The persistently rising ambient temperature is regarded as one of the most detrimental stresses among constantly changing environmental factors. It has been predicted that the global mean temperature could rise up to 2.0–4.5°C by the end of this century and the enhancement changes in minimum night temperature will more compare to maximum day temperature (IPCC, 2007). In china, climate conditions during rice growth seasons have changed dramatically, for instance, the particular area of Yangtze River Valley (YRV) is prone to high temperature especially in mid-growing season which may lead to huge yield loss, although the temperature may rarely increase more than 35°C (Matsui, 2009). Plants are sessile organisms, which cannot move to more favorable environments; consequently, plant growth and developmental processes are substantially affected, often lethally, by high temperature (HT) stress (Lobell and Field, 2007). Production of reactive oxygen species (ROS) in excesses amount is one of the major consequences of HT stress, which leads to oxidative stress (Hasanuzzaman et al., 2012, 2013). In response to HT, plants modify their metabolism in different ways particularly by producing compatible solutes, maintaining cell turgor by osmotic adjustment, and regulating the antioxidant system to re-establish the cellular redox balance and homeostasis (Valliyodan and Nguyen, 2006; Janská et al., 2010).

Although, rice possesses a comparatively higher tolerance to high temperature during vegetative stages, but on other sides its vulnerability rises to elevated temperature during the reproductive stage, particularly at flowering (Yoshida et al., 1981; Prasad et al., 2006; Jagadish et al., 2007, 2008, 2010a,b). In the past, the spatial analyses demonstrated the vulnerable periods of rice (i.e., flowering and early grain filling) overlapping with the mounting temperatures phases in countries like Bangladesh, eastern India, southern Myanmar, and northern Thailand (Maclean et al., 2002; Wassmann et al., 2009). Considering the predicted rate of increase in night temperature, the negative impact on rice production is likely to be higher in coming years with huge yield losses. High night temperatures (HNT) are generally related with increased respiration rates, reduced pollination, number of pollen germinated on the stigma, and increased spikelet sterility, which can lead to decrease in final grain yield (Mohammed and Tarpley, 2009a,b; Jagadish et al., 2010a). Likewise, high day temperatures can severely influence the photosynthesis activity, by bringing alterations in thylakoids structural organization and thus interrupting the whole cycle of photosynthetic system II (Karim et al., 1997; Zhang et al., 2005). Moreover, it results in production of excessive ROS, which affects the membrane integrity and may cause cell death (Schoffl et al., 1999; Howarth, 2005).

Various combinations of plant growth regulators (PGR) have been reported to improve the heat tolerance and stand establishment of rice (Mohammed and Tarpley, 2011; Shah et al., 2011; Fahad et al., 2015b) and no negative interaction among PGRs was observed in these studies. The ascorbic acid (vitamin C; Vc), alpha-tocopherol (vitamin E; Ve), brassinosteroids (Br), methyl jasmonates (MeJA), and triazoles (Tr) that were utilized in this experiment are pivotal in enhancing the agronomical and physiological attributes associated with thermo resistance and provide shelter against damaging influence of oxidative stress (Fletcher et al., 2000; Zhang et al., 2007; Beltagi, 2008; Mohammed and Tarpley, 2011). The Vc is a ubiquitous metabolite in plant tissues that performs a variety of functions. It plays an important role in the protection of cells and organelles from the oxidative damage by scavenging ROS, which are produced by environmental stresses such as heat. There is also growing evidence of the Vc participation in the regulation of cell division and elongation (Horemans et al., 2003). The Ve performs a vital role in the production of vital organic acids (L-tartaric, L-theronic, L-glyceric, and L-oxalic acids), and also take part in numerous processes such as flavonoid and phytohormone biosynthesis, and the xanthophyll cycle (Debolt et al., 2007). Furthermore, Ve diminishes the harmful effect of ROS and scavenges lipid peroxyl radicals (Munné-Bosch et al., 1999). Br are steroidal plant hormones, which perform a key role in various cellular and physiological processes like stem elongation, pollen tube growth, root inhibition, fruit development, ethylene biosynthesis, proton pump activity, xylem differentiation, photosynthesis, and gene expression (Yu et al., 2004; Fu et al., 2008). The Br can stimulate plant tolerance to a variety of abiotic stresses, such as high and low temperatures, drought, and salinity injury (Krishna, 2003; Kagale et al., 2007). It has been found that Br-induced increase in the basic thermo-tolerance was associated with increased heat shock protein synthesis and accumulation as well as increased expression of some components of translational machinery (Dhaubhadel et al., 2002).

The MeJA are crucial cellular regulators that are involved in several plant developmental processes in plants (Ueda and Saniewski, 2006; Norastehnia et al., 2007). Foliar application of MeJA brings alteration in numerous physiological responses and thus stimulates plant defense responses against a variety of biotic and abiotic stresses (Walia et al., 2007). Clarke et al. (2009) reported that exogenous application of MeJA in A. thaliana conferred basal thermo-tolerance and protected from the damaging influence of heat shock. Triazoles (Tr), as a plant growth regulating properties have the capability to cause changes in the balance of important plant hormones against stress including cytokinin augmentation, increased ABA and reduced ethylene (Kamountsis and Chronopoulon-Sereli, 1999; Fletcher et al., 2000; Gopi et al., 2007; Hajihashemi et al., 2007). Fletcher et al. (2000) observed that Tr-treated bean plants recorded less electrolyte leakage and were tolerant against heat stress.

Comparative responses of rice to either high-day time or night-time temperature stress in terms of physiological processes and reproductive function are not well understood. Furthermore, the different combinations of PGR (Ve, Vc, Br, MeJA, Tr) used in the present study have never been tested against high temperature stress in the past. Therefore, the present study was carried out to ascertain the morpho-physiological and yield responses of two rice cultivars to exogenously applied PGRs under high day and HNT stress. The findings of the present study will strengthen our understanding to improve the adaptation, survival and tolerance of rice to high temperature.

Wuhan (experiment site) is known as one of the major rice growing region in China, irrigated through Yangtze River. Average temperature for rice vegetative growth period in this region in 22–24°C while optimum temperature for grain filling period is 22–28°C (Li et al., 2011). In most areas, an elevated temperature up to 35–40°C is observed during grain filling period that is too harsh for normal grain development and filling. This rise in temperature interacts with normal functioning of plants through disturbance in many physiological processes and biochemical reactions (Larkindale and Knight, 2002). Peng et al. (2004) reported that high night time temperature increase is more fatal than increase in day time temperature. Variable observations regarding the intensity and effects of high temperature were previously reported for rice physiological processes. Alghabari et al. (2014) reported that HDT was more fatal to grain set and pollen fertility in cereals while Shah et al. (2011) observed poor rice performance under HNT compared with HDT. Narayanan et al. (2014) reported similar effect of HDT and HNT on plant physiology and growth when applied at anthesis stage of crop development. Our results depicted the variable effects of HDT and HNT for different studied parameters. The HDT resulted in greater reductions in A and WUE (Tables 1, 2). The IR-64 was more prone to high temperature stress as apparent by the enhanced level of E, gs, and LWP, whilst HHZ showed better level of tolerance to day and night thermal changes as apparent from higher rate of A and WUE under stress (Tables 1, 2).

Compared with AT, high temperature considerably reduced the morpho-physiological growth of rice (Tables 1–4). Reduction in morpho-physiological growth traits have been recorded to be accountable for decrease in rice yield production under high temperature (Li et al., 2011; Narayanan et al., 2014). Under heat stress, the thylakoid membrane becomes more permeable which increases proton leakage, affects normal synthesis of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), and consequently decreases the rate of A (Schrader et al., 2004; Narayanan et al., 2014). Loka and Oosterhuis (2010) reported that high temperature decreased A, sugar and starch content in rice while increased respiration and male sterility (Mohammed and Tarpley, 2009b). Guo et al. (2005) found that lower A under heat stress was due to reduction in leaf chlorophyll contents. Damage to electron transport chain especially at photosynthesis II or inhibition of CO₂ fixation under heat stress may also disturb photosynthesis which is the key process for photo-assimilate production (Havaux and Tardy, 1996; Yamane et al., 1997).

Due to high rate of E generated by HDT, the lower WUE was observed under HDT than HNT (Tables 1, 2). This could also be ascribed to reduced rate of A under HDT. The WUE defines the amount of photosynthate accumulation or biomass production based on amount of water used during E. Higher WUE resulted in higher plant biomass and yield. In the present study, decline in WUE could also be reason for grain yield reduction under HNT. Mohammed et al. (2014) reported similar results pertaining to WUE and noted significant decline in WUE under high temperature, which was associated with higher E. Under high temperature, higher Ci compared with AT (Tables 1, 2) suggested the inability of the leaf to fix the available C due to heat stress. We observed that higher Ci did not increase A but gs was higher, which indicated that both rice cultivars were efficiently able to enhance influx of CO₂ into leaves but due to high temperature induced detrimental effects on A, rice plants were not capable to fix that C into carbohydrates. In some earlier researches similar findings have been accounted which show that enhancement in gs or Ci did not affect A (Mohammed and Tarpley, 2009a).

Our study depicted slight improvement in plant height under HDT and HNT as compared with AT (Tables 3, 4). Heat-associated increment in plant height can be attributed to speedy elongation of internodes. This intermodal elongation may be linked to some internal hormones that were up-regulated under high temperature like ethylene and gibberellin (Qi et al., 2010). Our results are in consistent with the findings of Cheng et al. (2009) who reported considerable enhancement in rice plant height under HNT whereas results of some previous researchers are in contradictory with our findings who accounted HNT treatment did not significantly affect the morphological traits of rice including plant height (Mohammed and Tarpley, 2009a). High temperature reduced the leaf area and biomass accumulation in both rice cultivars as compared with AT (Tables 3, 4). Moreover, considerable decrease in leaf area guided to less A with associated reduction in the photosynthates assimilation in grain ultimately leading to a reduced grain yield. Mohammed and Tarpley (2014) authenticated that production area for total photosynthates was decreased due to leaf area reduction under high temperature stress, which resulted to considerable decline in grain yield. Additionally, higher night or day temperature activated photorespiration out coming in discrepancy between photosynthesis and respiration causing decline in grain yield (Venkatramanan and Singh, 2009).

Rice grain yield is dependent on number of panicles, number of spikelets per panicle, grain filling rate and total grain weight (Kim et al., 2009). High temperature particularly at night time decreased the grain numbers per spike, grain filling rate and total grain weight. Narayanan et al. (2014) also found considerable decrease in seed set, number of grains per spike and grain yield in wheat under high temperature and they attributed decreased grain number to poor seed set. Decrease in grain weight due to high temperature might be ascribed to reduced transfer of assimilates in grains and lower spikelet fertility under stress conditions (Tables 5, 6). Calderini et al. (2001) argued that reduced mobilization of stored pre-anthesis assimilates and reduced grain filling decreased the 1000-grain weight. Li et al. (2011) also reported that HNT was more harmful to grain weight in rice than HDT. Zakaria et al. (2002) reported that rate of grain growth was heat dependent and reduction in grain filling duration and dry matter accumulation led to smaller and imperfect grains. The apparent decrease in grain yield under high temperature suggested that HNT and HDT led to production of both smaller and imperfect grains. Our results also clarified pronounced differences between cultivars regarding growth and yield performance under high temperature (Tables 1–6). The HHZ performed better than IR-64 with better growth and higher grain yield under high temperature stress. These results are in confirmation with our previous reports (Fahad et al., 2015a,b, 2016), which revealed that HHZ is comparatively tolerant to high temperature compared with IR-64.

In the present work, application of PGRs was effective in alleviating the adverse effects of high temperature stress on rice. Exogenous application of PGRs considerably increased growth and yield of both rice cultivars as compared with NAC (Tables 1–6). Previously, Mohammed and Tarpley (2009b) reported that exogenous application of vitamin E, glycine betaine and salicylic acid improved rice yield and the ability of plants to tolerate high temperature stress. Better leaf gas exchange was observed when PGRs were exogenously applied as compared with NAC (Tables 1, 2). High rate of A in PGR-treated plants might be linked with higher WUE and lower E. In rice, application of PGR has been reported to increase leaf photosynthetic rates and decreases internal CO₂ concentration, respiration rates and membrane injury in rice (Farooq et al., 2008a; Khan et al., 2010). We observed better Ci in rice due to application of PGRs because of higher A and gs (Tables 1, 2), which resulted in higher C fixation and photosynthates production. Higher 1000-grain weight in PGR-applied plants might be due to enhanced mobilization of stored pre-anthesis assimilates and higher grain filling rate. Our results depicted that the combination of Vc+Ve+MeJA+Br outperformed than all other treatments for augmenting high temperature tolerance in both rice cultivars. The highest grain yield under AT as well as high temperature stress by this PGR combination might be attributed to higher A, WUE, spikelet fertility and grain filling than rest of the treatments.

The increase in spikelet fertility and 1000-grain weight by PGRs application (Tables 5, 6) might be attributed to augmented mobilization of stored pre-anthesis assimilates to the grain as a result of membrane stability and/or better pollen germination (Data not shown). Although the combination of various PGRs selected in current research have never been tested earlier, however, the individual function of Vc (Guo et al., 2005; Shah et al., 2011), Ve (Munné-Bosch and Alegre, 2002), MeJA (Tani et al., 2008; Clarke et al., 2009) and Br (Li et al., 2007; Zhang et al., 2007) against abiotic stresses is well apparent.

In summary, high temperature stress severely reduced the morpho-physiological growth and yield performance of rice cultivars in both years. The HDT posed more negative effects on rice physiological traits while HNT was more lethal for grain formation and rice yield. Exogenous application of PGRs was effective in alleviating the detrimental effects of heat stress. The combination of Vc+Ve+MejA+Br was more effective than all other PGR treatments for most of studied characteristics. The highest grain yield by this PGR combination was associated with enhanced A, spikelet fertility and grain filling, which compensated the adversities of high temperature stress. Taken together, these results may enhance our understanding regarding adaptation and survival mechanisms of rice to high temperature and advocate the usefulness of PGRs in alleviating the adversities of high temperature on rice growth and yield. However, further studies are required test the feasibility of these PGR combinations against different abiotic stresses in rice and other crops particularly in field conditions.

SF, JH initiated and designed the research, SF done the experiments, SF, SHu, SS, SHa, analyzed the data and wrote the manuscript, ZI, CW, AS, MY, WN, HA, FA, and JH revised and edited the manuscript and also provided advice on the experiments.