Physiological Evaluation of Wheat ( Triticum aestivum L.) Genotypes at Pre-Anthesis Stage under Heat Stress Conditions

Experiments were conducted to analyze effects of high temperature stress on wheat at pre-anthesis growth stage. Twenty four wheat genotypes exposed to a sub optimal temperature (>35˚C) which showed altered physiological, biochemical and agronomic characteristics. Accumulation of proline, presence of new protein bands and higher antioxidant enzyme activity in leaves of G.7 and G.17 reflects their better adaptive response under heat stress conditions. G.17 and G.19 showed least reduction in number of spikelets per spike, bio-logical yield and 100 grain weight. It was inferred that the genotypes G.7, G.17 and G.19 exhibited greater heat tolerance and could be recommended for cultivation under heat stress conditions.


Introduction
Gaseous emissions due to human activities are substantially adding to the existing concentrations of greenhouse gases, particularly CO 2 , methane, chlorofluorocarbons and nitrous oxides. Different global circulation models predict that greenhouse gases will gradually increase the world's average ambient temperature. According to a report of the Intergovernmental Panel on Climatic Change (IPCC), global mean temperature will rise 0.3˚C per decade [1] reaching ap-proximately 1˚C and 3˚C above the present value by years 2025 and 2100, respectively, and causing global warming. This may lead to altered geographical distribution and growing season of agricultural crops [2].
Heat stress is often defined as the rise in temperature beyond a threshold level for a period of time sufficient to cause irreversible damage to plant growth and is considered a serious threat to crop production worldwide [3]. Rise in temperature results in unexpected pronounced spatial and temporal variations leading to decline in plant growth and productivity [4]. Previously, it was reported [4] that a rise in temperature of 1˚C in the wheat growing season reduced wheat yield by 3% -10%. The optimum temperature for wheat germination is between 20˚C -22˚C whereas, the base temperature is 4˚C. During the grain development, the mean maximum temperature between 25˚C -32˚C is considered moderately high and temperature between 35˚C -40˚C is considered as very high temperature [5]. Temperature is one of the most important climatic factors which affect the growth, development and yield of the wheat. In wheat, the rate of grain filling can be decreased by high temperature (>31˚C) after anthesis [6]. High temperature episodes occurring near to anthesis can reduce the number of grains per ear and subsequently the rate of harvesting index resulting in smaller grain yields [7]. During recent years, wheat production faces very harsh environmental conditions, especially heat stress at pre-anthesis to physiological maturity. So, it is not sufficient to fulfill the needs of an ever increasing population.
The key adaptive mechanism to heat stress involves accumulation of a variety of osmolytes such as proline [8] [9] and antioxidant enzymes [10]. Under high temperature, free proline is involved in osmotic adjustment, protecting pollen and several plant enzymes from heat injury and serving as a source of nitrogen and other metabolites [11] [12]. Accumulation of proline under heat stress occurs in Arabidopsis [13], canola [14], cotton [15] and wheat [16]. Therefore, the current study was conducted to: 1) find out the physiological and biochemical basis of heat stress tolerance in wheat for breeding programs and 2) to analyze the impacts of high temperature stress on wheat genotypes at pre-anthesis stage.

Material and Methods
Twenty four wheat genotypes (Table 1)  The seeds were grown in pots (30 × 40 cm size) containing 10 kg sandy loam soil in a glass house with average day/night temperature 30˚C ± (8˚C) and 13˚C ± (5˚C) during the regular wheat crop season at National Agricultural Research Centre, Islamabad (latitude 33.38 N, longitude 73.00 E) from November to May respectively.
Plants were subjected to heat shocks at pre-anthesis (80 DAS) stage when the first anther extrusion occurred. The pots of four replications for each treatment,  each containing 4 -5 plants were moved to a controlled environment glasshouse for heat shocks where temperature was maintained at 40˚C -45˚C and 50%/70% relative humidity and illumination of 335 µmol•m −2 •s −2 . Plants were exposed to high temperature stress for 3 h consecutive ten days and then transferred to the greenhouse under natural daylight and 25˚C/20˚C day night temperature. Control and heat stressed plants were irrigated regularly as and when required.
Just after heat shock treatments, leaf sampling were done of the top fully emerged young leaves from control and stressed plants for analyses of proline, soluble protein, protein profiling, superoxide dismutase (SOD) and peroxidase (POD) activities.
For proline extraction [17], fresh plant tissue was extracted with 3% aqueous 5-sulfosalicylic acid and the filtrate was reacted with ninhydrin solution at 100˚C for 1 h. The reaction mixture was extracted with toluene and the absorbance of the chromophore containing toluene was read at 520 nm.
The POD activity was measured [18] following the assay mixture containing 0.1 ml enzyme extract, 1.35 ml of 100 mM MES (Methyl ethane sulphate) buffer (pH 5.5), 0.05% H 2 O 2 and 0.1% phenylene diamine. Change in absorbance was recorded at 485 nm for 3 min with a spectrophotometer. The activity of POD was presented as OD 485 nm/min mg protein.
SOD activity was determined by measuring inhibition of photochemical reduction of nitro blue tetrazolium (NBT) using method of Beauchamp and Fridovich [19]. The reaction mixture (3 ml) was composed of 13 mM methionine, 0.075 mM NBT, 0.1 mM EDTA, 0.002 mM riboflavin and 0.1 ml of enzyme extract in 50 mM phosphate buffer (pH 7.8). The mixture in tube was placed below light chamber for 15 min. The absorbance was read at 560 nm with a spectrophotometer. One unit of enzyme activity was taken as that quantity of enzyme, which reduces the absorbance reading to 50% in comparison with tube lacking enzyme.
For extraction of protein 5 to 7 cm long fresh leaves of wheat were grounded to fine powder using liquid Nitrogen. Four hundred micro-liter protein extraction buffer containing (0.6057 g Tris, 0.2 g SDS, 30 g urea, 70 ml distilled water + conc. HCl, 1 ml B-meracapto ethanol was added to 0.01 g of leaf powder and vortexed. The homogenized samples were centrifuged at 13,000 rpm for 10 min, at 4˚C in a refrigerated centrifuge. The extracted crud proteins were recovered as supernatant, transferred into a new 1.5 ml eppendorf tube and stored at 2˚C until they were run on polyacrylamide gel [20]. The electrophoresis procedure was carried out using SDS-PAGE (Bio-RAD, Model 600, UK) with 15% polyacrylamide gel. The molecular weight of the dis-

Result and Discussion
Heat stress is responsible for changes in physiological processes of plants. In this study heat stress significantly increased proline accumulation in leaves of all the wheat genotypes. The highest proline content was observed in genotype G.7 (90%) whereas; genotype G.16 has least proline content as compared to control (Table 1). Plants accumulate osmolytes like proline in response to abiotic stresses to protect the biomolecules of the cell [21]. Over accumulation of osmolytes such as proline may help plants to tolerate stress by improving their ability to maintain osmotic balance within the cell [22]. According to Wahid and Close [23], proline accumulation may buffer cellular redox potential under heat and other environmental stresses.
Heat stress significantly increased protein content in different wheat genotypes. However, highest protein content was observed in genotype G.7 (73%) whereas, least protein content was observed in G.10, as compared to control.
The protein content in genotype G.14 was approximate to G.7. However, the extent of increase in protein content was relatively higher in G.7 then G.14 ( Table   2). The effect of heat stress treatments on the change of protein patterns in 10 wheat genotypes grown under control and heat stressed conditions are presented in Figure 1(a) and Table 2. The SDS-PAGE showed the Band of 72 kDa appears under heat stress condition in 9 genotypes but the same polypeptide band was present in G.10 both in control and heat stressed treatment. A new protein of molecular weight 35 kDa appeared in genotype G.7 only. The protein profile of wheat genotypes G.11 -G.20 is presented in Figure 1 Accumulation of soluble proteins under heat stress is a common phenomenon in plants [24] [25] [26] [27]. Expression of stress proteins is an important adaptation to cope with environmental stresses. Most of the stress proteins are soluble in water and therefore contribute to stress tolerance presumably via hydration of cellular structures [28].
Under heat stress activities of superoxide dismutase and peroxidase were increased. However, the response of different genotypes to applied heat stress was different. The highest SOD activity was observed in genotype G.17 followed by G.11, G.16, and G.14. Least, SOD activity was observed in G.5. The genotype G.17 showed 79% increase in SOD activity under heat stress treatment imposed at pre-anthesis growth stage as compared to control.
The highest POD activity was observed in G.23 (65%) followed by G.1, G.22 and G.8 whereas; least POD activity was observed in G.17 as compared to control.
Genotypes having more SOD and POD activity become tolerant to heat stress. It has been reported that heat tolerance in crop plants is associated with an increase in antioxidant enzyme activity [29]   Heat stress markedly decreased plant biomass; maximum decrease was observed in G.18 (48%) while, minimum was observed in G.7, G.17, G.12 and G.24.
Heat stress significantly affected 100 grain weight in wheat genotypes. Maximum reduction (36%) in 100 grain weight was observed in G. 15. Whereas, G.7, G.12, G.17 and G.19 were observed with minimum reduction. Significantly higher reduction in number of spikelets/spike was recorded for G.22 (23%). Least reduction in the number of spikelets/spike was found in G.7 followed by G.17, G.19 and G.12 respectively. Maximum reduction (34%) number of florets per spike was shown by G.21 whereas; minimum decrease was shown by G.1 followed by G.7, G.17 and G.19 as compared to control (Table 2).
Heat stress is a common restriction during anthesis and grain filling stages in many cereal crops of temperate regions. At the reproductive phases, fertilization is one of the most sensitive stages to high temperature in various plants. During wheat grain filling it reduces kernel growth leading to loss in kernel density and weight [37] [38] [39] [40]. Behavior of different genotypes was different for heat stress imposed at the pre-anthesis stage. The maximum decrease in plant biomass and 100 grain weight was observed in G.18. However, some genotypes showed the minimum effects of heat stress. For example, G.7, G.12 and G.17 showed the minimum effect of heat stress on number of spikelets per spike, plant biomass and 100 grain weight, respectively. This behavior of genotypes indicates that these genotypes have the ability to tolerate heat stress. Ferris et al. [41] also found that imposition of heat stress in wheat at anthesis stage caused decrease in grain yield and biomass. Grain yield was negatively related to the thermal time accumulated above the base temperature of 31˚C [42].

Conclusion
Wheat is more sensitive to heat stress at the pre-anthesis growth stage. Under high temperature stress, activities of SOD and POD, protein and proline contents of different wheat genotypes increased, and new heat shock proteins were isolated from these genotypes which triggered the high temperature tolerance mechanism in these genotypes. Nevertheless, on the basis of yield, physiological and molecular attributes wheat genotypes G.7 and G.17 were declared tolerant to high temperature. The high temperature stress triggered antioxidant defensive mechanism in these two cultivars which helped to survive under heat stress environment. The information generated through present investigation will be helpful for plant breeders to include these traits in the breeding program for the development of heat tolerant wheat cultivars.