INFLUENCE OF HIGH TEMPERATURE AND BREEDING FOR HEAT TOLERANCE

2. DOCTORAL SEMINAR- I
On
INFLUENCE OF HIGH TEMPERATURE AND
BREEDING FOR HEAT TOLERANCE
Submitted by
VITNOR SUSHIL SARJERAO
Reg. No. 015/14
Submitted to
Dr. J. V. PATIL
Head,
Department of Agricultural Botany

3. Points to be covered:-
 Introduction
 Effects of high temperature
 Heat stress and heat tolerance
 Screening for heat tolerance traits
 Breeding for high temperature tolerance
 Summary and conclusion
 Case Study

4. 1. Introduction
 Heat stress is often defined as the rise in temperature beyond a threshold
level for a period of time sufficient to cause irreversible damage to plant
growth and development.
 At very high temperatures, severe cellular injury and even cell death may
occur within minutes, which could be attributed to a catastrophic collapse of
cellular organization
 Direct injuries due to high temperatures include protein denaturation and
aggregation, and increased fluidity of membrane lipids. Indirect or slower
heat injuries include inactivation of enzymes in chloroplast and
mitochondria, inhibition of protein synthesis, protein degradation and loss of
membrane integrity

5. II. EFFCTS OF HIGH TEMPERATURE
A. Morphological and yield traits
 At later stages, high temperature may adversely affect
photosynthesis, respiration, water relations and membrane
stability, and also modulate levels of hormones and
primary and secondary metabolites.
B. Morphological symptoms
High temperatures can cause considerable pre and
postharvest damages, including scorching of leaves and
twigs, sunburns on leaves, branches and stems, leaf
senescence and abscission, shoot and root growth inhibition,
fruit discoloration and damage, and reduced yield

6. B. Physiological and biochemical traits
1. Waters relations
Under field conditions, high temperature stress is frequently associated with
reduced water availability
2.Accumulation of compatible osmolytes
A key adaptive mechanism in many plants grown under abiotic stresses,
including salinity, water deficit and extreme temperatures, is accumulation of
certain organic compounds of low molecular mass, generally referred to as
compatible osmolytes
3 Photosynthesis
Any constraint in photosynthesis can limit plant growth at high
temperatures.
4. Assimilate partitioning
Under low to moderate heat stress, a reduction in source and sink activities
may occur leading to severe reductions in growth, economic yield and
harvest index. Assimilate partitioning, taking place via apoplastic and
symplastic pathways under high temperatures, has significant effects on
transport and transfer processes in plants

7. 5. Cell membrane thermostability
 Heat stress accelerates the kinetic energy and movement of
molecules across membranes thereby loosening chemical bonds
within molecules of biological membranes
6. Hormonal changes
 Hormones play an important role in this regard. Cross-talk in
hormone signaling reflects an organism’s ability to integrate
different inputs and respond appropriately.
 A gaseous hormone, ethylene regulates almost all growth and
developmental processes in plants, ranging from seed ger-
mination to flowering and fruiting as well as tolerance to
environmental stresses.

8. III. HEAT STRESS AND HEAT TOLERANCE
A.Definition of Heat Stress
Heat stress is often defined as the rise in temperature beyond a threshold level for
a period of time sufficient to cause irreversible damage to plant growth and
development.
B. Heat Tolerance
The adverse effects of heat stress can be mitigated by developing crop plants with
improved thermotolerance using various genetic approaches
Tolerance to this stress via knowledge of metabolic pathways will help us in
engineering heat tolerant plants.
A group of proteins called heat shock proteins are synthesized following stress
and their synthesis is regulated by transcription factors.
Under high temperature (HT), reactive oxygen species (ROS) are often induced
and can cause damage to lipids, proteins, and nucleic acids.

9. IV.SCREENING FOR HEAT TOLERANCE TRAITS
 it is imperative to use cost efficient and reliable techniques to screen the
available germplasm for various ecophysiological, morphological, and
reproductive traits to assist their utilization in crop breeding programs.
 A brief description of some new emerging ecological, morphological, and
physiological techniques, which are being used in many crop improvement
programs particularly at various international crop improvement centres
(mainly CIMMYT and IRRI) and other national research centres, are
discussed in this section.
 However, these controlled environments can be used for preliminary screening
but it will be important to also test the performance of the genotypes identified
under controlled condition in field conditions before they are used extensively
in the breeding programs.

10. PHYSIOLOGICAL AND/OR BIOCHEMICAL TRAITS
1. Cellular Membrane Thermo stability
High temperature modifies membrane composition and structure and can cause
leakage of ions. Membrane disruption also causes the inhibition of processes such
as photosynthesis and respiration.
2. Chlorophyll Content
exhibited physiological evidence indicating that
loss of chlorophyll during grain filling was associated with reduced yield in
the field .
They also established that while high chlorophyll content does not guarantee
heat tolerance.
3. Chlorophyll Fluorescence
Chlorophyll fluorescence emission kinetics from plants provides an indi
cator of plant photosynthetic performance
 The sensitivity of chlorophyll fluorescence to perturbations, in metabolism
coupled with the ease and speed of measuring chlorophyll fluorescence, makes
fluorescence a potentially useful for non invasive screening to identify metabolic
disturbances in leaves.

11. 4. Carbon Isotope Discrimination
There are two naturally occurring stable isotopes of carbon, 12
C and13
C
As water becomes limiting, stomatal closure occurs, therefore, discrimination
against 13
C decreases as water stress increases be-cause the ratio of 13
C:12
C increases
in stressed leaves of C3 plants, and Rubisco has less opportunity to discriminate
The carbon isotope discrimination has not been used to study the effects of high
temperature alone or in combination with water stress, despite the fact that heat
stress is an important component of drought stress
B. Ecophysiological Traits
1. Aerodynamic Resistance
If the canopy resistance to heat and water vapor diffusion is large, an increase in
stomatal conductance would tend to cool and humidify the air in the boundary
layer, thus lowering the leafair vapor pressure deficit (VPD); TE would then
increase
They observed maximum potential differences in evapotranspiration rates of
13%. The de-creased transpiration rate in the warmer environment should decrease
the rate of water uptake from the profile and increase the period of water
availability to the plant.

12. 2. Quantification of Stress Index Using Canopy Temperature
 Leaf, foliage, and canopy temperatures have excited plant physiologists
and atmospheric physicists alike for more than 100 years (Jackson, 1982).
 stated that plant temperature might be a valuable qualitative
index to differences in plant water regimes. In the last 25 years, there has
been rapid development in the use of foliage temperature to quantify plant
stress.
a. Canopy Temperature Depression.
 The difference between air and foliage temperature is referred to canopy
temperature depression (CTD). The ability of the plant to decrease temperature
through transpiration cooling will keep the plant cool and benefits plants at
above optimal stress conditions.
 demonstrated that canopy temperature of field grown cotton tracked air‐
temperature at night and became cooler than air temperature each morning
when the leaf temperature approached 27.5 C.

13. b. Crop Water Stress Index.
Initially stress degree day (SDD) was Ehrler (1973) concluded that using leaf‐
air temperature differences for scheduling irrigations in cotton was useful.
demonstrated that the difference in leaf and air temperature of well irrigated‐
cotton and wheat was linearly related to VPD of the atmosphere 1 m above the
crop canopy.
c. Thermal Stress Index.
These authors stressed that the impact of changing air temperature on plant
growth and performance can be understood only whenthe temperature providing
optimum enzyme function is known.
The temperature response curves for recovery of PSII fluorescence following
illumination compare favourably with the TKW in several crop species

14. C. Association Among Ecophysiological, Morphological, and Yield Traits
 reported a highly significant negative correlation (r2
¼ 0.79) between fruiting
height and yield. Empirically the first fruiting nodes number has been associated
with earliness of a particular genotype.
Again earliness has been found to be negatively correlated with yield in Pima
cotton. Temperature is an important factor modulating the interrelationship(s) of
the above parameters. Bhardwaj and Singh (1991)
Higher temperature can have significant negative impact on photosynthesis,
reduced photosynthetic rates, and the modulation of other metabolic factors, in
association with lower light intensities, may result in lower micronaire, fibre
strength, and yield
The micronaire reading of fiber produced in the warmest environment was
highest (Quisenberry and Kohel, 1975).

15. V. BREEDING FOR HIGH TEMPERATURE TOLERANCE‐
a.Genetic improvement for heat-stress tolerance
These responses accommodate short-term reaction or tolerance to specific
stresses. However, genome plasticity in plants, including genetic (e.g., directed
mutation) and epigenetic (e.g., methylation, chromatin remodeling, histone
acetylation) changes, allows long-term adaptation to environ-mental
changes/conditions
However, information regarding the genetic basis of heat tolerance is generally
scarce, though the use of traditional plant breeding protocols and contemporary
molecular biological techniques, including molecular marker technology and
genetic transformation, have resulted in genetic characterization and/or
development of plants with improved heat tolerance.

16. b. Conventional breeding strategies
Employing traditional breeding protocols to develop heat tolerant crop plants are
as follows:
1. Identification of genetic resources with heat tolerance attributes. In many
plant species, for example soybeans and tomatoes, limited genetic variations
exist within the cultivated species necessitating identification and use of wild
accessions. However, often there are great difficulties in both the identification
and successful use of wild accessions for stress tolerance breeding (Foolad,
2005).
2. When screening different genotypes (in particular wild accessions) for growth
under high temperatures, distinction must be made between heat tolerance and
growth potential. Often plants with higher growth potential perform better
regardless of the growing conditions.
3. When breeding for stress tolerance, often it is necessary that the derived
lines/cultivars be able to perform well under both stress and non-stress

17. A. TRAIT SELECTION
 emphasized that the selection of plants on a physiological and
genetic basis will make it possible to get varieties and hybrids with
high photosynthetic efficiency and a balanced ratio between source
and sink that will provide the maximum expression of yield
potential.
Plant breeders can use photosynthetic rate as a selection criterion
for improved lines. These improved lines in turn could be crossed
with other lines that possess suitable partitioning of photosynthates
between reproductive and vegetative growth
B. Isogenic lines to study individual trait performance
Ideally, several pairs of isogenic lines should be developed with
different genetic backgrounds, because the agronomic value of
a gene(s) can depend on the other genes present in the genome
Understanding the relationship between traits and yield is being
mediated by the identification and marking more of the controlling
genes and their alleles.

18. C. GENETIC VARIABILITY
(1)presence of suffcient genetic variability in its expression,
(2) the characters should be characterized genetically,
(3) the character must be related to agronomic benefit (e.g., yield, aspects of
quality, and production cost), and
(4) it must be measurable in large scale trials.
Therefore, in this section the results of various reports with regard to genetic
variability of different ecomorpho physiological traits related to high temperature‐
tolerance are presented and discussed.
This indicates that genes that allow the saprophyte to function at high
temperatures also allow the pollen to retain fertility after heat stress.
D. INHERITANCE STUDIES
It has been demonstrated that modifying membrane fluidity can influence gene
expression
reported that a soybean mutant deficient in fatty acid unsaturation showed strong
tolerance to high temperature.
the thylakoid membranes of two Arabidopsis mutants deficient in fatty acid
unsaturation (fad 5 and 6) showed increased stability to high temperature.

19. E. IMPACT OF HEAT TOLERANT GENES
 In cowpea, heat tolerant genes progressively enhanced
grain yield from first flush of flowers by increasing pod set on the main stem
nodes, and enhancing the overall partitioning of carbohydrates into grain with
increases in night time temperatures above 20 C
 Heat tolerant genes (or closely linked genes) also had a progressive dwarfing
effect, mainly resulting from shorter main stem internodes and involving
reduced shoot biomass production at night temperatures above 15 C.
 They concluded that heat tolerant (or associated) genes and the dwarfing and
reduced biomass production associated with the heat tolerant genes could have
negative effects in some environments.
 Transgressive segregation toward higher relative injury values in
progeny than in parents of wheat suggests that the parents contributed
different genes for high temperature tolerance and the trait is not simply
inherited
F. BREEDING FOR HIGH TEMPERATURE TOLERANCE
 The increase in productivity must be realized not by means of the vegetation
period but by activation of productive process by increased rate of
photosynthesis combined with higher number of bolls, increased boll weight,
and harvest index up to 50%

20. (1) conventional breeding and germplasm selection, especially of wild relatives of a
species;
(2) elucidation of the specific molecular control mechanisms in tolerant and
sensitive genotypes;
(3) biotechnology oriented improvement of selection and breeding procedures‐
through functional genomics analysis, use of molecular probes and markers for
selection among natural and bred populations, and transformation with specific
genes; and
(4) improvement and adaptation of current agricultural practices.
G. PRACTICAL ACHIEVEMENTS

21. VI.SUMMARY AND CONCLUSIONS
 Heat stress due to increased temperature is an agricultural problem in many
areas in the world.
 Transitory or constantly high temperatures cause an array of morpho-
anatomical, physiological and biochemical changes in plants, which affect plant
growth and development and may lead to a drastic reduction in economic yield.
 In order to cope with heat stress, plants implement various mechanisms,
including maintenance of membrane stability, scavenging of ROS, production
of antioxidants, accumulation and adjustment of compatible solutes, induction
of mitogen-activated protein kinase (MAPK) and calcium-dependent protein
kinase (CDPK) cascades, and, most importantly, chaperone signaling and
transcriptional activation.
 However, information regarding the genetic basis of heat tolerance is generally
scarce, though the use of traditional plant breeding protocols and contemporary
molecular biological techniques, including molecular marker technology and
genetic transformation, have resulted in genetic characterization and/or
development of plants with improved heat tolerance. In particular, the
application of quantitative trait locus (QTL) mapping has contributed to a better
understanding of the genetic relationship among tolerances to different stresses.

22. Case Study – I
1. High temperature stress in cotton Gossypium hirsutum L.
Jehanzeb Farooq1, Khalid Mahmood1, Muhammad Waseem akram2, Atiq Ur
Rehman2, Muhammad Imran Javaid2, I. Valentin Petrescu-Mag3,4, Bilal
Nawaz2
Abstract:-Heat stress is among one of the limiting and ever looming threats to
cotton productivity in Pakistan. This factor inflicted huge losses in the recent years.
In Pakistan genotypes developed for general cultivation face very high temperature
of about 50 °C during the month of June which is about 20 °C more than the
optimum temperature thus retard yield to greater extent. The plant parts like buds,
flowers, fiber quality traits are greatly influenced due to high temperature. This
mini review partially covers effect of heat stress on cotton fiber quality, plant parts,
screening procedures, genetics and biotechnological aspects related to heat stress.
All the information provided in the manuscript will help to better understand the
phenomena of heat stress tolerance thus will ultimately aid in the development of
heat tolerant cultivars in Pakistan.
Key words: Heat stress, cotton fiber, Pakistan, G. hirsutum, supra-optimal
temperature.

23. Case Study – II
2. The tolerance of durum wheat to high temperatures during grain filling
B. Maçãs, M.C. Gomes, A.S. Dias and J. Coutinho
SUMMARY – In South Portugal, rising temperatures during spring can be
considered an important factor limiting wheat yields. Heat stress assumes
particular importance when the wheat crop is under irrigation, where high yield
potential is needed. The main objective of this study is to evaluate, under field
conditions, the response of some wheat genotypes facing high temperatures
during and after anthesis. Nine durum and eight bread wheat genotypes were
exposed to two different sowing dates: normal and late sowing, to assure high
temperatures during and after anthesis, in 1997-1998 and 1998-1999. Grain yield
and individual grain weight were significantly affected by temperature increase in
1997-1998 season. Genotype x sowing date interaction was not observed
indicating that selection pressure must be applied to identify genotypes with
better resistance/tolerance to heat stress.
Key words: Durum wheat, heat stress, yield potential, grain filling.

24. Case Study – III
3. TERMINAL HEAT STRESS ADVERSELY AFFECTS CHICKPEA
PRODUCTIVITY IN NORTHERN INDIA STRATEGIES TO IMPROVE
THERMOTOLERANCE IN THE CROP UNDER CLIMATE CHANGE
P.S. Basu , Masood Ali and S.K. Chaturvedi∗
ABSTRACT:- Chickpea (Cicer arietinum L.) is a cool-season legume well
adapted within temperature range of 30/150C (day maximum and night minimum)
for optimum growth and pod filling. The northern plains of India once represented
a potential production zone for chickpea due to long winter favouring high
biomass production and pod filling. However, the crop in this region is now
adversely affected by climatic change, showing a trend of increasing minimum
night temperature more than that of maximum day temperature. The asymmetric
pattern of temperature rise resulted in a warmer winter, less dew precipitation and
heavy evapo-transpirational water loss. The crop often experiences abnormally
high temperature (>350 C) and atmospheric drought during reproductive stage.
The chickpea varieties are now gradually replaced by newly bred short duration
varieties escaping terminal heat, or breeding for heat tolerance has been initiated to
enhance productivity

25. A large number of germplasm were physiologically characterized for thermo
tolerance and screening techniques developed based on membrane stability,
photosynthetic efficiency (quantum yield, ratio of variable to maximal
chlorophyll fluorescence Fv/Fm) and pollen germinability. The foliar resistance
was much higher (above 400 C) than reproductive component like pollen
germination (usually occurs below 350C). The fluorescence inductions kinetics
showed a large differences in fluorescence peaks and quenching pattern when
leaves pretreated at 20, 30 40 and 460C with an irreversible damage of
photosynthetic systems at 460C. Membrane stability was significantly
correlated (R2= 0.7) with quantum yield (Fv/Fm) and proved to be viable
screening technique for thermo tolerance combined with pollen germinability at
high temperatures.

26. Case Study – IV
4. Screening wheat germplasm for heat tolerance at terminal growth stage
Aziz ur Rehman, Imran Habib, Nadeem Ahmad, Mumtaz Hussain, M. Arif
Khan, Jehanzeb Farooq and Muammad Amjad Ali*
Abstract:-The germplasm comprising of 442 wheat varieties/lines was sown in
one meter long row in a plastic sheet tunnel to screen the material for heat
tolerance during 2004-05 and 2005-06 at Wheat Research Institute, Faisalabad.
A set of the material was sown in the open adjacent to the tunnel. The material
was exposed to heat shock (>320C) by covering the tunnel with plastic sheet
during grain formation for two weeks in 2004-05 and for four weeks in 2005-
06. Data was recorded from 25 randomly selected heads from each row for
1000 grain weight, grains per spike and yield per spike during both the years.
The data regarding survival (ability to stay green under heat stress) was also
recorded. Heat effect was expressed as ratio of stressed / non stressed plants.
The effects of heat stress were lesser in shorter period exposure and more
drastic in prolonged exposure of the genotypes to heat. The ability of lines to
stay green for longer period in heat shock had no direct relationship with seed
setting.

27. Three entries CB-367 (BB#2/ PT// CC/ INIA /3/ ALD’S’) CB-333 (WL
711/3/KAL/BB//ALD ‘S’) and CB-335 (WL711/CROW ‘S’//ALD#1/CMH77A.
917/3/HI 666/PVN ‘S’ ) showed maximum grain development and survival. This
study revealed that these genotypes can be utilized in breeding programs for
development of wheat varieties having heat tolerance at terminal growth stage.
Keywords: Bread wheat; Germplasm; Tunnel; Heat stress; Survival

28. Case Study – IV
5. Effect of heat stress on proline, chlorophyll content, heat shock proteins
and antioxidant enzyme activity in sorghum (Sorghum bicolor) at seedlings
stage
G U Gosavi, A S Jadhav*, A A Kale, S R Gadakh, B D Pawar and V P Chimote
Abstract:- The effect of heat stress on various biochemical and physiological
parameters at seedling stage was investigated in drought tolerant, susceptible
and wild sorghum [Sorghum bicolour (L.) Moench] genotypes. Under heat
stress, susceptible genotypes showed higher reduction in total chlorophyll
content than tolerant genotypes. Significant increase in proline accumulation
and activities of d-1-pyrroline-5-carboxylate synthetase (P5CS), superoxide
dismutase (SOD), peroxidise (POD) and catalase (CAT) enzymes were
observed in stressed seedlings over control. Higher activities of antioxidant
enzymes under stress condition might be useful for sorghum seedlings to cope
up with oxidative damage by heat stress. Higher proline accumulation and
antioxidant activities were observed in wild sorghum genotypes and could be
used for gene mining for heat stress tolerance as well as in breeding
programmes for transfer of heat stress tolerance trait

29. . Many novel heat shock proteins (Hsps) were synthesized in drought tolerant
genotypes (19), followed by wild genotypes (8) and drought susceptible
genotypes (6). Thus, the above parameters studied would be useful as selection
criteria in identifying heat stress tolerant donors in future sorghum breeding
programmes. Pooled data from both RAPD and SSR analysis when subjected
to clustering analysis revealed wide divergence in wild genotypes for stress
resistant, while susceptible genotypes exhibited low divergence.
Keywords: Antioxidant enzymes, chlorophyll, heat stress, HSP, proline,
sorghum