Acta Botanica 1-2015 - za web.indd ACTA BOT. CROAT. 74 (1), 2015 109 Acta Bot. Croat. 74 (1), 109–121, 2015 CODEN: ABCRA 25 ISSN 0365-0588 eISSN 1847-8476 Heat tolerance indicators in Pakistani wheat (Triticum aestivum L.) genotypes SAMI U. KHAN1, JALAL U. DIN2, ABDUL QAYYUM1*, NOOR E. JAN2, MATTHEW A. JENKS3 1 Department of Agriculture Sciences, University of Haripur, 22620, Pakistan. 2 Plant Physiology Program, Crop Sciences Institute, National Agricultural Research Centre, Islamabad, 45500, Pakistan. 3 Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA. Abstract – The effect of high temperature stress on six wheat cultivars exposed to 35–40 °C for 3 h each day for fi ve consecutive days was examined. High temperature signifi - cantly affected total proline, soluble protein content, membrane stability index (MSI), yield, and various yield components, and had a direct effect on growth and other physio- logical attributes of wheat at anthesis and the milky seed stages. The wheat cultivar AS- 2002 achieved better osmotic adjustment by accumulating more leaf proline. Higher MSI was also observed in AS-2002, as well as Inqalab-91. The anthesis growth stage was found to be more sensitive to heat stress than seed development at the milky stage. Overall heat stress reduced yield 75% at anthesis and 40% at the milky stage. AS-2002 performed bet- ter on the basis of yield and yield components. Seed weight per spike was highest in AS- 2002, and lowest in SH-2002. The cumulative response of AS-2002 was better on the basis of physiological and yield attributes. In addition to yield, plant breeders should also in- clude proline and MSI as selection parameter in the breeding program for development of heat tolerant wheat cultivars. Most of the evaluated wheat cultivars/lines were developed for cultivation in the rainfed areas of Pakistan. Keywords: anthesis, grain yield, heat stress, membrane stability index, milky seed stage, proline, soluble proteins, Triticum aestivum L., wheat Introduction High temperature is a major problem in fi eld cropping systems world-wide, with unex- pected spatial and temporal variations causing reduced plant growth and productivity (PAR- ENT et al. 2010). It has been estimated that a rise in temperature of just 1 °C in wheat during the growing season reduces wheat yields by about 3–10% (YOU et al. 2009). Wheat is the * Corresponding author, e-mail: aqayyum@uoh.edu.pk Copyright® 2015 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. KHAN S. U., DIN J. U., QAYYUM A., JAN N. E., JENKS M. A. 110 ACTA BOT. CROAT. 74 (1), 2015 major staple food crop of Pakistan, where the estimated per capita consumption is about 124 kg year–1, among highest in the world. In order to meet the demand for food in Paki- stan, an increase in wheat production of at least 4% is required to keep up with population growth. In Pakistan, wheat yields are especially sensitive to heat stress during the fl owering to seed maturing stages. During this period, heat stress shortens the growth cycle and forces premature ripening, reduces the number of grains per spike, lowers grain weight, and ulti- mately results in grain yield and quality deterioration (KHAN et al. 2007, WAHID et al. 2007, DIN et al. 2010). Therefore, an urgent need exists to develop wheat cultivars which are bet- ter able to withstand heat stress during later growth stages, or else mature earlier to escape the heat stress typically occurring later in the growing season. Understanding the means by which crop plants like wheat can tolerate heat would lay the foundation for work to develop plants with improved heat tolerance. One of the most common responses of crop plants to high temperature stress is an increase in proline accu- mulation (AHMED and HASAN 2011). Under high temperature, free proline is involved in os- motic adjustment to protect pollen and plant enzymes from heat injury, and also provides a source of nitrogen and other metabolites (VERSLUES and SHARMA 2010). Accumulation of proline has been shown to occur under heat stress in arabidopsis (WEI-TAO et al. 2011), cot- ton (RONDE et al. 2001) and wheat (HASAN et al. 2007), and genotypic variation in proline accumulation have been reported for these species. Under high temperature certain heat shock genes are triggered, resulting in the synthesis of heat shock proteins, whereas other soluble and insoluble proteins have also been shown to exhibit changes in abundance under high temperature stress (SIMMONDS 1995, HE et al. 2005). High temperature can cause a loss of membrane integrity, damage to primary photosyn- thetic processes, and changes in lipid composition, and protein denaturation (WAHID et al. 2007). Membrane thermal stability due to heat stress, typically measured as ion leakage from the cell, has been used for screening wheat germplasm for thermal tolerance (YILDIRIM et al. 2009). BLUM et al. (2001) showed a higher yield in spring wheat lines having greater membrane-thermostability in fl ag leaves at anthesis. The study presented here was con- ducted to explore the physiological basis for heat stress tolerance during later growth stages in wheat, and recommend reliable screening strategies for heat tolerance that can be utilized in wheat breeding programs for Pakistan and elsewhere. Investigated genotypes were de- veloped for cultivation in rainfed areas of Pakistan so they could be further used as parent material for development of heat tolerant wheat cultivars in the domestic wheat breeding program. Materials and methods Six wheat genotypes AS-2002, Inqalab-91, Punjab-96, NR-234, Wafaq-2001 and SH- 2002 obtained from Wheat Program, Crop Sciences Institute, National Agricultural Re- search Centre (NARC), Pakistan, were used in the study. Wheat plants were grown in pots (30 × 40 cm size) containing 10 kg sandy loam soil in a greenhouse under natural daylight at NARC, Islamabad (latitude 33.38°N, longitude 73.00°E) during the winter/spring with average day/night temperature 30 ± 8 °C and 13 ± 5 °C, respectively. A recommended dose of NPK (120-100-60 kg ha–1) was applied as urea, diammonium phosphate and potassium sulphate. The pots were arranged in factorial, randomized, complete block design. Plants HEAT TOLERANCE INDICATORS IN WHEAT ACTA BOT. CROAT. 74 (1), 2015 111 were subjected to specifi c heat stress treatments immediately at anthesis (80 days after sow- ing) and at milky growth stages (120 days after sowing). At anthesis when the fi rst anther extrusion occurred, pots (three replications) each containing three plants were moved to a greenhouse where temperature was maintained at 35–40 °C and 14/10 h day/night, 50–70% relative humidity, and illumination of 335 μmol m–2 s–2. After high temperature treatment for 3 h daily for fi ve consecutive days, pots were moved back to normal temperature (aver- age day/night temperature 30 ± 8 °C and 13 ± 5 °C) conditions in open greenhouse atmo- sphere. These heat treatments were also applied to the second set of potted plants during the milky growth stage. After daily 3-h heat stress treatments on fi ve consecutive days, fl ag leaf from the control and stressed plants was sampled for analysis of proline, soluble protein, and membrane stability index (MSI). Total proline was determined using the method of BATES et al. (1973). Fresh plant tissue was extracted with 3% aqueous 5-sulfosalicylic acid and the fi ltrate was reacted with glacial acetic acid and ninhydrin solution at 100 °C for 1 h. The reaction mix- ture was extracted with toluene and the absorbance of the chromophore contain toluene was read at 520 nm. The leaf MSI was determined according to SAIRAM et al. (2002). Leaf strips (0.2 g) of uniform size were taken in a test tube containing 10 mL double distilled water in two sets. Test tubes in one set were kept at 40 °C in a water bath for 30 min and electrical conductiv- ity of the water containing the sample was measured (C1) using a conductivity bridge. Test tubes of the other set were incubated at 100 °C in boiling water for 15 min and their electri- cal conductivity was measured as above (C2). MSI was calculated using formula as below: MSI = [1 – (C1 / C2)] ×100 Soluble protein content was determined according to BRADFORD (1976). Fresh plant tis- sue was extracted with 0.15 M NaCl and the fi ltrate was reacted with Bradford reagent (Bio-Rad protein assay dye reagent). Protein concentration of the sample was calculated using the calibration curve of bovine serum albumin and expressed on a fresh weight basis. At physiological maturity, grain yield per plant, fl orets per spike, number of seeds, seed weight per spike, and number of sterile fl orets per spike of both the heat stressed and the control plants were recorded. The analysis of variance of the data for each attribute was carried out using Minitab ver- sion 13.1. The mean values were compared with Duncan’s multiple range (DMR) test at signifi cant differences (ANOVA) (P < 0.05) following SNEDECOR and COCHRAN 1980. Results Impact of heat on proline, total protein, and the membrane stability index (MSI) Heat stress imposed at anthesis and milky growth stages signifi cantly increased proline concentration in leaves of all the wheat genotypes in comparison to their control values. At anthesis, the highest increase was recorded in AS-2002 (89%) and lowest in Punjab-96 (76%) (Fig. 1a). At the milky stage, the trend of relative increase in proline concentration was lower in AS-2002 (76%) followed by Inqalab-91 (77%) and even higher in the rest of the genotypes (Fig. 1b). KHAN S. U., DIN J. U., QAYYUM A., JAN N. E., JENKS M. A. 112 ACTA BOT. CROAT. 74 (1), 2015 Under heat stress, increases in soluble protein content were recorded at both anthesis and milky growth stages (Figs. 2a, b). The increase in soluble protein concentration was comparable in all the genotypes, and there was only slight, non-signifi cant change in leaf protein content. High temperature decreased the MSI at both anthesis and milky growth stages in all wheat genotypes tested. At anthesis, the decrease in MSI was greater in SH-2002 (23%) and least in AS-2002 (11%) and Inqalab-91 (15%). A similar trend toward decreased MSI was exhibited by all the genotypes during the milky growth stage (Fig. 3). Impact of heat on grain yield, and the number of fl orets and seeds per spike The heat treatment signifi cantly decreased the grain yield per plant in all the tested wheat genotypes, at both anthesis and milky growth stages, and there was signifi cant varia- tion in grain yield within the genotypes, both at anthesis and milky growth stages (Tab. 1). Anthesis growth was more sensitive to heat stress, as heat treatments during this stage re- duced yield by 75%, as compared to milky stage treatments that decreased yield by only 0 250 500 750 1000 AS-2002 Inqalab-91 Punjab-96 NR-234 Wafaq-2001 SH-2002 P ro li n e c o n te n t (μ g g -1 F W ) Wheat genotypes Control Stresseda 0 250 500 750 1000 AS-2002 Inqalab-91 Punjab-96 NR-234 Wafaq-2001 SH-2002 P ro li n e c o n te n t (μ g g -1 F W ) Wheat genotypes Control Stressedb Fig. 1. Effect of heat stress on the leaf proline concentration (μg g–1 fresh weight): (a) at anthesis, and (b) milky growth stages of wheat genotypes. The vertical bars indicate standard error (± SE) of mean (n = 3). All means are signifi cantly different at p < 0.05. HEAT TOLERANCE INDICATORS IN WHEAT ACTA BOT. CROAT. 74 (1), 2015 113 40%, relative to non-treated controls. At anthesis, the magnitude of decrease in grain yield was least in AS-2002 (62.9%), whereas the decrease in grain yield in other genotypes was almost equal. At the milky stage, the smallest decrease in grain yield occurred for AS-2002 (16.2%), whereas the greatest decrease occurred for SH-2002 (57.5%). Similarly, heat treatment signifi cantly decreased seed weight per spike at anthesis growth stage, in all the tested wheat genotypes except cv. AS-2002 (Tab. 2). The highest seed weight per spike af- ter stress occurred with Inqalab-91 (1.06 g), whereas the lowest seed weight after stress treatment was found in NR-234 (0.54 g). At milky stage, seed weight decreased for all the genotypes, and their responses to the heat treatments were similar. However this effect was not statistically signifi cant among the tested wheat genotypes. The number of fl orets per spike decreased as a result of heat treatment for all the tested genotypes; however the effect was not statistically signifi cant. The decrease in number of fl orets was greater at anthesis and least at milky stage (Tab. 3). Heat stress signifi cantly in- creased the number of sterile fl orets per spike in all the tested wheat genotypes at the anthe- sis growth stage but not at the milky stage (Tab. 4). The highest and signifi cant number of 0 100 200 300 400 AS-2002 Inqalab-91 Punjab-96 NR-234 Wafaq-2001 SH-2002 S o lu b le p ro te in c o n te n t (μ g g - 1 F W ) Wheat genotypes Control Stressed b 0 200 400 600 AS-2002 Inqalab-91 Punjab-96 NR-234 Wafaq-2001 SH-2002S o lu b le p ro te in c o n te n t (μ g g -1 F W ) Wheat genotypes Control Stressed a Fig. 2. Effect of heat stress on the soluble leaf protein concentration (μg g–1 fresh weight): (a) at anthesis, and (b) milky growth stages of wheat genotypes. The vertical bars indicate standard error (± SE) of mean (n = 3). All means are signifi cantly different at p < 0.05. KHAN S. U., DIN J. U., QAYYUM A., JAN N. E., JENKS M. A. 114 ACTA BOT. CROAT. 74 (1), 2015 40 50 60 70 80 90 100 AS-2002 Inqalab-91 Punjab-96 NR-234 Wafaq-2001 SH-2002 M e m b ra n e s ta b il it y i n d e x ( % ) Wheat genotypes Control Stressed a 40 50 60 70 80 AS-2002 Inqalab-91 Punjab-96 NR-234 Wafaq-2001 SH-2002 M e m b ra n e s ta b il it y i n d e x ( % ) Wheat genotypes Control Stressed b Fig. 3. Effect of heat stress on the leaf membrane stability index (%): (a) at anthesis, and (b) milky growth stages of wheat genotypes. The vertical bars indicate standard error (± SE) of mean (n = 3). All means are signifi cantly different at p < 0.05. Tab. 1. Effect of heat stress applied at anthesis and milky growth stages on the grain yield per plant (g plant–1) of wheat genotypes. Values in columns having the same letter are not signifi cantly different at p > 0.05, Duncan’s multiple range test. Least signifi cant difference (LSD) value (0.05) for variety × treatment interaction (V × T) = 1.535 at anthesis, LSD value (0.05) for variety × treatment interaction (V × T) = 1.674 at milky stage. The data in parentheses indi- cate percent decrease in grain yield per plant (g plant–1) of wheat genotypes at anthesis and milky growth stages in comparison to their control values. Genotypes Anthesis stage Milky stage Control Heat stress Control Heat stress AS-2002 19.36±1.13 b 7.18±0.24 d (62.9) 19.36±1.13 b 16.23± 0.58 d (16.2) Inqalab-91 16.40±0.55 c 5.00±0.05 e (69.5) 16.40±0.55 cd 10.56± 0.33 f (35.9) Punjab-96 21.13±0.84 a 4.59±0.05 e (78.3) 21.13±0.84 a 14.26± 0.23 e (32.5) NR-234 22.20±0.86 a 4.42±0.01 e (80.0) 22.20±0.86 a 13.73± 0.26 e (38.2) Wafaq-2001 22.73±0.47 a 4.43±0.02 e (80.5) 22.73±0.47 a 9.83±0.15 f (56.8) SH-2002 18.00±0.15 b 4.62±0.04 e (74.3) 18.00±0.15 bc 7.66±0.33 g (57.4) Means 19.97 5.04 (75%) 19.97 12.05 (40%) HEAT TOLERANCE INDICATORS IN WHEAT ACTA BOT. CROAT. 74 (1), 2015 115 Tab. 2. Effect of heat stress applied at anthesis and milky growth stages on the seed weight per spike (g spike–1) of wheat genotypes. Values in columns having the same letter are not signifi cantly different at p > 0.05, Duncan’s multiple range test. There were non-signifi cant statistical dif- ferences for milky stage. Least signifi cant difference (LSD) value (0.05) for variety × treat- ment interaction (V × T) = 0.316 at anthesis, LSD value (0.05) for variety × treatment inter- action (V × T) = 0.801 at milky stage. Genotypes Anthesis stage Milky stage Control Heat stress Control Heat stress AS-2002 1.99±0.15 d 1.02±0.10 d 1.99±0.15 1.77±0.10 Inqalab-91 2.09±0.20 b 1.06±0.04 d 2.09±0.20 1.41±0.28 Punjab-96 2.46±0.11 a 0.83±0.18 de 2.46±0.11 1.12±0.04 NR-234 2.09±0.07 b 0.54±0.009 e 2.09±0.07 1.28±0.15 Wafaq-2001 1.93±0.07 bc 0.75±0.11 de 1.93±0.07 1.25±0.29 SH-2002 1.70±0.14 c 0.63±0.05 e 1.70±0.14 1.22±0.15 Tab. 3. Effect of heat stress applied at anthesis and milky growth stages on the number of fl orets per spike of wheat genotypes. There were non-signifi cant statistical differences for anthesis and milky stage. Least signifi cant difference (LSD) value (0.05) for variety × treatment interac- tion (V × T) = 10.28 at anthesis, LSD value (0.05) for variety × treatment interaction (V × T) = 8.312 at milky stage. Genotypes Anthesis stage Milky stage Control Heat stress Control Heat stress AS-2002 54.66±0.75 49.33±0.88 54.66±0.75 46.00±1.17 Inqalab-91 64.66±1.53 57.66±1.20 64.66±1.53 64.00±3.17 Punjab-96 46.66±0.75 32.33±1.85 46.66±0.75 54.30±3.33 NR-234 57.66±1.53 50.00±1.45 57.66±1.53 57.00±2.45 Wafaq-2001 58.66±0.75 44.00±1.60 58.66±0.75 49.00±1.05 SH-2002 55.66±0.96 42.00±2.08 55.66±0.96 46.00±1.97 Tab. 4. Effect of heat stress applied at anthesis and milky growth stages on the number of sterile fl orets per spike of wheat genotypes. Values in columns having the same letter are not sig- nifi cantly different at p > 0.05, Duncan’s multiple range test. There were non-signifi cant sta- tistical differences for milky stage. Least signifi cant difference (LSD) value (0.05) for vari- ety × treatment interaction (V × T) = 4.193 at anthesis, and LSD value (0.05) for variety × treatment interaction (V × T) = 6.649 at milky stage. Genotypes Anthesis stage Milky stage Control Heat stress Control Heat stress AS-2002 8.66±1.20 d 24.33±1.20 b 8.66±1.20 11.00±0.61 Inqalab-91 8.66±0.66 d 33.33±1.20 a 8.66±0.66 13.30±0.55 Punjab-96 7.66±0.29 d 26.00±1.52 b 7.66±0.29 7.00±0.96 NR-234 4.33±0.24 d 15.33±1.85 c 4.33±0.24 8.00±0.22 Wafaq-2001 4.66±0.41 d 23.66±1.45 b 4.66±0.41 9.23±0.90 SH-2002 6.00±1.00 d 17.33±2.72 c 6.00±1.00 11.20±0.77 Means 6.67 23.32 6.67 9.95 KHAN S. U., DIN J. U., QAYYUM A., JAN N. E., JENKS M. A. 116 ACTA BOT. CROAT. 74 (1), 2015 sterile fl orets was recorded in Inqalab-91 (33.33) and Punjab-96 (26.0), and least in NR-234 (15.33) at anthesis stage. At anthesis growth stage, the number of seeds per spike also de- creased signifi cantly in all the tested wheat genotypes, except cv. Punjab-96 (14.8) (Tab. 5). Signifi cant variations were observed among the genotypes. At anthesis stage, the quantity of decrease in number of seeds per spike was greater in NR-234 (41.7%) followed by SH- 2002 (38.8%). At milky stage all the tested genotypes exhibited similar trends of fl oret number and sterility as a result of applied heat stress. Tab. 5. Effect of heat stress applied at anthesis and milky growth stages on the number of seeds per spike of wheat genotypes. Values in columns having the same letter are not signifi cantly dif- ferent at p > 0.05, Duncan’s multiple range test. There were non-signifi cant statistical differ- ences for milky stage. Least signifi cant difference (LSD) value (0.05) for variety × treatment interaction (V × T) = 7.432 at anthesis, LSD value (0.05) for variety × treatment interaction (V × T) = 5.715 at milky stage. The data in parentheses indicate percent decrease in number of seeds spike–1 of wheat genotypes at anthesis and milky growth stages in comparison to their control values. Genotypes Anthesis stage Milky stage Control Heat stress Control Heat stress AS-2002 47.5±2.2 bcd 40.3±1.2 de (15.2) 47.5±2.2 44.7±0.9 (5.8) Inqalab-91 64.0±1.2 a0 51.7±0.6 bc (19.2) 64.0±1.2 59.3±3.1 (7.3) Punjab-96 51.3±0.9 bc 43.7±0.3 cd (14.8) 51.3±0.9 50.7±2.7 (1.1) NR-234 52.7±1.6 b0 30.7±0.6 f (41.7)0 52.7±1.6 48.7±2.3 (7.6) Wafaq-2001 50.7±0.4 bc 33.0±0.7 ef (34.9) 50.7±0.4 47.4±1.1 (6.5) SH-2002 49.0±1.2 bc 30.0±0.7 f (38.8)0 49.0±1.2 45.7±1.3 (6.7) Means 52.53 38.23 (27%) 52.53 49.42 (6%) Discussion In this study, heat stress imposed after anthesis and the milky stages resulted in changes in physiological attributes such as proline content, total soluble protein, MSI, yield param- eters. Susceptibility to high temperatures may vary with the stage of plant development, but all vegetative and reproductive stages are affected by heat stress to some extent (WAHID et al. 2007). Heat stress induced modifi cations in plants may be observed as changes in spe- cifi c physiological processes, or in varying effects on development, and these responses may vary from one growth stage to another. In the present study, heat stress application dur- ing reproductive stages, at either anthesis or the milky seed development stage, increased signifi cantly the proline concentration in fl ag leaves of all the wheat genotypes examined. At anthesis stage, the increase was greater in AS-2002 and lowest in SH-2002, whereas at the milky stage, the increase was lower in AS-2002 and Inqalab-2001, and higher in rest of the genotypes. One of the most common responses of many plant species exposed to abiot- ic stresses is the accumulation of compatible organic solutes such as proline. Proline has been suggested to play a protective role in plants acting as a cellular osmotic regulator be- tween cytoplasm and vacuole, and by its ability to detoxify reactive oxygen species (ROS) and thereby protecting membrane integrity and stabilizing antioxidant enzymes (ASHRAF and FOOLAD 2007). Under stress conditions, accumulation of proline in plants results either HEAT TOLERANCE INDICATORS IN WHEAT ACTA BOT. CROAT. 74 (1), 2015 117 from increased expression of proline synthetic enzymes or due to repressed activity of pro- line degradation (HONG et al. 2000). In our case, the increase in proline accumulation was greater in AS-2002 and lowest in SH-2002. Genotypic differences in proline accumulation under high temperatures were previously reported in 20 wheat genotypes (AHMED and HAS- SAN 2011). Leaf proline level is thought to serve as an effective index to screen wheat geno- types for relative differences in heat tolerance. In the present study, increase in total leaf protein under heat stress was observed at both reproductive growth stages. It seems likely that this increase in total soluble proteins under heat stress is due to the induction of stress proteins, as such stress induced protein expres- sion has been shown to be an important adaptive strategy of crop plants. Further, the major- ity of stress induced proteins is soluble in water and therefore contributes to stress tolerance presumably via hydration of cellular structures (WAHID and CLOSE 2007). In our study, in- creased protein concentration resulting from heat treatments was similar in all the geno- types. Likewise, DIN et al. (2011) observed no differences in leaf soluble protein concentra- tion in various canola cultivars under water stress. Further studies are needed to determine if other wheat genotypes show variation for total leaf protein content as a response to heat stress. Increased solute leakage is an indication of decreased cell membrane thermostability, and has long been used as an indirect measure of heat-stress tolerance in diverse plant spe- cies, including wheat (BLUM et al. 2001, WAHID et al. 2007). In the present investigation, high temperature decreased the MSI at both growth stages in all the tested wheat genotypes. At anthesis, the decrease in MSI was greater in SH-2002 (23%) and lowest in AS-2002 (11%) and Inqalab-91 (15%). A similar trend of decreased MSI was exhibited by all the genotypes at milky growth stage. YILDIRIM et al. (2009) and DHANDA and MUNJAL (2006) concluded from their fi ndings that membrane thermal stability was a useful selection crite- rion for heat stress tolerance in wheat, reporting differences in MSI among different wheat cultivars at various growth stages. As in that study, they also showed that the MSI of geno- types decreased towards later developmental stages, i.e. the milky stage. Membrane insta- bility at the milky growth stage might be associated with the beginning of senescence. SIK- DER et al. (2001) found signifi cant correlation between membrane stability of fl ag leaf and grain yield, and suggested that membrane thermostability can be used to determine the heat tolerance of wheat varieties under heat stress conditions. Heat stress is a common constraint during anthesis and grain fi lling stages in many ce- real crops in both arid and temperate regions. At the reproductive phases, fertilization has been shown to be highly sensitive to high temperatures in various plants, whereas heat stress during wheat grain fi lling is known to reduce kernel growth and cause a reduction in kernel density and weight (FOOLAD 2005, LAGHARI et al. 2012) In the present study, heat treatment signifi cantly increased the number of sterile fl orets per plant in all the tested wheat genotypes at anthesis growth stages. At anthesis, the highest number of sterile fl orets per spike after heat treatment occurred in Inqalab-91 (33.33), and the lowest in NR-234 (15.33) (Tab. 4). These results indicate that the genotypes examined here utilize unique re- sponses for heat tolerance, as the lines showing the best performance as measured in pro- line, total protein, and MSI response show different heat responses regarding sterility and other heat associated traits. Heat treatment decreased the grain yield per plant in all the tested wheat genotypes at both reproductive growth stages; however the most signifi cant decrease was observed by KHAN S. U., DIN J. U., QAYYUM A., JAN N. E., JENKS M. A. 118 ACTA BOT. CROAT. 74 (1), 2015 heat treatments during anthesis. There were signifi cant variations in grain yield per plant within the genotypes both at anthesis and milky growth stages. At anthesis, the highest de- cease in grain yield was observed in Wafaq-2001 (80.5%), NR-234 (80.0%) and Punjab-96 (78.3%), and the least effect was observed for AS-2002 (62.9%). KHAN and HUSSAIN (2006) tested AS-2002 for heat tolerance in fi eld studies, and found it had the best performance with respect to grain yield, followed by Inqalab-91. At the milky stage, the same cultivar also exhibited the least effect of heat stress on grain yield. Comparable studies show similar genotypic effects of heat on grain yield for wheat and other crop species (KHAN et al. 2007, NAHAR et al. 2010, BALOUCHI 2011). During the onset of meiosis in the male generative tis- sues until completion of anthesis, wheat grain setting is reduced by rises in temperature above optimum (FERRIS et al. 1998). Our results are consistent with these previous reports, showing the heat effects on yield for new genotypes in wheat. This study also shows that heat stress signifi cantly decreases seed number and weight per spike in these wheat genotypes, more during the anthesis growth stage than the milky stage. For heat stress applied during anthesis, the relative seed number reduction was low- est in Punjab-96 followed by AS-2002 and Inqalab-91, and highest in NR-234 followed closely by SH-2002 and Wafaq-2001. Similarly, for anthesis applied heat stress, the seed weight per spike was greater in Inqalab-91 and AS-2002, and lowest in NR-234. For heat stress applied during the milky stage, the seed number and weight per spike was reduced, but the effect was small and not signifi cant. In the present study, high temperature treatment at anthesis stage increased the number of sterile fl orets in all the tested genotypes, and most notably in the Inqalab-91, consequently reducing the number of grains per plant and the yield. Genotypic variation in grain yield for heat effects on the number of sterile fl orets was also observed. Among the reproductive stages, fertilization (1–3 days after anthesis) is one of the most sensitive to high temperatures (FOOLAD 2005). Pollen viability, patterns of as- similates partitioning, and growth and development of seed/grain, ear or spike are likewise highly affected by heat. Cereal crops can tolerate only narrow temperature ranges, and if these are exceeded during the fl owering stages they can damage fertilization and seed set, resulting in yield reduction (PORTER 2005). It has been shown for example that supra-opti- mal temperatures during grain fi lling decreased wheat yield by reducing kernel weight (GIBSON and PAULSEN 1999). In the current project, the decline in grain yield due to heat treatment at the milky stage is due to reduced seed weight per spike. In wheat, genotypic variation with respect to translocation of assimilates from source to sink under high tem- perature has already been reported (MOHAMMADI et al. 2009). The high grain yield and seed weight of the AS-2002 and Inqalab-91 cultivars may be due to their greater effi ciency in mobilizing reserves from leaves, stem or other plant parts towards sink (GUPTA et al. 2011). Furthermore, high temperatures can reduce the number of grains per spike, by causing ei- ther fl ower sterility or seed abortion, which reduces grain yield and ultimately harvest index causing smaller grain yield. WARDLAW (2002) found reductions in grain weight due to high temperature during both anthesis and milky growth stages under fi eld conditions. Conclusion Tolerance to heat stress is a complex phenomenon and is controlled by multiple genes imparting a number of physiological and biochemical changes. No single trait fully ex- plains why some wheat varieties are able to generate better yield under heat stress. Wide HEAT TOLERANCE INDICATORS IN WHEAT ACTA BOT. CROAT. 74 (1), 2015 119 variation in tolerance to heat stress existed in the tested wheat genotypes. Thus, there is a dire need for the development of heat tolerant wheat genotypes through a breeding program for cultivation under heat stress environments like those prevailing in Pakistan. The cumu- lative response of AS-2002 was better on the basis of physiological and yield attributes, in- dicating AS-2002 may represent an excellent parental material for a breeding program. Plant breeders should also use proline and MSI as selection markers in the breeding pro- gram for development of heat tolerant wheat cultivars, as these were shown here to be closely associated with yield in wheat. References AHMED, J. U., HASSAN, M. A., 2011: Evaluation of seedling proline content of wheat geno- types in relation to heat tolerance. 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