i Seasonal variations in some physiological responses of maize (Zea mays L.) under glasshouse conditions By Iqbal Hussain M.Sc. Botany (BZU) Regd. No. 2004-ag-504 A thesis submitted in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY IN BOTANY FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2009
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i
Seasonal variations in some physiological responses of maize (Zea mays L.) under glasshouse conditions
By
Iqbal Hussain M.Sc. Botany (BZU)
Regd. No. 2004-ag-504
A thesis submitted in partial fulfillment of the requirements for the Degree of
DOCTOR OF PHILOSOPHY
IN
BOTANY
FACULTY OF SCIENCES
UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN
2009
ii
DECLARATION
I hereby declare that the contents of the thesis, entitled “Seasonal variations in some
physiological responses of maize (Zea mays L.) under glasshouse conditions” are product of
my own research and no part has been copied from any published source (except the
references, standard mathematical or genetic models/equation/formulae/protocols etc.). I
further declare that this work has not been submitted for award of any other
diploma/degree. The University may take action if the information provided is found
inaccurate at any stage.
Iqbal Hussain Regd. No. 2004-ag-504
iii
The Controller of Examinations University of Agriculture Faisalabad
We, the Supervisory Committee, certify that the contents and form of thesis
submitted by Iqbal Hussain, 2004-ag-504 have been found satisfactory and
recommend that it be processed for evaluation by the External Examiners for the
award of degree.
SUPERVISORY COMMITTEE 1. Chairman ____________________________ Prof. Dr. Abdul Wahid 2. Member ___________________________ Prof. Dr. Muhammad Ashraf 3. Member ___________________________ Prof. Dr. Shahzad M.A. Basra
iv
Acknowledgement
I would like to express many sincere thanks to my supervisor Dr. Abdul Wahid Professor, Department of Botany, University of Agriculture, Faisalabad, who provided me encouragement, enthusiasm, guidance and support during my Ph.D research work, which enabled the production of this thesis and also helping me to overcome the difficulties of written language while writing this thesis
I also would like to acknowledge members supervisory committee supervisors, Dr. Muhammad Ashraf, Professor, Department of Botany and Dr. Shahzad M.A. Basra, Professor, Department of Crop Physiology, University of Agriculture, Faisalabad for reminding me of Physiological knowledge, their availability when needed, endless discussions during the research work
I also would like to acknowledge great cooperation rendered by Dr. Tadasi Sato and Dr. Atsushi Higashitani, Graduate School of life Sciences, Tohoku University, Sendai, Japan during my visit to their lab under IRSIP program of HEC, Islamabad, Pakistan. They brought me to a new fabulous area of molecular biology with modern quantitative RT-PCR techniques. Many thanks also go to Dr. Tadashi Sakata, Dr. Kazahiro Sasaki, Eiko Hanzawa, Yuri Sann, Lee Hyun Sook, Kuriyama Satohiro, Chihiro Mori, Tomabechi, Shishiki Sann, Takeshi Oshino, Miura Shinya and Takafami Kimura for their kind assistance while conducting molecular biology experiments and also helping me with preliminary inception of RT-PCR experiment
I would extend my profound gratitude to Dr. Muhammad Yasin Ashraf, NIAB Faisalabad for providing facilities for physiological studies. Thanks also go to Dr. Furrakh Javed, Assistant Professor, Department of Botany for allowing the use of spectrophotometer. My Thanks are due to those people listed hereafter who
v
have assisted me during difficult hours of work while I was conducting my Ph.D research: Dr. Sadia Gelani, Dr. Freeha Anjum, Mrs. Saqib Mahmood and Mr. Rizwan Rasheed. Many thanks are extended to Mr. Muhammad Saleem Anjum and Mr. Abid Mustafa for helping me setup glass canopies and helping with purchasing chemicals and equipment.
I am also grateful to all my friends especially Mr. Ejaz Hussain Siddiqi, Maqsood Iqbal Shami, Azam Zia, Abid Hussain, Zia Ullah, Aftab Allam, Irfan Azhar, Malik Munawar, Zafar Iqbal Javed, Munawer Ahmad Khan, Muhammad Arshad Dar, Mujahid Ahmad Qaiserani, Hasnat Abbas, Mukhtar Hussain, Zawar Hussain, Khadim Hussain, Abid Khan,Ejaz Ahmad,Ghulam Abbas Syed Seqlain Raza and Syed Muhammad Kazim Raza for their support and encouragement during the course of my studies and research.
My deep gratitude is given to the Government of Pakistan for having granted me doctoral scholarship under Indigenous 5000 fellowship program sponsored by Higher Education Commission, Pakistan (HEC) and my visit to Japan for six months training under International Research Support Initiative Program (IRSIP) because without this, it would not have been completed smoothly and rapidly.
I also pay gratitude to the anonymous external
examiners who greatly improved the thesis to bring it into a final shape.
I dedicate this work to my family for their
lifetime love and support. I owe my deepest gratitude to my parents, my dear mother Hayat BiBi and my father Muhammad Siddique, for nurturing me and inspiring to pursue doctorate education. Thanks are due to all my sisters and brother Altaf Hussain for their psychological encouragements. Finally, I wish to thanks my beloved wife Rubina Iqbal and children, Muhammad Muazzam Ali, Aemon Iqbal, Zoha Iqbal and Mahnoor Iqbal
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for their patience, continuously encouragement, inspiration and psychologicaly supported me to overcome the most desperate moments while completing this Ph.D progamme.
Iqbal Hussain
vii
TABLE OF CONTENTS
S. NO. TITLE PAGE
I INTRODUCTION 01
II REVIEW OF LITERATURE 06
III MATERIALS AND METHODS 28
IV RESULTS AND DISCUSSION 41
V SUMMARY 109
LITERATURE CITED 111
viii
ABSTRACT
In view of the changing climatic conditions mainly related to greenhouse effect, this study was
focused on determining the responses of two differentially heat tolerant maize varieties to
glasshouse condition. The parameters studies included growth, water relations, gas exchange,
photosynthetic pigments, oxidative damage and gene expression. Results revealed that prevailing
glasshouse conditions played a crucial role in affecting the maize growth across winter and
summer seasons. Despite differences in the growing seasons and varieties glasshouse conditions
were adverse for the photosynthetic systems in maize. Major yardsticks of sensitivity were loss
of chlorophyll and carotenoids in the light reactions, while reductions in the net photosynthesis
and stomatal conductance in the glasshouse grown maize. Prevailing glasshouse conditions were
greatly effective in hampering the leaf water relation particularly those of winter sown crop. The
glasshouse conditions in winter crop produced oxidative stress on the plants, which was explicit
from the increased synthesis of H2O2, MDA and increased permeability to the ion leakage.
Greater free proline accumulation in the tolerant variety not only presented itself as a major
amino acid accumulated in environmental stress tolerance but also indicated it as a reliable
indicator of tolerance to glasshouse condition in maize. With great varietal difference, changes of
temperature and relative humidity inside the glasshouse across the seasons were mainly
responsible for the observed changes in mineral nutrients. More distinct changes were evident in
K, Ca and nitrate nutrition, which were given greater credence in view of their closer association
to the seasonal changes in the environmental conditions inside the glasshouse. Maize seedlings
showed sensitivity to high temperature stress, which was recorded from morphological
(reduction in shoot fresh weight, dry weight of shoot and root and a reduction in fresh-to-dry
weight ratio) and gene expression patterns. The molecular studies suggested that the maize
sensitivity to high temperature was mainly due to enhanced coexpression of sag and dhn2 and
failure to express hsp70 and sgr2 during relatively long term exposure to high temperature.
1
CHAPTER-1 INTRODUCTION
Crop plants such as maize, sugarcane and sorghum are referred to as C4 plants. They have a
distinctive leaf anatomy and photosynthetic metabolism that concentrates carbon dioxide (CO2)
around rubisco as compared to rubisco of C3 plants such as cotton and soybean (Taiz and Zeiger,
2006). Plants with C4 anatomical and biochemical specialization show much less adverse effects
of elevated atmospheric CO2 (Ca) as compared to non-C4 plants. According to Ghannoum et al.
(2000), photosynthesis and growth of C4 plants positively respond to elevated Ca. The leaf
development and net photosynthesis in maize are maximum near 31oC (Tollenaar, 1989; Yan and
Hunt, 1999) and 34oC (Kim et al., 2007) at ambient Ca, which decreased at temperature above
37oC while complete inhibition took place near 45oC (Crafts-Brandner and Salvucci, 2002). High
temperature caused a reduction in shoot dry weight, relative growth rate and net assimilation rate
in maize (Ashraf and Hafeez, 2004). The rate of respiration, growth and dry matter yield varied
with temperature in maize (Tollenaar, 1989).
The plant growth, its development and leaf photosynthetic rate may not change at
elevated Ca in response to enrichment of CO2 in maize, but significantly change under high
temperature (Kim et al., 2007). Short-term studies by Drake et al. (1991) concluded that elevated
CO2 increase the net rate of photosynthesis and growth while long-term CO2 enrichment
decreases photosynthesis due to acclimation response in C4 plants (Kim et al., 2006; Leakey et
al., 2006). Recent climatic model predicts that ambient temperature around the globe may
increase 1.1 to 6.4oC with the doubling of atmospheric carbon dioxide (Kim et al., 2007). It is
believed that global climate is changing so exposure to high temperature is likely to increase.
According to Intergovernmental Panel on Climatic Change (IPCC), there was a 0.5oC increase
during the past 100 years and it is expected that our earth will be 0.2oC warmer per decade for
the next two decades and 1 to 3.4oC warmer in the year 2100 (IPCC, 2007), and this global
warming will have a great impact on agriculture.
High temperature that may interfere with pollen mother cells and microspores
development and causes male sterility in various plant species (Sakata et al., 2000; Abiko et al.,
2005). A gradual increase in temperature every year is likely to change cropping pattern, growth
2
and economic yield of many important crops (Wollenweber et al., 2003). Gradually increasing
temperature is a great threat to agricultural production all over the world. Heat stress is a serious
problem to agricultural productivity all over the world, particularly in tropical areas. In plants
optimum temperature is essential for reproduction and maximum yield (Cheikh and Jones, 1994;
Keeling et al., 1994). Some reports show that an increase in temperature by a single degree
above normal can lead to a significant reduction in growth and yield (Pastori and Foyer, 2002).
High temperature affects morphological, biochemical and physiological processes in plants and
the major effects entail scorching of aerial plant parts, sunburn of branches and stems, leaf death,
leaf abscission and senescence, and causes inhibition of shoot and root growth, and reduced yield
(Ismail and Hall, 1999; Wahid et al., 2007). High temperature stress leads to premature
development of anther and restricts cell proliferation (Oshino et al., 2007).
Heat stress has a harsh effect on many economically important cereals plants such as
wheat, rice, maize etc., and decreases their yield by affecting the reproductive stages of these
plants. It also decreased chlorophyll content, net photosynthetic rate and stomatal conductance
(Morales et al., 2003). Photosynthesis is of pivotal importance for carbon accumulation,
production of biomass in different plant species. Response of terrestrial plants to photosynthesis
can changed ecosystem balance and cycling of carbon under global warming (Gunderson et al.,
2000). Increased ambient temperature affects plant productivity by damaging photosynthesis
(Al-Khatib and Paulsen, 1990). Photosynthetic inhibition can be reversible when temperature is
slightly supra-optimal (Berry and Bjorkman 1980). The yield of PSII reaction centers and
amount of RUBP inhibit net photosynthesis at high temperature (Law and Crafts-Brandner,
1999).
Sudden heat stress may injure the membranes by denaturing membrane proteins or
increase in unsaturated fatty acids, leading to membrane rupture and loss of cellular contents
(Savchenko et al., 2002). Heat stress induces deterioration of biological membranes, resulting in
ion leakage and deactivation of membrane proteins, and loss of cellular functions. Membrane
damage occurs mainly due to (1) stress induced production of activated oxygen species (AOS)
and reactive oxygen species (ROS) and (2) dehydration induced changes in phase transitions
(Nishida and Murata, 1996; Liu and Huang, 2000). Electrolyte leakage is measured to detect
stress injury to cell membranes in order to assess the severity of existing stress (Foyer et al.,
1997; Al-Khatib and Paulsen, 1999). Electrolyte leakage varies in relation to membrane abilities
3
to take up and retain solutes and reflect stress induced changes in their potentials. Studies show
that search for genotypic variation in high temperature tolerance on the basis of leaf electrolyte
leakage may greatly influence the plants already exposed to this adversary (Li et al., 1991).
Membrane stability is positively associated with yield performance under heat stress (Rahman et
al., 2004). The AOS and ROS react with pigments, membranes, enzymes and nucleic acids, and
modify their functions (Smirnoff, 1993; Sairam et al., 2000). Heat stress decreases the activities
of antioxidant enzymes, leaf senescence and injury to cell membranes by increasing the level of
lipid peroxidation (Liu and Huang, 2000).
Heat stress has well marked effect on osmotic, turgor and water potential (Machado and
Paulsen 2001; Wahid and Close, 2007). Changes in plant processes like assimilate partitioning,
hampered water and nutritional relationships by the root has been observed under heat stress
(Morales et al., 2003; Ashraf and Hafeez, 2004; Wahid and Close, 2007). One of such
mechanisms is synthesis of specific proteins (Vierling, 1991) called heat shock proteins (HSPs).
All living organisms respond at the molecular level to thermal shock or other stresses by
inducing or enhancing the expression of a small number of specific heat shock protein (hsp)
genes and synthesis of their transcripts and showing the biosynthesis of stress proteins as a
stress-tolerance approach (Sun and Callis, 1997; Iba, 2002; Wahid and Close, 2007). Expression
of HSPs is an essential adaptive strategy for heat tolerance (Feder and Hoffman, 1999). The
HSPs, synthesized over a wide range size (10 kDa to 200 kDa) have chaperone-like functions
and are involved in signal transduction during heat stress (Schöffl et al., 1999). Rapid synthesis
of HSPs can be important for the protection of metabolic machinery of the cell (Camejo et al.,
2005; Wahid and Shabbir, 2005; Momcilovic and Ristic, 2007).
In Pakistan, maize (Zea mays L.) is the third most important cereal grain after wheat and
rice. Maize is essential for global food safety, since it is a multipurpose and commercial crop and
used as food, feed, fodder and agro-based industrial use (Dowswell et al., 1996; Herald et al.,
1996). It has high nutritive value and has been reported to contain about 72% starch, 10% protein
(Zein), 4.8% oil of good quality, 9.8% fiber, 3.0% sugar and 1.7% ashes (Choudhary, 1983;
Okoruwa, 1995). The oil of maize is of good quality. Maize is a short duration and fast growing
crop, having great adaptability to existing cropping systems and shows high yield potential. It is
an important summer crop in Pakistan and is grown on an area of 1.022 million hectares and its
total production is 3.560 million tons and an average grain yield of 3483 kg ha-1. Unfortunately,
4
the grain yield of maize is very low as compared to other countries (Anonymous, 2008). This is
mainly due to the physical constraints that plants usually encounter under field conditions such
as environmental stresses especially heat stress that may interfere with the crop productivity.
Heat stress readily changes the pattern of gene expression, as a significant part of
thermotolerance (Yang et al., 2006). It also weakens the mRNAs encoding non-heat-stress-
induced proteins (Gallie et al., 1995). The organisms suddenly exposed to high temperature;
synthesize a small amount of heat shock proteins (HSPs). Increased expression of HSPs is
mediated at multiple levels, i.e., mRNA synthesis and its stability, and translation efficiency. The
heat shock response is conserved reaction of cells and high temperature. It is found that plant
reproductive development and molecular response to stresses required specific gene expression
(Sorensen et al., 2002; Shinozaki et al., 2003; Chinnusamy et al., 2004). Specific heat shock
protein genes, transcripts accumulation biosynthesis of stress proteins is altered under several
stresses (Ozturk et al., 2002).
Maize pollen development and tube growth are more sensitive to high temperature
(Gagliardi et al., 1995; Mascarenhas and Crone, 1996). The appearance of heat shock protein 70
(HSP70) and heat shock factor (hsf) gene is high at the beginning of maize pollen exposure to a
heat shock. This defect is not associated to an alteration of hsf genes (Zmhsf a-c) expression at
the mRNA level (Gagliardi et al., 1995). The studies show that under heat stress, distinctive hsps
are not synthesized in germinating pollen. The hsp18 and hsp70 genes are transcribed in
response to heat shock but low level of the mRNA accumulation.
Photosynthesis is of fundamental importance for growth and biomass accumulation in
different plant species. High temperature accelerates the senescence, result decrease the
assimilation rate in grains (Spano et al., 2003). Leaf senescence is controlled by genetic and
abiotic factors (Nooden and Leopold, 1978). In leaf senescence, expression of gene is a complex
process. A large number of senescence associated genes (sags) and defensive genes has been
reported during the leaf senescence (Lee et al., 2001; Robatzek and Somssich, 2001, 2002;
Gepstein et al., 2003; Lin and Wu, 2004; Buchanan-Wollaston et al., 2005). Leaf sags have been
reported in some plants such as maize (Smart et al., 1995), barley (Kleber-Janke and Krupinska,
1997), rice (Lee et al., 2001), Arabidopsis thaliana (Lohman et al., 1994), tomato (John et al.,
1997).
5
Photosynthetic responses of annual plants can be improved by extending duration of heat
stress. This can be achieved by delaying the senescence leaf (Thomas and Howarth, 2000). Stay
green (SGR) proteins are responsible for the green-flesh and chlorophyll retainer during
senescence. Stay green (sgr) is upregulated that delays senescence (Buchanan-Wollaston, 1997;
Nam, 1997). In addition to Triticum durum, sgr expressed in other cereals such as sorghum,
maize, rice (Tao et al., 2000; Bekavac et al., 1995; Park et al., 2007; Buchanan-Wollaston,
1997). Stay-green regulates the loss of chlorophyll by disassembly of light-harvesting
chlorophyll-binding Protein (LHCP) during senescence (Park et al., 2007).
In view of the increasing adversaries due to global warming, it is necessary to find out
indicators and mechanisms of heat stress tolerance, which can be used to improve maize for
cultivation in warmer areas of the country. Ion homeostasis, relative membrane permeability and
production of H2O2, membrane per oxidation products and expression pattern of stress proteins
are important yardsticks to determine the heat stress tolerance of in maize. H2O2 accumulation is
also taken as the valid criterion for selection of high temperature stress tolerant materials.
Although many reports exists on the heat tolerance potential of maize, studies on the potential
indicators of heat tolerance under glasshouse conditions are lacking and need to be established
on firm grounds. These studies were conducted under the glass house conditions under the
following objectives:
1. Assessment of comparative heat stress response of maize at various phenological stages
during spring and autumn season
2. Determination of H2O2, accumulation of MDA, RMP and free amino acids in leaves as
indices of oxidative damage caused by heat stress
3. Establishment of possible relationships of the above attributes with heat stress tolerance
of maize genotypes
6
CHAPTER-2 REVIEW OF LITERATURE
In nature, plants growing under unfavorable conditions such as salinity, drought, high
temperatures, freezing, low temperature, flooding, UV light, heavy metal, pathogenicity and
nutrient deficiency may display delayed growth and development, reduced productivity and, in
extreme cases, plant death. Plants display distinct changes in the morphological features in
response to these abiotic stresses, including shortened of life cycle (Porter, 2005). In some plants
these morphological changes occur to overcome or reduce the harmful effect of stresses
(Ferguson, 2004; Wang et al., 2004).
High temperature stress is a great modulator of growth and productivity (Zhang et al.,
2000). Prevailing high temperature reduces crop yield and affect plant growth from germination
up to maturity. The mechanisms leading to the survival of a crop under heat stress entail changes
in physiology and accumulation of osmolytes like proline, glycinebetaine, soluble sugars and
proteins (Wahid and Close, 2007; Verbruggen and Hermans, 2008). In addition, recent studies
show that changes in the expression levels of an array of genes are major mechanisms that
contribute to our understanding of heat stress responses of plants (Hazen et al., 2003). An
account of pertinent literature reflecting the effect of high temperature on various aspects of plant
growth and development is given below:
2.1 Plant growth and phenology
High temperature is a major determinant of agricultural production throughout the world and its
effects are evident at all critical growth stages starting from seed germination to seed yield. An
account of changes in the phenology of plants is elaborated underneath.
2.1.1 Seed germination and seedling survival
Seeds subjected to germinate in hot condition show reduced or even inhibited germination. A lot
of work has been done for improvement of seeds vigor, seedling, and reduce germination rate in
some field crops (Basra et al., 2005). High temperature effects on seed storage process and
kernel quality like starch, protein metabolism by effecting it’s enzymes at grain filling stage of
7
maize (Wilhelm et al., 1999; Maestri et al., 2002). In studies on soybean, it was reported that
heat stress changed seed composition, seed protein expression profiles and reduce seed
germination and vigor, and thus appeared to determine the seed quality attributes (Egli et al.,
2005; Ren et al., 2009). Seed germination, seedling emergence and its establishment are
extremely affected by high temperature in a number of plant species (Grass and Burris, 1995;
Ashraf and Hafeez, 2004; Wahid et al., 2007). Columbo and Timmer (1992) demonstrated that
seedlings are more susceptible to high temperature stress than adult black spruce plant. Maize
shows climax germination and optimal growth at 20-30oC and 28-31oC, respectively (Hughes,
1979; Medany et al., 2007), and declines the coleoptiles growth in maize at 40oC and almost
ceases it at 45oC (Weaich et al., 1996; Akman, 2009). Many studies show that maize coleoptile
was more thermotolerant amongst all stages of seedling development (Venter et al., 1997;
Momcilovic and Ristic, 2007). Heat stress lowers the activity of specific enzymes and leads to
reduced synthesis of proteins in germinating maize embryos (Riley, 1981). The growth and
development of cotton (Gossypium hirsutum L.) seedling are reduced under high temperature
stress (Mahan and Mauget, 2005).
2.1.2 Vegetative growth and development
High temperature is a major environmental factor that determines the crop growth and yield in
some regions (Al-Khatib and Paulsen, 1999). Plant growth is often reduced at high temperature
(Blum, 1988). Plants grown under high temperature stress have lower biomass than those grown
at low temperature. Biomass, leaf area, photosynthesis and enzyme activities are decreased if
growth temperature is high (Kim et al., 2007). High temperature reduced stem elongation and
overall growth and limits plant survival (Ashraf and Hafeez, 2004). High temperature reduces
the plant growth by changing different plant mechanisms (Sibley et al., 1999). A steady increase
in temperature in each year may change cropping pattern, growth and economic yield of many
important crops (Wollenweber et al., 2003).
High temperature decreased the shoot dry weight, relative growth rate (RGR) and net
assimilation rate (NAR) in maize and millet (Ashraf and Hafeez, 2004) and sugarcane (Wahid,
2007). Heat shock affects the cell division in meristems and reduces the growth of various parts,
mainly the leaves (Salah and Tardieu, 1996). Maize leaf growth increased from 0 to 35oC, but
declined at 35 to 40oC. Above 40oC temperature, there was a severe reduction in photosynthesis
8
and alteration in protein metabolism such as protein denaturation, aggregation, enzyme
inactivation, inhibited protein synthesis and its degradation (Dubey, 2005). Heat stress halted cell
wall elongation, stimulated cell division and altered cell differentiation (Potters et al., 2007).
High temperature influenced the leaf expansion, internode elongation, motivate the flower bud
abortion in Brassica napus (Young et al., 2004), which may be due to limited supply of water
and nutrients (Hall, 1992). Increased temperature affects growth, metabolism, development
(Rawson, 1995). High temperature causes photosynthetic acclimation and alters physiological
processes directly, and changes the pattern of development indirectly (Downton and Slatyer,
1972). It increased the rate of development and shortened the growth period in annual species by
virtue of rapid CO2 fixation and biomass production before setting seed (Rawson, 1992;
Morison, 1996). High temperature decreased the growth and accumulation of starch in tubers
greater than shoot but did not affect the glucose in potato tubers (Lafta and Lorenzen, 1995).
2.1.3 Reproductive growth and yield attributes
High temperature greatly affects the reproductive growth by increasing flower abortion and
decreasing seed size (Talwar et al., 1999). Shah and Paulsen (2003) demonstrated that
photosynthesis and leaf area, shoot, grain biomass and sugar contents of kernels are rapidly
decrease under high temperature. Pollination, an important stage in reproductive development, is
especially sensitive to heat stress; the mature pollens being more sensitive failed to fertilize
(Dupuis and Dumas, 1990). High temperature stress causes premature development of anther and
arrests its cell proliferation (Oshino et al., 2007). It interferes with pollen and anther
development, and causes male sterility in certain plant species (Sakata et al., 2000; Sato et al.,
2006; Abiko et al., 2005). Heat stress affects kernel development in maize plant with the
accumulation of ZEIN transcript during cell division (Monjardino et al., 2006). It reduced the
rate of dry matter accumulation, kernel density and reproductive growth in maize, wheat and
Suneca during kernel development and its filling (Wilhelm et al., 1999; Maestri et al., 2002).
Kernel dry weight reduced from 79 to 95% in field conditions in B-73 inbred line of maize under
heat stress (Commuri and Jones, 2001). High temperature effects the endosperm development in
maize and reduces grain yield during endosperm cell division. These effects were due to dry
matter accumulation, interruption of cell division, aberrant sugar metabolism and starch
biosynthesis in endosperm of kernels (Monjardino et al., 2005).
9
2.2 High temperature and physiological phenomena
Long-term or even short-term exposure to high temperature culminates in changed metabolic
functions (Wahid et al., 2007). These alterations include water and nutrient uptake,
photosynthesis membrane properties, osmotic relations and gene expression. Accounts of these
phenomena are described underneath.
2.2.1 Water relations
Water is a basic need from seed germination to plant maturation. Heat stress leads to increased
evapo-transpiration of water from the plant surface and cause dehydration of aerial parts. High
temperature hampers the cell water relations and limits growth in many plant species (Machado
and Paulsen, 2001; Mazorra et al., 2002; Wahid and Close, 2007). Elevated temperature is
known to produce osmotic stress on the growing tissues due to reductions in root hydraulic
conductance and tissue water content (Jiang and Huang, 2001; Morales et al., 2003). Reddy et al.
(1991) demonstrated that reduction of water accessibility damage the growth and development of
cotton plants in sowing season. High temperature cause significant reduction in leaf growth of
sorghum (Sorghum bicolor) and water status and water potential in wheat leaves (Shah, 1992).
Low leaf water potential affects the nitrogen and protein metabolism to a great extent (Lawlor
and Cornic, 2002; Molnar et al., 2002).
High temperature hampers water, ions and organic solutes movement across the
biological membranes. It affects with photosynthetic and respiratory processes (Taiz and Zeiger,
2006), increases evapo-transpiration rate (Tsukaguchi et al., 2003) and reduces the leaf osmotic
potential and increases the leaf fluorescence (Huve et al., 2005). Heat stress induces the closure
of stomata and negatively influences leaf water status (Berry and Bjorkman, 1980). High
temperature induced water stress is closely associated to reduction of soil water contents (Talwar
et al., 1999).
2.2.2 Ionic and nutrient relationships
Mineral nutrition acquisition and assimilation is strongly influenced by high temperature stress in
plants (Taiz and Zeiger, 2006). Some essential nutrients such as carbon (C), nitrogen (N),
calcium (Ca), magnesium (Mg), phosphorus (P) and sulfur (S) are structurally important for the
proteins, nucleic acids, chlorophylls, certain secondary metabolites and defense related micro-
10
and macromolecules, while others have both structural and functional roles (Epstein and Bloom,
2005; Taiz and Zeiger, 2006). High temperature raised the rate of physiological processes of
plant growth, which determine nutrient absorption (Tollenaar, 1989). Under high temperature,
plants explore larger volumes of soils to absorb more P from the root (Fohse et al., 1988). K and
P uptake increased significantly with increase in temperature and maximum uptake of both was
evident at 32 and 38°C in maize roots, respectively (Bravo and Uribe, 1981). Under high
temperature stress, N concentration decreased sharply and S concentration decreased slightly,
while sodium (Na) was not affected (Muldoon et al., 1984).
Studies showed that the pattern of diurnal uptake of nitrate and dry matter accumulation
in maize seedling at varying day/night temperature of 30/20, 30/30, and 35/35°C. A greater
nitrate uptake took place at 30/30oC (Polisetty and Hageman, 1989). High temperature caused a
significant decrease in the shoot dry mass, RGR and NAR (Wahid, 2007). Significant increase in
the uptake of Ca and P and decrease one of N, S, Mg and Na was found at high temperature
(Ashraf and Hafeez, 2004). In sorghum, comparison of various temperatures for the uptake of
certain nutrients indicated that N and P was the highest at 27oC in whole plant, leaf, stem and
root (Ercoli, 1996).
High temperature affects the rate of biochemical reactions and enzyme denaturation,
resulting in decreased enzyme activities (Fukuokan and Enomoto, 2002). Nitrate reductase (NR)
is an important enzyme, the activity and stability of which increases by hardening (hyperthermia)
against a number of inactivating factors such as heating, proteolysis, in vitro and in vivo enzyme
degradation and enhances its ability to repair heat stress induced injury (Taiz and Zeiger, 2006).
The thermal stability of NR increased only in seedlings that were hardened at 40 and 44oC. A
short term exposure to heat stress led to recover the functional activity of NR without de novo
synthesis of the enzyme protein (Lyutova and Kamentseva, 2001). After absorbing nitrate, NR
controls the rate of protein synthesis (Srivastava and Naik, 1980). The activity of NR is affected
by light in several ways. Light activates one or both chloroplast photosystems in green tissues
and increases transport of stored NO3- from vacuole to cytosol where reduction of NR occurs
(Granstedt and Huffaker, 1982). Secondly, light activates the phytochromes, which increases the
potential of the ribosome to synthesize various proteins. Thirdly, light inactivates various
proteins, which act as inhibitors of NR activity. Finally light increases NR activity by increasing
11
the carbohydrate supply; NADH required for nitrate reduction is produced from these
carbohydrates when they are respired (Aslam and Huffaker, 1984).
2.2.3 Osmolytes accumulation
Certain organic osmotica such as leaf soluble proteins, proline and soluble sugar are important
adaptive components of heat tolerance in many plant species to prevent water loss and in
mediating osmotic adjustment (Ashraf et al., 1994; Sakamoto and Murata, 2002; Wahid and
Close, 2007). These osmolytes include nitrogenous compounds such as quaternary ammonium
compounds (QACs), and tertiary sulphonium compounds, other amino acids like proline, aspartic
acid and glutamic acid (Samuel et al., 2000; Wahid et al., 2007), polyamines, glycine betaine,
ectoine, polyols and soluble sugar (Ashraf et al., 1994; Wang et al., 2003; Chen and Murata,
2002; Rontein et al., 2002; Sairam and Tyagi, 2004; Wahid and Close, 2007). Accumulation of
proline occurs in many organisms subjected to abiotic stresses including heat stress (Saradhi et
al., 1995; Siripornadulsil et al., 2002; Verbruggen and Hermans, 2008).
Proline and QACs, in addition to being N-rich, accumulate in plants under variety of
environmental stresses, which may decrease stress-induced cellular acidification or primary
oxidative respiration to give required energy for survival. Accumulation of proline under stress
shows association with stress adaptation in higher plants (Lalk and Dorffling, 1985; Bartels and
Sunkar, 2005; Knipp and Honermeier, 2006). The proline accumulation is more important than
osmotic adjustment and stored carbon and nitrogen (Hare and Cress, 1997; Hare et al., 1999).
High level production of proline during stress may maintain NAD(P) + NAD(P)H ratios, which
matched well with metabolic steps under normal condition (Hare and Cress, 1997; Foreman et
al., 2003). At 35oC, there were high levels of proline and choline in tomato plants (Rivero et al.,
2004). Proline accumulation is controlled by tissue water status and unchanged by tissue
temperature up to 39°C in barley (Chu et al., 1974). Irigoyen et al. (1992) found a rapid
accumulation of proline with declined tissue water potential in alfalfa. High level of proline also
reduces the generation of free radical due to osmotic stress (Hong et al., 2000). Thus proline and
GB-synthesis may defend cellular redox potential under high temperature (Alia et al., 1998). A
rapid elevation of free proline was found in the sense-transforments that exhibited the smallest
amount of H2O loss, while the slowest elevation of proline levels was detected in antisense-
transforments that exhibited the greatest H2O loss during stress.
12
Genetic manipulations of osmolytes like proline levels also affect the stress induced
changes in the concentration of several other amino acids, which show the coordinated regulation
of metabolic pathways (Simon-Sarkadi et al., 2005). Combined effect of heat and drought stress
exhibited an increase in proline concentration in cotton (De Ronde et al., 2000). Accumulation of
free proline under stress conditions does not inhibit biochemical reaction and play an
osmoprotectant function during osmotic stress (Sawahel and Hassan, 2002). In root of maize
plant, solute potential declined primarily due to proline and soluble sugars accumulation
(Rodríguez et al., 1997). In A. thaliana, two pyrroline -5- carboxylate synthetase (P5CS) genes,
which are the rate limiting enzymes, play important roles in proline biosynthesis (Szekely et al.,
2008). These enzymes are regulated by transcriptional regulation and feedback inhibition in
plants (Zhang et al., 1995). Feedback regulation of P5CS plays a function in regulating the level
of proline in plants. Accumulation of proline is the component of stress signal that manipulate
the adaptive responses (Maggio et al., 2002), and in plant parts such as pollen grains, seeds and
roots, but is dependent upon the plant age, leaf part, leaf position on the plant and leaf age
(Chiang and Dandekar, 1995). Free proline accumulation may scavenge reactive oxygen species
(ROS) by enhancing photochemical electron transport activities (Smirnoff and Cumbes, 1989;
Pinheiro et al., 2001), maintaining enzyme structure and activity (Rajendrakumar et al., 1994;
Samuel et al., 2000) and protection of membrane integrity as an adaptation to water deficiency
(Hare, 1995; Bohnert and Jensen, 1996; Ashraf and Foolad, 2007).
Soluble sugar also plays important roles in osmotic regulation of cells under heat stress
(Bolarin et al., 1995). Expression of some seed germination genes is controlled by the sugars
levels (Reynolds and Smith 1995; Yu et al., 1996). Accumulation of sugars in mature seeds is
essential for the improvement of desiccation tolerance (Hoekstra et al., 2001). Study on 11
enzymes of carbohydrates metabolism from developing endosperm showed that ADP glucose
pyrophosphorylase, glucokinase, sucrose synthase and soluble starch synthase were highly
susceptible to the high temperature stress. Prolonged heat stress affects seed storage processes in
many maize inbreds (Wilhelm et al., 1999).
2.2.4 Leaf gas exchange
All aspects of photosynthesis are prone to episodes of high temperature. Increased ambient
temperature affects plant productivity by damaging photosynthesis (Al-Khatib and Paulsen,
13
1990). The maize seedling grown at 25oC and transferred to 35oC for 20 min led to 50%
inhibition in photosynthesis (Sinsawat et al., 2004). In maize, high temperature caused a distinct
decrease in growth, transpiration, respiration and photosynthesis (Karim et al., 2000), and final
yields (Stone, 2001). The net photosynthesis in maize was maximum near 31oC, which decreased
at temperature above 37oC while completely inhibited near 45oC (Crafts-Brandner and Salvucci,
2002). High temperature inhibited net photosynthetic (Pn) and stomatal conductance
significantly in many plant species (Ranney and Peet, 1994; Crafts-Brandner and Salvucci, 2002;
Morales et al., 2003; Ashraf and Hafeez, 2004). Net photosynthesis (Pn) of developed and nearly
developed leaves was more sensitive than developing leaves (Karim et al., 1997, 1999).
Photosynthetic apparatii are highly sensitive to high temperature and are inhibited when
leaf temperature exceed 38oC. This is because C4 plants have better ability to photosynthesize
under higher temperature than C3 plants (Berry and Bjorkman, 1980; Edwards and Walker, 1983;
Wahid and Rasul, 2005). It declines the activation of rubisco, a highly susceptible components of
the photosynthetic apparatus in C3 as well as C4 plants (Crafts-Brandner and Salvucci, 2000,
2002; Law and Crafts-Brandner, 1999; Salvucci and Crafts-Brandner, 2004). Photosystem (PS)
II, water splitting and oxygen evolving complex (OEC) in photosynthesis are more heat sensitive
components of photosynthesis (Edwards and Baker, 1993; Pastenes and Horton 1996a;
Heckathorn et al., 1998a). Leaf high temperature can disrupt the ultra structural characteristic of
chloroplast (Ristic et al., 2004). Thylakoid lamellae and stroma of chloroplast are very much
sensitive to high temperature (Wise et al., 2004). High temperature inhibits photosynthesis by
affecting the structure of thylakoid lamellae (Karim et al., 1997). Extensive studies show that
both PSI and PSII are damaged by increased high temperature. In barley and potato, heat stress
damaged PS-I and PS-II, and affected electron transport, which is very important during
photosynthesis (Havaux, 1998; Szilvia et al., 2005). Heat stress damaged the antenna complex of
PSII and reduced the respiratory and photosynthetic behavior (Carpentier, 1999; Zhang et al.,
2005). High temperature during greening led to the inactivation of PSI and PSII (Sasmita and
Narendranath, 2002). Heat stress inhibited the activity of PSII as determined from electron
transport measurement (Rokka et al., 2000).
The photosynthesis in C3 plants is more affected by high temperature than C4 plants
(Wahid and Rasul, 2005). Increased temperature reduced the activation of rubisco in the exposed
leaf tissue and increased the level of rubulose-1, 5-bisphosphate (Feller et al., 1998; Crafts-
14
Brandner and Law, 2000). High temperature altered the energy sharing by changing the action of
Calvin cycle and other metabolic processes such as photorespiration, and synthesis and stability
of the rubisco enzyme (Pastenes and Horton, 1996b), disruption of electron transport activity and
bound RUBP supply by heat stress (Ferrar et al., 1989). High temperature inhibited the activity
of rubisco by transfer of rubisco activase gene, which result inhibition of photosynthesis as
compared to control plants (Sharkey et al., 2001). High temperature enhanced chlorophyllase
activity and decreased photosynthetic pigments concentrations (Todorov et al., 2003). The loss
of chlorophyll is a good indicator of heat tolerance in wheat (Ristic et al., 2007; 2008). High
temperature enhanced chlorophyll a:b ratio and declined chlorophylls-to-carotenoids ratio in
sugarcane (Wahid, 2007).
2.2.5 Cell membrane thermostability
The cellular membranes play an important function in maintaining integrity of cell, by involving
in signal transduction and ion homeostasis under environmental stresses (Kaur and Gupta. 2005;
Tuteja and Sopory. 2008). High membrane stability, determined in terms of changes in ion-
leakage, is taken as an index of heat tolerance in several grain, forage and Pasteur crops
(Saadalla et al., 1990; Blum et al. 2001; Ashraf et al., 1994; Marcum, 1998; Ismail and Hall,
1999; Wahid and Shabbir, 2005). The membrane stability and its functions are susceptible to
high temperature (Nishida and Murata, 1996; Wahid et al., 2008). Maize showed a great
reduction in membrane stability under high temperature (Yang et al., 1996). Plant processes such
as photosynthesis, respiration, assimilate partitioning etc. are directly impinged by high ambient
temperature (Tsukaguchi et al., 2003; Iwaya-Inoue et al., 2004). Quite discernible changes occur
in the cellular membranes of organelle (Nash et al., 1982; Dionisio-Sese et al., 1999; Wahid et
al., 2007)
High temperature induced injury to thylakoids in winter wheat (Ristic et al., 2007),
resulting in the production of reactive oxygen species (Camejo et al., 2006; Guo et al., 2006).
Finding genotypic differences for heat tolerance based on leaf electrolyte leakage may be more
effective with plant subjected to heat stress (Li et al., 1991). High temperature damages
membrane by lipid peroxidation (Bhattacharjee and Mukherjee, 1998) making them more
permeable to ions (Wen-yue et al., 2001). Measurement of electrolyte leakage indicates the stress
damage to assess the harshness of existing stress (Foyer et al., 1997). Cell membrane
15
thermostability (CMT) is used to identify the genetic differences for high temperature tolerance
in sorghum and wheat germplasm (Sullivan and Ross, 1979; Ibrahim and Quick, 2001). Agarie et
al. (1998) used electrolyte leakage as an effective tool for measuring CMT in root tissues, which
exhibited a great sigmoid response to temperature for this tissue. Saadalla et al. (1990) found an
association in membrane thermostability (MT) between seedlings and flag leaves at anthesis for
genotypes of wheat grown under control and high temperature. Sullivan (1972) prepared a heat
tolerance analysis to demonstrate CMT by measuring the quantity of electrolyte leaked from leaf
after exposure to high temperature stress.
High temperature effect on cell MT is associated with yield performance (Rahman et al.,
2004). Agarie et al. (1995) used the cell membrane stability (CMS) as heat tolerance test in rice.
Injury to membranes from sudden heat shock may result from either denaturation of proteins or
increases unsaturation of fatty acids, leading to membrane rupture and loss of cellular solutes
(Savchenko et al., 2002). Genetic difference in cellular thermotolerance expressed by CMT of
membrane is a site of primary physiological injury with high temperature (Fokar et al., 1998;
Blum, 1988). MT is heritable and shows significant genetic association with yield (Fokar et al.,
1998). Relative cell injury (RCI) is a determinant of cellular and/or tissue heat tolerance. A lower
RCI reflects high CMT and vice versa (Rahman et al., 2004). Damage due to stress to plasma
membranes was much lesser in younger than the older maize leaves (Karim et al., 1999).
Excessive dehydration from the leaf surface due to heat stress leads to the disruption of cell
membranes by solublization and peroxidation of membrane lipids (Wen-yue et al., 2001; Jiang
and Haung, 2001; Iba, 2002).
2.2.6 Oxidative damage
Like other abiotic stresses, high temperature also induces oxidative stress as a result of an
imbalance in the formation and metabolism of ROS (Lee et al., 1983; Dat et al., 1998; Sairam
and Tyagi, 2004). Chloroplast, mitochondria, endoplasmic reticulum and microbodies are major
sites of ROS and malondialdehyde (MDA) production under abiotic stress (Foyer et al., 1997;
Dat et al., 1998; Breusegem et al., 2001; Sairam and Srivastava, 2002; Apel and Hirt, 2004;
Kukreja et al., 2005). Various roles have been assigned to ROS in plant development. There is
proof that ROS are necessary for growth of root hair, where they manage the activity of Ca2+
channels necessary for polar growth (Rachel and Dolan, 2006). H2O2 is a regulator of gene
16
expression in the cells such as gene encoding antioxidant, cell defense, signaling, stress protein
and transcription factors (Hernandez et al., 2000; Gabriela and Foyer, 2002; Wahid et al., 2007,
2008). The H2O2 breaks the seed dormancy in conifers; enhance heat and salt tolerance in rice
(Uchida et al., 2002). However, with the production of various ROS e.g., superoxide radical (O-
2), hydroxyl radical (OH-), H2O2, and singlet oxygen (O21) in higher quantities, peroxidation of
membrane lipids and damage to important molecules such as nucleic acids, proteins, chlorophyll
and other important macromolecules takes place (Smirnoff, 1993; Scandalios, 1993; Foyer et al.,
1997; Sairam et al., 2000; Hernandez et al., 2000 and Foyer and Fletcher, 2001; Wahid et al.,
2007). A well known effect of high temperatures induced oxidative damage in plants is
imbalance in photosynthesis and respiration (Fitter and Hay, 1987). H2O2, an important ROS,
causes injury to cellular processes, reduces photosynthesis and enhances senescence (Dhindsa et
al., 1981). Under combined effect of heat and drought stresses, turf quality, relative water
contents, chlorophyll and protein content were reduced because of decreased activities of
antioxidant enzymes and increase in electrolyte leakage and membrane lipid peroxidation
(Huang and Gao, 1999; Jiang and Huang, 2000; 2001; Xu and Huang, 2004).
AOS levels increase in stressed tissue due to reduced antioxidant activity (Fadzillah et
al., 1996). Plants have well developed several enzymatic and non enzymatic antioxidant defense
systems as a line of defense to remove and detoxify intracellular structures (Noctor and Foyer,
1998; Liu and Huang, 2000; Fu and Huang, 2001; Alscher et al., 2002; Sairam and Tyagi, 2004;
Farooq et al., 2008). The enzymatic antioxidants include catalase (CATs), superoxide dismutase
(SOD), glutathione peroxidase such as ascorbate peroxidase (APX), glutathione reductase (GSH)
and glutathione-synthesizing enzymes (Arora et al., 2002; Sairam et al., 2002; Gong et al.,
2005). SOD scavenges the superoxide radical to H2O2, which is converted to water and oxygen
by the action of glutathione peroxidase or CAT and GHS reductase in chloroplast and
All the salts were dissolved and mixed after autoclaving separately. Final pH of the solution was adjusted at 6.7.
30
Fig. 1: Plexiglass-fitted canopies used to create high temperature environment for maize. Light transmission index of the canopy was 75-80%.
Winter-2007
0
10
20
30
40
50
Mar-07 Apr-07 May-07 Jun-07
Outside canopyInside canopy
0
10
20
30
40
50
60
70
Mar-07 Apr-07 May-07 Jun-07
Outside canopyInside canopy
Summer-2007
0
10
20
30
40
50
Aug-07 Sep-07 Oct-07 Nov-07
Outside canopyInside canopy
01020304050607080
Aug-07 Sep-07 Oct-07 Nov-07
Outside canopyInside canopy
Temperature (oC) Relative humidity (%)
Fig. 2: Variation in the temperature and relative humidity inside and outside the plexiglass fitted canopy during experiment in Winter and Summer seasons in 2007
31
shoot and root were taken immediately after harvesting. For dry weights, the shoot and roots
were put in the paper bags and kept in an oven at 70oC for a week. For cob characteristics and
grain yield, the cobs were removed at maturity. The number of rows per cob and number of
grains per row were counted. The grains were extracted from the cobs and their yield was
assessed after weighing to express on per plant basis. The harvest index (HI) was calculated as:
HI (%) = (grain yield per plant) x 100/ (straw yield per plant)
The screening experiment led to the selection of two varieties; a heat tolerant (Sadaf) and
heat sensitive (Agatti-2002), on the basis of growth and yield characteristics at respective growth
stages in the above mentioned seasons.
3.2 Mechanism of heat tolerance in maize across the winter and summer seasons
The detailed mechanism of heat tolerance was studied at seedling, sikling and grain filling stages
of maize varieties Sadaf (tolerant) and Agatti 2002 (sensitive), selected from the screening
experiment.
3.2.1 Growth and yield determinations
The induction of treatments at all growth stages and growth measurements at seedling and
silking and grain yield characteristics at maturity were the same as given above. The design of
growth and yield experiments was completely randomized factorial with four replications.
3.2.2 Leaf water and osmotic relations
Third fully expanded leaf from top was excised to determine the leaf water potential (ψw) using
pressure chamber (Scholander Pressure Bomb, Arimad 2, Germany). The data was recorded
from 10:00 to 11:00 am. To determine the osmotic potential (ψs), the leaf from the same position
was excised, quickly frozen and kept in a freezer at -30oC. After about seven days, these leaf
samples were thawed at room temperature, put in a plastic syringe and sap expressed by applying
pressure and collected in a microfuge tube. The ψs was determined using osmometer (vapor
pressure based, Model-Wescor 5520, Utah). Turgor potential (ψp) was determined as difference
between the water potentials and osmotic potentials values as:
Ψp = ψw – ψs
32
For the determination of relative water contents (RWC), 10 leaf discs (1 cm square) cut
using cork-borer from third leaf from the top and weighed immediately for fresh weight (FW).
The discs were floated on water for overnight, weighed again next morning to obtained turgid
weight (TW). Dry weights (DW) were measured having dried the discs at 70oC in an oven for
one week. The RWC was calculated as given by Turner (1981):
% RWC = (FW-DW) × 100 / (TW-DW).
Total free amino acids were determined according to Hamilton and Van-Slyke (1943).
For this purpose, 1 mL of the aqueous extract of the sample was taken in test tube (25 mL
volume) and added 1 mL each of 10% aqueous pyridine solution and 2% ninhydrine solution
(prepared by dissolving 2 g ninhydrin in 100 mL distilled water). Then heated these test tubes in
boiling water in a water bath for about 30 min and transferred the solution to 50 mL test tube and
made the volume in each tube to 50 ml with distilled water. The absorbance of the colored
solution was taken at 570 nm using spectrophotometer. A standard curve was developed with
Lucine and calculated the free amino acids by the formula given below.
Total amino acids (mg/g Fresh weight) =
Graph reading of samples ×Volume of the sample × dilution factor
Weight of the tissue ×1000
Free proline was spectrophotometrically determined using the protocol of Bates et al.
(1973). Third leaf from the top (0.5 g) was homogenized in 5 mL of 3% of aqueous
sulphosalicylic acid and homogenate filtered through Whatman No.2 filter paper. One mL of
filtrate was taken and mixed with 1 mL of acid ninhydrin (1.25 g ninhydrine in 30 mL glacial
acetic acid) and 1 mL of glacial acetic acid in a test tube. The mixture was briefly vortexed and
heated at 100oC in a water bath for 1 h and then terminated the reaction in the ice bath. Four mL
of toluene was added to the solution and vortexed for 15-20 seconds while cool. The
chromophore containing proline was aspirated from aqueous phase in a test tube and warmed to
laboratory temperature. The absorbance was taken at 520 nm using spectrophotometer. The same
procedure was followed for blank using 2 mL of toluene. Standard curve was constructed using
proline (10 to 50 µg/2 mL). The amount of free proline was calculated by following formula:
Fig. 3: Changes in shoot length of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages. In this and subsequent figures, vertical lines on the bars are standard deviation of means
43
At silking stage, in winter season, data revealed significant difference in the varieties, but
a non-significant difference in the treatments with a significant interaction of these factors.
However, in summer a significant difference was noted in the varieties and treatments, but there
was no interaction of these factors. In winter, under glasshouse condition, Sadaf showed greater
shoot length than Agatti-2002 under both the conditions. Shoot length in the glasshouse grown
plants of Sadaf was higher, while a reverse trend was noted in Agatti-2002. However, in summer
season under glasshouse, Sadaf and Agatti-2002 showed greater shoot length than in control
condition. Plants grown in winter were longer than those in summer (Fig. 3).
At grain filling stage, statistical analysis of data revealed non-significant difference in the
varieties, a significant difference in the treatments but a non-significant interaction of these
factors in both the seasons. In both seasons, Sadaf performed better than Agatti-2002 both under
control and glasshouse conditions. In both the seasons, shoot length was greater under heat stress
than control in Sadaf, while in glasshouse grown plants of Agatti-2002, the value of this attribute
was lesser than control in both the seasons (Fig. 3).
b. Root length
At seedling stage, in winter season, there was no significant difference in the varieties, but a
significant difference was evident in the treatments and a no significant interaction of these
factors. However, in summer season, a significant difference was noted in the varieties but non-
significant one in the treatments, and a non-significant interaction of both the factors for root
length. During winter season, both the varieties indicated a similar pattern of changes in root
length. However, glasshouse grown plants displayed lesser root length as compared to respective
controls in both varieties. In summer season, although root length was greater in Sadaf, both the
varieties showed relatively increased root length than the corresponding controls (Fig. 4).
At silking stage, statistical analysis of data revealed significant differences in the varieties
and treatment, and with significant interaction of these factors in winter season. However, in
summer season, no significant difference in the varieties but a significant one in the treatments,
and a non-significant interaction of both the factors was noted. In winter season, Sadaf exhibited
longer roots than control, but reverse behavior was noted in Agatti-2002 under glasshouse
condition. Although much shorter than the winter season plants, glasshouse grown plants of both
44
the varieties in summer season indicated longer root length in both varieties as compared to
respective controls (Fig. 4).
At grain filling stage, statistical analysis of data revealed non-significant difference in the
varieties, while significant difference in treatments with non-significant interaction of these
factors during winter and summer seasons. In winter season, there was no difference in root
length of control and glasshouse grown plants, which decreased in latter condition in Agatti-
2002. Although much shorter than the winter season plants, control and glasshouse grown plants
of both the varieties in summer season indicated no specific difference in the root length (Fig. 4).
c. Shoot dry weight At seedling stage, in winter season, data showed significant difference
in the varieties and treatments with a significant interaction of both factors. However, in summer
Varieties (V) 1 85.56** 9.50ns Treatments (T) 1 52.57* 101.68** V × T 1 88.68** 2.50ns Error 12 8.32 5.99
Varieties (V) 1 71.11ns 56.77ns Treatments (T) 1 97.38** 74.70* V × T 1 12.78ns 6.78ns Error 12 7.34 27.67
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 4: Changes in root length of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
45
season there was significant difference in the varieties and treatments but no interaction of both
factors was evident. In winter, although shoot dry weight was quite low, glasshouse condition
further reduced it in both the varieties. In summer season, shoot dry weight was not affected
remarkably by glasshouse condition. Although Sadaf indicated a higher shoot dry weight than
Agatti-2002, glasshouse grown plants of both the varieties indicated greater value of this
attribute than Agatti-2002 (Fig. 5).
At silking stage, in winter season, data revealed significant difference in the varieties, and
treatments, also there was a significant interaction of both factors. Contrarily, in summer season
the varieties, but not the treatments, differed significantly as well as there was no interaction of
varieties and treatments for shoot dry weight. In winter, both the varieties had similar shoot dry
weight under control, while under glasshouse condition Agatti-2002 manifested significantly
Varieties (V) 1 100.17** 12.15* Treatments (T) 1 23.14* 2.10ns V × T 1 44.22** 3.63ns Error 12 2.94 1.78
Varieties (V) 1 14592.64** 924.16ns Treatments (T) 1 1375.67ns 5574.12** V × T 1 2668.76* 7951.29** Error 12 422.28 13.33
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 5: Changes in shoot dry weight of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
46
reduced shoot dry weight than Sadaf. In summer season, although lesser than winter, both the
varieties had similar shoot dry weight under control, while under glasshouse condition it was
greater than control in Sadaf but equal to control in Agatti-2002 (Fig. 5).
At grain filling stage, in winter season, data revealed significant difference among the
varieties but not the treatments, while there was a significant interaction of both factors.
However, in summer season, the varieties differed non-significantly while treatments indicated
significant difference with a significant interaction of varieties and treatments. In winter season,
although Sadaf indicated a higher shoot dry weight under either condition, glasshouse condition
greatly reduced this attribute in Agatti-2002. In summer season, under control condition although
shoot dry weight was relatively lesser in Sadaf than Agatti-2002, glasshouse condition did not
influence this variable in Sadaf but decreased remarkably in Agatti-2002 (Fig. 5).
d. Root dry weight
At seedling stage, in winter season, statistical analysis of data revealed no significant difference
among the varieties but a significant one in the treatments, with no significant interaction of both
factors. However, in summer season, there was a significant difference in the varieties, but non-
significant difference in the treatments, as well as there was no interaction of both these factors.
In winter season, both the varieties indicated similar root dry weight under control but a reduced
one under glasshouse condition. In summer season, on the other hand, both the varieties showed
similar response both under control and glasshouse condition for this parameter (Fig. 6).
At silking stage, in both the seasons, there was significant difference in the varieties and
treatments with a significant interaction of both the factors for this attribute. In winter, root dry
weight was greater in Sadaf, which increased further under glasshouse condition, while in
Agatti-2002 glasshouse condition reduced it compared to control. In summer, the root dry weight
increased in Sadaf under glasshouse condition as compared to control, but in Agatti-2002 root
dry weight was similar under both the conditions (Fig. 6).
At grain filling stage in winter season, there was no significant difference in the varieties
and treatments but there was significant interaction of both factors. However, in summer season,
a significant difference was notable in the varieties, while non-significant difference was evident
in the treatments with a non-significant interaction of both the factors. In winter season, root dry
weight was much greater in Sadaf, which increased further under glasshouse condition, while in
47
Agatti-2002, a greatly reduced root dry weight was noted under glasshouse condition. In
summer, although root dry weight was higher in both the varieties as compared to winter season,
the trend of changes did not differ much from that observed under winter season (Fig. 6).
e. Number of leaves per plant
At seedling stage, statistical analysis of data recorded from winter season crop revealed
significant difference in the varieties and treatments but there was no significant interaction of
both factors. However, in summer season crop, only the varieties differed significantly while no
difference was evident in the treatments, as well as an interaction of varieties and treatments was
missing. In winter season crop under control condition, number of leaves per plant was relatively
lesser in Sadaf than Agattti-2002, while in glasshouse both these varieties showed similar value
Varieties (V) 1 6.30** 5.25** Treatments (T) 1 0.67* 1.19** V × T 1 1.99** 2.49** Error 12 0.12 0.09
Varieties (V) 1 0.0638ns 11.672** Treatments (T) 1 0.0625ns 1.174ns V × T 1 5.842* 3.061ns Error 12 0.845 1.063
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 6: Changes in root dry weight of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
48
of this number. In summer season, this number was greater than that observed in winter season,
while glasshouse condition did not influence this attribute in both the varieties (Fig. 7).
At silking stage, in winter season, statistical treatment of data revealed significant
difference in the varieties, non-significant difference in the treatments and a significant
interaction of both the factors. On the other hand, in summer season, the varieties, treatments and
interaction of these factors were non-significant. In winter, number of leaves was greater in
Sadaf than Agatti-2002, which increased in the former and decreased in the latter variety under
glasshouse condition. In summer season, both varieties under either condition showed no big
difference in this number (Fig. 7).
At grain filling stage in winter and summer seasons, data revealed non-significant
difference in the varieties, a significant one in the treatments while there was no interaction of
both these factors. In winter season, Sadaf displayed lower number of leaves per plant than
Varieties (V) 1 58.78** 0.028ns Treatments (T) 1 0.25ns 1.361ns V × T 1 4.34* 0.001ns Error 12 0.858 0.514
Varieties (V) 1 11.11ns 106.77ns Treatments (T) 1 117.38** 164.70* V × T 1 2.78ns 1.78ns Error 12 7.34 27.67
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 7: Changes in number of leaves per plant of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
49
Agatti-2002 under control condition. However, under glasshouse condition, Sadaf produced
leaves in greater number than Agatti-2002. In summer season on the other hand, this number was
greater in Sadaf than Agatti-2002 under control condition. Under glasshouse condition, Sadaf
showed an increased while Agatti-2002 a decreased number of leaves per plant (Fig. 7).
f. Leaf area per plant
As shown in Fig. 8, at seedling stage in winter season, statistical analysis of data revealed a
significant difference in the varieties and treatments but a non-significant interaction of both
factors. However, in summer season, the varieties, but not the treatments, indicated significant
difference, and no interaction of varieties and treatments was evident. In winter season, leaf area
Varieties (V) 1 8226.99** 98.29ns Treatments (T) 1 161.46ns 76.15ns V × T 1 3854.02** 171.50** Error 12 97.40 32.37
Varieties (V) 1 511.11** 106.77** Treatments (T) 1 1117.38** 164.70** V × T 1 52.78** 61.78** Error 12 7.34 7.67
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 8: Changes in leaf area per plant of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
50
per plant was lower in Sadaf than Agatti-2002 under control condition, while glasshouse
condition decreased it in both the varieties. In summer season, although Sadaf had greater leaf
area per plant, glasshouse effect did not affect this attribute much in both varieties.
At silking stage, data showed significant difference in the varieties, non-significant
difference in the treatments while a significant interaction of both the factors was notable. In
summer season, varieties and treatments indicated no significant differences while there was a
significant interaction of both these factors. In winter season, Sadaf with greater leaf area under
control indicated an increase in it under glasshouse condition. In summer season, leaf area per
plant was similar in both the varieties under control condition, while glasshouse condition
increased it in Sadaf but decreased in Agatti-2002 (Fig. 8).
At grain filling stage in both the seasons, data indicated significant differences in the
varieties and treatments with a significant interaction of both factors. In winter season, both the
varieties had similar leaf area under control condition, which increased in Sadaf but decreased in
Agatti-2002 under glasshouse condition. In summer season, although both the varieties displayed
lesser leaf area compared with winter crop, the trend of changes in this season was similar to that
observed in winter season (Fig. 8).
g. Cob characteristics
Data for the number of cobs per plant indicated no significant difference in the varieties but a
significant one in treatments, although there was no interaction of these factors. However, in
summer season, the varieties indicated significant but treatments showed non-significant
differences, and a significant interaction of these factors was evident. In winter season, Agatti-
2002 had relatively greater number of cobs per plant than Sadaf under control condition in both
seasons. However, under glasshouse condition in winter season, both the varieties indicated a
reduction, while in summer season Sadaf showed an increase but Agatti-2002 a decrease in the
number of cobs per plant (Fig. 9).
For number of rows per cob, data revealed non-significant difference in the varieties, a
significant one in the treatments while there was no significant interaction of these factors. In
summer season, the difference in the varieties, treatments and an interaction of these factors were
non-significant. In winter season, both the varieties indicated a greater number of rows per cob
51
than summer season under control condition. However, under glasshouse condition, reduction in
this number was much higher in winter than in summer season (Fig. 9).
The data regarding number of grains per cob indicated significant difference in the
varieties and treatments with a significant interaction of these factors in winter season. However,
Varieties (V) 1 0.25ns 2.83ns Treatments (T) 1 8.03** 4.62ns V × T 1 0.69ns 4.77ns Error 12 0.92 2.39
Varieties (V) 1 1167.37** 207.83* Treatments (T) 1 3080.22** 2154.47** V × T 1 793.34* 291.87ns Error 12 88.71 31.73
Varieties (V) 1 387.62* 75.00ns Treatments (T) 1 4432.22** 1347.93** V × T 1 79.44ns 87.17ns Error 12 56.51 64.34
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 9: Changes in some cob characteristics of control and glasshouse grown maize varieties during winter and summer seasons at grain filling stages
52
in summer season, varieties and treatments showed significant difference while there was no
interaction of these factors. In winter season under control both the varieties indicated a similar
number of grains per cob, which reduced substantially in Agatti-2002 under glasshouse
condition. In summer season, although the trend of changes was similar to the winter season, but
the reduction in this attribute under glasshouse condition were lesser than those noted in the
winter season (Fig. 9).
The cob weight indicated significant differences in the varieties and treatments with a
non-significant interaction of these factors. However, in summer season crop only treatments
indicated significant difference. The cob weight did not differ much in both the varieties and
seasons under control condition. In the glasshouse grown plants, although there was a reduction
in this attribute in both the seasons, a greater reduction was evident in winter season (Fig. 9).
h. Grain yield characteristics
Statistical analysis of data on grain yield per cob indicated significant differences in the varieties
and treatments in both the seasons but there was a significant interaction of both these factors in
winter season, while a non-significant interaction in summer season. Under control condition in
both the seasons, grain yield per cob was similar in both the varieties. However, under
glasshouse condition although this attribute decreased in both the varieties, Agatti-2002 indicated
a greater reduction, which was well explicit in the winter season (Fig. 10).
For 100 grain weight, results revealed that in winter season varieties and treatments,
while in summer season only treatments indicated significant differences, while there was no
interaction of these factors in both the seasons. In winter and summer seasons, although 100
grain weight was greater in Sadaf than Agatti-2002, glasshouse condition produced a greater
reduction in the latter variety (Fig. 10).
For grain yield per plant, data revealed significant difference in the varieties and
treatments with a significant interaction of both these factors in both the seasons. Under control
condition, grain yield per plant was relatively greater in winter than summer season. Under
glasshouse condition, although grain yield per plant reduced in both the varieties, Agatti-2002
indicated a greater reduction than Sadaf, and glasshouse condition was more adverse ti this
attribute in winter than summer season (Fig. 10).
53
For harvest index, results revealed that in winter season, only treatments while in summer
season varieties and treatments indicated significant differences, while there was no interaction
of these factors in both the seasons. In winter season, although harvest index was higher in
Agatti-2002 than Sadaf under control condition, glasshouse condition greatly affected this
parameter in the former variety. In summer season, Sadaf had greater harvest index than Agatti-
Varieties (V) 1 210.35** 9.63ns Treatments (T) 1 1259.76** 21.13** V × T 1 7.52ns 0.28ns Error 12 30.89 4.81
Varieties (V) 1 571.66** 492.61** Treatments (T) 1 4959.95** 1513.98** V × T 1 479.90** 551.16** Error 12 20.36 29.43
Varieties (V) 1 0.31ns 49.55* Treatments (T) 1 633.11** 83.42** V × T 1 43.92ns 1.94ns Error 12 5.74 7.94
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 10: Changes in some grain yield characteristics of control and glasshouse grown maize varieties during winter and summer seasons at maturity
54
2002 under control condition, while glasshouse condition was almost equally detrimental to this
attribute (Fig. 10).
4.1.2 Discussion
In view of the changing environmental condition, mainly related to global warming, the plant
growing patterns are subject to rapid and continuous changes (Porter, 2005). Although maize is a
C4 plant and can withstand relatively higher ambient temperatures (Ashraf and Hafeez, 2004),
growth and yield response of this important cereal crop to glasshouse conditions have not been
comprehensively studies. Maize is a short duration crop and is grown in winter and summer
seasons in Pakistan. It shows differential growth and productivity in these seasons, as different
yields are obtained in both these seasons (Anonymous, 2008). In this study, determination made
for changes in some growth, cob and grain yield attributes of glass canopy (glasshouse) grown
varieties indicated great differences in the selected varieties and treatments. Most importantly,
some interactions of the varieties and treatments for various parameters present in one season
disappeared in the other season at various growth stages. This indicated that prevailing
glasshouse condition modulated the maize growth, although the effects were relatively lesser
evident on the high temperature tolerant variety (Sadaf) than the high temperature sensitive
variety (Agatti-2002) in the glasshouse.
Determination of growth responses at various critical phenological stages indicates the
specific responses of plants under study. This is because during transition from one growth phase
to the other, there is reprogramming of gene expression and sensitivity to changed environmental
conditions may be greater (Milligan et al., 2004; Qin et al., 2004; Wahid and Close, 2007).
These alterations in gene activities result in the developmental changes, as reflected from
changes in plant growth patterns (Srivastava, 2002; Taiz and Zeiger, 2006). In this study, the
determination were made at three phenological stages (seedling, silking and grain filling)
revealed that both the varieties behaved differently at all these growth stages under glasshouse
condition (Figs. 3-8). In addition, the influence of seasons was also well marked, as quite a few
interactions appearing in winter grown plants disappeared in summer grown plants. It is
important to note that at silking stage most of the interaction disappeared in summer. Silking
stage appears to be the most critical for final plant productivity because at this particular stage,
55
number of changes including success of fertilization, seed set and grain filling follow the
reception of pollen by the silk. Similar changes have been reported in maize and other plants
during fertilization (Le Deunff et al., 1993; Wahid et al., 2007).
Determination made for cob (Fig. 9) and grain yield and related (Fig. 10) characteristics
indicated that winter season produced more conspicuous changes than summer season.
Moreover, the effect of glasshouse was also a major factor in producing changes in these
attributes. Data revealed that for cob, most important differences observed across the seasons
were evident in number of grain rows per cob and number of grains per cob (Fig. 9), while for
grain and grain yield components, grain yield per cob, grain yield per plant and harvest index
were more important (Fig. 10). This revealed that glasshouse condition has definitive influence
on the growth and economic yield attributes of maize.
If the differences in the ambient temperature inside and outside the canopy are taken
together, it becomes clear that a rise in the temperature (in the months of May and June) of the
winter sown crop plays a crucial role in the occurrence of changes and producing interaction of
varieties and treatments. Under glasshouse condition in winter grown crop, where the
temperature rises further by 5-7oC and relative humidity declines. On the contrary, in summer
season at silking and grain filling stages there is a continuous decline in temperature and a rise in
relative humidity. These changing climatic conditions appeared to play a role in narrowing down
the differences in the varieties (Fig. 2), thus leading to the disappearance of interactions. In this
context, it is pointed out that a single degree change in ambient temperature is likely to produce a
set of changes in the plants (IPCC, 2007; Wahid et al., 2007).
In conclusion, changes in ambient temperature produce a lot of changes in growth and
yield of maize, and the prevailing glasshouse conditions play a crucial role in this regard across
winter and summer seasons. Investigations on the physiological and biochemical basis of these
changes (as reported in the next sections) will improve out understanding of the underlying
phenomena under the changing growth conditions in the glasshouse.
56
4.2 Leaf pigments and gas exchange properties
4.2.1 Results
a. Chlorophyll a
At seedling stage, in winter season, data revealed non-significant difference in the varieties and
treatments, with no interaction of both factors. In summer season, a non-significant difference in
the varieties and treatments was noted, but a significant interaction of both these factors was
evident for chlorophyll (Chl) a contents. During winter season, in glasshouse grown plants Chl a
contents decreased more in Agatti-2002 as compared to Sadaf. However, in summer season Chl a
contents increased over control in Sadaf but remarkably decreased in Agatti-2002 (Fig. 11).
Fig. 11: Changes in chlorophyll a concentration in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
57
At silking stage, in both the seasons, data indicated a non-significant difference in the
varieties and treatments, and there was no significant interaction of both factors for Chl a
contents. In winter and summer seasons, showing no change in control plants, Chl a contents did
not change in Sadaf but decreased in Agatti-2002 under glasshouse condition (Fig. 11).
At grain filling stage, data showed non-significant difference in the varieties and
treatments but there was a significant interaction of both factors in winter season. However, in
summer season, the varieties differed non-significantly, while a significant difference was seen
in the treatments, as well as there was non-significant interaction of varieties and treatments for
Chl a contents. The Chl a contents of winter season plants were greater than those of summer
grown plants. Glasshouse condition increased Chl a contents in Sadaf but decreased in Agatti-
2002. However, in summer season, no specific difference was recorded for changes in Chl a
contents in the varieties under both the conditions (Fig. 11).
b. Chlorophyll b
At seedling stage, statistical results revealed non-significant difference in the varieties and
treatments, as well as there was no significant interaction of both factors in both the seasons for
Chl b. In winter season, in glasshouse grown plants, Chl b contents increased in Sadaf and
reduced in Agatti-2002. However, in summer season the value of this attribute was increased in
Sadaf but did not change in Agatti-2002 under glasshouse condition (Fig. 12).
At silking stage, data analysis revealed non-significant difference in the varieties and
treatments and there was no significant interaction of both factors in both the growing seasons
for Chl b. Overall, the Chl b contents were greater in winter than summer grown plants. At this
stage, glasshouse grown plants of Sadaf displayed increased, while those of Agatti-2002
decreased Chl b contents. However, in summer season, the value of this attribute decreased both
in Sadaf and Agatti-2002 under glasshouse condition (Fig. 12).
At grain filling stage, data exhibited non-significant difference in the varieties but a
significant one in the treatments, while there was no interaction of both these factors in winter
season. However, in summer season, the varieties and treatments differed non-significantly, as
well as there was no interaction of varieties and treatments for Chl b contents. In winter season,
although Chl b contents reduced in both the varieties under under glasshouse condition, Agatti-
2002 was relatively more affected than Sadaf. In the summer season, although Chl b contents
58
were lesser than winter season plants, Sadaf showed increased while Agatti-2002 indicated no
change in Chl b under glasshouse condition (Fig. 12).
c. Total chlorophylls
At seedling stage, statistical treatment of data revealed no significant difference in the varieties,
treatments and no interaction of both factors was notable in both winter and summer season for
total chlorophylls. In winter grown plants, total chlorophylls did not change in Sadaf but
decreased in Agatti-2002 under glasshouse condition. In summer season, the total chlorophylls
increased in Sadaf and decreased in Agatti-2002 (Fig. 13).
Varieties (V) 1 0.815ns 0.035ns Treatments (T) 1 0.1060ns 0.374ns V × T 1 0.814ns 0.043ns Error 12 0.454 0.094
Varieties (V) 1 0.453ns 0.749ns Treatments (T) 1 1.353** 0.152ns V × T 1 0.129ns 0.476ns Error 12 0.10 0.168
Significant at: ** P<0.01, ns non-significant Seasons Fig. 12: Changes in chlorophyll b concentration of in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
59
At silking stage, in both the seasons, statistical analysis of data indicated no significant
difference in the varieties and treatments and there was no interaction of both factors. Winter
grown plants displayed greater total chlorophyll contents than summer grown plants. In winter
season, total chlorophyll increased in Sadaf but decreased in Agatti-2002 under glasshouse
condition. In summer season, glasshouse condition did not produced remarkable changes in the
total chlorophyll contents of both the varieties (Fig. 13).
At grain filling stage, results revealed significant difference in the varieties and
treatments, with a significant interaction of both factors in winter season. However, in summer
season, the varieties and treatments differed non-significantly and there was no interaction of
varieties and treatments for total chlorophylls. Like previous stage, winter grown plants
Varieties (V) 1 1.462ns 0.132ns Treatments (T) 1 2.298ns 0.032ns V × T 1 3.786ns 0.128ns Error 12 1.006 0.212
Varieties (V) 1 2.758** 0.520ns Treatments (T) 1 3.057** 0.012ns V × T 1 2.189* 0.067ns Error 12 0.29 0.17
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 13: Changes in total chlorophyll concentration of in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
60
manifested greater total chlorophyll contents than summer season plants. In winter season, Sadaf
exhibited no changes while Agatti-2002 indicated a decrease in total chlorophyll contents under
glasshouse condition. In summer season, on the other hand, total chlorophyll contents did not
differ much in the control and glasshouse condition in both the varieties (Fig. 13).
d. Chlorophyll a/b ratio
At seedling stage, for summer and winter seasons, results showed no significant difference in the
varieties and treatments and there was no interaction of both factors for this parameter Chl a/b
ratio. The winter grown plants showed a greater Chal a/b ratio than summer grown plants. In
winter season, this ratio was slightly reduced in Sadaf but increased in Agatti-2002. However, in
summer season, Chl a/b ratio reduced in both varieties, although greatly in Agatti-2002 (Fig. 14).
Fig. 14: Changes in chlorophyll a/b ratio of control in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
61
At silking stage, statistical analysis of data revealed non-significant difference in the
varieties, treatments and there was no significant interaction of both factors in winter season.
However, in summer season, the varieties differed non-significantly while a significant
difference was evident in the treatments, as well as no significant interaction of varieties and
treatments for Chl a/b ratio. Winter season plants displayed relatively reduced Chl a/b ratio than
the summer seaon plants. In winter season under glasshouse condition, although this ratio
reduced in both the varieties, but a greater reduction was observed in Agatti-2002. However, in
summer season, this ratio increased in both Sadaf and Agatti-2002, but a greater increase was
noted in the former variety (Fig. 14).
At grain filling stage, in winter season, results revealed non-significant difference in the
varieties and treatments, and there was no significant interaction of both factors. However, in
summer season, the varieties differed significantly while non-significant difference was present
in the treatments, and there was no interaction of varieties and treatments for Chl a/b ratio. In
winter season, although this ratio increased in both Sadaf and Agatti-2002 under glasshouse
condition, a greater increased was evident in Sadaf. In summer season, this ratio was reduced in
both varieties irrespective of the growth condition. Nevertheless, under glasshouse condition, this
ratio decreased more in Sadaf than Agatti-2002 (Fig. 14).
e. Total carotenoids
At seedling stage, in winter season, statistical analysis of results revealed significant difference
among the varieties but non-significant difference in the treatments with a non-significant
interaction of both these factors. However, in summer season, not the varieties but the treatments
indicated significant difference, but no interaction of both these factors was present for total
carotenoids (Car). In winter season, Car increased in Sadaf but decreased in Agatti-2002 under
glasshouse condition. In summer season, although glasshouse condition reduced Car, both the
varieties indicated a similar pattern of change (Fig. 15).
At silking stage, data revealed significant difference in the varieties, treatments and there
was significant interaction of varieties and treatments of both factors in winter season. However,
in summer season, the varieties differed non-significantly while a significant difference was
evident in the treatments, with a significant interaction of varieties and treatments for Car. In
winter season, under glasshouse condition, Sadaf indicated a smaller but Agatti-2002 a greater
62
decrease in Car. In summer season under glasshouse condition, Car increased substantially in
Sadaf but did not change much in Agatti-2002 (Fig. 15).
At grain filling stage, statistical analysis of results more indicated significant difference
in the varieties, while no significant difference was evident in the treatments, as well as there was
a significant interaction of varieties and treatments in winter season. However, in summer
season, the varieties and treatments differed significantly showing a significant interaction of
varieties and treatments for Car. In winter season, glasshouse grown plants of Sadaf indicated
increased Car over control, which decreased in case of Agatti-2002. In summer season, on the
Varieties (V) 1 0.052** 0.004ns Treatments (T) 1 0.068** 0.009* V × T 1 0.045** 0.016** Error 12 0.001 0.002
Varieties (V) 1 0.059** 0.034** Treatments (T) 1 0.003ns 0.027** V × T 1 0.054** 0.018** Error 12 0.01 0.001
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 15: Changes in total carotenoids concentration of control in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
63
contrary, control and glasshouse grown plants of Sadaf indicated similar Car while Agatti-2002
showed reduced Car (Fig. 15).
f. Net rate of photosynthesis
At seedling stage, for winter season, data revealed no significant difference in the varieties, while
significant difference in the treatments and a non-significant interaction of both these factors.
However, in summer season, the varieties differed non-significantly, while significant difference
was evident in treatments, and there was significant interaction of varieties and treatments for net
rate of photosynthesis (Pn). In both the seasons, Pn decreased in both the varieties under
glasshouse condition, although Agatti-2002 was more affected. Of the seasons, glasshouse
condition in winter season was more adverse than in summer season (Fig. 16).
At silking stage, in winter season, analysis of data revealed significant difference in the
Varieties (V) 1 49.21** 1.08ns Treatments (T) 1 78.32** 94.28** V × T 1 17.60* 3.65ns Error 12 2.35 3.64
Varieties (V) 1 2.576ns 14.861** Treatments (T) 1 206.928** 102.718** V × T 1 16.040** 14.100** Error 12 0.818 0.717
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 16: Changes in net rate of photosynthesis of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
64
varieties and treatments with a significant interaction of both factors. However, in summer
season, the varieties differed non-significantly while significant difference was evident in the
treatments. Furthermore, there was no interaction of varieties and treatments for Pn. In winter
season, Pn was lesser than that noted in summer season. Although glasshouse condition was
inhibitory to this character in both the varieties, Sadaf performed better than Agatti-2002.
Growing season had a great effect on this attribute of both the varieties (Fig. 16).
At grain filling stage, in winter season, there was a non-significant difference in the
varieties, but a significant difference was observed in the treatments, with a significant
interaction of both factors for Pn. In summer season, however, varieties and treatments exhibited
differences with a significant interaction of both the factors. In winter season, Pn was greater in
Agatti-2002 under control, but substantially decreased under glasshouse condition. In summer
season, both varieties showed similar Pn under control condition, but under glasshouse condition
Sadaf displayed greater Pn than Agatti-2002 (Fig. 16).
g. Transpiration rate
At seedling stage, in both the seasons, data showed non-significant difference in the varieties
and treatments with a non-significant interaction of both the factors for transpiration rate (E). In
winter season, E being similar in both varieties under control, decreased in Agatti-2002 under
glasshouse condition. Contrarily in summer season, this parameter decreased in Sadaf but
increased in Agatti-2002 under glasshouse condition (Fig. 17).
At silking stage, statistical analysis of data showed non-significant difference in the
varieties and treatments with a non-significant interaction of both these factors during winter
season. However, during summer season, there was difference in the varieties and treatments but
a non-significant interaction of both these factors. There was a significant influence of growing
season on this attribute. In winter season, Sadaf and Agatti-2002 showed similar value of E
under both conditions. Contrarily in summer, E was lower in Sadaf under both conditions as
compared to Agatti-2002, but growth condition did not affect E in Sadaf. However, E decreased
remarkably in glasshouse condition during summer in Agatti-2002 (Fig. 17).
At grain filling stage, data revealed non-significant difference in the varieties and
treatments, together with a non-significant interaction of both these factors in winter. However,
in summer season, the varieties and treatments differed significantly with significant interaction
65
of varieties and treatments for E. In winter season, E decreased in Sadaf and increased in Agatti-
2002 in glasshouse. However, in summer although both the varieties showed a decreased E but
this decrease was greater in Agatti-2002 under glasshouse condition (Fig. 17).
h. Water use efficiency
At seedling stage, in winter season, data revealed non-significant difference in the varieties but a
significant one in treatments, together with non-significant interaction of both factors for water
use efficiency (WUE). However, in summer season, the varieties differed non-significantly while
treatments differed significantly, with a significant interaction of varieties and treatments for this
parameter. In winter season, WUE reduced in both the varieties under glasshouse condition, but
Varieties (V) 1 0.006ns 4.45** Treatments (T) 1 0.000ns 0.87* V × T 1 0.0004ns 0.08ns Error 12 0.04 0.10
Varieties (V) 1 0.041ns 0.129** Treatments (T) 1 0.002ns 0.145** V × T 1 0.037ns 0.054** Error 12 0.018 0.004
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 17: Changes in transpiration rate of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
66
this decrease was greater in Agatti-2002. In summer, Sadaf showed similar WUE in both
conditions but Agatti-2002 exhibited reduced WUE in glasshouse (Fig. 18).
At silking, data revealed significant difference in the varieties and treatments but there
was no significant interaction of both factors during winter season. However, in summer season,
the varieties differed significantly but non-significant difference was noted in treatments, with no
interaction of these factors for WUE. In winter season, Sadaf indicated greater WUE than Agatti-
2002 under control. However, glasshouse condition reduced WUE in both varieties but greatly in
Agatti-2002. In summer season, again WUE was greater in Sadaf under control condition.
However, glasshouse increased this parameter in Sadaf but markedly reduced in Sadaf (Fig. 18).
Varieties (V) 1 16.26** 19.78** Treatments (T) 1 33.47** 0.51ns V × T 1 7.44ns 1.98ns Error 12 0.87 0.63
Varieties (V) 1 2.92** 1.80ns Treatments (T) 1 80.27** 39.22** V × T 1 10.51** 5.08* Error 12 0.21 0.74
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 18: Changes in water use efficiency of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
67
At grain filling stage, data revealed significant difference in the varieties and treatments,
with a significant interaction of both factors in winter season. In summer season, on the contrary,
varieties showed non-significant but treatments indicated significant difference with a significant
interaction of varieties and treatments for WUE. Under control condition, WUE was similar in
both the varieties across the seasons. However, glasshouse condition in both the seasons was
damaging to this attribute, although Sadaf performed better than Agatti-2002 in both the seasons
for WUE (Fig. 18).
i. Stomatal conductance
As evident from Fig. 19, at seedling stage, in winter season, data analysis showed no significant
difference in the varieties but a significant one in the treatments, with non-significant interaction
of both the factors. However, in summer season, the varieties and treatments differed
significantly, showing significant interaction of varieties and treatments for stomatal
conductance (gs). In winter season, both the varieties indicated greater gs than those grown in
summer irrespective of the growth condition. Although glasshouse condition reduced this
attribute in both the seasons, Agatti-2002 was more affected than Sadaf.
At silking stage in summer season, data showed significant difference in the varieties but
non-significant difference in the treatments, with a non-significant interaction of both factors for
gs. However, in summer season, there was non-significant difference in the varieties and
treatments with non-significant interaction of both these factors. In winter season, gs was greater
in both the varieties than summer. In winter, Sadaf under glasshouse condition showed greater gs
than controls, which was reduced in Agatti-2002. However, in summer season both the varieties
indicated a reduction in gs, which was proportionately greater in Agatti-2002 (Fig. 19).
At grain filling stage, analysis of data for gs, in winter season, showed significant
difference in the varieties and treatments, but there was no interaction of both factors. However,
in summer season, a significant difference was evident in varieties, a non-significant one in
treatments but a significant interaction of both these factors for gs. At this stage, gs was much
lower in winter than in summer under both the conditions. Glasshouse condition reduced this
attribute more in Agatti-2002 than Sadaf. In summer season, Sadaf showed increased gs over
control in glasshouse condition, which decreased remarkably in Agatti-2002 (Fig. 19).
68
j. Sub-stomatal CO2 concentration
At seedling stage, for winter season plants, statistical analysis of data revealed no significant
difference among the varieties and treatments, and no significant interaction of both these
factors. However, in summer season, the varieties and treatments differed significantly showing
a significant interaction of varieties and treatments for sub-stomatal CO2 concentration (Ci).
Both varieties, showing similar Ci under control condition across the seasons, indicated an
increased Ci under glasshouse condition. However, Agatti-2002 showed a relatively greater
increase than in Ci than Sadaf in both the seasons (Fig. 20).
Fig. 19: Changes in stomatal conductance of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
69
At silking stage in winter season, data for Ci showed significant difference in the
varieties and treatments, although there was no significant interaction of both factors. However,
in summer season, the varieties and treatments differed non-significantly with no interaction
these factors for this attribute. In winter season, Ci of control plants was lower than those of
summer season plants. Glasshouse condition increased Ci in both the varieties in both seasons,
although such an increase was significantly greater in Agatti-2002 (Fig. 20).
At grain filling stage, in both the seasons, data revealed no significant difference in the
varieties but a significant difference in the treatments, and there was no interaction of both
factors for Ci. In both winter and summer season, Ci was similar in control plants of both the
varieties. Glasshouse condition led to an increase in Ci in both the seasons, although this increase
Varieties (V) 1 3326.41* 273.90ns Treatments (T) 1 5886.73** 2631.69ns V × T 1 1070.93ns 637.56ns Error 12 425.13 725.98
Varieties (V) 1 852.64ns 368.84ns Treatments (T) 1 5062.32* 7421.82** V × T 1 145.20ns 49.70ns Error 12 549.78 598.44
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 20: Changes in substomatal CO2 concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
70
4.2.2 Discussion
Plant photosynthesis comprises two processes; light reaction and dark reactions. Light reactions
are involved in the generation of reducing powers for the dark reaction. Photosynthetic pigments
(primarily chlorophylls and secondarily carotenoids) are important components of light
harvesting centers in light reactions (Taiz and Zeiger, 2006). Studies show that these
photosynthetic pigments are highly prone to the changes in the environmental conditions
(Pastenes and Horton, 1996a, b), which have been taken as stress sensitivity criteria in wheat
(Ristic et al., 2007, 2008). Quite a few studies report the influences of sub-optimal
environmental conditions on the photosynthetic pigments but those describing the changes in the
photosynthetic pigments in glasshouse grown plant throughout the plant ontogeny are few.
Results of this experiment revealed that both the maize varieties indicated quite a lot
changes in Chl a, Chl b, their total, Chl a/b ratio and Car contents in both the growing seasons
and growth conditions (Figs. 11-15). In general, with remarkable differences in the varieties,
winter grown plants indicated more explicit differences than the summer grown plants at all
phenological stages. Of two chlorophyll species, Chl b was more damaged by prevailing high
temperature condition in the glasshouse (Fig. 12) than Chl a (Fig. 11), leading to an overall loss
of chlorophyll (Fig. 13), thereby causing more yellowing of leaves in Agatti-2002 than Sadaf.
These changes resulted in increased chlorophyll a/b ratio (Fig. 14), which was notably higher in
the winter grown plants. It has been shown that high temperature enhances chlorophyllase
activity that degrades the chlorophylls and reduces their contents (Todorov et al., 2003; Wahid et
al., 2007). From the changes in the chlorophyll concentrations, it can be deduced that sensitivity
of Chl b to glasshouse condition is mainly responsible for the yellowing of leaves, particularly in
winter grown plant. Perusing the prevailing temperature conditions in the canopy grown plants in
winter compared to summer, it can be seen that plants sown in winter months had to face more
adverse temperatures at later growth stages (silking and grain filling), than the summer grown
plants, which do not experience such a high temperature during these growth stages. Thus, it can
be inferred that glasshouse conditions are more detrimental to photosynthetic machinery of the
winter sown plants in the warmers months.
Carotenoids have dual roles in plants. By acting as accessory light harvesting pigments,
they harvest the light and funnel onto the photosystems. The other important role of carotenoids
71
remains the alleviation of oxidative damage on the biological membranes via xanthophylls cycle
(Havaux, 1998). Environmentally stressed tolerant plants, are reported to show greater Car as
compared to respective control plants, which suggest their role in the stress tolerance
(Haldimann, 1997; Wahid and Ghazanfar, 2006; Wahid, 2007). In the present research, it was
noted that tolerant maize variety (Sadaf) under glasshouse condition either showed increased,
steady state or minimal decrease in Car during both the seasons as compared to sensitive variety
(Agatti-2002), which displayed decreased Car contents in both the seasons under glasshouse
condition. However, these changes were more remarkable in the winter than summer season
sown maize plants (Fig. 15). Thus, in line with the previous information (Wahid et al., 2007),
this also substantiated a crucial and profound role of Car in the relatively adverse condition like
glasshouse, where increased temperature is a main determinant of growth.
Plant productivity is assessed on the basis of efficiency of a plant to fix CO2 and
production of photoassimilates by the leaves (source tissue) for export to various sinks for
utilization and storage (Rajcan and Tollenaar, 1999). Maize, like a number of other crop plants,
also shows great changes in CO2 fixation under suboptimal growth conditions (Tollenaar, 1989;
Sinsawat et al., 2004). In this study, the gas exchange properties of maize leaves were studied in
terms of changes in net photosynthetic rate (Pn), transpiration rate (E), water use efficiency
(A/E) stomatal conductance and substomatal CO2 concentration (Ci). Present studies revealed
that growing season and glasshouse condition had a great influence on the gas exchange
attributes of both the varieties. Leaf Pn, E and gs were lowly affected at seedling, reduced more
at silking and reduced the most at grain filling stage under glasshouse condition, whilst Agatti-
2002 showed greater sensitivity to glasshouse condition (Fig. 16, 17 and 19). WUE, derived as a
ratio of Pn and E, indicated an increase in winter grown plants than summer grown plants (Fig.
18), which is contrary to many previous reports. However, Ci indicated a decreased value in
winter than during summer (Fig. 20), which is in contrast to the earlier studies (Ranney and Peet,
1994; Morales et al., 2003). A critical perusal of data indicated that declined in the gas exchange
and CO2 fixation by the maize varieties was mainly due to reduced the conductance of CO2 by
stomata to absorb CO2 and its fixation in the Calvin cycle.
As mentioned above, both photosynthetic pigments and gas exchange parameters are
fundamental processes involved in dry matter yield. Thus, optimal operation of reactions in both
these processes is important. Studies highlighting the proportionate changes in these processes
72
are scanty (Tardy and Havaux, 1999). In this study, it was noted that the pattern of changes in
Chl b was more closely related to that those of gs and Ci. Despite the fact that both systems are
entirely different in nature and composition, these results show that parallel changes in both are
important determinants of maize growth.
In conclusion, despite differences in the growing seasons and varieties glasshouse
conditions were adverse for the photosynthetic systems in maize. Major yardsticks of sensitivity
were loss of chlorophyll and carotenoids in the light reactions, while reductions in the stomatal
conductance and substomatal CO2 concentration in dark reaction of the glasshouse grown maize.
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4.3 Leaf water, osmotic and membrane characteristics
4.3.1 Results
a. Relative water contents
As presented in Fig. 21, at seedling stage, in winter season, there was non-significant difference
in the varieties and treatments, with a non-significant interaction of both factors. However, in
summer season, the varieties and treatments differed significantly, although there was no
interaction of these factors for relative water contents (RWC). In winter season, the varieties
indicated similar RWC under control, which decreased in more in Agatti-2002 as compared to
Sadaf under glasshouse condition. In summer season, Sadaf displayed greater RWC in both the
conditions, although glasshouse condition reduced this character more in Agatti-2002.
Varieties (V) 1 82.96* 552.86** Treatments (T) 1 590.33** 758.55** V × T 1 13.12ns 97.74ns Error 12 14.83 28.91
Varieties (V) 1 71.61* 390.26** Treatments (T) 1 327.34** 225.30** V × T 1 34.69ns 13.65ns Error 12 8.16 15.94
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 21: Changes in leaf relative water contents of control and glasshouse grown maize varieties during winter and summer seasons at seedling and silking stages
74
At silking stage, in both the summer seasons, results revealed significant difference in the
varieties and treatments but there was no significant interaction of both factors for RWC. RWC
was not much different in both the varieties under control condition in both the seasons.
However, glasshouse effect was evident on both the varieties indicated reduction in RWC,
although Agatti-2002 was affected more than Sadaf in both the seasons (Fig. 21).
At grain filling stage too, during both the seasons, data revealed significant difference in
the varieties and treatments with a non-significant interaction of these factors for RWC. At this
stage, RWC was slightly higher in Agatti-2002 than Sadaf in both the seasons. However,
glasshouse condition was almost equally adverse for this attribute in both the varieties (Fig. 21).
b. Leaf water potential
At seedling stage, statistical analysis of data showed no significant difference in the varieties but
a significant difference in the treatments, together with a significant interaction of both the
factors. Contrarily, in summer season the varieties and treatments differed significantly but there
was no interaction of varieties and treatments for leaf water potential. In winter season, the leaf
water potential was similar in Sadaf under both the conditions, but increased under glasshouse
condition in Agatti-2002. Contrarily, in summer season, leaf water potential was increased in
both the varieties in a similar manner under glasshouse condition. Winter grown plants had more
negative leaf water potential than those grown in summer season (Fig. 22).
At silking stage, analysis of data showed no significant difference in the varieties and the
treatments with a non-significant interaction of both the factors during winter season. However,
in summer season, a non-significant difference in the varieties but a significant one in the
treatments was noted, but there was no significant interaction of these factors. In winter season,
the leaf water potential was almost similar in Sadaf and Agatti-2002 under both the conditions.
Contrarily, in summer season, leaf water potential increased in both the varieties in a similar
manner under glasshouse condition as that in the control. Summer grown plants had more
negative leaf water potential than those grown in winter season (Fig. 22).
At grain filling stage, results revealed no significant difference in the varieties and
treatments, and a non-significant interaction of both these factors in both the seasons. In winter
season under glasshouse condition, Sadaf indicated an increase while Agatti-2002 indicated a
decrease in this character under glasshouse condition. However, in summer season Sadaf
75
indicated a decrease while Agatti-2002 an increase in this attribute under glasshouse condition
(Fig. 22).
c. Leaf osmotic potential
At seedling stage, analysis of data showed significant difference in the varieties, treatments
together with more significant interaction of both the factors in winter season. However, in
summer season, the difference in varieties and treatments and their interaction was non-
significant. In winter season, the leaf osmotic potential was similar in Sadaf under both the
conditions, but increased under heat stress in Agatti-2002. Contrarily, in summer season, leaf
osmotic potential decreased in Agatti-2002 but Sadaf showed similar trend in both conditions.
Winter grown plants had more negative leaf osmotic potential than those grown in summer
Varieties (V) 1 0.000ns 0.003ns Treatments (T) 1 0.001ns 0.200** V × T 1 0.000ns 0.005ns Error 12 0.004 0.002
Varieties (V) 1 0.000ns 0.000ns Treatments (T) 1 0.005ns 0.005ns V × T 1 0.016ns 0.016ns Error 12 0.051 0.004
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 22: Changes in leaf water potential of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
76
At silking stage, in both the seasons, data showed non-significant difference in the
varieties, treatments together with non-significant interaction of both these factors. In winter
season, the leaf osmotic potential was better in Sadaf than Agatti-2002 under heat stress
condition. In summer season, leaf osmotic potential decreased a little in both the varieties under
heat stress than control condition (Fig. 23).
At grain filling stage, data revealed non-significant difference in the varieties, but a
significant one in the treatments, although there was no interaction of these factors in the winter
season. However, in summer season there was no significant difference in varieties and
treatments, and no interaction of these factors was evident. In winter season plants, leaf osmotic
potential decreased almost equally both in Sadaf and Agatti-2002 under glasshouse condition.
However, in summer season, under glasshouse condition, Sadaf indicated a decrease while
Agatti-2002 a small increase in this parameter (Fig. 23).
Varieties (V) 1 0.0038ns 0.0012ns Treatments (T) 1 0.0078ns 0.0042ns V × T 1 0.0153ns 0.0001ns Error 12 0.0312 0.0076
Varieties (V) 1 0.0100ns 0.007ns Treatments (T) 1 0.042* 0.010ns V × T 1 0.002ns 0.026ns Error 12 0.005 0.008
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 23: Changes in leaf osmotic potential of control and glasshouse grown maize varieties during winter and summer seasons at seedling and silking stages
77
d. Leaf turgor potential
At seedling stage, data analysis revealed significant differed in the varieties but non-significant
difference in the treatments, with no interaction of both factors in winter season. In summer
season, on the other hand, the varieties differed non-significantly while the treatments indicated
significant difference with a significant interaction of varieties and treatments. In both winter and
summer, although turgor potential was reduced in both the varieties under glasshouse condition,
Agatti-2002 was affected lesser than Sadaf in winter season but more than Sadaf in summer
season. Growing season had great effect on this attribute in both the varieties (Fig. 24).
At silking stage, statistical analysis of data indicated non-significant difference in the
varieties and treatments with no interaction of both these factors in winter season. However, in
summer season, a non significant difference was noted in the varieties but a significant
Varieties (V) 1 0.001ns 0.008ns Treatments (T) 1 0.015ns 0.262** V × T 1 0.017ns 0.006ns Error 12 0.045 0.009
Varieties (V) 1 0.000ns 0.004ns Treatments (T) 1 0.039** 0.030** V × T 1 0.000ns 0.001ns Error 12 0.001 0.002
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 24: Changes in leaf turgor potential of control and glasshouse grown maize varieties during winter and summer seasons at seedling and silking stages
78
difference was evident in the treatments, with non-significant interaction of both these factors.
During winter season, glasshouse condition did not influence this attribute in Sadaf but decreased
in Agatti-2002. In winter season, leaf turgor potential was not affected in Sadaf but reduced in
Agatti-2002. In summer, both the varieties showed a reduction in this attribute (Fig. 24).
At grain filling stage, data revealed non-significant difference in the varieties, but a
significant one in treatments, while there was no interaction of both these factors during summer
and winter seasons. In winter and summer season plants, leaf turgor potential decreased equally
in Sadaf and Agatti-2002 under glasshouse condition (Fig. 24).
e. Hydrogen peroxide concentration
At seedling stage, results revealed significant difference in the varieties and treatments but there
was no interaction of both factors in winter season. However, in summer season, the varieties
differed non-significantly, while the treatments differed significantly, while there was no
interaction of varieties and treatments for this parameter. In winter season, although H2O2
concentration was increased in both the varieties in glasshouse grown plants, Agatti-2002
produced substantially higher H2O2. However, in summer season, the increase in H2O2
concentration in glasshouse grown plants was smaller than winter season plants (Fig. 25).
At silking stage, analysis of data revealed significant difference in the varieties and
treatments with a significant interaction of both the factors in winter season. However, in
summer season, the varieties differed non-significantly, treatments significantly, and there was
no interaction of varieties and treatments for this attributes. In winter season, H2O2 concentration
was greater in Agatti-2002 than Sadaf in glasshouse condition. However, in summer season, both
the varieties showed an increased H2O2 synthesis in both the growth conditions, although,
glasshouse condition was effective in increasing its concentration in both the varieties (Fig. 25).
At grain filling stage, statistical analysis of data revealed significant difference in the
varieties and treatments but there was no interaction of both factors in winter as well as summer
seasons. In winter and summer seasons, Sadaf manifested a lower H2O2 production in both
control and glasshouse grown plants as compared to Agatti-2002. Growing season had a
significant effect on this attribute in both the varieties (Fig. 25).
79
f. MDA concentration
At seedling stage, results showed significant difference in the varieties and treatments with a
significant interaction of both these factors during winter season. However, during summer
season, the varieties and treatments differed significantly while there was no interaction of these
factors for malondialdehyde (MDA) accumulation. Maize plants in both the seasons under
glasshouse condition, indicated an accumulation of MDA, but this tendency was much greater in
the winter season plants. More importantly, Agatti-2002 showed greater MDA accumulation
than Sadaf in winter under glasshouse condition (Fig. 26).
Varieties (V) 1 118.58** 14.84ns Treatments (T) 1 328.25** 36.79** V × T 1 71.65** 4.92ns Error 12 1.84 3.92
Varieties (V) 1 112.29** 328.23** Treatments (T) 1 61.68* 15.73* V × T 1 24.73ns 0.47ns Error 12 7.69 3.14
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 25: Changes in hydrogen peroxide concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
80
At silking stage, data showed significant difference in the varieties and treatments
together with a significant interaction of both the factors during both winter and summer seasons.
In winter season, Sadaf did not show the accumulation of MDA, which increased remarkably in
Agatti-2002 under glasshouse condition. Contrarily, in summer season, MDA increased in both
the varieties, but Agatti-2002 indicated markedly greater MDA accumulation than Sadaf under
glasshouse condition (Fig. 26).
At grain filling stage, statistical treatment of data showed non-significant difference in
the varieties, but a significant difference in the treatments together with a significant interaction
of both the factors in winter season. However, in summer season, the varieties and treatments
differed significantly while there was no interaction of varieties and treatments for MDA. In
Varieties (V) 1 101.46** 48.77** Treatments (T) 1 54.45** 215.90** V × T 1 62.33** 15.87* Error 12 1.84 1.82
Varieties (V) 1 17.45ns 32.60** Treatments (T) 1 178.35** 65.04** V × T 1 45.24* 0.55ns Error 12 6.11 1.56
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 26: Changes in MDA concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
81
winter season, both the varieties accumulated greater MDA than in the summer season. The
prevailing glasshouse condition although increased MDA in both the varieties, but this increase
was greater in Agatti-2002. In summer season too, MDA increased in both the varieties, but was
greater in Agatti-2002 (Fig. 26).
g. Relative membrane permeability
As presented in Fig. 27, at seedling stage, in winter season, statistical analysis of data showed
significant difference in the varieties and treatments, but there was no interaction of both the
factors. However, in summer season, varieties and treatments showed significant difference
Varieties (V) 1 428.38** 7.84ns Treatments (T) 1 1783.80** 1065.79** V × T 1 210.41** 44.90* Error 12 6.18 6.10
Varieties (V) 1 32.51** 37.22ns Treatments (T) 1 184.21** 424.44** V × T 1 16.03* 32.093ns Error 12 2.25 13.138
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 27: Changes in relative membrane permeability (RMP) of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
82
together with a significant interaction of both these factors for relative membrane permeability
(RMP). In winter season, the RMP increased in Sadaf as well as in Agatti-2002 under glasshouse
condition, but Agatti-2002 showed higher RMP. Contrarily, in summer season, RMP was
nominally increased in Sadaf but greatly in Agatti-2002 under glasshouse condition (Fig. 27).
At silking stage, analysis of data showed significant differences in the varieties and
treatments with a significant interaction of both the factors in the winter season. However, in
summer season, the varieties differed non-significantly, but the treatments differed significantly
and there was a significant interaction of varieties and treatments for RMP. In both winter and
summer season, the RMP was increased both in Sadaf and in Agatti-2002 under glasshouse
condition, but a substantial increase was observed in the latter variety (Fig. 27).
At grain filling stage, in winter season, data showed significant difference in the varieties
and treatments with a significant interaction of both these factors in the winter season. However,
in summer season, the varieties differed non-significantly, while treatment differed significantly,
although there was non-significant interaction of varieties and treatments for RMP. Winter
grown plants, irrespective of the growth condition, indicated reduced RMP than summer grown
plants. In winter and summer seasons, the RMP was increased in both Sadaf and Agatti-2002
under glasshouse condition. Nonetheless, Agatti-2002 showed relatively higher RMP than Sadaf
in both the seasons (Fig. 27).
h. Leaf free proline
At seedling stage, statistical analysis of results revealed a significant difference in the varieties
and treatments and there was a significant interaction of both factors in winter season. However,
in summer season, the varieties differed significantly, treatments non-significantly, while there
was no interaction of varieties and treatments for this parameter. Leaf free proline accumulation
was greater in winter than summer season, irrespective of the growth condition. Nonetheless, in
winter season, leaf free proline was similar in both the varieties under control but increased more
in Sadaf under glasshouse condition. In summer season, free proline increased in Sadaf but
decreased in Agatti-2002 under glasshouse condition (Fig. 28).
At silking stage, data revealed non-significant difference in the varieties but a significant
difference in the treatments, and there was significant interaction of both factors in winter
season. However, in summer season, the varieties differed significantly while no significant
83
difference in the treatments, as well as there was no interaction of varieties and treatments for
free proline. In winter season, free proline increased in Sadaf, but decreased in Agatti-2002
under glasshouse condition. In summer season, Sadaf showed a substantial increase in free
proline while Agatti-2002 remained at par with its control value (Fig. 28).
At grain filling stage, analysis of data revealed non-significant difference in the varieties,
a significant difference in the treatments, while there was no interaction of both the factors in
winter season. However, in summer season, the varieties differed significantly while no
significant difference was observed in treatments, but there was significant interaction of
varieties and treatments. In winter season, under glasshouse condition although free proline
increased in both the varieties, Agatti-2002 indicated a greater increase than Sadaf. Contrarily in
Varieties (V) 1 3.25ns 233.71* Treatments (T) 1 5.90* 104.16ns V × T 1 33.42** 106.84ns Error 12 1.12 50.34
Varieties (V) 1 74.64ns 20.62* Treatments (T) 1 345.38** 1.99ns V × T 1 61.22ns 68.64** Error 12 25.27 2.25
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 28: Changes in free proline concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
84
summer season, glasshouse condition increased leaf free proline in Sadaf, while reduced it in
Agatti-2002. Growing season had a great effect on this attribute in both the varieties (Fig. 28).
i. Total free amino acids
At seedling stage, in both winter and summer seasons, data showed significant difference in the
varieties and treatments, but with a non-significant interaction of both the factors. In both winter
and summer seasons, the amount of free amino acids accumulation was almost similar in both
the varities, although glasshouse condition caused a substantial increase in their levels in both
Varieties (V) 1 1347.73** 245.93** Treatments (T) 1 4492.15** 4290.25** V × T 1 9.43ns 83.46ns Error 12 22.05 31.86
Varieties (V) 1 415.60** 219.31* Treatments (T) 1 1271.57** 1863.88** V × T 1 497.09** 478.42** Error 12 10.81 41.53
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 29: Changes in total free amino acids of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
85
At silking stage, during winter and summer seasons, data showed significant difference in
the varieties and treatments, while there was non-significant interaction of both the factors for
total free amino acids. In winter season, the amount of total free amino acids was lesser in both
the varieties than summer season plants. However, glasshouse condition produced a great
increase in their levels in both the seasons, although this accumulation was greater in Sadaf (Fig.
29).
At grain filling stage, statistical analysis of results showed significant difference in the
varieties, treatments and there was significant interaction of these factors in both winter and
summer season. In both the seasons, free amino acids accumulation was similar in both varieties
under control condition. However, glasshouse condition increased them in both the varieties, but
this increase was substantially greater in Sadaf in both the seasons (Fig. 29).
4.3.2 Discussion
An important consequence of high temperature stress is the evapo-transpirative loss of water
from plant surface, resulting in the dehydration and consequently hampering the cell water status
(Machado and Paulsen, 2001; Mazorra et al., 2002). Under dehydration stress, plants show quite
a lot of metabolic changes; the accumulation of compatible solutes for osmotic adjustment are
the most important one (Zhu, 2003; Farooq et al., 2009). Excessive dehydration from the leaf
surface, due to prevailing high temperature or drought conditions, leads to the disruption of cell
membranes by peroxidation and solublization of membrane lipids (Wen-yue et al., 2001; Jiang
and Huang, 2001; Iba, 2002). Results of these experiments on maize showed that the glasshouse
condition reduced leaf RWC in both the seasons, although varietal difference was evident (Fig.
21). This reduction led to the altered leaf water potential (Fig. 22), osmotic potential (Fig. 23)
and reduced turgor potential (Fig. 24). This revealed that glasshouse conditions were greatly
effective in modulating the plant water relations.
It is important to note that both the varieties behaved differentially in glasshouse
conditions in both the seasons. This appeared to be related to the prevailing environmental
conditions of temperature and humidity in the glass canopies (Fig. 2). In winter grown plants
(sown in February), at the time of harvesting at seedling, silking and grain filling stages, the
temperature sufficiently increased and relative humidity decreased to substantially affect the leaf
86
water status. However, in summer season (sown in August) crop, although the temperature was
rather higher at the sowing time, at the aforesaid critical stages, the temperature inside the
canopy was reduced and relative humidity increased (Fig. 2), which did not influence much the
leaf water relations.
In a number of plant species, there has been an enhanced production of H2O2, which is a
strong reactive oxygen species (Wahid et al., 2007). Although there is the production of H2O2 in
the normally functioning cells (Taiz and Zeiger, 2006), its production beyond limits, particularly
under stress effects leads to the peroxidation of membrane lipids, and as a result enhanced
production of thiobarbituric acid reactive substances (TBARS) is observed; predominant
amongst those is MDA (Wahid et al., 2007). As a result of these changes, there is the loss of
integrity of cellular membranes and enhanced ion-leakage is observed (Yang et al., 1996). In this
study, it was noted that in both the varieties there was a high production of H2O2 under
glasshouse condition in winter grown plants (Fig. 25), which also exhibited the production of
MDA (Fig. 26). These changes consequently led the membranes to become permeable, as
evident from the ion-leakage measured in terms of relative membrane permeability (Fig. 27). All
these changes paralleled well in winter and summer season crops with the prevailing
environmental conditions. Thus, it is convincing that glasshouse conditions during warmer
months of the years are more damaging to the plant functions than in relatively cool months.
Plants exposed to dehydration or osmotic stress conditions show the accumulation of
compatible solutes. The accumulation of free proline and a number of other amino acids has been
shown to play roles in the osmotic adjustment and stabilization of membrane structures under
stressful conditions (Taiz and Zeiger, 2006; Wahid et al., 2007; Ashraf and Foolad, 2007). In
view of the importance of amino acids to the growth of maize (a C4 plant), the determinations
were carried out for free proline alone (Fig. 28) and total free amino acids (Fig. 29). The results
revealed that under glasshouse condition, tolerant maize variety (Sadaf) accumulated a higher
free proline in higher amounts, while there was a general tendency of both the varieties to
accumulate free amino acids under glasshouse (high temperature) condition. A critical view of
the data revealed that in some cases, free proline accumulation constituted 25-40% of the total
free amino acids accumulated, which further substantiates the role of free proline accumulation,
as reported in a number of heat stressed plants (Chiang and Dandekar, 1995; Wahid, 2007;
87
Wahid et al., 2007; Verbruggen and Hermans, 2008). Thus, the accumulation of free proline can
be taken as a reliable criterion of tolerance to glasshouse conditions.
In conclusion, prevailing glasshouse conditions particularly were greatly effective in
hampering the leaf water relation particularly those of winter sown crop. The glasshouse
conditions in winter crop produced oxidative stress on the plants, which was evident from the
increased synthesis of H2O2, MDA and increased permeability to the ion leakage. Greater free
proline accumulation in the tolerant variety not only presented itself as a major amino acid
accumulated in environmental stress tolerance but also indicated it as a reliable criterion of
tolerance to glasshouse condition in maize.
88
4.4 Nutritional relationships
4.4.1 Results
a. Shoot K contents
At seedling stage, in both the seasons, statistical analysis of data revealed significant differences
in the varieties and treatments but there was no significant interaction of both factors for shoot K.
In both the seasons varieties exhibited differential response to accumulation of K in the shoot
tissues. In glasshouse condition during winter season, Sadaf indicated a greater K accumulation
while in winter season Agatti-2002 excelled Sadaf in accumulating this ion (Fig. 30).
Fig. 30: Changes in shoot potassium concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
89
At silking stage, analysis of results revealed non-significant difference among the
varieties and treatments in both winter and summer season, while there was no significant
interaction of these factors in winter but a significant interaction in summer season. In both
winter and summer season, shoot K was increased in Sadaf and decreased in Agatti-2002 under
glasshouse condition (Fig. 30).
At grain filling stage, statistical analysis of data revealed non-significant difference in the
varieties, while significant difference in the treatments and a significant interaction of both
factors was seen in winter season. However, in summer season, the varieties and treatments
differed non-significantly, but there was significant interaction of varieties and treatments for
shoot K. In winter season, although shoot K was decreased in both varieties under glasshouse
condition, Agatti-2002 underwent a greater reduction. Contrarily, in summer season shoot K
increased in Sadaf and decreased in Agatti-2002 under glasshouse condition (Fig. 30).
b. Shoot Ca contents
At seedling stage, statistical analysis of data revealed non-significant difference in the varieties
while a significant difference in the treatments with a non-significant interaction of both factors
in winter season. However, in summer season, the differences among the varieties, treatments
and their interaction were significant for shoot Ca. In winter season, this attribute was increased
in Sadaf and decreased in Agatti-2002 under glasshouse condition. In summer season, however,
glasshouse condition did not change shoot Ca much in Sadaf but increased under glasshouse
condition (Fig. 31).
At silking stage, analysis of data revealed non-significant difference in the varieties,
treatments and as well as there was no interaction of varieties and treatments for shoot Ca in
winter season. However, in summer season, the varieties differed significantly while non-
significant difference was evident in treatments and there was no interaction of varieties and
treatments for Ca. Comparison of seasons indicated that shoot Ca was higher in winter than in
summer grown plants irrespective of the growth conditions. In winter season, Ca contents
decreased more in Agatti-2002 under glasshouse condition. In summer season, glasshouse
condition increased Ca in Sadaf but did not remarkably change in Agatti-2002 (Fig. 31).
At grain filling stage, statistical analysis of data revealed non-significant difference in the
varieties, treatments, and there was no interaction of varieties and treatments in winter season.
90
However, in summer season, the varieties and treatments differed significantly, but no significant
interaction was evident in them for shoot Ca. In winter season, shoot Ca increased in Sadaf and
decreased in Agatti-2002 under glasshouse condition. However, in summer season, glasshouse
condition increased the shoot Ca more greatly in Sadaf than Agatti-2002. Growth season had an
effect on this attribute (Fig. 31).
c. Shoot Mg contents
At seedling stage, analysis of data revealed no significant difference in the varieties, a significant
difference in the treatments, but there was no interaction of both these factors in winter season.
However, in summer season, the varieties differed significantly while no difference was seen in
the treatments, and there was a significant interaction of varieties and treatments for shoot Mg.
Varieties (V) 1 4.34ns 4.75** Treatments (T) 1 3.15ns 0.81ns V × T 1 0.77ns 0.15ns Error 12 2.12 0.84
Varieties (V) 1 2.86ns 20.14* Treatments (T) 1 1.27ns 20.14* V × T 1 7.25ns 3.70ns Error 12 1.55 2.18
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 31: Changes in shoot calcium concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
91
At this stage shoot Mg contents were reduced in winter season grown plants as compared to
summer season plants. In winter season, shoot Mg decreased almost equally in Sadaf and Agatti-
2002 under glasshouse condition. In summer season, although Sadaf showed no change but
Agatti-2002 showed a great increase in shoot Mg contents under glasshouse condition (Fig. 32).
At silking stage, data revealed significant difference in the varieties and treatments and
there was significant interaction of both factors in winter season. In summer season, the varieties
and treatments differed non-significantly, with no interaction of varieties and treatments for
shoot Mg. Winter grown plants showed greater shoot Mg content than summer grown plants. In
winter season, shoot Mg decreased in both the varieties, but a greater decrease was noticeable in
Varieties (V) 1 17.41** 0.49ns Treatments (T) 1 18.38** 0.18ns V × T 1 3.49** 0.10ns Error 12 0.18 0.10
Varieties (V) 1 5.41* 4.27** Treatments (T) 1 1.08ns 7.67** V × T 1 3.84ns 0.17ns Error 12 0.99 0.30
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 32: Changes in shoot magnesium concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
92
Agatti-2002 under glasshouse condition. In summer season, Sadaf indicated no difference whilst
Agatti-2002 indicated a decrease in shoot Mg contents under glasshouse condition (Fig. 32).
At grain filling stage, data revealed significant difference in the varieties but no
difference was noted in the treatments, while there was no interaction of these factors in winter
season. In summer season, the varieties and treatments differed significantly, while there was no
interaction of both these factors. Shoot Mg was greater in winter than summer season plants. In
winter season, shoot Mg increased in Sadaf while decreased in Agatti-2002 under glasshouse
condition. In summer season, shoot Mg decreased equally in both the varieties (Fig. 32).
d. Shoot phosphate contents
As can be seen in Fig 33, at seedling stage, statistical analysis of results in both the seasons
Varieties (V) 1 3.03ns 0.35ns Treatments (T) 1 19.25* 11.68** V × T 1 0.082ns 2.73ns Error 12 2.73 1.12
Varieties (V) 1 56.32** 0.68ns Treatments (T) 1 9.20ns 18.01** V × T 1 16.89* 0.75ns Error 12 3.47 1.73
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 33: Changes in shoot soluble phosphate contents of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
93
revealed a significant difference in the varieties and treatments but there was no significant
interaction of both these factors. In winter season, shoot phosphate decreased, while in summer
season, this attribute increased in both varieties.
At silking stage, data analysis revealed non-significant difference in the varieties but a
significant difference in the treatments with a non-significant interaction of these factors in both
the seasons. In both winter and summer season, shoot phosphate increased in both varieties under
glasshouse condition, although this increase was greater in winter grown plants (Fig. 33).
At grain filling stage, data revealed significant difference in the varieties while non-
significant difference in the treatments, and there was a significant interaction of both factors in
winter season. However, in summer season, the varieties differed non-significantly, while the
treatments differed significantly, although there was no interaction of varieties and treatments for
shoot phosphate. In winter season, shoot phosphate increased remarkably in Sadaf but did not
change much in Agatti-2002 under glasshouse condition. In summer season this attribute was
increased almost equally in both the varieties (Fig. 33).
e. Shoot nitrate contents
At seedling stage, in winter season, data analysis revealed significant difference in the varieties,
while non-significant difference in the treatments and with a non-significant interaction of these
factors. However, in summer season, varieties and treatments indicated non-significant
difference, as well as no interaction of both these factors was observed for shoot nitrate. In
winter season, although glasshouse condition increased shoot nitrate in both Sadaf and Agatti-
2002, a greater increase was experienced in the latter variety. In summer season, shoot nitrate
declined in both varieties but this decline was well explicit in Sadaf (Fig. 34).
At silking stage, statistical analysis of data revealed non-significant difference in the
varieties and treatments and there was no significant interaction of both factors in winter season.
However, in summer season, the varieties differed significantly while no difference was evident
in the treatments, but there was a significant interaction of varieties and treatments for shoot
nitrate. In winter, shoot nitrate increased in Sadaf and decreased in Agatti-2002 under glasshouse
condition. In summer season, glasshouse condition decreased this parameter in Sadaf but
increased in Agatti-2002 (Fig. 34).
94
At grain filling stage, statistical analysis of data revealed non-significant difference in the
varieties and treatments, with no significant interaction of both factors in winter season.
However, in summer season the varieties and treatments differed non-significantly, whilst there
was a significant interaction of varieties and treatments for shoot nitrate. In both winter and
summer season, shoot nitrate increased in Agatti-2002 while decreased in Sadaf under
Varieties (V) 1 0.117ns 0.008** Treatments (T) 1 0.453ns 0.002ns V × T 1 0.102ns 0.007** Error 12 0.212 0.001
Varieties (V) 1 0.002ns 0.0041ns Treatments (T) 1 0.0001ns 0.0002ns V × T 1 0.001ns 0.010* Error 12 0.0009 0.0015
Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 34: Changes in shoot soluble nitrate of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages
95
4.4.2 Discussion
Among the multifarious effects of high temperature stress, changes in the mineral nutrition of the
plant have been regarded as the crucial one. It has been shown that maize, millet and many other
plants grown in heat stress environment show great changes in the mineral nutrition including K,
Ca, Mg, N and P (Ashraf and Hafeez, 2004; Wahid et al., 2007). It is important to mention that
most of these studies have been conducted in artificial hot environment. However, studies
conducted under glasshouse environment, which produce high temperature stress along with low
relative humidity are few.
In this part of the manuscript, determinations were made for the changes in major
nutrients including K, Ca, Mg, phosphate and nitrate. All these have been grouped as major
nutrient for plant growth, since they are either the structural or functional constituents (Epstein
and Bloom, 2005). Furthermore, they play multiple roles in physiological and biochemical
phenomena including osmoregulation, action as cofactors in the enzyme activities, metabolites
synthesis and signal transduction (Taiz and Zeiger, 2006). It was noted that, though not too great,
glasshouse condition across the seasons adversely affected the tissue contents of K (Fig. 30), Ca
(Fig.31), Mg (Fig. 32), phosphate (Fig. 33) and nitrate (Fig. 34) in the shoot at three growth
stages. Nonetheless, varietal difference was evident across the seasons; Sadaf appeared to
maintain greater shoot tissue nutrients than Agatti-2002 in most cases. Plants grown in the winter
season indicated more conspicuous changes than those grown in the summer. It is important to
note further that shoot tissue contents of K, Ca and, to some extent, nitrate indicated more clear
trends of changes across the seasons, which suggested a greater role of these nutrients in the
tolerance to glasshouse condition.
As mentioned previously, growth conditions of temperature and relative humidity inside
the glasshouse are more adverse in the summer instead of winter months. In summer months, the
temperature inside the glass canopy was 5-7oC higher while relative humidity was 5-10% lower
than the outside environment (Fig. 2). Heat stress acts as a dehydrative force, and change of
ambient temperature by 1oC can produce considerable changes in the metabolic phenomena
(IPCC, 2007). The root acquisition and shoot transport of the mineral nutrient is greatly
dependent on the prevailing climatic condition, mainly related to evapo-transpirational load on
the plant leaves. In case of glasshouse, where both the temperature and relative humidity are
96
great modulating factors, great changes in the mineral nutrient concentrations in the shoot are
anticipated. These results therefore confirm the assertion that glasshouse conditions are great
growth modulating factors, and plant growth under such conditions is also affected, at least in
part, by the changes in the tissue nutrient concentrations.
In crux, with great varietal difference, changes of temperature and relative humidity
inside the glasshouse across the seasons were mainly responsible for the observed changes in
mineral nutrients. More distinctly, changes in K, Ca and nitrate nutrition were given greater
credence in view of their closer association to the seasonal changes in the environmental
conditions inside the glasshouse.
97
STUDIES AT SENDAI, JAPAN
The studies at Sendai, Japan were carried out to determine the patterns of gene expression in
maize. Although maize is a C4 plant species, it shows reduced growth at supra-optimal
temperatures. The changes in temperature lead to the altered expression of gene. In view of this,
studies were initiated at the Tohoku University, Sendai Japan to determine the changes in the
expression of heat shock protein 70 (hsp70), dehydrin2 (dhn2), stay green gene (sgr2) and
senescence associated gene (sag) in maize at seedling stage in a time course manner (at 1, 3, 6,
254, 48 and 72 h time interval). Results recorded for the time course changes in the growth
attributes and expression of the abovementioned genes are explained below.
98
4.5 Growth and gene expression in maize under heat stress
4.5.1 Results
a. Plant growth attributes
For shoot growth, data indicated no significant difference in the temperature treatments, but a
significant one in the time points with a significant interaction of both these factors. For shoot
fresh and dry weight, temperature treatments and time points indicated significant difference
while there was no interaction of both these factors, while for shoot fresh-to-dry weight ratio
only temperature treatments indicated significant difference (Table 5). As compared to control,
shoot length in the high temperature treated plants indicated a decrease at 1 h time point, was
equal at 3, 6 and 24 h but increased at 48 and 72 h time points. Shoot fresh and dry weight, and
shoot fresh-to-dry weight ratio indicated a consistent decline as compared to controls, although
fresh weight was more sharply decreased at all time points (Fig. 35).
For root growth, data indicated no significant difference in the temperature treatments,
but a significant one in the time points with a non-significant interaction of both these factors.
For shoot fresh and dry weight and shoot fresh-to-dry weight ratio, temperature treatments and
Table 5: Analysis of variance (mean squares) of some growth and expression analysis attributes of maize under control and high temperature stress (42oC) in a time course manner Parameters Temperature
and dehydrin gene 2 (dhn2). A high level expression of the transcriptional levels all these genes
at 1 h after exposure to high temperature stress (Fig. 36) indicated the possibility of elicitation of
Fig. 36: Gene expression in maize under heat stress: Agarose gel showing the molecular weight of the senescence associated gene (sag), stay green gene (sgr2), heat shock protein 70 (hsp70) gene and dehydrin 2 (dhn2) gene. Bar charts show the transcriptional levels of sag, sgr2, hsp70 and dhn2 genes, respectively studies in a time course manner
Elec
troph
retic
exp
ress
ion
of g
enes
b b b b b b
a
b bb
ab
b
0
2
4
6
8
10
12
1 3 6 24 48 72
sag
Time (h)
ControlHigh temperaturePoly. (High temperature)
b b b b b b
a
abb b
bb
02468
101214
1 3 6 24 48 72
srg2
Time (h)
b b b b b b
aab
b b b b
-505
1015202530
1 3 6 24 48 72
hsp7
0
Time (h)
ab ab ab ab ab ab
a
bb b
ab ab
0.0
0.5
1.0
1.5
2.0
2.5
dhn2
Tran
scrip
tion
leve
ls o
f gen
es (r
elat
ive
ratio
to re
spec
tive
cont
rols
)
102
stress response triggered by certain signals under heat stress (Bohnert et al., 2006; Wahid et al.,
2007). In view of the fact that heat stress has multiple effects on the plant growth and
development, the expression of sag and dhn2 is a direct high temperature stress response. Heat
stress, among the others, has two major effects; senescence and dehydration of living tissues (Liu
and Huang, 2000; Spano et al., 2003). However, induction of sgr2 a stay green response gene
(Harris et al., 2007) and hsp70 (Wahid et al., 2007) appear to be a protective strategy under high
temperature. It is important to notice that both sag and dhn2 again showed an increase in the
transcriptional level, while sgr2 and hsp70 failed to accomplish this tendency. This was further
evident from a negative correlation of transcriptional level of hsp70 with shoot length (r = -
0.871; P<0.05), root length (r = -0.833; P<0.05) and shoot dry weight (r = -0.893; P<0.05) and
that of sgr2 with shoot length (r = -0.919; P<0.01). This revealed that sensitivity of maize to high
temperature stress can be, at least, partly assigned to failure to express hsp70 and sgr2 under
prolonged high temperature spell.
Studies show the coexpression of various genes, as stronger response to prevailing stress
conditions (Weston et al., 2008). The expression of four genes in this study was more or less
similar, which indicated the possibility of their coexpression. To substantiate this possibility,
regression analysis and correlation coefficient of all the genes was carried out. It was noted that
dhn2 was strongly regressed with sag, while hsp70 and sag were only weakly regressed with
sgr2 gene (Fig. 37). This finding further confirmed the assertion that both dhn2 and sag were
coexpressed to display the high temperature sensitivity response in maize.
y = 9.26x - 1.23r = 0.533ns
05
10152025
0.0 1.0 2.0
hsp7
0
dhn2
y = 4.50x - 0.84r = 0.934**
02468
0.0 1.0 2.0
sag
dhn2
y = 4.30x - 0.99r = 0.776ns
02468
10
0.0 1.0 2.0
srg2
dhn2
y = 0.15x + 2.01r = 0.533ns
02468
0 20 40
sag
hsp70
y = 0.28x + 0.81r = 0.874*
02468
10
0 10 20 30
sgr2
hsp70
y = 0.94x - 0.15r = 0.820*
02468
10
0 5 10
srg2
sag Fig. 37: Trendlines and interrelationships of transcriptional levels of four genes under high temperature stress in maize at seedling stage (Significant at **, P<0.01; *, P<0.05 and ns, non-significant)
103
In summary, maize seedlings showed sensitivity to high temperature stress, which was
evident in terms of morphological (reduction in shoot fresh weight, dry weight of shoot and root
and a reduction in fresh-to-dry weight ratio) and molecular responses. The molecular studies
suggested that the maize sensitivity to high temperature was mainly due to enhanced
coexpression of sag and dhn2 and failure to express hsp70 and sgr2 during relatively long term
exposure to heat stress.
104
GENERAL DISCUSSION
Stress has the potential to produce injury, which occurs as a result of aberrant metabolism and
leading to reduction in growth, yield or even death of the plant or plant parts. Plants undergo a
depression in visual growth and development, but the extent of reduction depends greatly upon
the type of stress, its severity and duration (Zhu, 2003; Taiz and Zeiger, 2006). Greater dry
weight results from the available photosynthetic area together with enhanced capacity of leaves
to photosynthesize (Karim et al., 2000; Huve et al., 2006; Suárez and Medina, 2006). The
observed changes in the maize varieties across the seasons in growth attribute in this study
indicate the possibility of greater effects of heat in restricting the photosynthetic capacity of
maize in dry matter production at various growth stages.
In this study, the determination made at three phenological stages (seedling, silking and
grain filling) showed differential behavior of both the varieties at all growth stages under
glasshouse condition. Appearance/disappearance of quite a few interactions of the parameters
indicated the influences of seasonal changes on these attributes. In this respect, silking stage was
the most important, where most of the interaction appearing during winter season disappeared
during summer. Silking stage is more critical for final plant productivity because at this
particular stage, number of changes including success of fertilization, seed set and grain filling
follow the reception of pollen by the silk (Le Deunff et al., 1993), which ultimately determine
the final plant productivity. These data substantiated that the effect of glasshouse to be major
determinant of changes in various attributes.
It has been established that both light and dark reactions of photosynthesis are prone to
adversaries of environmental stresses (Wahid and Rasul, 2005). Photosynthetic pigments have a
considerable significant, as any reduction/loss in their content is likely to substantially influence
the dry matter production (Wahid, 2007). Likewise, enhanced gaseous exchange capacity of
plants is a real determinant of productivity under stressful conditions (Lawlor and Cornic, 2002).
Among the various gas exchange parameters, net rate of photosynthesis, transpiration rate,
photosynthetic water use efficiency, stomatal conductance and substomatal CO2 concentration
are of great significance in the exhibition of dry matter and grain yields under stress (Morales et
al., 2003; Omami and Hammes 2006). In the present study, although both varieties showed a
reduction in chlorophyll, a, b and their total, and carotenoids, Sadaf showed either maintained or
105
increased amounts of chlorophyll and carotenoids content under glasshouse conditions as
compared to Agatti-2002. Among the gas exchange attributes, net rate of photosynthesis,
transpiration rate and substomatal CO2 concentrations were of greater importance in tolerance to
glasshouse condition. Tardy and Havaux (1999) showed that chlorophyll loss in Syrian barley
had no relationship with loss of the photosynthetic activity of leaves. From this study it emerges
that tolerance to glasshouse conditions is strongly associated with increased content of
photosynthetic pigments. Increased content of carotenoids is of greater significance, since they
have dual role in the plant leaves; light harvesting and stabilization of photosynthetic membranes
(Havaux, 1998; Rmiki et al., 1999; Wahid et al., 2007).
Leaf water relations are hampered under any type of stress condition, which has a great
influence on the dry matter production by the plants (Machado and Paulsen, 2001; Wahid and
Close, 2007). In this research, both varieties behaved differentially to glasshouse conditions
across the seasons, mainly because of prevailing environmental conditions of temperature and
humidity in the glass canopies. The glasshouse conditions greatly hampered the leaf water,
osmotic and turgor potentials. Leaf osmotic potential on the other hand was much more reduced
under glasshouse condition, which displayed remarkable changes in the leaf turgor. Response of
Sadaf under glasshouse condition was better as revealed from higher RWC and maintenance of
greater turgor under glasshouse condition.
Apart from other effects, there is a consensus over one common facet of heat stress
regarding the loss of membrane integrity making them more permeable due to peroxidation of
lipid component, reduced production of antioxidants and a concomitant enhanced production of
reactive oxygen species (Blum and Ebercon, 1981; Yang et al., 1996; Hernandez et al., 2000;
Rizhsky et al., 2002; Rahman et al., 2004; Rachel and Dolan, 2006). Amongst various reactive
oxygen species (ROS), hydrogen peroxide (H2O2) is the most stable and long-lived molecule,
which damages the cellular membranes (Dat et al., 2000; Uchida et al., 2002; Steven et al.,
2003). Enhanced membrane stability and reduced generation of ROS are taken as important
criteria of stress tolerance (Wahid et al., 2007). In the present investigation although varietal
differences were noted for increased relative membrane permeability, RMP (a measure of
membrane stability) and H2O2 content, the varieties with reduced RMP and H2O2 content
displayed better tolerance to the prevailing glasshouse condition. This revealed that stress
tolerance in maize is associated to the improved stabilization of membranes in addition to other
106
factors. It is further pointed out that accumulation of free proline, as noted here comprised about
30-40% of the total free amino acids. This accumulation appeared to play pivotal role in
producing tolerance of winter crop to glasshouse condition. The roles played by free proline
accumulation appear to be related to the osmotic adjustments and stabilization of membrane
structure (Wahid et al., 2007; Verbruggen and Hermans, 2008).
Plant mineral nutrition is another important aspect of plant growth and development
which is greatly influenced by the prevailing conditions, especially those of temperature and
relative humidity (Epstein and Bloom, 2005; Farooq et al., 2009). Many studies conducted under
artificial heat stress showed great changes in the mineral nutrient contents in different plants
(Epstein and Bloom, 2005; Ashraf and Hafeez, 2004; Wahid et al., 2007). However, studies
reporting the changes in plant mineral nutrition under glasshouse conditions are scarce. Here
experiments conducted on five major nutrients (K, Ca, Mg, phosphate and nitrate) showed that,
though not too great, glasshouse condition across the seasons adversely affected the tissue
contents of all these nutrients in the shoot at three growth stages. However, varietal difference
was evident across the seasons; Sadaf maintained greater shoot tissue nutrients than Agatti-2002
in most instances. Winter grown plants indicated more conspicuous changes than those in grown
the summer. Among the nutrients, K, Ca and nitrate indicated more clear trends of changes
across the seasons, suggesting their greater role in the tolerance to glasshouse conditions.
It has been established that stress tolerance is a multigenic phenomenon, involving the
up- or down-regulation of a number of genes (Bohnert et al., 2006; Walia et al., 2006;
Tomassami et al., 2008). This study reporting the short term (hourly) response of maize
indicated differential growth responses of shoot and root to high temperature, primarily related to
the evapo-transpirational loss of water (Wahid et al., 2007). These quantitative changes are the
outcome of modulated activities of various genes. Thus the determinations were made on the
time course changes in the transcriptional levels of four stress-related genes; senescence
associated gene (sag), stay green2 gene (sgr2), heat shock protein gene 70 (hsp70) and dehydrin
gene 2 (dhn2). A high level expression of the transcriptional levels of all these genes at 1 h after
exposure to high temperature stress indicated the possibility of elicitation of stress response
triggered by certain signals under heat stress (Bohnert et al., 2006; Wahid et al., 2007). Among
these genes, the coexpression of sag and dhn2 appears to be a direct high temperature stress
107
response, since senescence and dehydration of living tissues are the direct consequences of heat
stress response (Liu and Huang, 2000; Spano et al., 2003).
In essence, maize is generally sensitive to environmental stresses and these affects are
evident on the photosynthetic pigments, gas exchange and water relations. With great varietal
difference, changes of temperature and relative humidity inside the glasshouse across the seasons
were mainly responsible for the observed changes in mineral nutrients. More distinctly, changes
in K, Ca and nitrate nutrition were given greater credence in view of their closer association to
the seasonal changes in the environmental conditions inside the glasshouse. At molecular level,
coexpression of dhn2 and sag determined the heat sensitivity response in maize. Future research
aimed at large scale gene expression and crop modeling for growing maize in glasshouse
conditions are likely to increase our understanding for growing maize in the upcoming climatic
changes.
108
FUTURE PROSPECTS
It is well accepted that greenhouse effect is changing the crop growth patterns throughout the
globe. Thus is a firm need to carry out concerted efforts to get requisite crop productivity from
the available resources under changing climatic conditions. These studies provided baseline
information on the growth and physiological responses of maize, a C4 plant species, to
glasshouse conditions. Further studies on the metabolic changes and gene expression patterns of
the glasshouse grown plants at various critical growth stages and modeling the crop productivity
on would be imperative.
109
CHAPTER-5 SUMMARY
Environmental stresses are serious threats to agricultural productivity around the globe. The
increasing menace of environmental stresses necessitates finding strategies to cope with their
ever-increasing adversaries. In view of the changing climatic conditions mainly related to
greenhouse effect, this study was focused on determining the responses of two differentially heat
tolerant maize varieties to glasshouse condition. The parameters studied included growth, water
relations, gas exchange, photosynthetic pigments, oxidative damage and gene expression.
Results revealed that changes in ambient temperature produce an array of changes in the
growth and yield of maize, and the prevailing glasshouse conditions play a crucial role in this
regard across winter and summer seasons. Investigations on the physiological and biochemical
basis of these changes (as reported in the next sections) will improve our understanding of the
underlying phenomena. Despite differences in the growing seasons and varieties glasshouse
conditions were adverse effects on the photosynthetic systems in maize. Major indicators of
sensitivity were loss of chlorophylls and carotenoids in the light reactions, while reductions in
the net photosynthesis and stomatal conductance in the dark reaction of the glasshouse grown
maize leaves. Prevailing glasshouse conditions particularly were greatly effective in hampering
the leaf water relation particularly those of winter sown crop. The glasshouse conditions in
winter crop produced oxidative stress on the plants, which was evident from the increased
synthesis of H2O2, MDA and increased permeability to the ion leakage. Greater free proline
accumulation in the tolerant variety not only presented itself as a major amino acid accumulated
in environmental stress tolerance but also indicated it as a reliable criterion of tolerance to
glasshouse condition in maize. With great varietal difference, changes of temperature and
relative humidity inside the glasshouse across the seasons were mainly responsible for the
observed changes in mineral nutrients. More distinctly, changes in K, Ca and nitrate nutrition
were given greater credence in view of their closer association to the seasonal changes in the
environmental conditions inside the glasshouse. Maize seedlings showed sensitivity to high
temperature stress, which was evident in terms of reduction in fresh and dry weight of shoot and
root and a reduction in fresh-to-dry weight ratio, and molecular responses. The molecular studies
110
suggested that the maize sensitivity to high temperature was mainly due to enhanced expression
of sag and dhn2 and failure to express hsp70 and sgr2 during relatively long term exposure.
In crux, although the varieties showed decreasing trend in growth and physiological
attributes response of Sadaf was better to heat stress. These studies indicated that changes in
physiological attributes are genetically related in maize, which provide a great room for
improvement of maize for enhanced net rate of photosynthesis, water use efficiency,
photosynthetic pigments reduced membrane permeability and production of reactive oxygen
species. Selection of varieties showing enhanced expression of heat shock protein and stay green
genes could be a promising approach for successfully growing maize in the upcoming
environmental conditions, when high temperature and low humidity could be likely threats to
maize production.
111
LITERATURE CITED Abiko, M., K. Akibayashi, T. Sakata, M. Kimura, M. Kihara, K. Itoh, E. Asamizu, S. Sato, H.
Takahashi and A. Higashitani. 2005. High-temperature induction of male sterility during
barley (Hordeum vulgare L.) anther development is mediated by transcriptional
inhibition. Sex Plant Rep., 18: 91-100.
Acevedo, E., M. Nachit and G.O. Ferrar. 1991. Effects of heat stress on wheat and possible
selection tools for use in breeding for tolerant. D.A. Saunders (ed.). Wheat for the