Vegetative growth can be assessed as plant height to distinguish heat-stress tolerant genotypes (Debnath et al., 2016). In Brassica juncea L., high temperature (34°C) after the induction of flowering significantly affected plant height when grown in the field, declining by 18.9–30.5% (mean 22.3%), relative to the control. Genotypes BPR-538-10, NRCDR-2, RH-0216 had lower heat susceptibility, based on plant height, than genotypes RGN193, NPJ112 and SKM531 (Chauhan et al., 2009). Heat stress (>40°C) reduced plant height in 20 maize genotypes in the field (Debnath et al., 2016), with the most heat-tolerant genotype (DTPYC9F119) declining by 2.31% compared with a 72.2% reduction in the most heat-sensitive genotype LM13. Assessment of five potato cultivars (L1: 84.194.30; L2:86.61.26; L3: 87HW13.7, L4: DG81-68, and L5: Desiree) under controlled environment of combined heat (30 + 1°C) and drought stress (PEG 8000) for 21 days revealed severe effects all the cultivars on plant height, when both the stresses were together, except L2 and L3 (Handayani and Watanabe, 2020).

Seedling growth could be a potentially useful trait for early screening against heat stress. For instance, in some tropical parts of Africa, surface temperatures of tropical soils at planting time can exceed 50°C for hours to restrict the germination and seedling growth (Setimela et al., 2007). Hence, seedling heat tolerance is critical for adequate crop establishment in the semi-arid tropics. In a study carried out on sorghum (Sorghum bicolor) in Zimbabwe, seedling tolerance was estimated as heat tolerance index (HTI; defined as a ratio of resumed coleoptile growth after a controlled heat shock, compared to normal growth). Genetic parameters of HTI were assessed by crossing four sorghum lines having varying HTI, with three tester lines, and deriving F1, F2, F3, BC1, and BC11 families for generation means analysis. HTI was found to be highest (0.71) in Line IS20969 from Egypt, while an experimental line (290R), from the University of Nebraska, had the lowest at 0.51. The study revealed that additive and dominance effects contributed to coleoptile elongation under normal conditions, but only additive effects were significant in recovery growth. Epistatic effects were observed in both normal and heat-stressed environment. General combining ability (GCA) effects for HTI were highly marked in both conditions, but specific combining ability (SCA) effects were negligible. These results showed that it is achievable to enhance seedling heat tolerance and, thus, improve plant populations of sorghum in tropical areas where hot soil temperatures occur.

The structure, spatial, and temporal configuration of the plant root system is called root system architecture (RSA) (de Dorlodot et al., 2007). The organization of primary and secondary roots is determined by RSA at the macroscale (Smith and De Smet, 2012). Root microstructure, such as fine root hairs, root tips and their interactions with soil and soil microorganisms responsible for water and mineral uptake, is determined by RSA at the microscale (Wu et al., 2018). Most resources are heterogeneously distributed in the soil, and the spatial and temporal distribution of roots determines the crop’s ability to exploit resources (Brussaard et al., 2007). Better understanding of RSA allows us to determine the impact of environmental conditions and management practices on crops, which can help to reduce the difference between genetic potential and actual average yields (Garnett et al., 2009; Judd et al., 2015; Ryan et al., 2016). RSA has a vital role in plant–soil–microbe interactions and resolves the crosstalk with beneficial soil microbes in the rhizosphere (Ryan et al., 2016).

Across fluctuating environments, RSA is an important characteristic for adaptability. Therefore, we can improve crop performance in terms of increased root traits, such as allocation, morphological, anatomical, or developmental plasticity (Sultan, 2000). There is a direct relationship between individual RSA plasticity and yield, which is related to more stable plant performance across changing environments in various species (Sadras, 2009; Niones et al., 2012, 2013). Root branching is important for improving the root surface area, enabling the plant to reach more distant reserves of water and nutrients and improve soil anchorage. In plants, heat stress generally reduces primary root length, lateral root density (number of lateral roots per unit primary root length) and angle of emergence of lateral roots from the primary root, but has little effect on average lateral root length (McMichael and Quisenberry, 1993; Nagel et al., 2009). Root growth has a lower optimal growing temperature than shoot growth and is thus more sensitive to rising temperatures (Huang and Gao, 2000; Xu and Huang, 2000). Plant heat tolerance is directly influenced by root morphological features. Among Kentucky bluegrass (Poa pratensis) cultivars, increased root number and root length contributed to variations in heat tolerance (Lehman and Engelke, 1993). Root phenotyping of 577 common bean (Phaseolus vulgaris L.) genotypes in variable environments of heat, drought, and nutrient deficiency revealed significant relationships between seed yield and seedling basal root number, seedling adventitious root abundance, and seedling taproot length. Under heat stress, adventitious root number had a positive relationship (13%) with seed yield. Mesoamerican genotypes of common bean yielded higher than Andean genotypes under heat stress (Strock et al., 2019). In canola, heat stress reduced stem diameter by 8.4%, cross-sectional area by 17.3%, and aboveground biomass by 11.5% in two genotypes; genotype 13C204 (heat-sensitive) had smaller stem diameter, cross-sectional area, root length, root surface area, root biomass, and root volume than Invigor 5440 (heat-tolerant) (Wu et al., 2017). In another study, heat stress reduced lateral root elongation (–38%), number of axile roots (–30%), elongation rate of primary root (–26%), root dry weight (–39%), leaf water potential (–59%) and leaf area (19%) in heat-sensitive maize lines K64R, Ac7643, and Ac7729TZSRW when the temperature increased from 28 to 37°C. The heat-tolerant lines H16, CML444, and SC-Malawi were least affected by high temperature (Trachsel et al., 2010). In maize, screening of 10 genotypes for combined heat and drought stress (40°C/60%) revealed DK 6789, NT 6621 as tolerant and genotypes FH 988 and FH 1137 as sensitive on the basis of root tolerance indices (Ayub et al., 2020).

Wheat seedlings grown for 7 days under normal conditions and then subjected to heat stress (42°C for 2 h) in a growth chamber revealed growth differences between genotypes (Gupta et al., 2013). In general, heat stress reduced growth (shoot/root dry weight and shoot/root length). Heat-tolerant genotypes (Raj-4037 and PBW590) retained more shoot and root length and dry weight than heat-sensitive genotypes (PBW502, DBW16, DBW17, WH1021, and PBW550) (Gupta et al., 2013). In a field experiment, heat stress (30/20°C) reduced wheat biomass, relative to optimum conditions (25/15°C), but heat-tolerant genotypes (MW-8, BW-4, and BW-3) maintained more biomass than heat-sensitive genotypes (MW-7, MW-6, and BW-5) (Rahman et al., 2009). Heat stress (>32/20°C) significantly reduced chickpea biomass by 22–30%, relative to those grown under normal temperatures; heat stress had a smaller effect on the biomass of heat-tolerant genotypes (ICC15614 and ICCV92944) than heat-sensitive genotypes (ICC5912, ICC10685) (Kaushal et al., 2013). Similarly, in a field experiment on alfalfa (Medicago sativa), heat stress (38/35°C) reduced plant biomass, relative to the control (25°C), more so in heat-sensitive Wl712 than heat-tolerant Bara310SC (Wassie et al., 2019). Field studies on lentil revealed that heat stress (>32/20°C) significantly reduced plant biomass, relative to the control (Sita et al., 2017a); genotypes IG3263, IG2507, IG3297, IG3312, IGG3327, IG3330, IG3546, IG3745, IG4258, and FLIP2009 retained more biomass (termed heat-tolerant) than genotypes IG2519, IG2802, IG2506, IG2849, IG2821, IG2878, IG3326, IG3290, IG3973, IG3964, IG4242, DPL15, DP315, IG4221, and IG3568 (termed heat-sensitive). Likewise, heat stress (>40/28°C) significantly reduced mungbean biomass (up to 76%), relative to the control (34/16°C) in the field, due to the inhibition of vegetative growth and acceleration in reproductive growth. Genotypes EC693357, EC693358, EC693369, Harsha, and ML 1299 produced more biomass under heat stress (heat-tolerant) than genotypes EC693363, EC693361, KPS1, EC693370, and IPM02-3 (heat-sensitive) (Sharma et al., 2016). A study on potato (Solanum tuberosum) revealed that warmer temperatures (31/29°C) severely affected plant biomass in two genotypes—Norchip (heat-tolerant) and Up-to-date (heat-sensitive)—grown in controlled environment chambers (Lafta and Lorenzen, 1995). Both genotypes had similar total dry mass under controlled conditions (19/17°C), but heat stress (31/29°C) reduced total dry mass by up to 44% in Norchip and 72% in Up-to-date. Leaf, stem, shoot, and tuber dry and fresh weights followed the same trend under high temperature in both genotypes. At Niger, West Africa (ICRISAT Sahelian Centre research farm (13° 29≪ N, 2° 10≫ E; 221 m above sea level), field experiments were performed to evaluate heat tolerance of groundnut (Arachis hypogaea L.) using physiological traits identified in a yield model [crop growth rate (C), reproductive duration (Dr) and partitioning (p)]. After screening 625 diverse groundnut genotypes, under irrigation during the hottest months (February to May), 16 contrasting genotypes, selected on the basis of combination of high pod yield and partitioning coefficient, revealed that crop growth rate was a powerful factor affecting pod yield. Pod yield of most genotypes decreased by more than 50% because of heat stress (40°C) at the time of flowering and pod formation. The findings showed that estimates of p would be vital as a dependable selection criterion, compared to yield, for identification of heat tolerant genotypes. The breeders should explore strategies to maximize the crop growth rate and partitioning in genotypes growing under warm environments (Ntare et al., 2001). Under combined heat and drought (36/26°C without irrigation) stresses, Biomass was used as a trait for evaluation of 3 tomato cultivars (Arvento and two heat tolerant; LA1994 and LA2093) (Zhou et al., 2017) resulting in identification of “Areventro” cultivar as more tolerant than “LA1994” and “LA2093” genotypes (Zhou et al., 2017).

Tissue damage can be assessed by measuring membrane damage based on electrolyte leakage, which is a reliable indicator of heat sensitivity in several crop species. The primary target of environmental stress is the cell membrane (Chen et al., 2014; Sita et al., 2017b). Heat stress loosens chemical bonds within the molecules of biological membranes by accelerating the kinetic energy and movement of molecules across membranes, which results in membrane fluidity by protein denaturation or increased unsaturated fatty acids (Savchenko et al., 2002). Under high temperature, protein denaturation, increased membrane fluidity, and enzyme inactivation decreases protein synthesis and degradation, and alters membrane integrity (Howarth, 2005). The tertiary and quaternary structure of membrane proteins changes with heat stress and enhances membrane permeability, as evident from increased ionic leakage, which is an indicator of decreased cell membrane thermostability (CMT) (Wahid et al., 2007). Damage to leaf membranes occurs due to the direct effects of high temperature, photo-oxidation of chlorophyll pigments, impaired electron flow, inhibition of carbon fixation, and water loss from leaves. Damage to membranes impairs photo-assimilate production in leaves (Prasad et al., 2017). Under high temperature, the relationship between CMT and crop yield varies from plant to plant. CMT has been used as an indirect measure of heat stress tolerance in plant species, such as sorghum (Sullivan, 1997; Marcum, 1998), soybean (Martineau et al., 1979), potato and tomato (Chen et al., 1982), sorghum, wheat (Blum et al., 2001), cotton (Ashraf et al., 1994; Cottee et al., 2010), lentil (Sita et al., 2017a), chickpea (Kaushal et al., 2013), mungbean (Sharma et al., 2016), and barley (Wahid and Shabbir, 2005). Abro et al. (2015) identified several heat-tolerant cotton genotypes with high membrane thermostability at 50°C in the laboratory environment, which could be used for breeding purposes to develop heat-tolerant genotypes. During the late developmental phase of plants, membrane stability tends to decrease (Ahmad and Prasad, 2011). For breeding purposes, significant variations in membrane thermostability among genotypes could be used to improve selection (Hemantaranjan et al., 2014).

In wheat grown under high temperatures (45°C for 2 h), genotypes (Raj4037, PBW373) with high CMT (58.20, 55.43) during grain filling performed better than those (Raj4083, DBW16, PBW550) with low membrane thermostability (48.15, 50, 51.96). Under controlled conditions, membrane thermostability was maximum in WH1021 (64.13) and minimum in DBW16 (51.11) (Gupta et al., 2013). Similarly, CMT was markedly higher in heat-tolerant (56.83%) than heat-sensitive (31.43%) wheat genotypes during grain filling. Based on CMT, Bala and Sikder (2017) identified heat-tolerant wheat genotypes BAW-1143, BARI Gom-25, BARI Gom-26, and Prodip. At the seedling stage in wheat, CMT had a positive correlation with grain yield, grain weight (Saadalla et al., 1990), and biomass (Blum et al., 2001), indicating the effectiveness of this trait for assessing heat tolerance. In rice at 40°C, thermostability was closely related to crop yield potential (Maavimani and Saraswathi, 2014). In a comparative study of rice and maize grown under controlled high temperatures (40/35°C and 45/40°C), the rice genotypes (PR116, PR118) had greater electrolyte leakage (27.4–40.2%) than the maize genotypes (PMH1, PMH2) (19.2–26.2%) (Kumar et al., 2012). Similarly, among three rice cultivars, F60 and F733 were more heat-susceptible than F473 when grown at 40°C, with greater electrolyte leakage (20 and 15%) (Sanchez-Reinoso et al., 2014). Likewise, Yadav et al. (2014) used CMT as an effective screening parameters for selecting heat tolerant lines in Pearl millet. From the same study, the authors also identified H77/29-2 × CVJ-2-5-3-1-3 hybrid as heat tolerance based on seedling thermotolerance index. Under combined stresses (drought-42–45% of irrigated conditions) and heat (> 32/20°C), the drought tolerant chickpea genotypes were found to tolerate the two stresses more effectively than heat tolerant genotypes. For instance, genotypes ICC1356 (drought-tolerant) showed less damage to membranes than genotype ICC3776 (drought-sensitive), when subjected to both the stresses (Awasthi et al., 2017).

In legumes, a few studies have identified heat-tolerant and heat-sensitive genotypes. Based on the membrane stability test, chickpea was most sensitive to heat stress, relative to other legumes such as pigeon pea, groundnut, and soybean (Devasirvatham et al., 2012). Contrasting chickpea genotypes exposed to high temperatures (40/30°C and 45/35°C) varied markedly, with heat-tolerant genotypes (ICCV07110, ICCV92944) showing less membrane damage (22.6, 20.6%) than heat-sensitive genotypes (ICC14183, ICC5912) (30.4, 33.3%) (Kumar et al., 2013). A similar test conducted at 37/27°C reported up to 25% electrolyte leakage in chickpea seedlings (Pareek et al., 2019). A heat-tolerant genotype (ICC1205) had low electrolyte leakage (13–14%), indicating better cell membrane integrity. Screening of cowpea genotypes exposed to heat stress also revealed less leaf electrolyte leakage (35.8–36.7%) in heat-tolerant genotypes (H36, H8-9, DLS99) during flowering and pod set than heat-susceptible genotypes (CB5, CB3, DLS127) (66.2–79%) (Ismail and Hall, 1999). In lentil, heat tolerance was related to less membrane damage (<20%) in heat-tolerant genotypes (IG2507, IG3263, IG3745, IG4258, and FLIP2009) than heat-sensitive genotypes (IG2821, IG2849, IG4242, IG3973, IG3964) (> 30%) at 38/28°C and 40/30°C in a controlled environment (Sita et al., 2017a). In another study, lentil genotypes (Ranjan, Moitree, 14-4-1, IC201710, and IC208329) were reported as heat-tolerant based on cell membrane stability under field and growth chamber studies at 34°C (Choudhury et al., 2012). Barghi et al. (2013) reported the highest CMT in genotype Qazvin (98.13%) and regarded it as heat-tolerant, whereas genotype B4400 (33.19%) had the lowest CMT (heat-sensitive). Under high temperature (38/35°C), screening of 15 Medicago cultivars for CMT identified Bara310SC and WL712 as heat-tolerant (24.07%) and heat-sensitive (53.2%) cultivars, respectively, having minimum and maximum electrolyte leakage, respectively (Wassie et al., 2019).

Cotton displays heat sensitivity at various growth stages. Cotton genotypes grown in a controlled environment under optimal conditions (35/21 ± 2°C) for 30 days and then exposed to high temperature (46/30 ± 2°C) at the reproductive stage, by gradually increasing temperature by 2°C per day, were screened for CMT—cultivars FH-900, MNH-552, CRIS-19, and Karishma emerged as relatively heat-tolerant (thermostable) and FH-634, CIM-448, HR109-RT, and CIM-443 as heat-susceptible (Rahman et al., 2004). In a similar study at > 32°C, cotton genotypes B557 and NIAB-78 showed minimum electrolyte leakage (<40%) and were regarded as tolerant compared to genotypes MNH-554, FH682 and FH900 which showed maximum electrolyte leakage (>50%) (Rana et al., 2011). Abro et al. (2015) reported cotton varieties NIA-80, NIA-81, NIA-83, NIA-84, NIA-M-30, NIA-M31, NIA-HM-48, NIA-HM-327, NIA-H-32, NIA-HM-2-1, NIA-Bt1, NIA-Bt2, NIA-Perkh, CRIS-342, CRIS-134, and NIAB-111 and check variety Sadori as heat-tolerant using CMT as a screening parameter in both heat-stressed (44°C) and non-stressed (32°C) temperature regimes. Other similar studies where cotton genotypes were differentiated by CMT into heat-tolerant and heat-sensitive were conducted by Karademir et al. (2012); 15 genotypes; > 40°C) and Singh K. et al. (2018); 37 genotypes; > 40°C).

Likewise, in cucumber, contrasting genotypes were identified based on membrane stability under heat stress (40/32°C)—L3466 and Desi cucumber as heat-tolerant and Suyo Long and Poinsett as heat-sensitive (Ali et al., 2019). In tomato, 2 h exposure to high temperature (45°C) altered CMT more in heat-sensitive variety Campbell-28 (> 45%) than heat-tolerant variety Nagcarlang (<20%) (Camejo et al., 2005). In another study on 44 tomato lines, exposure to 44°C for 4 h after 1 week of vegetative stage increased electrolyte leakage in heat-sensitive genotypes (32.92 μmhos/cm) more than heat-tolerant genotypes (22.2 μmhos/cm) (Hameed et al., 2015). Similar studies have screened tomato genotypes for heat tolerance using membrane thermostability (Sangu et al., 2015; Alsamir et al., 2017). Thus, CMT is an effective trait for identifying stable and heat-tolerant genotypes.

At the whole crop level, leaf temperatures decrease below air temperature when water evaporates. Canopy temperature depression (CTD)—the difference between air temperature (T_a) and canopy temperature (T_c)—acts as an indirect measure of transpiration (Reynolds et al., 2001) and plant water status (Araus et al., 2003). A positive CTD value is observed when the canopy is cooler than the air (CTD = T_a–T_c) (Balota et al., 2008). CTD is a heritable trait that can be measured on cloudless days with an infrared thermometer (Reynolds et al., 1998). Plants transpire through open stomata to maintain canopy temperature in a metabolically comfortable range. Under stress, plants close their stomata for some period, which increases the canopy temperature (Kashiwagi et al., 2008). Canopy temperature is affected by soil water status, wind, evapotranspiration, cloudiness, conduction systems, plant metabolism, air temperature, relative humidity, and continuous radiation (Reynolds et al., 2001). To assess heat tolerance, many traits can be used as selection criteria, but, CTD is considered to be best as a single reading integrates scores of leaves (Reynolds et al., 1994, 1998; Fischer et al., 1998). Yield potential and the metabolic fitness of crop plants under specific environmental conditions are determined by CTD (Kumari et al., 2013). A study on barley revealed a strong link between epicuticular leaf wax QTL and CTD, and that wax load influences plant canopy temperature (Awika et al., 2017). Based on phenotypic variation, CTD can act as a desirable criterion for heat-tolerant genotype selection (Mason and Singh, 2014). CTD is a mechanism of heat escape and has a strong genetic correlation with yield (Reynolds et al., 2001). Heat-tolerant genotypes of wheat had higher CTD than heat-sensitive genotypes, indicating their greater ability to maintain a cooler canopy environment (Gare et al., 2018). In another study, the CTD value in wheat was correlated with heat resilience (Pradhan et al., 2012). In 102 durum wheat genotypes tested under late-sown conditions, CTD had a strong positive correlation with days to maturity (Gautam et al., 2015), confirming that CTD is an effective selection criterion in plant breeding (Seema et al., 2014). Leaf area having more greenness and CTD are strongly interrelated in wheat and with grain yield, grain-filling duration, and biomass (Kumari et al., 2013). Stay-green genotypes have high CTD values due to transpirational cooling, resulting in lower canopy temperatures (Reynolds et al., 1994; Fischer et al., 1998). In stay-green lines, low CTD values delayed senescence (Kumari et al., 2013). Leaf width in wheat had a high correlation with canopy temperature under heat stress (Mohammadi et al., 2012). In durum wheat, CTD had a positive correlation with biological yield and spike number/m² at first spikelet emergence and 50% inflorescence stages. At three growth stages (first spikelet emergence, 50% inflorescence, and completion of anthesis), harvest index had a negative correlation with CTD (Bahar et al., 2008). Screening of Indian and CIMMYT wheat germplasm for the stay-green trait and CTD revealed higher CTD values in the stay-green genotypes due to transpirational cooling and lower canopy temperatures (Kumari et al., 2013). In wheat (Triticum aestivum), heat stress reduced CTD by 39.7% at the grain-filling stage (Joshi et al., 2016). Timely sown wheat had higher CTD than late-sown wheat (Saxena et al., 2016), with genotypes HD2932, HD2864, HD3095, HI8703, and HUW234 identified as heat-tolerant due to their higher net photosynthesis, relative water content, membrane stability index and CTD than the other tested genotypes (Saxena et al., 2016). Additional management factors, such as the use of farmyard manure and NPK, improved physiological traits (light interception, CTD, and flag leaf chlorophyll content) in wheat (Badaruddin et al., 1999). In seven rice varieties, CTD was closely related to stomatal conductance and leaf photosynthetic rate (Takai et al., 2010). Rice varieties Takanari and TUAT1-5-6a had lower leaf temperatures and higher stomatal conductance and leaf photosynthetic rates than the other varieties tested under cloudy conditions. Infrared thermography, as a simple method of evaluating varietal differences in stomatal conductance via CTD, is feasible even under cloudy conditions. In chickpea, water potential, osmotic pressure, relative leaf water content, and seed yield had a negative correlation with CTD (Sharma D. K. et al., 2015). Heat-tolerant chickpea genotypes ICCVs 95311, 98902, 07109, and 92944 had higher CTD than sensitive genotypes ICCVs 07116, 07117, and 14592, which had negative CTD values (Devasirvatham et al., 2015). In mungbean, CTD had a significant positive correlation with seed yield, and a negative correlation with root traits, such as lateral branch number and dry root weight (Raina et al., 2019). Greater pod number and pod to node ratio was associated with CTD in pea (Tafesse et al., 2019). In cotton, the involvement of CTD in heat tolerance was indicated (Cornish et al., 1991), with additive, dominance, and epistatic components involved in its inheritance (Khan et al., 2014). In another study on cotton, crop development stage had no effect on CTD, which was significantly correlated with seed yield (Karademir et al., 2018). Canopy temperature in cotton increased under combined heat and drought stress treatment (>36°C and 35% irrigation) (Carmo-Silva et al., 2012), as compared to control. Low canopy temperature was noticed in cotton cultivar Pima S-6 (S6), which was reported as tolerant, unlike high canopy temperature in Monseratt Sea Island (MS), termed as sensitive, under combined stress.

Under heat stress, regulating the transpirational mechanisms is a possible strategy for selecting heat-tolerant varieties (Condon et al., 2007). As leaves open their stomata, the rate of gaseous exchange may create differences in stomatal behavior that can be recorded by a leaf porometer (Chandra et al., 2017; Priya et al., 2018). Fully opened stomata increase the diffusion of CO₂ and, at the same time, increase transpiration and photosynthetic efficiency in wheat (Condon et al., 2007). Consequently, stomatal regulation is an important factor that governs plant growth and survival. Therefore, stomatal conductance (g_S) is a useful trait for determining photosynthetic and transpiration rates. Stomatal conductance increases with rising temperature (Urban et al., 2017). Crawford et al. (2012) suggested that plants acclimatize to high temperatures by evaporating more water, thereby keeping their canopies cool despite the presence of fewer stomata. Similarly, semi-dwarf spring wheat cultivars had strong positive correlations between g_S and photosynthetic rate, cooler canopies and yield (Fischer et al., 1998). Heat-tolerant advanced cotton lines (e.g., Pima S-6) developed by Cornish et al. (1991) had higher stomatal conductance and photosynthetic rates under heat stress, which was possibly due to cooling effect of plants through stomata. The stomatal conductance of 50 cotton genotypes was measured under high temperature (45–50°C/20–30°C day/night) in a glasshouse, and identified five heat-tolerant genotypes (NIAB-111/2, BH-160, MNH-554, N-313, BH-163, Mutant-94) (Khan et al., 2008). Similarly, 41 wheat lines of different origin were screened for higher g_S, which was associated with heat tolerance (36/30°C for 1 week) (Sharma K. D. et al., 2015). Heat-tolerant genotypes with high g_S also had higher photosynthetic efficiency under severe heat stress; therefore, this trait acts as a useful genetic tool for developing heat tolerance. Stomatal conductance increased in heat-stressed tomato plants, relative to control conditions (Camejo et al., 2005). In another study, heat-tolerant tomato genotypes maintained higher stomatal conductance under stressed conditions (36/28°C), relative to the control (26/18°C). Further, heat stress severely affected stomatal anatomy and stomatal number in heat-sensitive genotypes, relative to heat-tolerant genotypes (Zhou et al., 2015).

Multiple screening parameters, including stomatal conductance, were used to screen 15 common bean genotypes for heat tolerance in a greenhouse chamber (Traub et al., 2018). Five genotypes—SB761, SB776, SB781, Jaguar, and TB1—were screened at three temperature regimes (35/30, 40/35, 45/40°C). Stomatal conductance increased with increasing temperature until 40/35°C—after which, it declined—genotype TB1 had the highest values for stomatal conductance. In mungbean genotypes, g_S increased up to 40/30°C but declined significantly under heat stress at 43/30°C and 45/32°C, contributing to a rise in leaf temperature (Kaur et al., 2015). In another study on mungbean, g_S was used to differentiate between heat-tolerant and heat-sensitive genotypes (Sharma et al., 2016). Using a similar approach, Sita et al. (2017a) identified heat-tolerant lentil genotypes (IG2507, IG3263, IG3745, IG4258, and FLIP2009) on the basis of stomatal conductance, with g_S increasing with increasing temperature up to 38/28°C in heat-tolerant genotypes. Heat-tolerant genotypes also had higher g_S values under late-sown than normal-sown conditions; in contrast, heat-sensitive genotypes were unable to maintain higher g_S under heat stress. In chickpea, heat-tolerant (ICC1356, ICC15614) and heat-sensitive genotypes (ICC4567, ICC5912) genotypes were selected on the basis of leaf and seed traits (Awasthi et al., 2014)—heat-tolerant genotypes maintained higher stomatal conductance and photosynthetic function than heat-sensitive genotypes under similar conditions and produced more seed yield. Evaluation of three varieties of tomato (Nagcarlang, Hybrid 61 and Moskvich) against combined heat and drought stresses (25–45°C; 20% irrigation; 2 days), revealed that genotype Hybrid 61 performed better by maintaining higher stomatal conductance and having lower leaf temperature than other two varieties (Nankishore and Farrell, 2016), suggesting this trait to be useful even under stress combinations.

Carbon isotope discrimination has become an important tool for interpreting photosynthetic rate and water use efficiency (WUE) in plant species (Sheshshayee et al., 2003; Bindumadhava et al., 2011). ¹²C (98.89%) and ¹³C (1.11%) are the two stable carbon isotopes (non-radioactive) in the global carbon pool. Small but significant amount of ¹³C (heavy isotope) incorporated in the organic and inorganic matter during CO₂ fixation by carboxylating enzymes. These small differences in ¹³C abundance are expressed as Carbon isotope ratio and analyzed with isotope ratio mass spectrometer (IRMS) (Farquhar et al., 1989). Composition of carbon isotopes in plant tissue samples show photosynthetic ability governed by RuBisCO in mesophyll tissues (Bindumadhava et al., 2005, 2011, Impa et al., 2005). Lower values of CID represent lower stomatal conductance (limited diffusion of CO₂) and vice versa (Bindumadhava et al., 2011). Further, under high temperature, leaf water status declines due to reduced root hydraulic conductivity, resulting in stomatal closure (Hairat and Khurana, 2016). Therefore, lower CID values at high temperature can be ascribed to indicate declined root absorption and stomatal closure. In barley, carbon-13 discrimination is a useful indicator of high yield (Craufurd et al., 1999), and could be a sound screening parameter for identifying heat-tolerant genotypes. Heat-tolerant (C306, K7903) and heat-sensitive (HD2329) wheat genotypes were identified from CID values and other physiological traits. The heat-tolerant genotypes had higher mean CID values at high temperature (42°C) than the heat-sensitive genotypes. This study demonstrated that the heat-tolerant genotype maintained stomatal opening by accumulating osmolytes, such as proline, to maintain osmotic pressure for water absorption (Hairat and Khurana, 2016).

Heat stress negatively affects photosynthesis by decreasing leaf pigment content and damaging leaf ultrastructure. Chloroplasts play a vital role in photosynthesis as one of the most heat-sensitive organelles (Krause and Santarius, 1975; Ogweno et al., 2008; Abdelmageed and Gruda, 2009). Decreases in total chlorophyll content and changes in the chlorophyll a/b ratio have been correlated with reductions in photosynthesis during heat stress, due to reduced “antenna (pigment units)” size and thus reduced light-harvesting (Blum, 1986; Harding et al., 1990; Shanmugam et al., 2013). The stay-green (SGR) trait, or delayed leaf senescence, is a crucial trait that allows plants to retain leaves in an active photosynthetic state under high temperature to maintain the assimilation process and increase crop yield (Gregersen et al., 2013; Kumari et al., 2013). Stay-green rice genotypes exhibited high photosynthetic activities under heat stress, resulting in high yields (Jagadish et al., 2015).

Chlorophyll content is an integrative trait that is correlated with stomatal conductance, photosynthetic rate, and transpiration (Del Blanco et al., 2000; Netto et al., 2005), and considered a good criterion for screening for heat-stress tolerance. In the current era of global climate change, introduction of the SGR trait is vital for developing heat-resistant cultivars (Kumari et al., 2013). The SGR trait has been linked to increased yield production in many crops under heat stress, including wheat, barley, rice, maize, and cowpea (Kumari et al., 2007; Borrell et al., 2014; Kobata et al., 2015; Gous et al., 2016; Abdelrahman et al., 2017). The stay-green trait has helped to identify heat-tolerant wheat cultivars that maintain yields at high temperatures (Vijayalakshmi et al., 2010). A significant correlation was detected between heat tolerance and the stay-green trait in 936 elite wheat genotypes (Kumari et al., 2007), suggesting that delayed senescence is an essential selection criterion for heat adaptability. The stay-green characteristic of wheat cultivar Mairaj-2008 was correlated with higher grain yield under heat stress than other lines that lacked the stay-green trait (Nawaz et al., 2013). Genotypes with delayed leaf senescence or stay-green traits have been associated with thermotolerance, due to the longer grain-filling period and thus higher yields, relative to genotypes lacking these traits (Reynolds et al., 1997; Vijayalakshmi et al., 2010). Delayed leaf senescence enhances the transpiration use efficiency, resulting in higher yields. Thus, the stay-green trait is beneficial for retaining active photosynthesis under heat stress (Bavei et al., 2011).

The stay-green trait was used to identify three promising heat-tolerant wheat genotypes [CB-367 (BB#2/PT//CC/INIA/3/ALD“S”), CB-333 (WL 711/3/KAL/BB//ALD “S”), and CB-335 (WL711/CROW “S”//ALD#1/CMH 77A] based on maximum grain development and survival under heat stress (32°C for 4 weeks) (Rehman et al., 2009). Two recombinant inbred lines (RILs) of wheat, SB062 and SB003, were exposed to 7-day heat shocks (32.7/21.6°C day/night) in a growth chamber during the vegetative or reproductive stage. SB062 maintained leaf greenness for longer than SB003 under heat stress and identified as heat-tolerant; in addition, delayed leaf senescence appeared to play a role in maintaining grain size in SB062 under heat stress (Ullah and Chenu, 2019). Lu et al. (1997) suggested that higher stomatal conductance and photosynthetic rate are functionally important for higher heat tolerance and yields. A high temperature (38/28°C) treatment for 6 days under controlled conditions in a greenhouse modified chlorophyll content in two contrasting maize genotypes; DTPYC9F119 maintained higher leaf chlorophyll content (identified as heat-stress tolerant) than K64R (identified as heat-stress susceptible) (Debnath et al., 2016; Singh et al., 2020). In another study, 12 barley genotypes were exposed to heat stress (> 40°C) for 107–119 days in the field—genotypes L3, L6, L8, and L10 had longer stay-green duration and higher yields under heat stress than the other genotypes. Fifteen cotton genotypes were screened for thermotolerance (40°C) in the field—genotypes AGC375 and AGC208 were identified as heat-tolerant based on their chlorophyll content (Karademir et al., 2012). In a similar study, cotton genotype Sicot 53 had higher thermotolerance than Sicala 45 (Cottee et al., 2007). In rice, cultivar N44 was identified as heat-tolerant (exposed to 38°C for 25 days in the field during the reproductive stage), with its higher chlorophyll content under heat stress than N-22 (Bahuguna et al., 2015).

Chlorophyll content was used to screen for heat tolerance in several lentil genotypes after exposure to heat stress (>32/20°C) in a growth chamber at the vegetative and reproductive stage. Heat-tolerant genotypes IG3263 and IG2507 had more chlorophyll than heat-sensitive genotypes IG4242 and IG3964, which was positively correlated with yield (Sita et al., 2017a). In chickpea, genotypes were selected for heat tolerance based on the SGR trait; plants were exposed to gradual increasing temperatures (2°C per day) from 27/18°C to 42/25°C day/night for 8 days in a growth chamber; at which time, genotype ICC16374 (heat-sensitive) had lower leaf chlorophyll content than JG14 (heat-tolerant) (Parankusam et al., 2017). Likewise, Kaushal et al. (2013) identified two heat-tolerant (ICC15614, ICCV92944) and two heat-sensitive (ICC10685, ICC5912) chickpea genotypes based on chlorophyll content, after exposure to heat stress (>32°C/20°C) in the field during reproductive development. The stay-green trait could be used as a morphological indicator for thermotolerance in tomato, as in wheat (Sharma D. K. et al., 2015; Zhou et al., 2015). The stay-green trait contributes to high yield in tomato exposed to heat stress (Zhou et al., 2015). Tomato’s ability to stay-green and maintain photosynthesis during heat stress at different developmental stages, especially anthesis, could be vital for reproductive growth and yield (Zhou et al., 2017). Heat-sensitive tomato genotypes do not stay-green under heat stress due to the decline in chlorophyll and carotenoid contents, and show early chlorosis and withered leaves (Vijayalakshmi et al., 2010; Zhou et al., 2015).

Chlorophyll fluorescence (F_v/F_m ratio) is a relatively sensitive indicator of direct or indirect effects of abiotic stress on photosynthesis (Schreiber and Bilger, 1993). The relationships between primary photosynthetic reactions and chlorophyll fluorescence are crucial as they provide information on the plant’s photosynthetic capability and its acclimation capacity under stressful environmental conditions (Lichtenthaler, 1987; Kalaji et al., 2018). Of the photosynthetic apparatus, photosystem II (PSII) is the most heat-labile cell structure (Vacha et al., 2007). As damage to PSII is often the first response when plants are subjected to heat stress, PSII response studies can reveal the primary effects of heat stress on plants (Mathur et al., 2011; Van der Tol et al., 2014); measuring chlorophyll a fluorescence is an effective and non-invasive technique to identify damage to PSII efficiency (Baker and Rosenqvist, 2004; Baker, 2008). The ratio between variable fluorescence (F_v) and maximum fluorescence (F_m), or F_v/F_m, reflects the maximum quantum efficiency of PSII (Butler, 1978), and is one of the most heat-affected fluorescence parameters. A decline in F_v/F_m is frequently observed when plants are subjected to abiotic stress, including heat (Willits and Peet, 2001; Molina-Bravo et al., 2011; Sharma et al., 2012). There is a negative linear correlation between F_v/F_m and the maximum quantum yield of photosynthesis, when measured as O₂ evolution (Demmig and Björkman, 1987; Kao and Forseth, 1992) and CO₂ fixation (Ogren and Sjostrom, 1990). Screening methodologies using chlorophyll fluorescence to detect and quantify damage in photosystem II (PSII) and thylakoid membranes in response to temperature stress have been used in several cereal crops, including barley (Rizza et al., 2011), wheat (Balouchi, 2010), maize (Sinsawat et al., 2004), legume crops [chickpea, groundnut, pigeon pea (Cajnus cajan), and soybean] (Srinivasan et al., 1996; Herzog and Chai-Arree, 2012), and horticultural crops, including strawberry (Fragaria ananassa) (Ledesma et al., 2004; Kadir et al., 2006), tomato (Willits and Peet, 2001), grapes (Vitis vinifera) (Kadir et al., 2007), and various tropical and subtropical fruits (Yamada et al., 1996; Weng and Lai, 2005). Therefore, chlorophyll fluorescence is a promising tool for detecting stress-induced injuries and thermotolerance (Méthy et al., 1994) but its successful implementation in crop breeding programs requires careful selection of suitable fluorescence parameters (Malaspina et al., 2014).

Heat-tolerant wheat lines with tolerance to high temperatures during grain filling had greater F_v/F_m ratios than heat-sensitive lines in warmer irrigated environments, which were linked to higher grain yield (Shefazadeh et al., 2012). The physiological state of thylakoid membranes, as determined by chlorophyll a fluorescence, identified heat-tolerant wheat cultivars with high chlorophyll fluorescence (Ristic et al., 2007). Various wheat lines were exposed to heat stress for 3 days at 40°C in controlled conditions; the lines having high chlorophyll fluorescence (F_v/F_m 0.836)—830, 1313, 1039, 1223—were less sensitive to heat in terms of growth and photosynthesis than the other lines, and were identified as heat-tolerant (Sharma et al., 2014). Similarly, genotypic variation for chlorophyll fluorescence parameters exists in rice under heat stress (29°C for 25 days at anthesis) in a growth chamber; N22 genotype maintained high F_v/F_m (0.75) under heat stress, and was identified as heat-tolerant, relative to the low F_v/F_m (0.70) in Vandana (Sailaja et al., 2015). Modified chlorophyll fluorescence imaging was used to screen 20 wild barley (Hordeum spontaneum) genotypes exposed to heat stress (45°C, 1 h) in growth chambers, and identified HOR10478 as the most heat-sensitive and HOR12818 as the most heat-tolerant genotypes (Jedmowski and Brüggemann, 2015). Oukarroum et al. (2016) also differentiated heat tolerance in 10 varieties of barley. After 2 weeks of growth, detached leaves were exposed to a short-term heat treatment at 45°C for 10 min in a growth chamber, which decreased chlorophyll fluorescence; notably, varieties Ig, Im, and Tz had high chlorophyll fluorescence (heat-tolerant) and Ma, Ra and I_gr had low chlorophyll fluorescence (heat-sensitive).

In many legumes, chlorophyll fluorescence has been used to identify genotypes that tolerate heat stress. In lentil, photosynthetic efficiency was measured as PSII function (F_v/F_m ratio) in a natural environment by exposing plants to heat stress (above 32/20°C) during the reproductive stage. Heat-tolerant genotypes—IG2507, IG3263, IG3297, IG3312, IG3327, IG3546, IG3330, IG3745, IG4258, and FLIP2009—maintained high chlorophyll fluorescence (F_v/F_m 0.71) under heat stress, relative to heat-sensitive genotypes—IG2821, IG2849, IG4242, IG3973, IG3964—which had the lowest F_v/F_m values (0.58) (Sita et al., 2017a). Nine common bean lines were measured for changes in chlorophyll fluorescence under heat stress at flowering (2 h at 45°C) in a greenhouse; thermotolerant lines 83201007 and RRR46 had higher F_v/F_m values under heat stress than the heat-sensitive line Secuntsa (Petkova et al., 2009). In another study, 12 varieties and lines of common bean were exposed to 42°C in the field during the reproductive period; two genotypes (Ranit and Nerine) maintained F_v/F_m values at 42°C, relative to the controls at 26°C, and were considered heat-tolerant. These two genotypes also showed good productivity and quality and can be used as parental lines in bean breeding programs (Petkova et al., 2007). Likewise, 41 mungbean lines were grown outdoors and exposed to high temperatures (>40/28°C) during the reproductive stage; several promising heat-tolerant lines (EC693358, EC693357, EC693369, Harsha, and ML1299) were identified, with high F_v/F_m ratios (0.73–0.75 units) compared to sensitive lines (0.61–0.67 units), which would not only serve as useful donor/s for breeding programs, but also as suitable base plant source to gain insight into heat-stress-induced effects in cell metabolism (Sharma et al., 2016). In chickpea, heat stress (>30°C) in the field during the reproductive stage reduced F_v/F_m more (0.48, 0.41) in two heat-sensitive genotypes ICC10685 and ICC5912, than in two heat-tolerant genotypes ICC15614 and ICCV92944 (0.64, 0.60) (Kaushal et al., 2013; Awasthi et al., 2014). A field experiment conducted in two winter seasons at three locations with known differences in temperature in NE South Africa, involving four chickpea genotypes, showed. that two genotypes, which were tolerant to heat stress had chlorophyll fluorescence (Fv/Fm) of 0.83–0.85 at the warmer site, while the two sensitive genotypes showed lower Fv/Fm of 0.78–0.80; these values correlated positively with grain yield. The two tolerant genotypes had higher photosynthetic rates, starch, sucrose and grain yield than the sensitive genotypes at the warmer site. The observation revealed that chlorophyll fluorescence and leaf carbohydrates are suitable tools for selection of heat tolerant chickpea genotypes under field conditions (Makonya et al., 2019). Screening of 15 alfalfa (Medicago sativa L.) genotypes by exposing seedlings to 38/35°C day/night for 7 days in a growth chamber identified Bara310SC (F_v/F_m 0.79) and WL712 (F_v/F_m < 0.79) as heat-tolerant and heat-sensitive cultivars, respectively (Wassie et al., 2019), showing that F_v/F_m is an effective tool for phenotyping contrasting genotypes for heat tolerance.

The heat susceptibilities of 67 tomato genotypes were evaluated in a climate chamber—the genotypes with higher F_v/F_m under heat stress (36/28°C for 4 days or 40°C for 7 h), maintained their physiological status, relative to genotypes with lower F_v/F_m (Zhou et al., 2015). The two genotypes with the highest F_v/F_m ratios (heat-tolerant group; T1, T2; 0.82, 0.80 units) and two with the lowest F_v/F_m ratios (heat-sensitive group; S1 and S2; 0.74, 0.77 units) were selected for further study (Zhou et al., 2015). Another study screened wild genotypes and cultivars of tomato in a growth chamber at 33°C—wild tomato varieties Pe and Pr1 had the highest temperature stress tolerance with high F_v/F_m ratios (0.56, 0.58), while the cultivated species were more sensitive to temperature stress with lower F_v/F_m ratios (0. 28, 0.38) (Zhou et al., 2018).

Chlorophyll fluorescence was used to screen cotton landraces—6-week-old cotton plants were subjected to heat stress at 45°C in a growth chamber to determine thermotolerance in terms of photosynthetic ability, independent of agronomic yield and productivity. Three genotypes (TX2287, TX2285, and TX761) maintained high photosynthetic efficiency (F_v/F_m 0.57), relative to sensitive genotype (F_v/F_m 0.46) (Wu et al., 2014). In another growth chamber study, a commercial set of eight cotton genotypes was screened for heat tolerance by subjecting to heat stress (>35°C); four genotypes (SG215BR, ST474, and DP444BG/RR) had relatively high F_v/F_m indicating that they suffered less from stress, while Sphinx and Acala Riata had low F_v/F_m, indicating temperature sensitivity (Bibi et al., 2004). In a related study, screening of 15 cotton genotypes for thermotolerance (40°C) in the field identified genotypes AGC375 and AGC208 as heat-tolerant, based on their superior chlorophyll fluorescence (Karademir et al., 2012). Imposing combined drought and heat stress significantly affected the photosynthetic efficiency of chickpea (Cicer arietinum) genotypes, in a study conducted in outdoor conditions at two different sowing times [November (<32–20°C at the time of reproductive stage; control) and in February (>32–20°C at the time of reproductive stage; heat stress during pod filling)], while drought was applied during both sowing times during pod filling (at ∼75% podding) by withholding water until maturity. The photosynthetic efficiency (Fv/Fm) of the leaves decreased more in plants subjected to drought stress (54–74%) than to heat stress alone (9–46%) and the combined heat + drought stress treatment showed the greatest reduction in photosynthetic efficiency (68–83%), with the smallest reduction occurring in the drought-tolerant genotype (ICC8950), compared to drought-and heat sensitive genotypes (Awasthi et al., 2017).

Heat stress affects plant characteristics such as the stay-green trait, chlorophyll content, and chlorophyll fluorescence, which influences the photosynthetic rate (Sharkey, 2005). Hence, photosynthetic rate can be used as a screening parameter for the selection of heat-tolerant genotypes. Variation in photosynthetic rate among plant species in response to heat stress has been well-documented. For example, a heat-shock treatment (45°C for 2 h at the fourth true leaf stage) reduced the net photosynthetic rate (P_n) of two tomato cultivars, more so in Campbell-28 (heat-sensitive) than wild Nagcarlang (heat-tolerant) (Camejo et al., 2005). High temperature deactivates RuBisCo, which could be involved in reducing photosynthetic rate (Sharkey, 2005). Another study on tomato compared the P_n of one cultivated (Ly from Solanum lycopersicum) and six wild (Ha from S. habrochaites, Pe from S. pennellii, Pi1 and Pi2 from S. pimpinellifolium, Pr1 and Pr2 from S. peruvianum) genotypes grown at high temperature (33°C) in a growth chamber—Ly, Ha, Pi1, and Pi2 had lower P_n than the control, while Pe, Pr1, and Pr2 showed higher P_n indicating their heat tolerance (Zhou et al., 2018). Plants of the tomato cultivar “Liaoyuanduoli” grown in greenhouse exposed to heat stress (35°C after 15 DAS led to a significant change in photosynthetic apparatus as damage of chloroplast membrane and at the same time, the thylakoids loosely distributed with lesser grana, thus, changed chloroplast ultrastructure might have declined the P_n (Zhang et al., 2014). In rice, heat tolerant genotype (N22) could maintain photosynthetic activity for a longer time after anthesis and thus could produce higher grain weights, compared to heat-sensitive genotypes (IR20, IR53, IR46) (Gesch et al., 2003).

Soybean cultivars (IA3023 and KS4694) and PI lines (PI393540 and PI588026A) expressed heat tolerance and susceptibility with high and low P_n, respectively (Djanaguiraman et al., 2019). The soybean cultivars had less thylakoid membrane damage than the PI lines. In an earlier study on soybean genotype K03−2897, high-temperature stress (38/28°C) for 14 days at the flowering stage significantly decreased leaf P_n, due to anatomical and structural changes (increased thickness of palisade and spongy layers and lower epidermis) in cells and cell organelles, particularly damage to chloroplasts and mitochondria (Djanaguiraman and Prasad, 2010). Two heat-tolerant chickpea genotypes (Acc#RR-3, Acc#7) had higher P_n than two heat-sensitive genotypes (Acc#2, Acc#8) at high temperature (35/30°C), which may have been due to increased RuBisCo activity (Makonya et al., 2019). In another chickpea study, 56 genotypes were exposed to high temperatures in the field from the flowering stage to crop maturity (maximum temperatures 25–40°C)—the tolerant genotypes (PUSA1103, PUSA1003, KWR108, BGM408, BG240, PG95333, JG14, BG) had higher Pn than the sensitive genotypes (ICC1882, PUSA372, PUSA2024) (Kumar et al., 2017). Similarly, the response of four chickpea genotypes to a natural temperature gradient in the field at the flowering stage identified two heat-tolerant genotypes (Acc#RR-3, Acc#7) with high P_n and two heat-sensitive genotypes (Acc#2, Acc#8) with lower P_n; these results were validated in a climate chamber experiment set at 30/25°C and 35/30°C (Makonya et al., 2019). Improvement of heat stress tolerance by stabilizing PSII system through introducing IbOr gene in transgenic potato (Goo et al., 2015), sweet potato (Kang et al., 2017), and in alfalfa (Wang et al., 2015) is worth mentioning. Heat, drought and their combination limited the Photosynthetic rate of lentil (Lens culinaris Medikus), particularly during reproductive growth and seed filling. In recent study eight lentil genotypes two drought-tolerant (DT; DPL53 and JL1), two drought-sensitive (DS; ILL 2150 and ILL 4345), two heat-tolerant (HT; 1G 2507 and 1G 4258) and two heat-sensitive (HS; 1G 3973 and 1G 3964) sown at the normal time (November), at the time of seed filling (30/20°C), or sown late (February) to impose heat stress (> 30/20°C (day/night) and drought maintained by water withheld (50% of field capacity) from the start of seed filling to maturity. The photosynthetic rate (Pn) decreased significantly more under drought stress (33.4–56.6%) than heat stress (13.3–43%), as compared to the control plants. Under the combined stress, Pn declined more (57–82% reduction), less so in the heat and drought tolerant genotypes compared to sensitive (Sehgal et al., 2017).

Leaf photosynthates are largely transported to sink organs in the form of sucrose, and sucrose synthase (SS) is a key enzyme for sucrose to enter a variety of metabolic pathways (Lu et al., 2005). Down-regulation of SS indirectly inhibits carbohydrate production, eventually reducing yield and quality. Maintaining sucrose levels is vital during stressed conditions, which depend on its synthesis and hydrolysis. Heat-stressed plants had significant reductions in the activity of key enzymes—sucrose phosphate synthase (SPS) and SS—involved in sucrose synthesis. The availability of sucrose to reproductive organs is crucial for sustaining their function (Kaushal et al., 2013). Heat-tolerant genotypes are expected to stabilize the photosynthetic process better than heat-sensitive genotypes. Measuring sucrose concentrations reveals the photosynthetic status of plants under heat stress (Awasthi et al., 2014). A large core-collection of chickpea genotypes screened or heat tolerance (32/20°C) in a natural environment identified two heat-tolerant (ICC15614, ICCV92944) and two heat-sensitive (ICC10685, ICC5912) genotypes. The heat-sensitive genotypes had significantly greater inhibition of RuBisCo (carbon-fixing enzyme), SPS, and SS than the heat-tolerant genotypes, and thus produced less sucrose than the tolerant genotypes (Kaushal et al., 2013). Heat-sensitive genotypes produced far less leaf sucrose than heat-tolerant genotypes, which impaired its supply to developing reproductive organs (flowers, pods, and seeds) in sorghum (Prasad and Djanaguiraman, 2011), tomato (Li et al., 2012), and chickpea (Kaushal et al., 2013).

In wheat, heat-tolerant genotypes (PBW343 and C306) exposed to heat stress (>25°C) in the field had higher SS activity and thus higher sucrose contents in grain than heat-sensitive genotypes (PBW521, PBW522) (Bavita et al., 2012). Limitations in sucrose supply may disrupt the development and function of reproductive organs (Prasad and Djanaguiraman, 2011; Snider et al., 2011). In lentil, sucrose production is vital for leaf and anther function, and has been correlated with SPS activity in natural high-temperature environments (> 32/20°C). Heat-tolerant lentil genotypes (IG2507, IG3263, IG3297, IG3312, IG3327, IG3546, IG3330, IG3745, IG4258, and FLIP2009) produced more sucrose in their leaves (65–73%) and anthers (35–78%), than heat-sensitive genotypes (IG2821, IG2849, IG4242, IG3973, IG3964), which was associated with superior reproductive function and nodulation in tolerant genotypes (Sita et al., 2017a). Thus, heat stress negatively affects sucrose metabolism due to the inhibition of carbon fixation and assimilation (Awasthi et al., 2014). Sucrose concentrations in leaves and anthers and SS and SPS activities declined significantly in two mungbean genotypes (SML832 and SML668) exposed to heat stress (>40/25°C day/night) outdoors and in a controlled environment, more so in SML668 (heat-tolerant) than SML832 (heat-susceptible) (Kaur et al., 2015). Tomato cultivars exposed to heat stress in growth chambers (31/25°C day/night) or greenhouses (32/26°C day/night) revealed four genotypes (FLA7516, Hazera3018, Hazera3042, and Saladate) as heat-tolerant with high sucrose contents in the mature pollen grains, and three genotypes (Grace, NC8288, and Hazera3017) as heat-sensitive, with 50% less sucrose than the tolerant genotypes (Firon et al., 2006).

Expression of the sucrose transporter gene, OsSUT1, is important for maintaining photo-assimilate supply to grains. In rice exposed to high-temperature stress (31/26°C) in a glasshouse, cultivar Genkitsukushi (heat-tolerant) had higher expression of OsSUT1 in stems than Tsukushiroman (heat-sensitive), indicating that sugar transport is more effective in Genkitsukushi than Tsukushiroman under heat stress, which improves grain quality (Miyazaki et al., 2013).

Heat stress negatively affects cellular metabolism due to extensive ROS production that can severely damage lipids, proteins, and nucleic acids (Bita and Gerats, 2013). Plants protect themselves from ROS production by activating enzymatic and non-enzymatic processes (Bita and Gerats, 2013). The main ROS-scavenging enzymes are superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR), and the non-enzymatic system includes ascorbic acid (ASC) and glutathione (GSH) (Suzuki et al., 2012). Genotypes can be selected based on their enzyme expression level, with more prominent activities among heat-tolerant than heat-sensitive genotypes (Kumar et al., 2013). Genotypes respond differently to heat stress due to variation in their antioxidant systems. Hence, this trait is useful for identifying heat-tolerant genotypes.

Two tomato cultivars differing in heat sensitivity (Sufen14, Jinlingmeiyu) were raised in a greenhouse in optimum temperature (26/18°C) and heat-stressed (38/30°C for 6 days with 2 days recovery). Jinlingmeiyu had lower activities of SOD, POD, APX, and MDA (malondialdehyde) and lower proline content than Sufen14, suggesting the involvement of these enzymes in imparting heat tolerance in Sufen14 (Zhou et al., 2019). Categorization of 50 Brassica juncea genotypes into tolerant, moderately tolerant and susceptible genotypes after exposure to 45°C was based on oxidative damage tolerant genotypes had less lipid peroxidation and higher POD, CAT, and GR activities than moderately tolerant and susceptible genotypes (Wilson et al., 2014). In contrast, Brassica juncea seedlings grown under optimum (25°C) and high (45°C) temperatures had higher MDA and lipoxygenase (LOX) activities of antioxidants (SOD, CAT, POX, APX, and GR) in the thermosensitive genotype (NPJ-119) than the thermotolerant genotype (NRCDR-02) suggesting variations in the response of antioxidatnts, which might be stage-or plant-specific (Rani et al., 2012). Wheat genotypes were differentiated into heat-tolerant (C306), intermediate heat-tolerant (HD2285), and heat-sensitive genotype (HD2329) by subjecting them to heat stress (8 and 23 days after anthesis) by delaying the sowing time: C306 had higher relative water content, ASC, APO, CAT, and SOD and lower lipid peroxidation and H₂O₂ content than HD2285 and HD2329 (Sairam and Srivastava, 2000).

In chickpea plants raised under natural conditions and heat stressed at 50% flowering (30/20, 35/25, 40/30, and 45/35°C) in growth chambers, tolerant genotypes (ICCV07110, ICCV92944) had lower MDA concentration and H₂O₂ content than sensitive genotypes (ICC14183, ICC5912), which was attributed to their higher activity levels of APX, GR, and ASC (Kumar et al., 2013). Forty-one mungbean genotypes exposed to heat stress (>40/28°C) in the field revealed that heat-tolerant genotypes (EC693357, EC693358, EC693369, Harsha, and ML1299) suffered less oxidative damage (1.52–2.0-fold increase MDA; 1.59–1.96-fold increase H₂O₂) than sensitive genotypes (2.2–2.4-fold increase MDA; 2.21–2.93-fold H₂O₂) (Sharma et al., 2016). The heat-tolerant genotypes also significantly increased APX activity (by 1.48–1.77-fold), relative to susceptible genotypes (1.27–1.37-fold) and similar response was observed for GR activity. However, heat-tolerant and heat-sensitive genotypes had similar increases in CAT activity. Similarly, 38 lentil accessions screened for heat tolerance (>35/20°C) during the reproductive stage revealed less oxidative damage (MDA and H₂O₂ contents increased) and higher SOD, CAT, APX, and GR activities—involved in detoxification—in heat-tolerant genotypes (IG2507, IG3263, IG3745, IG4258 and FLIP2009) than heat-sensitive genotypes (IG2821, IG2849, IG4242, IG3973, IG3964 (Sita et al., 2017a). Concurrence of heat and drought stress will do more damage at the biochemical level. Oxidative damage and antioxidant mechanisms responding toward combined stress were reported in tomato cultivars. Two cultivars of tomato (CV1; Sufen14 and CV2; Jinlingmeiyu) were raised in green house conditions to compare the cultivar difference. Treatment (Heat stress-38/30°C, and drought stress-no irrigation) were given to 28 days old seedlings for six days. Significant increase in ROS such as H₂O₂ and O^2– were reported in both the cultivars than control (26/18°C). Their studies showed that CV2 had lower activity of enzymes-peroxidase, ascrobate peroxidase, superoxide dismutase, malondialdehyde (MDA) and proline content than CV1, under combined stress on day 6, clearly depicting cultivar differences with respect to antioxidant activity (Zhou et al., 2019).

Plant metabolites are low molecular weight compounds involved in stress tolerance. They play a crucial role in maintaining the redox homeostasis of cells and stabilizing cell membranes and proteins (Wahid et al., 2007) through various intermediate/precursor compounds, such as compatible solutes, signaling agents, and antioxidants (Kaplan et al., 2004). Metabolites are categorized into primary and secondary metabolites. Primary metabolites that are specifically upregulated in response to abiotic stress are amino acids (proline), polyamines (spermidine, spermine, putrescine), carbohydrates (sucrose, hexoses, polyhydric alcohols), and glycine betaine. Similarly, secondary metabolites include phenolic compounds (flavonoids, isoflavonoids, anthocyanins), terpenoids (saponins, tocopherols), and nitrogen-containing metabolites (alkaloids and glucosinolates) (Rodziewicz et al., 2014). Under heat stress, plants restructure their metabolites to help the cells to maintain homeostasis via the production of stress-induced compounds (Serrano et al., 2019). Activation of heat-shock factors, such as HSFA2 and HSFA3, increases the level of metabolites such as galactinol and its derivatives in response to heat stress (Song et al., 2016). Therefore, metabolites may serve as a useful tool for selecting heat-tolerant varieties under high-temperature stress. Comparing heat-tolerant and heat-sensitive genotypes can identify metabolite markers that are constitutively expressed and allow selection of superior germplasm.

Seed metabolomic analysis performed on contrasting soybean genotypes (PI587982A, heat-tolerant; A5279 and DP3478, heat-sensitive) revealed 25 metabolites that differed between genotypes, including tocopherol isoforms, ascorbate precursors, flavonoids, two amino acids, and amino acid derivatives (Chebrolu et al., 2016). At 36°C, 10 flavonoids were more abundant in the seeds of the heat-tolerant genotype than the heat-sensitive genotypes, along with several tocopherols (major antioxidants). Moreover, the heat-tolerant genotype had higher levels of a precursor of L-ascorbic acid biosynthesis—gulono-1,4-lactone—than the heat-tolerant genotypes. Overexpression of these stress-induced compounds provides thermotolerance to soybean seeds, which ultimately perform better in terms of seed vigor, seed germination, seed weight, and oil content. Metabolomic analysis of rice spikelets in a heat-tolerant (N22) and heat-sensitive (Moroberekan) genotype revealed that N22 accumulated more metabolites than Moroberekan, including carbohydrates (glucose 6-phosphate, fructose 6-phosphate, glucose, maltose, and other sugars), compatible solutes, and amino acids (leucine, isoleucine, and valine). N22 had lower levels of trehalose, sugar phosphatases, malic acid, and galactaric acid than Moroberekan under heat stress (Li X. et al., 2015). In wheat, a comparative analysis of metabolites in transgenic wheat (PC₂₇ and PC₅) and its wild type (varying in heat sensitivity), exposed to heat stress (40°C for 4 h) during heading revealed 25 metabolites that were highly expressed in transgenic wheat, including proline, three sugar alcohols (inositol, mannitol, and xylitol), pyruvic acid, and other amino acids (glycine, alanine, serine, valine, and tyrosine) (Qi et al., 2017). The metabolite profiling approach is an effective way to accurately screen and select the best-performing genotypes.

Proline is a multifunctional amino acid with diverse roles in maintaining cellular redox balance by dissipating excess of reducing potential (Rivero et al., 2004). Proline levels are upregulated under stress conditions as its biosynthesis is an adaptive response to reduce excess NADPH produced in response to the halt in CO₂ fixation in the Calvin cycle due to stomatal closure (Berry and Bjorkman, 1980). Moreover, under stress conditions, proline is involved in osmotic adjustment, ROS scavenging, and as an energy source. Therefore, high proline contents under high-temperature stress can be used to screen heat-tolerant genotypes. Twenty wheat genotypes were screened for heat tolerance by exposing them to 25 or 35°C, and measuring proline content and membrane damage (Ahmed and Hasan, 2011). Heat-tolerant genotypes (Bijoy, Sufi, Kanchan, Fang 60, BAW 1059, BL 1883, BL 1022, IVT 7, IVT 8, IVT 9, IVT 10, and BAW 917) had higher proline contents (>200%) and less membrane damage (<50%) than heat-sensitive genotypes (Shatabdi, PRODIP, BAW 1064, Gourab, Pavon 76, Sonara, Kalyansona, and IVT 6). Thirty-five-day-old seedlings of different cabbage cultivars, including Chinese cabbage and their hybrids, were exposed to two temperature regimes (25 or 35°C) at the flower bud stage and assessed for heat tolerance based on proline contents in stalks, flower buds, and leaves—heat-tolerant cultivars (Yoshin, Kenshin, and full white) had higher proline levels than heat-sensitive cultivars (YR Kinshun, Chihiri 70, and Large leaf) (Hossain et al., 1995). Six cotton cultivars (Sicala, Acala 1517-88, Molopo, Alpha, Delta Pine Acala90, and OR19) were tested for genetic variability against combined heat and drought stress. Stress treatment (Heat stress; 40°C without irrigation for 15 days) were imposed on 3 weeks old seedlings. Stress treatment were increased the proline content in all the genotypes but the accumulation was more in tolerant genotypes (Alpha, Delta Pine Acala90, and OR19) compared to sensitive genotypes (Sicala, Acala 1517-88, Molopo) (De Ronde et al., 2000).

During rapid heat stress, plants synthesize and accumulate specific proteins called heat-shock proteins (HSPs) (Howarth, 1991); this is a universal response to high-temperature stress in all organisms (Vierling, 1991). Heat-shock genes are upregulated during stress to encode HSPs which are vital for plant survival under such conditions (Chang et al., 2007). Three classes of HSPs are distinguished, according to molecular weight—HSP90, HSP70, and low molecular weight proteins. HSPs provide stress-related chaperone functions in plants under stress conditions (Reddy et al., 2010, 2016). Chaperones have a role in protein synthesis, maturation, targeting, degradation, renaturation, and membrane stabilization (Reddy et al., 2014, 2016). HSPs are located in the cytoplasm, nucleus, mitochondria, chloroplasts, and endoplasmic reticulum (Waters et al., 1996). Heat-stress transcription factors (HSFs), located in the cytoplasm in an inactive state, control HSP gene transcription and play a vital role in plant thermotolerance. Specific HSPs have been identified in response to high temperature, including HSP68 in the mitochondria of potato, maize, soybean, and barley (Neumann et al., 1994). The expression profiles of HSPs have been compared in plant species/genotypes contrasting in heat sensitivity. For instance, the higher heat tolerance of maize than wheat and rye at 42°C is correlated with the expression of five mitochondrial low molecular weight HSPs (28, 23, 22, 20, and 19 kDa), as opposed to only 20 kDa in wheat and rye (Korotaeva et al., 2001). According to Sharma-Natu et al. (2010), HSP18 was upregulated in developing grains of heat-tolerant wheat exposed to 3.2–3.6°C higher temperatures than normal. In other studies, HSP100 increased with heat stress in a tolerant wheat variety (Sumesh et al., 2008). Similarly, HSP26 increased in heat-tolerant wheat genotypes (K7903, C306) at 42°C, relative to heat-sensitive genotypes (PBW343, HD2329) (Hairat and Khurana, 2016). At 42°C, the expression levels of five Hsps—Hsp26.7, Hsp23.2, Hsp17.9A, Hsp17.4, and Hsp16.9A—were upregulated in the heat-tolerant rice cultivar Co39, relative to the heat-sensitive rice cultivar Azucena, and regarded as biomarkers for screening rice cultivars for heat tolerance (Chen et al., 2014). At 40°C, potato cultivar Norchip synthesized small (sm) Hsps for longer than other cultivars. In Norchip and Desiree, an 18 kDa small (sm)HSP increased for up to 24 h, while in cultivars Russet Burbank and Atlantic, the levels started to decline after 4 and 12 h respectively (Ahn et al., 2004). Anthers of a heat-tolerant tomato cultivar had higher constitutive levels of HSP100 than a heat-sensitive cultivar (Pressman et al., 2007). In chickpea, HSP levels increased in genotype JG14 (heat-tolerant) more than genotype ICC16374 (Heat-sensitive) when exposed to 42/25°C at anthesis (Parankusam et al., 2017). Likewise, in peanut, the best-characterized aspect of acquired thermotolerance is HSP production, with ICGS76, COC038, and COC068 selected as heat-tolerant genotypes and COC812, COC166, Tamrun OL 02, and Spanco selected as heat-sensitive (Selvaraj et al., 2011). In another study, heat-tolerant peanut genotype ICGS 44 showed higher HSP expression throughout the stress period than heat-sensitive genotypes AK 159 and DRG 1 (Chakraborty et al., 2018). Comparison of expression of heat shock proteins in wheat cultivars (Katya and Sadovo) under combined heat and drought stress (40°C/56%) reported 100% elevation of HSP100 and HSP70 as compared to 60 and 10% elevation under individual drought and heat stress in tolerant cultivar Katya (Grigorova et al., 2011).

Heat stress hastens the seed filling rate and reduces the duration of seed filling. In cowpea, raising the temperature from 15.5 to 26.6°C shortened the seed filling duration by 14–21 days (Nielsen and Hall, 1985). Heat stress impaired the growth of the cotyledons, and reduced the number of endosperm cells and cell expansion in the embryo, which had a negative effect on photosynthate translocation in developing seeds and resulted in shriveled seeds in maize (Jones et al., 1985; Munier-Jolain and Ney, 1998). A heat-stressed environment (>32/20°C) during seed development increased the seed filling rate in six chickpea genotypes, relative to the optimum temperature (Awasthi et al., 2014). The same study revealed that heat stress decreased the duration of seed filling more in heat-sensitive (ICC 4567) than heat-tolerant (ICC1356, ICC15614) genotypes. High temperature (25/20°C) reduced the duration of grain filling by 30% and increased the grain-filling rate by 20% in six wheat genotypes (G1, G2, G3, G4, G5, G6), relative to the control (20/15°C), more so in heat-sensitive (G6) than heat-tolerant (G4) genotypes (Yin et al., 2009).

Heat stress leads to poor pollination and fertilization, which reduces seed number. In faba bean (Vicia faba L), seed number declined with increasing temperature (Bishop et al., 2016). In mungbean, heat-tolerant genotype (SML 832) produced more seeds than heat-sensitive genotype (SML 668) under high temperature (45/32°C) in the field (Kaur et al., 2015). While testing 24 genotypes of common bean in the greenhouse under different temperature regimes (24/21°C, 27/24°C, 30/27°C, 33/30°C), 33/30°C was the most damaging to plants with respect to seed number and seeds/pod, with the reductions more prominent in heat-sensitive genotypes (–66%; A55, Labrador, Majestic, IJR) than heat-tolerant genotypes (–31%; Brio, Carson, G122, HB1880, HT38, Venture) (Rainey and Griffiths, 2005). Heat stress (36/27°C) reduced seed number/pod in 46 of 48 lines of cowpea (Vigna unguiculata) evaluated for heat tolerance in a greenhouse; two heat-tolerant lines (B89-600 and TN88-63) did not exhibit reduced seed numbers/pod (Ehlers and Hall, 1998). The average number of seeds/pod varied in the heat-sensitive genotypes (e.g., 3.3 in IT82E-60, 2.9 in Bambey 21 and 3.6 in IT84S-2049), while those of the heat-tolerant genotypes had 6.3 in B89-600 and 8.1 in TN88-63 compared to control values (e.g., 11 in IT84S-2049, 9.6 in IT82E-60, 7.4 in B89-600 and 6.4 in TN88-63).

Seed weight represents the ultimate yield of the crop; hence it has been reliably used as a trait to screen for heat tolerance (Sehgal et al., 2018). Chickpea yields declined when genotypes were exposed to various temperature ranges (35/25°C, 40/30°C, and 45/35°C) in a growth chamber, relative to the control (30/20°C) (Kumar et al., 2013). At 40/30°C, the seed weight of heat-sensitive genotypes (ICC14183, ICC5912) declined by 37–45% compared with heat-tolerant genotypes (ICCV07110, ICCV92944). At 45/35°C, heat-tolerant genotypes also experienced a decline in seed weight but heat-sensitive genotypes did not set any pods. Similarly, mungbean genotypes grown outdoors in April, with high temperatures (45/32°C) coinciding with reproductive phase, reduced seed weight by 48.3% in the heat-sensitive genotype (SML668) and 35.1% in the heat-tolerant genotype (SML832), relative to control (Sharma et al., 2016). Likewise, seed weight of lentil grown at high temperature (>32/20°C) in field declined drastically compared to control plants (Bhandari et al., 2016), more so in heat-sensitive genotypes (–50%; LL699 and LL1122) than the heat-tolerant genotype (–33%; LL931). In common bean, heat stress (33/30°C) under field conditions was significant for the selection of heat-tolerant (Brio, Carson, G122, HB1880, HT38, Venture) and heat-sensitive genotypes (A55, Labrador, Majestic, IJR), based on seed weight. At this temperature, seed weight declined by 47% across genotypes, more so in heat-sensitive genotypes (–88%) than heat-tolerant genotypes (–25%) (Rainey and Griffiths, 2005). In cowpea, studies at two locations with varying temperatures (Coachella (41/25°C) and Riverside (36/17°C) assessed the effect of high temperature on the yield of contrasting genotypes (Ismail and Hall, 1999). Yield parameters such as seed weight and seeds/pod reduced drastically, as the temperature increased, however, heat-tolerant genotypes (H36, H8-9, DLS99) at higher temperature (41/25°C) retained more seed weight (193 mg/seed) than heat-sensitive genotypes (CB5, CB3, DLS127), which had smaller seeds with an average weight of 168 mg. Screening experiments on Pearl millet, conducted over a period of 3–4 years (2009–2012) at ICRISAT, India, involving 221 hybrid parental lines (both B- and R-lines), 53 germplasm accessions and 4 improved populations over 4-year period showed large genetic variability in seed set at daily maximum air-temperature of ≥ 42 °C during flowering. Five hybrid seed parents (ICMB 92777, ICMB 05666, ICMB 00333, ICMB 02333, and ICMB 03555) and a germplasm accession IP 19877 with 61–69% seed set as compared to 71% seed set in a heat tolerant commercial hybrid 9444 (used as a control) was identified. A comparative study on 23 hybrids and their parents for seed set at high air temperature (>42°C) showed heat tolerance as a dominant trait, indicating that heat tolerance in one parent would be ample to generate heat tolerant hybrids in pearl millet (Gupta et al., 2015). In sub-Saharan Africa, 24 elite durum wheat breeding lines and cultivars were tested for adaptation to warm environments at two stations: Kaedi, Mauritania and Fanaye, Senegal. Top grain yield was recorded at 5,330 kg ha^–1 and the average yield at 2,484 kg ha^–1. Biomass and spike fertility (i.e., number of seeds produced per spike) were found to be the most vital adaptive traits to warm environments. The study showed three genotypes (“Bani Suef 5,” “DAWRyT118,” and “DAWRyT123”) as the most stable and high yielding; while the last two genotypes were the best performers (Sall et al., 2018).

Combined drought and heat stress were found to be greatly detrimental for production potential of crops. Thus, lentil genotypes were evaluated for their response to impacts of combined drought and heat stress (drought tolerant: DPL53 and drought sensitive: LL699) (Sehgal et al., 2019). The heat and drought (33/28°C with 50% field capacity) treatments were imposed to determine to effects on yield traits (seed filling duration, seed filling rate, seed number/plant, and seed weight/plant). Under combined stress, a decline in seed filling duration by 5.4–8.9 days, seed growth rate by 44–60.2%, seed number/plant by 35–48.7%, seed weight/plant by 47–59% compared to control. This reduction pattern was more drastic in heat sensitive genotype than heat tolerant genotype. A field experiment on 300 maize inbred lines test-crossed to CML539 was conducted at multiple locations (Tlaltizapán, México (18°41/c N, 99°07/c W, and 940 m asl), Kiboko, Kenya (2°21/c S, 37°72/c E, and 975 m asl), Chiredzi, Zimbabwe (21°01/c S, 31°34/c E, and 430 m asl), at the Nakhonsawan Field Crops Research Center in Takfa, Thailand (15°21/c N, 100°30/c E, and 87 m asl), and at the ICRISAT experimental station in Hyderabad, India) to evaluate their response to reproductive stage drought stress, heat stress, and combined drought and heat stress. The study identified few lines (notably La posta Sequia C7-F64-2-6-2-2 and DTpYC9-F46-1-2-1-2) having higher tolerance to drought and combined drought and heat stress. The findings indicated that tolerance to individual stresses was genetically distinct from tolerance to combined stresses. The assessment indicated that most of the current drought donors and key inbreds used in widely grown African hybrids were sensitive combined drought and heat stresses. The identified lines, as mentioned above, need to be introduced into breeding programs for maize (Cairns et al., 2013).