Shahwani, Muhammad Naeem (2011) Studies on abiotic stress tolerance in Hordeum vulgare L ...

388 Pages·2011·37.33 MB·English

by  Jan Muhammad

Shahwani, Muhammad Naeem (2011) Studies on abiotic stress tolerance
in Hordeum vulgare L. genotypes from arid and temperate regions. PhD
thesis.

http://theses.gla.ac.uk/2775/

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Studies on Abiotic Stress Tolerance in
Hordeum vulgare L. Genotypes from Arid
and Temperate Regions

Muhammad Naeem Shahwani

Thesis Submitted for the Degree of
Doctor of Philosophy

College of Medical, Veterinary and Life Sciences,
School of Life Sciences
University of Glasgow
U.K.

July, 2011

© M. N. Shahwani
ABSTRACT
Plants growing in arid regions often suffer from high shoot temperatures, low shoot
water concentrations, low turgor pressures, and high salinity in the rhizosphere. To
investigate which traits confer tolerance on plants in these areas a range of genotypes
was studied. These included Local (an uncharacterized landrace grown in south western
Pakistan), Soorab-96 and Awaran-2002 (both elite cultivars developed by ICARDA and
commercially grown in Pakistan), and Optic (an elite European cultivar). Measurements
on germination, growth, and yield suggested that landrace Local is significantly least
affected by high salinity (p<0.05) compared with lines Soorab-96, Awaran-2002 and
Optic. Further investigations on ion profiling established that landrace Local could
maintain low Na+/K+ ratios. This appeared to arise from Local’s ability to prevent Na+
accumulation in the roots and shoots by enhanced exclusion or efflux or both. Probably
this unique characteristic of landrace Local helped in maintaining its photosynthetic
efficiency, plant water status, and stomatal conductance, which resulted in its better
performance and survival in high salinity. There was no evidence that high tissue solute
concentrations, high proline levels or life cycle strategies played a role in salt stress
tolerance. In addition, there was no evidence that osmotic stress was responsible for the
observed suppression of growth in any of the genotypes. The main conclusion from this
study is that for glycophytes (which do not complete a full life cycle above 100 mM
NaCl; this includes all of the world’s major crops), it is the ionic component of salinity
stress that impairs growth processes and yield, not the osmotic component. Further
research on salinity stress in crops should focus on understanding the processes that
control ionic balance rather than osmoregulation. There is some evidence that long term
exposure of plants in the preceding generations to moderately high salt concentrations
(e.g. 100 mM NaCl) improves barley halotolerance in succeeding generations, i.e.
halotolerance has a transgenerational, epigenetic basis, but there was also evidence that
the improved halotolerance in the Local genotype was partly genetic.
In another series of experiments the importance of short periods of high leaf
temperatures (T ) on photosynthetic efficiency of barley genotypes Local, Optic, and
leaf
Soorab-96 was investigated. In all three genotypes light saturated carbon dioxide
assimilation rates (A ) and the carboxylation coefficients (Φ , a measure of the
sat CO2
efficiency of CO fixation) in the fourth fully expanded leaves were equally suppressed
2
to approximately 20 % of their pre-treatment levels immediately after a short period of
i
heat stress (T > 40.0 ± 0.5 °C for 20 minutes). Parallel measurements using a range of
leaf
techniques confirmed that the suppression of A and Φ was not attributable to
sat CO2
changes in the light harvesting capacity (leaf absorptance and chla excitation spectra),
maximum quantum efficiency of PSII (Φ , Fv/Fm), and to stomatal conductance (g ).
PSII s
It is unlikely that the suppression arose from damage to the electron transport chain¸ or
to the capacity to develop or maintain non-photochemical quenching (NPQ, which is
dependent on the transthylakoid ΔpH), but these possibilities cannot be dismissed. LC-
MS and enzymic analysis of leaf metabolite levels showed that the pools of metabolites
feeding into RuBisCO are not affected by heat stress whilst those of the metabolites
flowing away from RuBisCO were significantly depleted. The implication is that short
periods of heat stress severely impairs RuBisCO, RuBisCO Activase, or processes close
to the carboxylation step. Five days after heat stress A and ΦCO had significantly
sat 2
recovered to approximately 40 % (p<0.05) of their pre-stress levels in landrace Local,
but no significant recovery was observed in any of the elite lines including those
distributed by ICARDA for arid land production. These findings provide evidence that
thermal damage may play a significant role in yield suppressions in arid regions and
that there is a genetic basis for thermotolerance in barley1.

1 Some of the work presented in this thesis has been given as oral presentations at two international
scientific meetings.
1. International Conference on Food Security and Climate Change in the Dry Areas, by ICARDA
in Amman-Jordan from 1-4th February 2010.
2. SEB Annual Conference, Prague, 30th June-3rd July 2010.
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In the Name of Almighty Allah (God), Most Gracious, Most Merciful
ACKNOWLEDGEMENTS:
First and foremost countless thanks and praises to Almighty Allah (Subhana-u-
watahala) for his granted bounties (i.e. observe, analyse and interpret). Then to His
beloved Messenger Muhammad (PBUH) for prioritizing the knowledge for the benefit
of all humanity.
I am indebted to my supervisor Dr. Peter Dominy, for his unfailing support and
encouragement, the kindness, the opportunity and help to allow me independence ‘no
words can describe his assistance’. He also helped me a lot to believe in my self and in
possibility to reach my goals. I have not only learned material facts, but also focus and
organization. His supervision has provided an ideal intellectual environment to learn
science for which I will always be grateful and his enthusiasm and encouragement have
persisted and sustained me over the last three years. Special thanks to all staff members
and scholars of the Dominy group, in particular, and other groups from the Plant
Sciences Discipline in general, for the warmth and kindness shown to me. It will be an
injustice if I do not pay thanks to Dr. Stéphanie Arrivault (The Max Planck Institute of
Molecular Plant Physiology, Am Muehlenberg, Germany) for her incredible help in
analyzing my plant samples for C3 cycle metabolite pools.
I thank all friends specially Dr. Peter Meadows and Dr. Azra Meadows for their
incredible help and support during my studies. I would like to thank the people of
Islamic Republic of Pakistan, the administration of BUITEMS, and the Higher
Education Commission of Pakistan for their continuous encouragement, well wishes,
and financial support.
Last but not least. I would like to express my sincere thanks to my Parents for their
sincere love and good wishes, to my wife Saiqa for playing a great role during my
scholastic studies. I also thank my children Tayeba, Anam, Iqra, Momina, and Malaika
for their patience during my long absence from home and for creating a warm and
rewarding environment when I returned home tired. Finally, I am extremely grateful to
my brothers, sisters, and close relatives for their encouragement and support. Without
the help of Almighty Allah and the help of all these people mentioned above this work
would not have been completed.
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TABLE OF CONTENTS
Sections....................................................................................................................... Page
Title Page...........................................................................................................................0
Abstract...............................................................................................................................i
Acknowledgements..........................................................................................................iii
Table of Contents.............................................................................................................iv
List of Figures..................................................................................................................xi
List of Tables..................................................................................................................xiv
Abbreviations..................................................................................................................xv
Declaration....................................................................................................................xvii

1 Chapter 1: Introduction.........................................................................................1
1.1 Food Supply and Population Growth in the 21st Century.........................1
1.2 Abiotic Stress: A Worldwide Problem in Agriculture..............................2
1.2.1 Soil Salinity...............................................................................................3
1.2.1.1 The Effect of Salinity on Agriculture...................................................4
1.2.1.2 Classification of Plants According to Their Tolerance of Salinity.......4
1.2.1.3 The Deleterious Effects of High Salinity on Plants..............................5
1.2.1.4 Strategies for Coping with High Salinity..............................................6
1.2.1.5 The Importance of Turgor.....................................................................7
1.2.1.6 Na+ Uptake Mechanisms.......................................................................8
1.2.1.7 Salt Stress Sensing in Plants...............................................................10
1.2.1.8 Na+ Sequestration................................................................................10
1.2.2 Thermal Stress.........................................................................................12
1.2.2.1 Effects of Heat Stress on Plants..........................................................13
1.2.2.2 Plant Responses to Heat Stress...........................................................14
1.2.2.2.1 Morpho-Anatomical Responses....................................................14
1.2.2.2.2 Physiological Responses...............................................................15
1.2.2.3 Mechanisms of Thermotolerance........................................................18
1.3 Thermotolerance Studies in Barley.........................................................21
1.4 Background of the Study.........................................................................22
1.4.1 Plant Material and Area..........................................................................22
1.4.2 Hypothesis Behind Current Study...........................................................23
1.4.3 Aims and Objectives of the Study...........................................................25
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2 Chapter 2: Materials and Methods.....................................................................26
2.1 Halotolerance..........................................................................................26
2.1.1 Plant Material..........................................................................................26
2.1.2 Agronomic Characterization of Barley Genotypes Under High Salt
Concentrations........................................................................................................28
2.1.2.1 Germination........................................................................................28
2.1.2.1.1 Experimental Design.....................................................................28
2.1.2.2 Growth of Mature Plants.....................................................................28
2.1.2.2.1 Plant Material................................................................................28
2.1.2.2.2 Experimental Design for Growth and Yield.................................30
2.1.2.2.3 Shoot and Root Length, Fresh and Dry Weight............................30
2.1.2.2.4 Tillers, Ears, Grains and Grain Weight Per Plant.........................30
2.1.2.3 Assessment of Development...............................................................30
2.1.2.3.1 Assessment of Ppd-H1 Flowering Locus......................................31
2.1.3 Physiological Characterization of Barley Genotypes under high salt
concentrations.........................................................................................................33
2.1.3.1 Photosynthesis.....................................................................................33
2.1.3.1.1 CO Response Curve.....................................................................33
2
2.1.3.1.2 Light Response Curve...................................................................37
2.1.3.2 Water Potential....................................................................................40
2.1.3.3 Solute Potential...................................................................................40
2.1.3.3.1 Solute Potential of Tissue Cell Sap (Tissue ψ )................................40
s
2.1.3.3.2 Solute Potential of Exuded Xylem Sap (xylem ψ )..........................40
s
2.1.3.3.3 Proline Concentration...................................................................41
2.1.4 Ion Analysis............................................................................................41
2.1.4.1 Preparation of Barley Material for ICP-OES Analysis.......................41
2.1.4.1.1 Standard Solution..........................................................................42
2.1.4.1.2 Assessment of Ion Concentration in Plant Material......................42
2.2 High Temperature Thermotolerance.......................................................43
2.2.1 Plant Material..........................................................................................43
2.2.2 Exposure to Heat Stress..........................................................................43
2.2.2.1 ‘Incremental’ Heat Stress Experiments...............................................43
2.2.2.2 ‘Temperature Jump’ Heat Stress Experiments....................................43
2.2.3 Leaf Absorptance and Fluorescence Measurements...............................48
2.2.3.1 Measurement of Leaf Absorptance and Light harvesting Capacity....48
2.2.3.2 Measurement of Photosystem-II Quantum Yield (ΦPS-II) and
Electron Transport Rates (ETR).............................................................48
2.2.4 Gas Exchange Measurements.................................................................50
2.2.5 Metabolite Pools Analysis......................................................................50
2.2.5.1 Preparation of Plant Extracts...............................................................50
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2.2.5.2 Sample Extraction...............................................................................52
2.2.5.3 Analysis of Metabolite Pools..............................................................52
2.2.5.4 Synthesis of Deuterated G6P and G3P...............................................52
2.2.5.5 Ion Pair Chromatography – Triple Quadrupole MS...........................53
2.2.5.6 Detection and Validation of Signals in Plant Extracts........................54
2.2.5.7 Other Metabolite Assays.....................................................................54
2.2.6 Statistical Analysis..................................................................................56
3 Chapter 3: Characterization of Halotolerance in Barley Genotypes:
Germination, Growth, Yield, and Development........................................................57
3.1 Germination............................................................................................57
3.2 Growth Parameters..................................................................................62
3.2.1 Phenotypic Representation of Barley Genotypes under High Salt
Concentrations........................................................................................................62
3.2.2 Shoot and Root Length...........................................................................65
3.2.3 Shoot and Root Fresh Weight.................................................................65
3.2.4 Shoot and Root Dry Weight....................................................................69
3.2.4.1 Tillering...............................................................................................71
3.3 Yield Parameters.....................................................................................74
3.3.1 Ears and Grains per Plant........................................................................74
3.3.2 Grain Weight per Plant and 1000 Grain Weight.....................................76
3.4 The Effects of Salt Stress on the Development of Barley Genotypes ....78
3.4.1 The Effects of Salt Stress on Four Key Developmental Stages..............81
3.4.2 Assessment of Ppd-H1 Flowering Locus................................................84
3.5 Discussion...............................................................................................86
4 Chapter 4: Characterization of Halotolerance in Barley Genotypes:
Physiological Responses................................................................................................90
4.1 Carbon Dioxide Supply and Photosynthesis...........................................90
4.1.1 Stomatal Conductance (gs).....................................................................91
4.1.2 CO Assimilation Rates and Quantum Efficiency..................................91
2
4.1.3 Carboxylation Efficiency (Φ CO )..........................................................95
2
4.1.4 Apparent Photorespiration (V ) and Dark Respiration (mitRd)...............95
o
4.1.5 Ratio of Internal CO (C) to Atmospheric CO Concentration (C ), and
2 i 2 a
Assimilation versus Stomatal Conductance............................................................99
4.2 Plant Water Status.................................................................................102
4.2.1 Relative Water Content and Water Use Efficiency..............................102
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4.2.2 Leaf Water Potential (ψ ), Xylem Solute Potential (Xylemψ ) and Turgor
H2O s
Pressure ...............................................................................................................104
4.2.3 Leaf Tissue Solute Potential and Proline Concentrations.....................107
4.3 Ion Content Assessment........................................................................109
4.3.1 K+, Na+ Content and Na+/K+ Ratio of Barley Stem..............................109
4.3.2 K+, Na+ Content and Na+/K+ Ratio of Barley Root...............................113
4.3.3 K+, Na+ Content and Na+/K+ Ratio of Barley Green Leaves................116
4.3.4 K+, Na+ Content and Na+/K+ Ratio of Barley Desiccated Leaves........119
4.3.5 Distribution of K+ and Na+ Content in Shoots and Roots.....................119
4.4 Assessment of Sodium Uptake.............................................................123
4.4.1 Na+ and K+ Uptake by Barley Shoots....................................................123
4.4.2 Na+ and K+ Uptake by Barley Roots.....................................................125
4.4.3 Distribution of K+ and Na+ Content in Shoots and Roots.....................125
4.5 Mapping Loci Conferring Tolerance in Landrace Local......................128
4.6 Epigenetic Studies in Barley Genotypes...............................................130
4.6.1 The Effects of Parental Salt Stress on Seedling Growth.......................130
4.7 Discussion.............................................................................................134
5 Chapter 5: Characterization of Thermotolerance in Barley...........................139
5.1 ‘Incremental’ Heat Stress......................................................................139
5.1.1 Gas Exchange Measurements...............................................................139
5.1.1.1 Light Saturated CO Assimilation Rates (A ).................................140
2 sat
5.1.1.2 Transpiration Rates (E) and Stomatal Conductance (gs)..................140
5.1.1.3 Relationship of CO Assimilation (A) with Stomatal Conductance (gs)
2
under Incremental Heat Stress Events..................................................140
5.1.2 Leaf Absorptance..................................................................................145
5.1.2.1 Light Absorptance.............................................................................145
5.1.2.2 Chlorophyll Fluorescence Excitation Spectra...................................145
5.2 ‘Temperature Jump’ Heat Stress Experiments......................................148
5.2.1 Gas Exchange Measurements...............................................................148
5.2.1.1 Light Saturated CO Assimilation Rates (A ) and Carboxylation
2 sat
Efficiency (Φ CO )...............................................................................148
2
5.2.1.2 Phenotypic Differences in Leaves After Heat Stress........................149
5.2.1.3 Transpiration (E) and stomatal Conductance (gs).............................155
5.2.1.4 CO Assimilation and Photorespiration vs. Stomatal Conductance..156
2
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5.2.1.5 Relationship of CO Assimilation (A) And Apparent Photorespiration
2
with Stomatal Conductance (gs) under Temperature Jump Heat Stress
Events....................................................................................................157
5.2.1.6 Effects of Temperature Jump Heat Stress on the Ratio of Stomatal
Conductance Before, Immediately After and 5 Days after...................159
5.2.1.7 Stomatal Densities in Leaves of Local and Optic.............................159
5.2.2 Fluorescence Measurements.................................................................161
5.2.2.1 Quantum Efficiency (Fv/Fm) and Non Photochemical Quenching
(NPQ)....................................................................................................161
5.2.2.2 Steady State and Maximum Electron Transport Rates.....................165
5.2.3 C3 Cycle Metabolites............................................................................168
5.2.3.1 Effect of Heat Stress on Metabolite Pool Levels of Cytosol,
Mitochondria and Plastid......................................................................168
5.3 Discussion.............................................................................................173
6 Chapter 6: General Discussion..........................................................................176
6.1 Comparison of Salt Stress in Barley Genotypes...................................176
6.1.1 Epigenetic Base of Halotolerance in Barley.........................................183
6.2 Comparison of Thermal Stress in Barley Genotypes............................184
6.3 Conclusions and Future Work...............................................................190
6.3.1 Salt Stress..............................................................................................190
6.3.2 Thermal Stress.......................................................................................190
6.3.3 General Conclusions.............................................................................191
7 References............................................................................................................192
8 Appendix....................................................................Error! Bookmark not defined.

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