Agronomic and genetic approaches for enhancing tolerance to heat stress in rice: a review

Rice is an important cereal crop worldwide that serves as a dietary component for half of the world’s population. Climate change, especially global warming is a rising threat to crop production and food security. Therefore, enhancing rice growth and yield is a crucial challenge in stress-prone environments. Frequent episodes of heat stress threaten rice production all over the world. Breeders and agronomists undertake several techniques to ameliorate the adverse effects of heat stress to safeguard global rice production. The selection of suitable sowing time application of plant hormones, osmoprotectants and utilization of appropriate fertilizers and signaling molecules are essential agronomic practices to mitigate the adverse effects of heat stress on rice. Likewise, developing genotypes with improved morphological, biochemical, and genetic attributes is feasible and practical way to respond to this challenge. The creation of more genetic recombinants and the identification of traits responsible for heat tolerance could allow the selection of early-flowering cultivars with resistance to heat stress. This review details the integration of several agronomic, conventional breeding, and AcademicPres Notulae Botanicae Horti Cluj-Napoca Agrobotanici Rasheed A et al. (2021). Not Bot Horti Agrobo 49(4):12501 2 molecular approaches like hybridization, pure line selection, master-assisted-selection (MAS), transgenic breeding and CRRISPR/Cas9 that promise rapid and efficient development and selection of heat-tolerant rice genotypes. Such information’s could be used to determine the future research directions for rice breeders and other researchers working to improve the heat tolerance in rice.

3 Dingkuhn, 2013). Heat tolerance can be gained by altering several morphological, molecular, and biochemical traits. The heat-tolerant wild genotype (Oryza meridionalis) maintained a high photosynthesis rate under HS conditions owing to better maintenance of the enzyme Rubisco (Qu et al., 2021). The chlorophyll content and electrolyte leakage from leaves and roots increased during HS and can be utilized as marker to investigate heat tolerance.
Different agronomic and breeding approaches could be used to enhance the heat tolerance in rice crops. Agronomic approaches involve early sowing, spraying of signalling molecules, and hormones and osmoprotectants to mitigate the adverse effects of heat stress. The early sowing ensures the plant's survival at high temperatures, increasing overall productivity and quality (Krishnan et al., 2011). The application of plant hormones (auxins, salicylic acid, ascorbic acid, methyl jasmonate, alpha-tocopherol, and brassinosteroids) can also alleviate the adverse effect of heat stress and ensures better productivity (Khan et al., 2019). Likewise, the application of signalling molecules reduced the heat-induced adverse effects in rice crop by increasing the PS II efficiency, water use efficiency, and activity of anti-oxidants (Chandrakala et al., 2013). Additionally, the application of variable osmolytes (proline, glycine-betaine, and spermidine) is also considered a critical approach to improving the robust approach to improving rice crops heat tolerance (Khan et al., 2019;Sakamoto and Murata, 2000).
Breeding techniques are considered a long-term solution to solve the heat stress problem in rice crop. Conventional breeding approaches, including the selection of heat-tolerant cultivars, can help to improve the heat tolerance of rice crops. Likewise, recently developed molecular approaches have adopted the omics technique to develop transgenic plants by manipulating targeted genes, which can also help to improve the heat tolerance of rice crops (Duque et al., 2013;Kosová et al., 2011). Additionally, the identification of heat-tolerant QTLs and the use of proteomics and transcriptomics approaches may help identify underlying molecular heat tolerance processes, providing development to the heat-tolerant crop genotypes. Therefore, the current review reports on the agronomic approaches, conventional, and molecular approaches that can improve heat stress tolerance in rice. We discuss the heat stress mechanisms, agronomic approaches, conventional approaches, molecular approaches, heat tolerance QTL and genes, and the future outlook for heat tolerance in rice.

Effects of heat stress on rice
In the future, rice production may face massive challenges due to an increase in extreme climatic events (Sun et al., 2021). Heat stress can cause permanent injury to plants (Wahid et al., 2007) by impairing growth, metabolic processes, and seed setting rate as well as causing pollen infertility , therefore leads to severe yield reduction (Hasanuzzaman et al., 2013;Zafar et al., 2017). Extreme heat stress rapidly decreases plants photosynthesis rate, grain weight (Table 1), leaf area, and water use efficiency (WUE) (Shah and Paulsen, 2003). High levels of heat stress may hinder vegetative and reproductive growth (Katiyar-Agarwal et al., 2003). The booting and flowering stages are the most crucial stages of the rice life cycle, and exposure to HS at these stages can cause complete infertility in rice crop (Shah et al., 2011).
Effects of heat stress on photosynthesis, growth, and yield Heat stress (HS) significantly decreases rice growth during the initial stages ( Figure 1) and, seedling mortality is considered as a common effect of HS (Abd El-Daim et al., 2014;Xiao et al., 2011). Depending on the genotype, heat stress reduces rice growth and final production by decreasing relative water content (RWC), photosynthetic pigment concentration, and assimilating production (Fahad et al., 2016a;Ihsan et al., 2016).
The process of photosynthesis is vulnerable to HS, and it is adversely affected by high-temperature stress. Heat stress (30 °C and 35 °C) decreased photosynthesis, stomatal conductance, and transpiration, substantially reducing the final yield and quality (Fahad et al., 2016b). Other impacts of HS are reductions in carbon dioxide pathways, electron transport chain and rubisco activity. Heat stress remarkably reduces photosynthetic 4 processes and causes membrane instability and damage (Zheng et al., 2016). However, the consequences of this stress might be different among plant species (Alghabari et al., 2016). Heat stress decreases the concentrations of proteins and lipids; however, reducing lipids is relatively more significant (Johnston et al., 2007). The presence of HS at the flowering stage has been shown to spikelet sterility ( Figure 1) by stopping another dehiscence and germination of pollen on the stigma (Coast et al., 2016). Heat stress also leads to a poor seed filling rate and poor seed development (Sehgal et al., 2018). High temperatures cause low root vigor, and the selection of root base traits becomes tedious . In conclusion, HS reduces the chlorophyll content, rubisco activity, water use efficiency and induces oxidative damage and stomata closure, therefore causing significant reductions in the growth and yield of rice plants. The influences of heat stress on rice are given in Table 1.

2014) 35°C
Heat stress reduced yield traits, grain size, and rice yield (Wu et al., 2016) 29 °C Heat stress reduced growth and increased kernel chalkiness (Shi et al., 2016) 34 °C Reduced Pollen fertility (Wada et al., 2020) (Chidambaranathan et al., 2021) Effect of heat stress on water usage efficiency and nutrient uptake Heat stress reduces the water use efficiency in rice  by increasing transpiration rate (Topbjerg et al., 2015). Rice plant showed reduction in WUE under extreme heat stress and it leads to lower photosynthesis (Piveta et al., 2021). HS also disrupts plant minerals and their translocation. Likewise, Cabral et al. (2016) noted that higher nitrogen (N) concentration was allocated to grains than tillers under HS, and the phosphorus (P) concentration was also decreased in wheat plants subjected to the HS. High temperature (>2 °C above the average level) was shown to enhance the C: N (Figure 1) ratio and decreased the concentration of nutrients in maize plants (Zhang et al., 2013a). In another study the effects of heat stress on nutrients uptake were studied. The heat stress significantly reduced the nitrogen application rate in Boro rice variety (Hossain et al., 2021). In general, few studies have investigated the impacts of HS on the mineral status of plants. More extensive investigation could help to establish nutrient absorption pathways for heat-stressed plants.
Effects of heat stress on the production of reactive oxygen species and antioxidant enzymes Oxidative stress is also accompanied by heat stress (Figure 1) and occurs due to the accumulation of ROS in plants (Pucciariello et al., 2012), which cause substantial damage to the significant molecules, including the DNA, proteins, sugars, and carbohydrates, and can induce cell death (Gill and Tuteja, 2010). In crops, reactive oxygen species exist in molecular forms. Their ionic form is superoxide anions, and their molecular state is singlet oxygen (Mittler et al., 2004). Numerous enzymes, such as NADPH oxidases, polyamine, and a large 5 family of class III peroxidases present on the cell surface, create ROS (Cosio and Dunand, 2009). The overproduction of ROS during the HS is highly toxic to lipids, proteins, and nucleic acids, eventually, damaging cells and leading to cell death (Gill and Tuteja, 2010). Plants use different types of antioxidant enzymes, like catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR), to scavenge ROS. The antioxidant molecules are positioned in plant cells and work in groups to detoxify ROS (You and Chan, 2015). In conclusion, HS induces the production of ROS, which cause damage to significant molecules and eventually leads to cell death. Therefore, proper measures should be adopted to reduce ROS production under HS conditions to ensure better rice production. Plants have different mechanisms, including escape, avoidance, and survival, to cope with HS. Plants undergo several types of modifications to avoid heat stress. Rice varieties have covered panicles to reduce HS by decreasing the evaporation rate (Shah et al., 2011). Early flowering varieties have a better capability to tolerate heat stress by using a heat avoidance mechanism (Bheemanahalli et al., 2017;Ishimaru et al., 2012) . Genetic variations in rice plants can be exploited to screen the germplasm for HS tolerance (Ishimaru et al., 2012).

Size of anther and basal pore
The length of anther varies among genotypes. Genotypes associated with longer anthers are considered more heat tolerant than those associated with short anthers (Matsui and Omasa, 2002). Under heat stress conditions, floret sterility is a direct cause of reduced pollen grain germination on the stigma owing to poor anther dehiscence (Ishimaru et al., 2012) . Anther size has a positive association with the number of pollen grains per anther. It is considered that cultivars with more prominent anthers have more pollen grains, allowing them to compensate for temperature-induced effects (Matsui and Omasa, 2002). Likewise, the size of basal pores varies among cultivars. The large anther size can minimize the effects of heat stress and could be use as tolerance criteria in rice under extreme heat stress (Santiago et al., 2021). Large basal pores induce the release of pollen grains to the stigmata during another dehiscence; thus, the number of pollen grains in the plant sigma depends on the basal pore size (Matsui, 2005). Conversely, pollen grains remain inside the anther in plants with tiny basal pores until the floret opens. Then, the anther bends and spreads pollen. Thus, genotypes with tiny basal pores undergo self-pollination and are more likely to cross-pollinate (Matsui and Kagata, 2003). Large basal pores facilitate the release of pollen from the anther and increase the chance of pollination. Basal pore can also be used as tolerance criteria under condition of extreme heat stress and pollination rate can be enhanced . In conclusion, cultivars with large anthers and basal pores should be cultivated to reduce the effects of heat stress.

Photosynthesis and the carbohydrate content
The selection of genotypes that can accumulate a high concentration of nonstructural carbohydrates produce more biomass, and maintain a higher photosynthesis rate under extreme heat stress could be used to develop the heat-tolerant cultivars to minimize yield loss induced by heat stress. Genotypes tolerant to heat stress at the anthesis and reproductive stages can maintain a higher rate of photosynthesis for a long time and thus produce more grains (Egeh, 1991). To attain heat tolerance and maximum growth, retention of a high photosynthetic rate is critical. The response of photosynthetic parameters to HS was observed in two rice cultivars (IR46 and IR53) under high heat stress conditions during the day-time. It was noted that photosynthetic traits like the chlorophyll content were more prominent in cultivar N22 than another cultivar. It was found that all photosynthetic traits of N22 were more prominent than in the other genotypes at elevated temperatures, showing the greater tolerance of N22. The rate of photosynthesis was first improved with temperature up to an optimal temperature of 32 °C, which then reduced when the temperature continued to increase to 42 °C (Gesch et al., 2003). Thus, we should develop rice cultivars with improved photosynthesis and carbohydrate synthesis capacities to achieve the maximum yield potential under HS conditions.

Heat shock protein content
Heat shock proteins (HSPs) are considered significant molecular chaperons that help in folding protein assembly and the maintenance of homeostasis under both ideal and adverse conditions (Lin et al., 2014). In a previous study, Jagadish et al. (2010) observed the changes in protein expression under HS conditions and showed that the tolerant genotype N22 expressed more HSPs. Hence, it was proven that HSPs enhance HS tolerance. The gene expression analysis showed a variation in the expression levels of genes encoding HSPs in rice leaves. Higher expression of HSPs was found in the heat-tolerant rice cultivars R-1389 and N22 (Chandel et al., 2013). The HSPs (OsHsfA7 and OsHsfA2a) were strongly upregulated in plants with the N22 genotype under HS conditions (42 o C) at the flower opening phase. The functions of Hsfs, OsHsfA2e and OsHsfA7 were also upregulated in the Vandana variety, but the increase in function was lower than in N22 (Sailaja et al., 2015). In another experiment, Lin et al. (2014) tested the heat tolerance of the japonica and indica N22 varieties of rice. Both studies analyzed the heat-stress tolerance of different rice varieties to allow a clear comparison between varieties to be made. These cultivars exhibited conflicting levels of heat tolerance. N22 showed a rapid decline in HSP101 compared with japonica, which might have been due to environmental alterations. The Nipponbare genotype attained HS tolerance over the long term, while N22 showed greater basal HS tolerance (Katiyar-Agarwal et al., 2003). The high expression of genes related to the heat shock transcription factor (Hspf) is one of the important factors in the reaction of plants to heat waves (Liu et al., 2009). Consequently, HSPs are associated with higher heat-stress tolerance in rice crops as they reduce the permanent accumulation and degradation of mis-folded proteins and maintain cellular homeostasis.
Thermostability of the cell membrane and chlorophyll fluorescence Genetic variations have been identified in rice cultivars regarding chlorophyll fluorescence traits under HS (Sailaja et al., 2015). The rice genotype N22, tolerant to heat-stress, demonstrated a high Fv/Fm ratio when exposed to HS (42 o C) (Bahuguna et al., 2015). Heat stress declined the chlorophyll contents, and the decline was more prominent in heat-sensitive cultivars (Sailaja et al., 2015;Zhou et al., 2007). Moreover, membrane thermostability (MTS) is a trustworthy feature that could be exploited to select tolerant genotypes. It has been 7 shown to have a, more significant association with yield under HS conditions (Sailaja et al., 2015). Heat stress was shown to increase electrolyte leakage, leading to a significant reduction in the final yield (Mohammed and Tarpley, 2009). Zhang et al. (2005) examined the influence of HS on the physiological and biochemical features of rice at the flowering and heading stages. They noted that the sensitive variety (4628) had lower membrane permeability than the heat-tolerant (line-96) under HS conditions. In another study, membrane thermostability was found to have a significant association with the grain yield/plant. Earlier studies showed that early-morning flowering could minimize the effects of HS on rice and be used as a selection criteria. In this study, a set of diverse rice genotypes were exposed to HS and the flowering time was observed (Bheemanahalli et al., 2017). Membrane and chlorophyll fluorescence must be increased in rice cultivars under changing HS scenarios to ensure better rice production.
Spikelet fertility and yield traits There are genetic differences in the sensitivity of spikelets to HS (Buu et al., 2021). Spikelet's from heattolerant genotypes show better results than sensitive ones under HS (Jagadish et al., 2010;Prasad et al., 2006). Two traits, spikelet fertility and yield/plant could be selected to attain HS tolerance. For instance, N22, a highly heat-tolerant rice cultivar, maintains spikelet fertility of 71%. In contrast, cultivars with moderate or poor heat tolerance (IR 64 and Moroberekan) retained 48% and 18% spikelet fertility levels, respectively. Nevertheless, Prasanth et al. (2016) stated that spikelet fertility is not a significant criteria for determining heat tolerance after excluding the yield/plant.

Agronomic approaches to enhancing heat tolerance in rice
Early planting Determining an appropriate sowing time is an imperative agronomic strategy for reducing the damaging effects of heat stress. Most agronomic practices focus on the early sowing of rice crop, the adjustment of irrigation systems, and the adaptation of early and late maturing cultivars to mitigate the adverse effects of heat stress (Krishnan et al., 2011) . Early sowing of rice has significant role in avoiding stress in rice. Ding et al. (2020) adjusted the sowing date of rice under adverse climatic conditions and concluded that shifting of sowing date showed promising results in terms of rice yield. In this way yield loss can be compensated. Jagadish et al. (2015) also presented a detailed review in which they showed that management of sowing date in rice could protect rice from extreme heat stress. Appropriately timed sowing of rice is crucial to reduce the effects of heat stress at critical growth stages. Setiyono et al. (2018) found significant reductions in the rice yield with heat stress at the plant reproductive stage due to a high rate of spikelet sterility. They also suggested that yield losses in rice crops can be reduced substantially by early sowing. The use of optimum sowing dates also reduces the unfavorable adverse effects of heat stress on the grain yield and quality. Zhu et al. (2013) studied the impacts of various sowing dates on the rice yield and quality. They found that adjusting the sowing time can reduce the effects of heat stress on the rice yield and quality. However, they also suggested that adjusting the sowing time is very difficult because it affects the proceeding crops. In a flooded anaerobic system, methane and nitrous oxide emissions are the main factors responsible for global warming. Therefore, adjusting the irrigation system, for example, using alternate wetting and drying periods, can help to decrease the effects of heat stress by reducing greenhouse gas emissions ( Yu et al., 2004;Aamer et al., 2021). Additionally, covering the soil surface with crop residue and modifying the microclimate by shading can reduce the effects of heat stress in rice crops (Krishnan et al., 2011) . In conclusion, optimizing the sowing time can help reduce the harmful effects of heat stress in rice crops.

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Employing plant hormones to increase heat tolerance in rice It is well known that the five classical phytohormones have drawn the interest of many scientists and have been investigated for decades . Plant hormones may be targeted to enhance abiotic stress tolerance and the growth and yield of rice (Ciura and Kruk, 2018). Increasing the auxin level during panicle formation increases heat tolerance in rice (Sarwar, 2019). The gaseous hormone ethylene is developed in response to heat stress (Wu and Yang, 2019), and ethylene-mediated signaling induces heat tolerance in rice seedlings. In a previous experiment, ethylene-responsive mutants were identified and characterized in rice. It was concluded that ethylene significantly increases heat tolerance in rice seedlings (Wu and Yang, 2019) by maintaining the seed ripening rate during extreme heat waves.
Abscisic acid is an important plant hormone that contributes to the response to diverse stress conditions including HS, drought, and cold stress (Zou et al., 2017). Many studies have revealed that ABA improves thermos-tolerance in various plant species (Claeys et al., 2014). Amino acids also play key roles in HS tolerance in rice crops. Amino acids are involved in metal binding, cell signaling, and the antioxidant defense system. Therefore, they play essential roles in plant defenses when exposed to different stresses (Sharma and Dietz, 2006). Proline is vital amino acid that protects the plants from stressful conditions (Verbruggen and Hermans, 2008). It plays a significant role in different mechanisms, including antioxidant defense, turgor production, N, and carbon assimilation (Verbruggen and Hermans, 2008), and protein stabilization (Maggio et al., 2002).
Proline minimizes the negative impacts of HS by lowering the ROS concentration and increasing the activity of antioxidants and the accumulation of different metabolites, including proline, ascorbic acid, and glutathione . Spermidine is a natural polyamine that is involved in the adaptation of plants to various abiotic stresses, such as heat (Tian et al., 2012), cold (Yamamoto et al., 2012), heavy metals (Xu et al., 2011), anddrought (Fu et al., 2019). Spermidine was shown to increase, therefore, antioxidants activity, therefore increasing plant survival under stressful conditions (Tian et al., 2012).
Ethylene and cytokinin also play a significant role under heat stress in plants as well as rice. In response to extreme heat stress in rice, the gaseous hormone like, ethylene is produced in plants and its manipulation under heat stress brings promising results (Poór et al., 2021). Likewise, cytokinin (CK) a plant growth promoting hormone also protects plants under heat stress. In rice, CK governs several biological features like, growth of shoot and increase spikelets number under heat stress (Wu et al., 2017).
Foliar spraying of spermidine has been shown to improve the tolerance of rice to HS by reducing oxidative damage and increasing photosynthetic and antioxidant activity under HS conditions (42 °C) (Mostofa et al., 2014). Moreover, spermidine was found to increase plant growth and the chlorophyll content under HS conditions (Murkowski, 2001;Zain et al., 2017). Salicylic acid is a type of phytohormones with an abundant distribution in plants. It governs the response of a large number of physiological features to abiotic stresses. The exogenous application of SA to rice seedlings was found to minimize the adverse effects of high heat waves at a up to 32 °C and enhance dry matter portioning at up to 16% (Mohammed and Tarpley, 2009).
The induction of a class 11 HSP, Oshsp18.0, by SA in rice demonstrated the role of SA in response to heat waves (Chang et al., 2007). Methyl jasmonates play a crucial role in alleviating heat stress, and their application increased early flowering under heat stress conditions (Kobayasi and Atsuta, 2010). Zhang et al. (2018) revealed the consequences of spraying auxin on the elongation of pollen tubes of heat-tolerant and susceptible plant varieties. They stated that spraying naphthalene acetic acid reduced and upturned the spikelet sterility in heat susceptible and tolerant rice plant genotypes by obstructing the reduction of pollen tube growth. These findings suggest that plant hormones could increase growth and improve HS tolerance in rice crops.
Utilizing fertilizers and signaling molecules to increase heat tolerance in rice The application of signaling molecules and fertilizers can significantly reduce the negative impacts of heat stress in rice. Likewise, the application of CaCl2 (10 mM) was shown to mitigate the adverse effects of heat stress in rice by improving the PS-II efficiency and water use efficiency and increasing the chlorophyll content and spikelet fertility (Chandrakala et al., 2013). Similarly, nitric oxide regulated different processes in plants and was shown to improve flowering, fertilization, and high-stress tolerance (Hasanuzzaman et al., 2013). Rice seedlings treated with hydrogen peroxide showed significant improvements in PS-II efficiency, antioxidant activity, and gene expression, which increased rice heat tolerance. The basal application of boron mitigated the adverse effects of heat stress in rice by improving the membrane stability and spikelet fertility (Shahid et al., 2018). The soil application of potassium fertilizers remarkably reduced the effects of drought stress by increasing the photosynthetic efficiency and antioxidant activity and decreasing the MDA content. Thus, the application of fertilizers and signaling molecules can reduce the adverse impacts of heat stress by increasing the photosynthetic efficiency, membrane stability, and activity antioxidants.
Using osmoprotectants to increase heat tolerance in rice The accumulation of various osmoprotectants in response to different abiotic stresses can alleviate the adverse impacts of stressful conditions (Hassan et al., 2020). Osmoprotectants protect plant metabolic processes by stabilizing cellular membranes and increasing different antioxidants' photosynthetic efficiency and activity. Glycine betaine (GB) is an essential osmolyte that accumulates in plants under heat stress conditions (Hassan et al., 2020). It induces heat tolerance by protecting different enzymes (Rubisco and citrate synthase) from heat degradation (Quan et al., 2004). Many plant species such as maize and sugarcane accumulate higher concentrations of GB under heat stress conditions, whereas rice, Arabidopsis, and mustard do not accumulate GB (Annunziata et al., 2019). Thus, the exogenous application of GB increases the rice yield under heat stress conditions by promoting the activity of antioxidants which, in turn, protects the membrane, enzymes, and major molecules from the damaging effects of heat stress (Mohammed and Tarpley, 2009). The application of GB prevents the degradation of rubisco due to heat stress which, in turn, improves the photosynthetic efficiency and, consequently, the rice yield (Dionisio-Sese et al., 2000).
Proline also accumulates in plants in response to heat stress and protects the plants from the damaging effects of heat stress. Proline protects the rubisco enzyme from degradation due to heat stress which, in turn, increases the overall photosynthetic efficiency and rice yield (Dionisio-Sese et al., 2000). Spermidine is also a critical osmoprotectant that plays a vital role in increasing the tolerance to different stresses, including heat stress . Spermidine-induced tolerance to heat stress in rice is attributed to an increase in antioxidant, starch, and polyamine metabolism. Spermidine increases the expression of the starch synthesis enzyme, favouring an increase in starch accumulation (Tang et al., 2018).
Moreover, spermidine reduces the hydrogen peroxide content in japonica rice by modulating the glutathione and glyoxalase systems (Mostofa et al., 2014;Tang et al., 2018). The application of spermidine also increase the grain yield and grain filling rate in rice by increasing antioxidant activity, photosynthetic activity, the efficiency of PS-II, and the sugar content and modulating starch and polyamine metabolism (Fu et al., 2019). It is concluded that osmoprotectants are important contributing factor in heat stress tolerance in rice. These osmoprotectants can be used in different modified forms and significant heat tolerance can be achieved in rice. Their role can be studied by exposing rice under different heat stress conditions.

Breeding approaches for enhancing heat tolerance in rice
Phenotypic selection of tolerant cultivars Developing breeding methods that promote HS tolerance in rice crops requires a proper understanding of HS tolerance mechanisms (Karwa et al., 2020). Considerable and significant genotypic variation is present in rice cultivars subjected to HS, and the selection of heat-tolerant genotypes will ensure sustainable production under HS conditions (Prasad et al., 2006;Shah et al., 2011). Rice genotypes show varying responses to heat stress, and this increases the possibility of identifying heat-tolerant genotypes. With the changing climate, the selection of tolerant genotypes will become a greater focus. In a previous experiment, 1217 rice genotypes collected from different areas were tested for heat tolerance, and it was observed that only 2% of cultivars were 10 heat tolerant under changing environments (Masuduzzaman et al., 2016). Heat-tolerant genotypes should be investigated to identify the heat stress tolerance mechanism present in rice crop. A schematic display of increasing heat tolerance in rice using several factors and development of heat tolerant cultivars using breeding methods is provided in Figure 2 and Figure 3.  Loci with a putative quantitative trait confer heat tolerance in rice QTL mapping is an effective technique that can be used to locate genomic regions controlling several traits (Rasheed et al., 2020a(Rasheed et al., , 2020bRasheed et al., 2020;Rasheed et al., 2021b). Multiple QTLs have been mapped for HS tolerance (Xu et al., 2021), especially for rice flowerings (Ye et al., 2012;Ye et al., 2015a). Such markers can be used to initiate MAS for a pyramid of genes to promote the breeding of plants with greater HS tolerance (Cheng et al., 2012;Ye et al., 2015b). Nonetheless, before the use of these QTLs in MAS, the potential of subsequent populations to be used in large germplasm must be determined after initial mapping (Ye et al., 2015a).
Nowadays, rice breeders use double haploid (DH), backcross inbred lines (BIL), and RIL to unfold the genetic backgrounds of HS-tolerant rice plants (Qingquan et al., 2008;Tao et al., 2008). A previous study used a set of introgression lines (ILs) developed from a cross of Oryza officinalis and Koshihikari. The lines showed earlier flowering and improvement in the spikelet fertility and yield/plant compared with late-flowering genotypes (Ishimaru et al., 2010). Heat-stress tolerance is a polygenic feature governed by multiple genes that varies by stage and between plants species (Ashraf and Harris, 2005;Bohnert et al., 2006). Due to the advancement of marker technology, identifying QTLs associated with heat tolerance is an important approach. QTL identification assists in unfolding genetic mechanisms and the cloning of QTL. Many QTLs have been identified in previous studies Xiao et al., 2011). The majority of these QTL were identified in rice genotypes at the flowering stage. QTLs governing spikelet fertility and the yield/plant were mainly mapped on chromosomes, 1, 4, 10, and all 12 linkage groups (Qingquan et al., 2008;Tao et al., 2008). In a previous study, Zhao et al. (2016) evaluated chromosome segment substitution lines (CSSL) from Habataki (heat-tolerant) and Sasanishiki (heat-sensitive) cultivars. Two QTLs (qSFht2 and qSFht4.2) correlated with spikelet fertility, and two (qDFT3 and qDFT10.1) correlated with flowering time were mapped. SL412 presented a considerably higher spikelet fertility level in Habakati than Sasanishiki, and 6 CSSL exhibited a high pollen detachment level. Zhang et al. (2009) identified two SSR markers (RM3586 and RM3735) accountable for 3% and 17% of the difference in HS tolerance among plants. They recommended the exploitation of genetic loci using MAS to develop HS-tolerant cultivars. Jagadish et al. (2010) used the F6 progeny of RIL and documented eight QTLs related to spikelet fertility in HS on dissimilar chromosomes. A QTL accountable for 18% of the phenotypic deviation in tolerance to heat stress was identified on chromosome 1. Xiao et al. (2011) used pollen sterility to indicate HS tolerance and recognized two QTLs (qPF4 and qPF6) that improved pollen fertility. Ye et al. (2015a) identified several QTLs with a deviation in spikelet fertility in HS. In another investigation, Shanmugavadivel et al. (2017) crossed the heat-tolerant genotype (Nagina 22) with the heat-susceptible genotype (IR64) to investigate the QTL responsible for heat tolerance. Huang et al. (2012) recognized 32 novel loci linked to the flowering time. Lafarge et al. (2017) used the GWAS technique to identify the QTL associated with preserving spikelet fertility under high HS conditions. They selected 167 indica lines with 13,162 single nucleotide polymorphisms (SNPs). A total of 14 loci were linked with spikelet sterility, and 8 of these were consistent with previously identified QTLs. Genes at loci related to the fertility of spikelets were linked with the response of plants to HS conditions. N22 and some Indian and Taiwanese genotypes are active contributors of HS tolerance in rice (Lafarge et al., 2017). Kushwah et al. (2021) identified a QTL Qdg-01 for days to germination under heat stress using recombinant inbred lines population (RIL). A list of QTLs related to heat-stress tolerance is given in Table 2.  (Kushwah et al., 2021) Development of transgenic rice tolerant cultivars Heat shock proteins are synthesized by many genetic factors (Buu et al., 2021) that switch on when exposed to HS and play critical roles in the recovery of plants after HS (Liu et al., 2006;Nakamoto and Hiyama, 1999). Alterations in transgenic rice associated with HSPs have the potential to increase HS tolerance in rice . The genetic enhancement of rice cultivars is a reliable approach to sustain rice production under a changing environment . Heat tolerance in rice using the transgenic approach has rarely been reported. The overexpression of HSPs in rice has been associated with heat tolerance (Katiyar-Agarwal et al., 2003). HSPs improve HS tolerance in mutant and transgenic rice species (Katiyar-Agarwal et al., 2003). Earlier, Katiyar-Agarwal et al. (2003) used Arabidopsis thaliana and transformed HSP (AtHSP101 cDNA) into the indica variety of rice (Pusa basmati 1). Compared with typical plants, the existence and development of T2 lines were enriched with proteinaceous material under HS.
In a previous study, a transgenic rice variety (Hoshinoyume) showed overexpression of HSP (sHSP17.7), which was confirmed to be associated with greater tolerance to HS (Murakami et al., 2004). Qi et al. (2011) described that, in transgenic rice cultivars, the overexpression of mitochondrial genetic factors associated with mtHsp70 enhanced HS by decreasing programmed cell death, improving the stability of the mitochondrial membrane, and inhibiting ROS production. WRKY genetic factors are recognized as the encoding for many transcription factors, contributing to numerous abiotic factors. Wu et al. (2009) merged cDNA from OsWRKY11 with the HSP101 promoter and transferred it to rice d plants grown under HS conditions. The WRKY genes e overexpressed in rice were associated with improved HS tolerance and growth and growth traits under HS. Proteomic investigations can help us to understand the molecular foundations of HS tolerance in rice plants. Lee et al. (2007) examined the proteomes of rice leaves grown under HS conditions and identified nearly 73 low molecular mass proteins, and these were mostly linked to HSPs. In conclusion transgenic cultivars can play significant roles in increasing HS tolerance in rice crops. However, future studies must be conducted to develop cultivars and tested these cultivars in a wide range of field conditions to ensure their availability across the globe. List of genes related to heat tolerance in transgenic rice and normal rice is given in Tables 3 and 4. 13  (Jia et al., 2015) DPB3-1 Rice Arabidopsis Overexpression of DPB3, upregulation of stress-related genes (Sato et al., 2016) rcbs Rice Oryza australiensis Due to overexpression of the yield (Scafaro et al., 2018) UGT73B3

OsBiP2
Rice Rice Improved dehydration tolerance (Raza et al., 2020) OsNTL3 Rice Rice NAC transcription factor improved thermal tolerance in rice (Liu et al., 2020b) AtPLC9 Rice Rice Expression of transcription factors (Liu et al., 2020b) OsWRKY Rice Rice Improved thermal tolerance (Jeyasri et al., 2021) OsIAA13, Os IAA20 Rice Rice Improved heat tolerance  LOC_Os08g 07010 Rice Rice Decreased extreme heat stress    (Liu et al., 2020b) OsBiP2, OsMed37_1 Controls heat tolerance = (Raza et al., 2020) OsHTAS Controls heat tolerance (Jan et al., 2021) Proteomics and transcriptomics approaches for increasing heat tolerance in rice Comprehensive proteomic surveys of metabolic enzymes, storage and structural proteins, and different allergens found in rice grains have been done using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and gel-free-based shotgun technologies (Koller et al., 2002;Lee and Koh, 2011;Lin et al., 2005). Lee and Koh (2011) identified 4172 non-redundant proteins with a range of pI (pH 2.9-12.6) and molecular weights (5.2-611 kDa) in developing and maturing grains of rice. The analysis of the expression of different protein groups linked with diverse functional categories showed dynamic changes in metabolism during rice grain development. A switch from carbon metabolism to alcohol fermentation is imperative for the synthesis and accumulation of starch during the development process (Xu et al., 2008). It was also noticed that proteins involved in the citric acid cycle, lipid metabolism, glycolysis, and proteolysis were accumulated more in mature grains than developing grains (Lee and Koh, 2011). Proteomic studies also revealed that all classes of storage proteins increased considerably at the early ripening stages, whereas the polyamine concentration decreased considerably at the maturation and desiccation stages (Lin et al., 2010). Moreover, Li et al. (2011) noted that pullulanase (PUL) was downregulated, whereas pyruvate phosphate dikinase (PPDK) was upregulated in a grain filling study. Thus, the proteomic approach can help us improve protein expression in rice to increase heat tolerance.
Transcriptomics has been widely used to study the molecular mechanisms associated with heat tolerance in wheat, tomato, and potato (Bita et al., 2011;Ginzberg et al., 2009;Qin et al., 2008) and different pathways and genes have been identified as being heat-responsive. In rice crops, few transcriptomic analyses have been conducted to determine the heat response at the flowering stage (Endo et al., 2009;Zhang et al., 2013b). Most of these studies were conducted on spikelets and flag leaves, with limited studies conducted on anthers or pistils (González-Schain et al., 2016;Li et al., 2015). Liu et al. (2020a) found a stable anther structure in rice line SDWG005 under heat stress conditions. Their transcriptomic analysis found 3559 differentially expressed genes in anthers of SDWG005 plants under heat stress at the anthesis stage. They also stated that the agmatinecoumarin-acyltransferase gene is involved in heat tolerance in SDWG005 plants (Liu et al., 2020a).
Mutation for improving heat tolerance in rice A mutation is an essential tool that can create genetic variability (Mba et al., 2010). Over the last century, physical mutagens, including ultraviolet rays, X-ray and chemical rays, and chemical mutagens such as Nmethyl-N-nitrosourea (MNU), sodium azide, hydrogen fluoride (HF), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS), have been used to create mutations in plants (Krishnan et al., 2009).
The EMS-induced mutation is considered very effective, and it is commonly used in a diverse range of breeding programs to develop improved crop genotypes. The application of EMS improves agronomic traits and the rate of photosynthesis in rice leaves while reduces the concentration of mesophyll interveinal cells (Feldman et al., 2017;Feldman et al., 2014). Moreover, MNU is another important mutagen that is mainly used to create mutations in rice. The application of MNU is a more efficient way to create mutations in developing rice cells than in seeds (Satoh et al., 2010). MNU-induced mutagenesis affects various physiological processes and leads to discovering gene functions and increased genetic variability in rice (Satoh et al., 2010). Therefore, mutagens can be used to change the genetic makeup of rice to develop genotypes with desired traits.
Role of CRISPR/Cas9 in improving the heat tolerance in rice Conventional breeding techniques also bring undesirable genes along with desirable genes (Rasheed et al., 2021c). These techniques are time-consuming and hence they are not suitable to increase the rice production for rapidly growing world population. In addition, hybridization is possible among two plants of the same species, limiting new traits and genes (Jiang et al., 2012). Therefore, in these scenarios, the novel genome editing techniques (GET) can tackle the limitations of conventional breeding by improving the desirable traits in any species in a short time (Jiang et al., 2012). Nonetheless, information related to gene sequencing, genes function, and QTL responsible for traits of interest is vital for GET application. The application of GET modifies the particular gene of the desired trait by DNA cutting via target-specific nucleases. Different site-specific endonucleases (SSE), i.e., zinc finger nucleases, transcription activator-like effector nucleases introduced in the last decade, have been widely used in gene editing tools (Chen and Gao, 2014). Different studies were conducted in which several genes were knock out using CRISPR/Cas9 technique to improve heat stress tolerance in rice. The CRISPR/Cas based editing tool to edit genes responsible for heat tolerance has been widely used in rice . CRISPR/Cas9 application can help to develop the heat-tolerant cultivars of rice by changing plant and panicle architecture, leaf morphology, and ABA signalling pathway by modifying desired genes (Jiang et al., 2012). The knockout of OsNAC006 gene significantly increased heat stress tolerance in rice . Likewise in another study, OsProDH gene was mutated using CRISPR/Cas9 technique and it was concluded that this gene negatively regulates the heat tolerance in rice by scavenging of ROS. Recent advances in GET involve developing a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system. Multiple Cas proteins, such as Cas8, Cas9, Cas12a, or Cpf1 are identified, used in genome editing by CRISPR technology to improve the diverse traits in plants (Cebrian-Serrano and Davies, 2017;Naeem et al., 2020). CRISPR/Cas9 is considered as an easier, reliable, and efficient system used for improving stress resistance, grain yield, herbicide resistance, and product quality in many crops such as sativa, barley, maize, cucumber, soybean, wheat, and rice (Komor et al., 2016).

Conclusions
As a complex polygenic trait, heat stress tolerance is difficult to improve by using conventional breeding methods. Genetic factors which are governing the heat stress tolerance in rice and least influenced by the environment can be explored to improve heat stress tolerance in rice. Cultivars with strong and stable genetic makeups can maintain a higher rate of spikelet fertility, early flowering, and a higher yield under frequent heat episodes. The complex genetic architecture of this polygenic characteristic has still not been fully explored. Investigations are underway to detect the roles of morphological, physiological and biochemical features in sustainable rice production under exposure to heatwaves. Integrating several molecular techniques, including genomics, proteomics, and transcriptomics, is critical to develop highly heat-tolerant genotypes in rice. The high yielding genotypes are specially used because they can maintain high yield under heat stress. Hormonal applications significantly increased heat stress tolerance in rice. QTL pyramiding technique is very effective to transfer multiple heat tolerant QTL in genotypes and we can bring durable tolerance in rice genotypes. Hence, we have concluded that there are several factors needed to study to improve heat stress tolerance in rice. The novel breeding techniques like improved hybridization and molecular breeding methods can be effectively used to enhance heat stress tolerance in rice. The CRISPR/Cas9 and its variants need to use to edit targeted gene in rice responsible for heat stress tolerance in rice.
A recent period of extreme heat stress had damaging effects on crop production, and it is expected that this situation will continue to occur in the near future. This will threaten the global food supply chain. To overcome this and sustain rice production, effective management strategies must be implemented, and tolerant rice cultivars must be produced. We must improve the tolerance of rice to heat stress at the physiological, molecular, and biochemical levels through the development of tolerant cultivars to maintain the quality and quantity of rice across changing environments. Heat stress affects grain filling and pollen fertility, disturb plant water relation, lead to lipid peroxidation, and cause oxidative stress in rice. Plant hormones and inorganic osmolytes can induce an acclimation response in plants. The use of these hormones and osmolytes is an excellent solution to reduce the consequences of heat stress. Few studies have described the roles of these regulators in plant responses to heat stress, and further studies are needed. In the era of modernized genetics, high throughput phenotypic, and genotyping approaches like GWAS to identify phenotypic diversity can contribute to the development of heat-tolerant genotypes. For unique trait such as heat tolerance, the recent development of advanced gene-editing technique, like CRISPR-Cas9 will further speed up crop improvement.

Authors' Contributions
AR conceptualized and prepared the draft; MFS, MN, AM, MRA, MAA, MA, MAE, EHE, and MA, and MUH reviewed the manuscript; MB improved the scientific figures; ZU and HL supervised the study. All authors read and approved the manuscript. All authors read and approved the final manuscript.