that activities of G6PDH,
FK, PGM and HXK showed distinct pattern in both source and sink organs. Activity of
these enzymes increased under e [CO2] in irrigated and drought stressed plants. These
results are similar to (Nakamura et al., 1997) who reported that in pollen cells, FK was
chief component whereas; the activity of HXK was comparatively low. It has been
126
found that part of the HXK protein is most important particulate, principally in
mitochondrial membranes, and this outward appearance is strongly introverted by
micro molar ADP concentrations (Yang et al., 2001).FK and HXK in the anther of
flower that present near the pollen may also have a key role to determine the
development of pollen cells and in turn germination capacity, given that the anther
walls be in charge of sugar nutrition as was found in Lilium pollen grains.
In our experiment, the PGI, PFK activities enhanced under e[CO2] while aldolase
and PFK activities showed decreasing pattern. In some previous studies no significant
variations were found for the actions of aldolase or PGI between optimum [CO2] treated
cells, whereas PFK and PFP activities decreased more than 40% under high CO2. A decline
of PFK as well as PFP activities under elevated CO2 conditions could be effected from an
inhibition or inactivation of preexisting PFK and PFP. However, it was reported that
regulating glycolysis by formation of PFK affected to a great extent on pH (Turner et al.,
1980).
4.8.5 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on key Enzymes
(a)activities for Carbohydrate Metabolism
Symbols denoted E and A was seeds from previous generation while a and e was
levels of CO2. Activities of different antioxidant enzymes were shown in (Fig. 4.58).
127
A and E= seeds from previous generation, a and e= current level of CO2. W= irrigation, D= drought,
L=Leaf. S=Spike
Figure-4.58 Key (A) enzymes activities of primary carbohydrate metabolism under drought and
wellwatered within source (L) and sink (s).
In recent study, superoxide dismutase (SOD) activity was significantly affected
under the exposure of [CO2]. This activity was increased in plants grown under Ea[CO2]
and Ee[CO2] in leaves. In spikes SOD activity remained unaffected in all plants under all
CO2 treatments. Before drought treatment, in leaf higher catalase (CAT) activity was
observed under Ee[CO2] and similar trend was estimated under remaining treatments. In
terms of spike CAT activity was significant affected under CO2. In contrast for drought
stressed plants CAT activity increased under Aa[CO2] and Ae[CO2](Fig.4.59-60).
128
Ascorbate peroxidase (APX) activity was increased in various species grown under
Ae[CO2] while decreased under Ee[CO2] in leaf. APX activity was significantly affected
in spikes under e[CO2] had greater APX activity. Glutathione reeducates (GR) activity
129
increased under Aa[CO2] and Ea[CO2] in leaf and spike respectively. After drought in leaf
and spike GR activity increased under e[CO2] (Fig. 4.61-62).
130
Peroxidase, dialysed extract (DPOX) activity increased under Ea, Ee[CO2] in
leaves while DPOX activity remained unchanged in spike. ZPOX activity increased under
Ea[CO2], Ee[CO2] in leaves. Peroxidase, Z extract (ZPOX) activity increased under
Aa[CO2] and Ae[CO2] in spike. Similarly, in drought stressed plants ZPOX activity
increased in leaf under Aa[CO2] and Ae[CO2] while in spike increased under Aa[CO2]
(Fig. 4.63-64).
Total antioxidant potential (TAP) showed similar trend in all plants grown under all
level of [CO2]. However, in spike significant variation was observed. Total antioxidant
potential was high in plants species that grown under Ae[CO2]. Under drought stressed
conditions plants TAP was dramatically increased under Ea [CO2] and Ee[CO2].
131
However, in spikes higher TA value was calculated for the plants grown under
Ae[CO2] (Fig. 4.65).Previous literature supported our findings that catalase under stress
has the ability of many reversible proteins in leaf and its activity is reduced under drought
condition. Due to the inhibition of photorespiration under elevated [CO2] enzyme activity
and catalase gene expression have been decreased in wheat, however, under drought they
increased (Vicente-Suarez et al., 2015).In literature, SOD activity was increased after 4
days of water stress in barley (Acar et al., 2001), and wheat (Sairam and Tyagi, 2004). (Lin
and Wang, 2002) observed that during the activities represented by SOD and CAT were
specifically much higher in CO2 enriched wheat than its ambient level.
132
The APX genes present in mitochondria, chloroplast and cytosol showed
differential inflection by large number of abiotic stresses in plants (Caverzan et al., 2014).
The activity of GR was higher in leaf under a[CO2] while in spikes, reverse effect was
observed and this activity was significantly higher under e[CO2]. Similarly, D-POX and
Z-POX activity increased under e[CO2]. Few previous reports about the activity of
antioxidants suggest that the activities of Catalase, supeoxide dismutase, Ascorbate
peroxidase, Peroxidase and Glutathione reductase decreased, in leaves of soybean genotype
grown at e[CO2]. Studies of (Zinta et al., 2014) supported that antioxidant level or activities
increased under e[CO2]. Some Previous findings supported our results and suggested that
e[CO2] had little or no effect on antioxidant and sometimes it even decrease the level of
these enzymes (Mishra et al., 2013). In most studies, about 50% of the observations of
some key antioxidant enzymes (ASC, APX and CAT) were seems to remain unchanged
under stress and elevated CO2 or even decreased for 28% of the observation (Glutathione
peroxidase). This showed that the under stress-mitigating conditions, effects of elevated
CO2 cannot be found all around the world to attributed to enhanced antioxidant defenses.
133
The concentrations of sucrose, glucose and fructose and starch were examined. In
wheat, significant increases were found for fructose and glucose in the high-CO2 treatment.
The concentration of sucrose was increased and decreased during different treatments.
However, CO2 effects were also statistically significant on starch. As reported previously,
the increased starch accumulation under elevated CO2 conditions affects sucrose
metabolism and causes the decrease in glucose content (Walter and Schurr, 2005). The
present study find that under elevated CO2 starch increased while in drought decreased.
(Woodhams and Kozlowski, 1954) and found the similar results. During photosynthesis
leaf soluble sugars formed which export for plant growth from the region of source that are
in most conditions are leaves into the area of phloem (Dietze et al., 2014). Consequently,
due to the assimilation of large amount of carbon or low concentration of carbon export
these sugars are a temporary pool and starch is a short-term storage induced. Meanwhile,
drought may limit the sink action, and sore strain the non-soluble carbohydrates export
from leaves and use, primary growth reduction is linked with leaf starch accretion (Lemoine
et al., 2013). During drought, photosynthesis is often decreased which is compensate by
e[CO2] (Bencze et al., 2014), which ultimately improved the levels of carbohydrates for
new tissue formation and also grain filling (Wall et al., 2006). Though, all theprevious
studies was not achieved the positive effects of e[CO2] for stress tolerance (Pleijel et al.,
2000). (Bencze et al., 2014) reported that, in bread wheat drought along e[CO2] result in
enhancement of the antioxidant enzyme which led to a high level of oxidative stress.
Previous studies that was done on durum wheat confirmed the approachability to e[CO2]
(Aranjuelo et al., 2013) , drought (Aprile et al., 2013;Habash et al., 2014) and the
combination of elevated carbon and water stress(Erice et al., 2007) is genotype specific.
Furthermore, different growth stages of durum wheat respond differently to e[CO2]
(Vicente et al., 2015). Some studies have shown the positive effect ofe[CO2] on water stress
134
tolerance (Harnos et al., 2002)(Robredo et al., 2007)(Bencze et al., 2014). It can be
summarized that Increased CO2 concentration along with availability of water contents may
retard the photosynthetic process (Robredo et al., 2007). Tha resultant reduced rate of
transpiration may alter or sometimes enhances the affect of stress induced by drought
(Kirkham, 2016; Miranda‐Apodaca et al., 2015; Kadam et al., 2015).
At the germination and development stages of crop, drought stress was foundin
winter months in previous studies (Russo et al., 2015). Response of plant to e[CO2] or
drought are correlated with growth stage, as well as environmental factors duration, level
and the genetic variability.
Wheat is being grown at the diverse climatic regions of the world. Due to wider
adaptability this crop has to face certain stresses and drought is one of them. Numbers of
strategies are being utilized to mitigate this stress however; e[CO2] has proved more
helpful. Nevertheless, the exact mechanism of this stress tolerance is still unknown. All
these studied attributes have major contribution towards starch synthesis, accumulation and
utilization. Photosynthesis has major contribution towards starch synthesis through these
enzymes activities. Likewise, accumulation of high starch within plants is always
rewarding in term of yield. However, better quality is always necessary alongwith high
yield and in our later study from same experiment we will also focus on quality of grain.
4.8.6 Conclusion
The change in atmospheric CO2 concentration affects the balance of carbohydrate
metabolism. However, more detailed analysis about gene determination is necessary to
relate carbohydrate accumulation with changes in the photosynthetic apparatus. In this
study we found that elevated e[(CO2)] provokes different enzymes in source (leaf) and sink
(spike) which are helpful to tolerate drought stress. It was observed that in source
135
CytInvertase, Sucrose synthase (Susy) along with catalase and ascorbate peroxidase was
decreased however, the activity CWInvertase was increased under drought and high CO2.
Similarly, within the sink the action of Glucose-6 phosphate dehydrogenase (G6PDH),
superoxide dismutase, ascorbate peroxidase and dialysed peroxidase was decreased under
drought while the activity of aldolase and frutokinase were increased. An interesting path
of activity of G6PDH within the source was observed as it remained unchanged before
drought but its activity was reduced under drought and increased under re-watering.
Similarly, the grain number was reduced under drought stress which indicates that aldolase,
G6PDH and susy are actually responsible to lower this number. Our results suggest that
high activity of Susy within the source and high aldolase and G6PDH activity within the
sink may be helpful to mitigate the drought stress under high value of CO2.
Under elevated CO2 conditions alongwith drought physiological processes can be
inhibited and promoted by photosynthesis and water use efficiency. Dissolved organic
carbon in soil is retained for a long time under elevated CO2 as a result mineralization
improved water use. Microbial carbon (biomass) and C:N ratio was decreased under
drought but drought along with CO2 is not as significant as alone. Plant root exudationcan
be stimulated under elevated CO2 as a result in more activities by invertase and catalase
activities. Findings suggest that soil carbon concentration, activities mediated by soil
enzyme and plant physiology are directly correlated with drought however elevated
CO2 reduced negative effects of drought (Yuhui et al., 2017).
136
SUMMARY
Two experiments were conducted during current study. First experiment was
carried out in University of (AJ&K) Pakistan and effect of drought stress on five wheat
cultivars viz. AARI-11, Chakwal-50, Shahkar, Pakistan-13 and FSD-08 was studied.
Hormonal seed priming (SA, GA) were used as a short term approach to reduce drought
stress. Different morpho-physiological, traits were studied from flag leaf while quality as
well as seed bio physiological changes were after harvesting.
Priming with SA and GA improved the activities of oxidative and antioxidant
enzymes in seeds like amylase, protease, catalase, SOD and POD. Genotypic difference
varied in term of treatments. Priming with both the growth regulators increased yield under
normal and drought conditions and it was observed that higher yield was due to rapid
emergence and vigorous seedlings. Pakistan-13 and FSD-08 were ranked higher for overall
grain. The study of soluble compounds, lipid peroxidation, relative water contents,
membrane stability, minerals, high proline, antioxidant and maintenance of cell membrane
integrity contributed toward osmotic adjustment which increased the yield under stress
conditions. These findings suggest that adverse effect of water stress on wheatcan be
overcome by using GA and SA. Wheat grain quality attributes were also studied and it was
observed that drought had significant effect. However, hormonal seed priming also help in
maintaining the undoing adverse effects of drought. The FSD-08 was maintainedquality of
grain under normal and stressed conditions. Seed protein profiling indicated that many new
peptides were appeared after priming and it can be hypothesized that these proteins may be
helpful to cope stress.
137
Second experiment was conducted at University of Copenhagen, Denmark to find
out the transgenerational effect of elevated e[CO2] in winter wheat at anthesis drought
stress. It was observed that, photosynthesis and activities of antioxidant enzymes was
increased under e[CO2]. Resultantly, all these traits contributed to increase the yield
especially under e[CO2]. Trans-generational effect indicated that seeds had stress memory
and thus maintained the effect of previous generation. Activities of all enzymes varied
within source and sink. UDP- glucose pyrophosphorylase (UGPase) activity was increased
under Ae[CO2], while ADP- glucose pyrophosphorylase (AGPase) activity increased under
both level of CO2.
Elevated [CO2] can increase the carbohydrates within the source, mainly in the form
of starch. These results indicate that high metabolism of Sucrose synthase, aldolase and
Glucose- 6 phosphate dehydrogenase might be helpful to mitigate the drought stress under
elevated CO2. Based on these results we can report that enzymes involved in carbohydrate
metabolism are affected under elevated CO2 and drought. Hence, studied cultivar had very
good yield under e[CO2] even in drought stress.
138
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APPENDIX
APPENDIX 1
Description of wheat genotypes used in current study
S.
No
Genotypes Properties
1 FSD-2008
Stem rust resistant; Smut resistant; recommended for irrigated
areas but also have very good performance in drought areas.
2 AARI-2011
For irrigated areas / Punjab
3 CHAKWAL-
50
For arid climate, drought and heat tolerant
4 Pakistan-13 For arid climate, drought and heat Resistant
5 SHAHKAR
2013
Bold seed, high yielding variety for irrigated areas
169
APPENDIX 2
Multiple ANOVA for seed biochemistry attributes
Estrase Protease Amylase SOD POD CAT
R² 0.929 0.990 0.950 0.855 0.933 0.913
F 13.944 106.768 20.567 6.322 14.966 11.206
Pr > F < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 < 0.0001
Geotypes 9.311 252.468 21.260 2.614 9.225 14.449
0.001 < 0.0001 < 0.0001 0.077 0.001 < 0.0001
Treatment 28.004 24.617 29.029 6.715 55.108 8.320
< 0.0001 < 0.0001 < 0.0001 0.008 < 0.0001 0.004
Geotypes*Treatment 12.639 50.715 17.699 7.070 6.150 9.671
< 0.0001 < 0.0001 < 0.0001 0.001 0.001 0.000
170
APPENDIX 3
Multiple ANOVA for oxidative behavior
Protease Estrase Amylase SOD POD Cat ASP
R² 0.861 0.528 0.785 0.857 0.840 0.479 0.837
F 11.169 2.023 6.620 10.838 9.490 2.973 16.596
Pr > F < 0.0001 0.029 < 0.0001 < 0.0001 < 0.0001 0.000 < 0.0001
Genotype 5.830 2.664 2.909 2.562 1.957 7.404 8.879
0.001 0.047 0.034 0.054 0.121 < 0.0001 < 0.0001
Stress 20.898 0.689 13.705 0.858 3.794 11.042 278.997
< 0.0001 0.508 < 0.0001 0.432 0.031 0.001 < 0.0001
Treatmet 94.233 0.001 41.412 188.379 99.774 0.856 0.794
< 0.0001 0.979 < 0.0001 < 0.0001 < 0.0001 0.429 0.456
Genotype*Stress 8.390 0.654 3.704 1.506 4.615 3.401 6.401
< 0.0001 0.728 0.003 0.188 0.001 0.014 0.000
Genotype*Treatmet 0.447 2.680 1.079 2.630 7.989 0.741 0.565
0.774 0.046 0.380 0.049 < 0.0001 0.656 0.803
Stress*Treatmet 2.520 7.884 9.516 0.458 4.421 0.266 1.144
0.094 0.001 0.000 0.636 0.019 0.767 0.325
171
APPENDIX 4
Multiple ANOVA for physiological parametere
TOS MDA CMT RWC Chla Chlb Cartnid Antho protn Sugar Prol K+ Ca+
R² 0.384 0.462 0.837 0.862 0.733 0.567 0.593 0.514 0.514 0.857 0.960 0.956 0.870
F 1.127 1.551 16.594 20.200 8.906 4.243 4.709 3.422 3.423 19.447 77.501 69.641 21.636
Pr > F 0.365 0.117 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Geotypes 1.794 0.978 48.187 61.631 8.248 1.889 7.029 8.689 11.327 78.552 251.630 219.610 68.670
0.150 0.431 < 0.0001 < 0.0001 < 0.0001 0.122 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Stress 0.534 2.252 96.596 105.298 96.550 32.325 3.000 6.645 3.619 9.086 418.111 109.580 2.260
0.590 0.119 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.088 0.012 0.061 0.004 < 0.0001 < 0.0001 0.137
Treatmet 1.404 0.006 0.312 4.473 17.820 17.499 31.329 13.646 2.014 4.960 8.447 3.153 14.630
0.243 0.940 0.733 0.015 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.141 0.010 0.001 0.049 < 0.0001
Geotypes*Stress 1.509 1.269 11.835 3.643 0.340 0.328 0.619 0.104 1.208 5.649 33.940 14.583 2.306
0.187 0.288 < 0.0001 0.010 0.850 0.858 0.651 0.981 0.316 0.001 < 0.0001 < 0.0001 0.067
Geotypes*Treatmet 0.398 1.370 1.378 3.307 0.808 0.723 0.244 0.274 1.238 5.786 5.922 48.915 12.819
0.809 0.262 0.222 0.003 0.598 0.671 0.981 0.972 0.291 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Stress*Treatmet 0.173 4.073 0.072 11.197 7.011 3.565 0.343 0.283 2.093 3.147 1.428 9.236 18.186
0.842 0.025 0.931 < 0.0001 0.002 0.034 0.711 0.755 0.131 0.049 0.247 0.000 < 0.0001
172
APPENDIX 5
Multiple ANOVA for Morphology Parameters
Plant
height Tillar Spiklength Spiklts
Extrusin
length
Peduncl
length Grains
Grain
weight Grainyield Bioyield
Harvest
Index
R² 0.892 0.331 0.829 0.699 0.514 0.467 0.817 0.641 0.829 0.781 0.751
F 62.328 3.728 36.404 17.513 7.966 6.596 33.644 13.410 36.411 26.758 22.641
Pr > F < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Geotypes 44.773 6.242 60.298 16.815 5.546 4.081 74.738 8.824 106.606 36.148 57.442
< 0.0001 0.000 < 0.0001 < 0.0001 0.000 0.004 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Stress 1075.433 41.100 445.663 225.630 103.541 83.735 267.451 227.914 313.053 363.827 193.977
< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Treatmet 15.191 1.542 12.691 22.129 15.731 10.042 55.348 7.072 7.131 1.114 0.734
< 0.0001 0.217 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 0.001 0.331 0.482
Geotypes*Stress 1.916 1.544 6.560 2.608 0.784 0.675 0.234 0.523 0.880 8.794 4.138
0.110 0.192 < 0.0001 0.038 0.537 0.610 0.919 0.719 0.477 < 0.0001 0.003
Geotypes*Treatmet 1.536 0.348 2.621 2.434 0.240 1.588 2.950 0.267 0.661 1.944 4.124
0.149 0.945 0.010 0.016 0.983 0.132 0.004 0.976 0.725 0.057 0.000
Stress*Treatmet 2.014 0.082 2.522 0.355 2.523 1.480 2.447 0.018 1.041 0.276 0.354
0.137 0.921 0.084 0.702 0.083 0.231 0.090 0.982 0.356 0.760 0.703
173
APPENDIX 6
Multiple ANOVA for Quality parameters
Wglutn GlIndx F No Protein Moistr Strch
R² 0.654 0.867 0.942 0.543 0.830 0.802
F 6.116 21.040 52.828 3.849 15.768 13.137
Pr > F < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Geotypes 8.809 47.491 209.443 9.805 25.768 7.493
< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Stress 2.119 87.321 1.582 3.429 112.591 90.171
0.150 < 0.0001 0.213 0.068 < 0.0001 < 0.0001
Treatmets 1.912 10.836 30.195 3.563 3.765 17.050
0.156 < 0.0001 < 0.0001 0.034 0.028 < 0.0001
Geotypes*Stress 17.617 13.146 36.133 3.369 11.771 5.502
< 0.0001 < 0.0001 < 0.0001 0.014 < 0.0001 0.001
Geotypes*Treatmets 1.603 4.419 7.397 2.052 6.297 8.904
0.140 0.000 < 0.0001 0.053 < 0.0001 < 0.0001
Stress*Treatmets 1.981 27.472 2.962 0.584 5.241 14.200
0.146 < 0.0001 0.058 0.560 0.008 < 0.0001