How Rice Responds to Temperature Changes and Defeats Heat Stress

Abstract

With the intensification of the greenhouse effect, a series of natural phenomena, such as global warming, are gradually recognized; when the ambient temperature increases to the extent that it causes heat stress in plants, agricultural production will inevitably be affected. Therefore, several issues associated with heat stress in crops urgently need to be solved. Rice is one of the momentous food crops for humans, widely planted in tropical and subtropical monsoon regions. It is prone to high temperature stress in summer, leading to a decrease in yield and quality. Understanding how rice can tolerate heat stress through genetic effects is particularly vital. This article reviews how rice respond to rising temperature by integrating the molecular regulatory pathways and introduce its physiological mechanisms of tolerance to heat stress from the perspective of molecular biology. In addition, genome selection and genetic engineering for rice heat tolerance were emphasized to provide a theoretical basis for the sustainability and stability of crop yield-quality structures under high temperatures from the point of view of molecular breeding.

Introduction

Rice (Oryza sativa L.) as one of the most predominant food crops in the world, has always been of great concern by agricultural scientists for its yield and quality issues. In recent years, the local high temperature climate in the world has brought a certain impact on the rice industry, and the abiotic stress factor of high temperature has had to be highly valued.

High temperature has many effects on plants, including but not limited to shortening the plant growth cycle, reducing photosynthetic efficiency, increasing respiration, and accelerating transpiration and other physiological processes, all of which lead to the accumulation of reactive oxygen species (ROS), decrease in biomass and damage to the fixation of organic matter (Seth and Sebastian 2024). In particular, some crops encounter high temperatures at the reproductive growth stage, which leads to a decline in floret fertility, gametophyte sterility and severe fertilization abortion, resulting in a poor harvest. When high-temperature stress occurs in the early stage of seed/fruit development, it causes an imbalance in energy transfer from the source to the sink, leading to dysplasia of the embryo structure and reducing the accumulation of nutrients such as sugars, proteins and trace elements. In turn, the quality worsens (Janni et al. 2020; Seth and Sebastian 2024).

When the average temperature increases by 1 ℃ in the growing season, the rice yield decreases by 6.2%, the total brown rice yield decreases by 7.1% and 8.0%, the whole milled rice yield decreases by 9.0% and 13.8%, and the total income from rice milling decreases by 8.1% and 11.0%, respectively (Lyman et al. 2013). If rice is subjected to extreme high temperature during the vegetative growth period, it will not be conducive to its morphogenesis. The critical value of high temperature response for rice seedlings is 35 ℃, and exceeding this temperature may cause rice plants to die during the seedling stage (Aryan et al. 2022). When suffering to high temperatures above 33 ℃ during the tillering stage, rice tillering will be affected, and even the number of panicles will decrease (Mohanty and Hembram 2024; Ahmed et al. 2024). Exposure to high temperature stress during the reproductive growth period, such as short-term heat stress at the booting stage significantly decreases the panicle size, seed setting rate, grain size, chalky grain rate, milling characteristics and amylose content. Severe heat stress also decreases the peak viscosity and breakdown value and increases the gelatinization temperature of rice (Zhen et al. 2019). Rice is particularly sensitive to high temperatures at the heading and flowering stages. During these reproductive stages, high-temperature damage leads to fewer spikelets, deformed flower organs, and pollen abortion, seriously affects the processes of pollination and fertilization, and greatly reduces the seed setting rate. When rice experiences high temperatures during the filling period, it accelerates the filling rate and shortens the filling time, which are not conducive to the formation of a seed embryo structure, increasing grain chalkiness and deteriorating grain quality, taste, grain size and yield (Xu et al. 2021). Hence, heat damage to rice at any growth stage is not conducive to the formation of the final yield–quality structure.

The perception and response of plants to external temperature involves a complex network of multilevel signal connections formed by the intersection of multiple pathways, which reflects the integration of the activities of hundreds to thousands of different molecules in cells (Kerbler and Wigge 2023). Kan et al. (2023) consider that most of the identified thermosensory genes in plants are related to thermomorphogenesis, and that heat perception and response are always interrelated and synergistic. This understanding has made some progress in Arabidopsis. So how does rice perceive the increase in temperature and respond to it to achieve positive thermomorphogenesis, as well as defeat high temperatures to stabilize yield and quality? This review will integrate new discoveries on the molecular mechanisms of high temperature tolerance in rice in recent years to reply this issue. And on this basis, genome selection and genetic engineering for rice heat tolerance were supplemented, hoping to provide some theoretical guidance for rice heat-resistant breeding.

Thermosensing in Rice

Macromolecules are typically affected by heat, therefore they have the potential to serve as thermosensors (Hayes et al. 2021). As one of the most thermosensitive macromolecular structures, the plasma membrane has long been considered as a major candidate for plant thermosensing. The increase of membrane fluidity as well as the changes in structure and function at high temperature will strongly influence the folding, migration and activity of integral proteins or membrane-associated proteins (Hayes et al. 2021; Kerbler and Wigge 2023). And as a medium, membrane-associated proteins transmit signals to downstream pathways, regulating the expression of target genes to respond high temperature.

Transcriptional Regulation of NAC TFs

NAC family TFs proteins target different categories of genes involved in various stress responsive processes through membrane-nuclear translocation, completing responses to abiotic stress factors. Heat stress induces ONAC023 nuclear translocation for thermosensing. The dephosphorylation of the remorin protein OsREM1.5 is indispensable in this transversion. The nuclear ONAC023 targets and promotes the expression of OsFKBP20-1b and OsSF3B1 involved in alternative splicing. OsANN1, which has been proven to regulate rice heat tolerance, is accurately spliced downstream of ONAC023-OsFKBP20-1b/OsSF3B1 to perform its biological functions normally (Chang et al. 2024). The deletion of the membrane-associated NAC transcription factor OsNTL3 makes rice heat sensitive because it migrates from the plasma membrane to the nucleus under high-temperature stress, directly binds the promoter of OsbZIP74, promotes OsbZIP74 expression under heat stress, reduces H₂O₂ accumulation and weakens the leakage of electrolytes. In turn, the upregulation of OsNTL3 expression depends on OsbZIP74, and the interaction between these genes constitutes an excellent heat-tolerant circuit in rice (Liu et al. 2019a). ONAC127 and ONAC129 can form a heterodimer to respond heat stress. OsMSR2 and OsEATB may be directly regulated by them, while OsHSP101 and OsHCI1 are indirectly regulated (Ren et al. 2021). Overexpression of SNAC3 enhances the stability of the cell membrane and targets several ROS scavenging genes to positively regulate heat tolerance (Fang et al. 2015; Fig. 1).

Transduction of Ca²+ Signal

OsANN1 mentioned above is a member of the annexins family, carrying Ca²⁺ binding ability and ATPase activity. OsANN1 located on the periphery of cells probably form Ca²⁺ channels, regulating intracellular Ca²⁺ levels to modulate downstream signaling events. And OsANN1 can increase SOD and CAT activity to regulate H₂O₂ content and redox homeostasis under high temperature. It may directly act on OsCDPK24 to confront abiotic stress (Qiao et al. 2015). Cyclic nucleotide-gated ion channels (CNGCs) mediate heat-induced Ca²⁺ influx, which can be used as primary thermosensors to affect Hsf and HSP genes expression to improve heat tolerance. OsCNGC14 and OsCNGC16 are involved in the early signaling events of thermosensing and are essential modulators of Ca²⁺ signaling transduction (Cui et al. 2020a). In addition, HTS1 is believed to be associated with heat stress signaling. HTS1 encodes a thylakoid membrane-localized β-ketoacyl carrier protein reductase involved in de novo fatty acid biosynthesis, and it is a prerequisite for stabilizing the cell membrane. Compared to the wild type, the hts1 mutant exhibits disorganized stress signal transduction, larger Ca²⁺ influx to mesophyll cells, and transcriptional activation of HsfA2s and downstream target genes was inhibited (Chen et al. 2021a; Fig. 1).

Ubiquitination Pathway

TT3.1 is a thermosensor in rice that can sense extremely high temperatures. It encodes an E3 ubiquitin ligase located on the plasma membrane, whereas TT3.2 encodes a chloroplast precursor protein. The excessive accumulation of TT3.2 in chloroplasts caused by a high-temperature environment disrupts the normal structural morphology of chloroplasts. In the upstream and downstream regulatory pathways of TT3.1-TT3.2, TT3.1 is transferred from the cell membrane to the multivesicular body (MVB) after sensing a high temperature, and TT3.2 is introduced into the MVB and ubiquitinated, ultimately degrading TT3.2 in vacuoles, weakening its accumulation in chloroplasts, and helping to improve the heat tolerance of rice (Zhang et al. 2022a; Fig. 1).

OsHCI1, as a RING E3 ligase, is supposed to play an important role in thermal signal regulation. Although it is Golgi-localized, it can be induced by heat shock to enter the nucleus along the cytoskeletal tracts. OsHCI1 interacts with six substrate proteins, including OsbHLH065, and transports nuclear substrate proteins into the cytoplasm through monoubiquitination (Lim et al. 2013; Fig. 1).

Fig. 1

Molecular regulatory pathways in which plants perceive elevated temperature

Thermomorphogenesis in Rice

As the ambient temperature fluctuates, plants detect these shifts and dispatch signaling molecules to activate downstream pathways. They subsequently rely on a cascade of intricate molecular mechanisms to orchestrate physiological and biochemical adjustments, thereby responding to temperature variations. This adaptive process to guarantee the normal growth of tissues and the development of organs and to synchronize their morphogenesis is termed thermomorphogenesis (Casal and Balasubramanian 2019). In the face of rising temperature and oscillation of circadian clock, plants enhance their resilience to heat stress by undergoing macroscopic phenotypic alterations, which include the elongation of roots, hypocotyls, and petioles, increase in leaf height above soil level, as well as early flowering (Casal and Balasubramanian 2019; Zhang et al. 2021; Zhu et al. 2023). The adverse effects of high temperatures at night on rice have also been a hot research topic in recent years. For example, the transduction of nighttime temperature signals by the evening complex (EC) regulates the thermomorphogenesis in plants (Zhang et al. 2021). Yang et al. (2024) detect that OsGRP3/OsGRP162 transmits diurnal signals downstream of EC, regulating the diurnal thermotolerance of rice. In order to better explain the thermomorphogenesis of rice, we extend this concept to the morphogenesis of plant architecture, floral organs, and caryopses under heat stress.

Plant Architecture of Rice

Plant architecture is a significant set of agronomic traits that determine rice yield, mainly influenced by factors including tillering and plant height (Wang and Li 2005). The DEAD-box RNA helicase TOGR1, located in the nucleus, is regulated by both temperature and circadian clock, can serve as an intrinsic companion to promote effective pre-rRNA processing required for normal cell division, ensuring the morphogenesis of rice at high temperatures (Wang et al. 2016). TOGR2 encodes the auxiliary subunit of N-terminal acetyltransferase A, referred to as OsNAA15. Its two splicing variants, OsNAA15.1 and OsNAA15.2, coordinate glycolate-mediated ROS homeostasis in a temperature dependent manner, antagonizing the regulation of thermoresponsive growth in rice (Li et al. 2024). AET1 advantages rice adapt to high temperatures and is an essential guanosine transferase for pre-tRNA^His maturation. It can interact with RACK1A and eIF3h in the endoplasmic reticulum to regulate the translation of OsARFs (Chen et al. 2019). The mutation of Ptd1 causes the loss of disulfide bonds Cys⁵⁶-Cys¹⁰² and Cys⁷⁶-Cys¹¹⁶ in the eight-cysteine motif of its encoded non-specific lipid transfer protein, resulting in a change in protein conformation and causing rice to exhibit thermosensitive dwarfism (Deng et al. 2020). OsSPL7 was induced by heat stress, and the mutant spl7ko showed delayed growth and development, reduced culm length and plant height, while overexpression of SPL7OX-M resulted in the opposite effect (Hoang et al. 2019). A single-nucleotide substitution in GRY3 disrupts the production of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, affecting the pathway of methylerythritol 4-phosphate in chlorophyll synthesis. Under high temperature, ROS accumulation increases, photosynthetic protein degradation occurs, then plant height decreases (Jiang et al. 2023). The functional loss mutation of HSA1 creates delayed chloroplast biogenesis in rice under high temperature, bringing out a decrease in plant height and effective tillers (Qiu et al. 2018b). Stvb-i can protect meristematic tissues from heat stress, and the RNAi lines of Stvb-i show delayed growth and reduced tillering under heat wave (Hayano-Saito and Hayashi 2020; Fig. 2). The srl10 mutant is sensitive to high temperatures and exhibits semi-rolled leaves. The double-stranded RNA-binding protein encoded by SRL10 can alter the biogenesis of miRNA and interact with catalase isozyme B, regulating leaf morphology and heat tolerance (Wang et al. 2023).

Morphogenesis of Rice Floral Organs

Ensuring high seed setting rate under high temperature is particularly vital, as it requires guaranteeing the normal development of rice floral organs to accurately complete fertilization (Liu et al. 2023b). Spikelet fertility is determined by a combination of pistil and pollen viability. In contrast, male gametophytes are considered more sensitive than female gametophytes. But the development of the pistil under heat stress is equally important (Shi et al. 2022; Jagadish 2020). OsSGS3-tasiRNA-OsARF3 is an integral module for the development of rice floret under high temperature conditions. A single-nucleotide deletion causes the truncation of OsSGS3a protein, rejecting the interaction of OsSGS3a-OsSGS3b, resulting in the fault of the aforementioned module. Repeated heat waves during the booting stage disarray the development of fibrous sclerenchyma and spongy parenchymatous cells, eventuating in palea defects (Gu et al. 2023). The recessive mutation of SLG1 obstructs tRNA 2-thiolation, affecting the protein homeostasis of stress response. Under high temperature stress, pollen fertility deteriorates and pollen tubes are difficult to elongate (Xu et al. 2020b). EG1 protects the expression of floral organ characteristic genes OsMADS1, OsMADS6, and OsG1 through the high-temperature mediated mitochondrial lipid pathway, further promoting floral organ homeostasis (Zhang et al. 2016a). TSD1, as a transcription factor, physically interacts with YABBY protein to regulate the morphogenesis of spikelets to adapt to high temperatures (Cai et al. 2023). RINO2 regulates Ca²⁺ signaling and actin filaments in the inositol-dependent manner, which is crucial for heat tolerance related changes in pollen germination and pollen tube growth (Zhou et al. 2024a; Fig. 2).

OsTMS15 encodes an LRR-RLK protein MULTIPLE SPOROCYTE1, which is an necessary determinant for the normal development of the tapetum to form pollen. Its point mutation causes damage to the function of the tapetum under high temperature, leading to male infertility (Han et al. 2023). OsTMS19 is similarly a key gene for pollen formation under high temperatures. When the pentatricopeptide repeat protein encoded by it mutates, heat stress forces excessive ROS accumulation in the anthers during pollen mitosis, resulting to pollen sterility (Zhou et al. 2024b). Mutation of ERS1 leads to severe delays in pollen development and meiosis under high temperature, a relatively negative antioxidant system, and excessive ROS accumulation results in male infertility (Liu et al. 2024). OsRHS is a negative regulator of heat tolerance during rice flowering stage and can interact with cHSP70-4. The knockout of OsRHS and cHSP70-4 resulted in higher pollen fertility under heat stress compared to the wild type (Mao et al. 2024). The novel mutation of OsGRF4 at the GS2 loci allows it to evade miR396-mediated degradation, leading to a decrease in rice anthers heat tolerance (Mo et al. 2023). The OsHSP60-3B-FLO6 module is essential for pollen heat tolerance (Lin et al. 2023a; Fig. 2).

A study on the damage of female reproductive organs in rice under high temperature stress found that the thermosensitive genotype IR64 exhibited a higher frequency of embryo sac abnormalities, sugar starvation, higher ROS accumulation and altered TCA cycle and amino acid metabolites under heat stress. The heat-resistant genotype N22 can exhibit better metabolite maintenance and faster recovery by reducing H₂O₂, MDA, and increasing the supply of sugar and starch (Shi et al. 2022). A point mutation of TSF1 prevents the normal production of its encoded AGO7 protein, thereby impairing its ability to load miR390/miR390* duplex and eject miR390* during RNA-Induced Silencing Complex (RISC) formation. This damages the miR390-TAS3-ARF module at high temperatures, causing female infertility due to hindered pollen tube growth (Li et al. 2022). The HEI10 encoded by TFS2 can interact with RPT4 and SRFP1, ensuring average chromosome segregation during meiosis under heat stress. A point mutation causes female infertility in HEI10^tfs2 because when ZH11 is used as the male parent to pollinate HEI10^tfs2, it still does not fructify at high temperatures, while when HEI10^tfs2 is used as the male parent, the opposite is true (Zhang et al. 2024). OsMADS8 is crucial for the growth of rice pistils. The osmads8 mutant exhibits an initiation defect in the ovule at high temperatures, bringing about failed fertilization (Shen et al. 2024; Fig. 2).

Early flower opening to avoid diurnal heat stress is also a manifestation of heat tolerance during the flowering period (Liu et al. 2023b). DFOT1, also known as EMF1, located on the cell walls and can interact with multiple members of the pectin methylesterase (PME) family as well as OsGLN2, affecting the synthesis of pectin and cellulose in the cell walls of lodicule. In the dfot1 mutant, PME activity decreases, and the level of pectin methylation in the cell walls increases, promoting the absorption of water by the lodicule cell walls and swelling, pushing away the glume, and premature flowering to escape the high temperature during the daytime (Xu et al. 2022; Wang et al. 2022). qEMF3 originates from Oryza officinalis can equally assist rice escape daytime heat stress by early flower opening (Hirabayashi et al. 2015; Fig. 2).

Morphogenesis of Rice Caryopses

The morphology of rice caryopses determines yield and quality. The matter of starch synthesis in stable endosperm under high temperature urgently needs to be addressed. OsMADS7 participates in the stabilization of rice amylose content under heat stress. The expression of GBSSI, the main enzyme responsible for amylose biosynthesis, is elevated in OsMADS7-RNAi plants. Increased chalkiness occurs at high temperatures (Zhang et al. 2017). LOGL1 encodes a putative cytokinin-activation enzyme that regulates the biosynthesis pathway of thiamin regulated by circadian rhythms. The High nighttime temperature causes the grain weight of the logl1 mutant to be higher than that of the wild type (Sandhu et al. 2024). Under high temperatures, OsbZIP58 promotes expression of many seed storage protein genes and starch synthesis genes, while inhibiting expression of some starch hydrolyzing α-amylase genes. The loss of OsbZIP58 function leads to floury and shrunken endosperms, and a sharp decrease in storage materials in seeds under high temperatures (Xu et al. 2020a). FLR3 and FLR14 are positive regulators of grain quality that maintain redox homeostasis in the endosperm, and can reduce grain chalkiness by alleviating heat-induced oxidative stress in rice endosperm (He et al. 2023). HSP70cp-2 encoded by FLO11-2 mediates the regulation of temperature dependent chalkiness in grains, and high temperature causes severe chalkiness in flo11-2 mutants (Tabassum et al. 2020). FLO24 encodes HSP101, which can physically interact with enzymes related to rice starch biosynthesis and synergistically regulate rice starch biosynthesis and endosperm development under high temperature with HSP70cp-2 (Wu et al. 2024). Milled rice of the oslea1b mutant showed a significant decrease in starch content and structural abnormalities under high temperature conditions, with significantly higher chalkiness and chalky grain rate than the wild type (Li et al. 2023a; Fig. 2).

Fig. 2

The genes related to rice thermomorphogenesis are divided into three different modules that regulate rice architecture, floral organs and caryopses morphology. The regulation of floral organs is further divided into female and male fertility. Genes within the red box indicate negative regulatory effects on thermomorphogenesis, while the rest have positive regulatory effects

Heat Shock Transcription Factors and Heat Shock Proteins

Heat shock transcription factors (Hsfs) are conserved members of the transcription factor family. Their N-terminal DNA binding domain (DBD) can recognize and bind heat shock elements (HSEs) consisting of a repeated palindrome motif (AGAAnnTTCT3’), thus activating the expression of downstream heat shock response (HSR) genes, of which the heat shock proteins (HSPs) genes are the most specific class of HSR genes. HSPs can function as molecular chaperones to help some proteins avoid the formation of terminal aggregates due to misfolding and to regulate protein homeostasis so that plants can face various abiotic stresses (Fragkostefanakis et al. 2014; Guo et al. 2016).

Twenty-six Hsfs genes have been identified in rice, and a transcriptome analysis revealed that 22 Hsfs genes were induced by high temperature, of which OsHsfA2a was the most significantly induced gene (Mittal et al. 2009). OsHsfA2a can produce six transcriptional isomers through variable splicing, and all the other isomers except OsHsfA2a-5 can be significantly upregulated in the early stage of heat stress (Wang et al. 2012). OsHsfA2d encodes two main splicing variant proteins: OsHsfA2dII, which is dominant but transcriptionally inactive under normal conditions, and OsHsfA2dI, which is located in the nucleus and is induced by heat stress. OsHsfA2dI can upregulate the expression of the unfolded protein response sensors OsIRE1 and OsbZIP39/OsbZIP60 and the unfolded protein response marker OsBiP1 to re-establish cellular protein homeostasis (Cheng et al. 2015). OsHsfA6a interacts physically with HSP16.9 A-CI and HSP18.0-CII in the cytosolic region and with cHSP70-1 in the nucleus, synergistically mediating transcriptional regulation of HSP101 (Singh et al. 2021).

It is known that the ClpB1/Hsp101 gene is crucial for plant heat tolerance, and this gene is 100% distributed in rice varieties. However, compared to japonica rice, most indica and aus rice HSP101 proteins insert glutamic acid at 907th position. This may be one of the reasons for the difference in heat resistance between them (Kumar et al. 2023b). The positive feedback loop formed by HSP101 and HSA32 at the post transcriptional level prolongs the heat acclimation effect of rice seedlings and also affects the basal heat tolerance of rice seeds (Lin et al. 2014). The OsClpB/HSP100 molecular chaperone protein is regulated by OsHsfA2c and OsHsfB4b at high temperatures, which mediate the decomposition of denatured proteins. Three ClpB/HSP100 subtypes have been identified in rice: the cytoplasmic subtype (ClpB-cyt)/HSP100, the chloroplast subtype (ClpB-c)/HSP100 and the mitochondrial subtype (ClpB-m)/HSP100. The transcripts of cytoplasmic subtypes are induced by heat, and those of chloroplast subtypes and mitochondrial subtypes are present constitutively and upregulated under heat stress (Singh et al. 2012). 5’UTR of ClpB-C/Hsp100 aids in loading of transcripts onto polysomes, resulting in an uninterrupted and enhanced translation during heat stress, and it is an obligate requirement for ClpB-C/Hsp100 expression (Mishra et al. 2016). OsHSP60-3B is a member of the HSP60 family, and the heat shock protein 60-3b (oshsp60-3b) mutant is male sterile at high temperature. In-depth studies have revealed that high temperature is not conducive to the formation of starch granules in the pollen of the oshsp60-3b mutant or the clearance of ROS in male gametophyte cells, which leads to pollen abortion, the accumulation of ROS, and cell death, whereas the overexpression of OsHSP60-3B enhances the heat tolerance of the pollen of transgenic plants. Because the OsHSP60-3B protein interacts with FLOURYENDOSPERM6 (FLO6), the key component of rice pollen starch grain formation in plastids, it stabilizes the expression of FLO6 at high temperatures and ensures the normal development of rice male gametophytes (Lin et al. 2023a).

Small Heat Shock Proteins

Small heat shock proteins (sHSPs) are HSPs (15–30 kDa) with low molecular weights that exist widely in prokaryotes and eukaryotes and can play a role in combating various abiotic stresses (Wang et al. 2004; Zhang et al. 2016b). Under heat stress, HSP26.7, HSP23.2, HSP17.9 A, HSP17.4 and HSP16.9 A are upregulated at the seedling stage and flowering stage. Chen et al. (2014b) analyzed the sHSP complex in the heat-tolerant variety Co39 and the heat-sensitive varieties Azucena and Nipponbare. A protein complex named C1 was found to be composed of HSP18.1, HSP17.9 A, HSP17.7 and HSP16.9 A, and after exposure to heat stress for a certain period of time, the expression levels of these four sHSPs in Co39 were significantly higher than those in the other two rice varieties. The overexpression of OsHSP16.9 A in rice increased the seed and pollen germination rates and the seedling survival rate at high temperatures. Mass spectrometry analysis of the seeds of the OsHSP16.9 A-overexpressing lines revealed that the OsHSP101 and OsHSP16.9 A proteins accumulated together and formed a stable protein complex under heat stress, whereas the overexpression of OsHSP16.9 A in the oshsp101-silenced mutant did not increase the seed germination rate under heat stress, making the interaction of the OsHSP16.9 A-OsHSP101 protein more persuasive (Liu et al. 2023d). OsHSP16.9 C was highly expressed in rice panicles and leaves under heat stress, which was similar to its homologous gene AtHSP18.2 in Arabidopsis. A promoter deletion analysis revealed an incomplete but indispensable HSE fragment upstream of -539 to -523 bp, which was identified as the key cis-acting element of OsHSP16.9 C (Zhang et al. 2016b).

Thermotolerant loci

Heat tolerance in rice is a very complex quantitative trait that is regulated by many minor-effect loci (Liu et al. 2023a). Excavating heat-tolerant germplasm resources, locating heat-tolerant quantitative trait loci (QTLs) and cloning heat-tolerant genes are very helpful for understanding the molecular mechanism of heat tolerance and provide some theoretical guidance for heat-tolerant rice breeding. Plant scientists have created a series of mapping populations, including temporary populations such as F₂ and backcross (BC) populations, and permanent segregated populations, such as NIL, RIL and DH populations, to locate and clone heat-tolerant genes.

Mapping of Heat-Tolerant QTLs

Through a SNP marker analysis of N22/IR64 hybrid progeny BC₁F₁ and F₂ populations, Ye et al. (2011, 2015) mapped two major thermostable QTLs, qHTSF1.1 and qHTSF4.1, on rice chromosome 1 and chromosome 4 that control spikelet fertility under high-temperature stress, which could explain 12.6% and 17.6% of the phenotypic variation, respectively, and then finely mapped qHTSF4.1 to the interval of 1.2 Mb using the BC₅F₂ population. Stephen et al. (2023) used an F₂ population crossed between the heat-tolerant variety NERICA-L44 and the heat-sensitive variety Uma and identified a new marker, RM337, linked to spikelet fertility under high-temperature stress via bulked segregant analysis (BSA) combined with SSR markers, which were between those of OsNAC31 (LOC_Os08g01330) and OsMOT1 (LOC_Os08g01120). The subsequent expression data suggest that OsNAC31 is a very likely candidate gene. Chen et al. (2021b) mapped the locus qHTT8, which is related to heat tolerance at the flowering stage, to chromosome 8 by BSA-seq, and the F_2:3 population was crossed between the heat-sensitive variety 9311 and the relatively heat-tolerant variety Huang Huazhan (HHZ) as materials. LOC_Os08g07010 and LOC_Os08g07440 in this region were much more highly expressed in HHZ than in 9311 under heat stress, suggesting that these two genes might be candidate genes for qHTT8. Zhao et al. (2016) used chromosome segment substitution lines (CSSLs) between Nipponbare and 9311 to map two QTLs, qBDL2-2 and qBDL10, that control the basal dehiscence length of rice anthers at high temperatures. Hirabayashi et al. (2015) developed NILs of the medicinal wild rice Oryza officinalis on the genetic background of indica and mapped the heat-tolerant QTL qEMF3 at the flowering stage on chromosome 3. Flowering in the early morning could allow rice to escape heat stress during the daytime and reduce spikelet sterility caused by heat at the flowering stage. Fan et al. (2023) created 146 backcrossed inbred lines (BILs) between Oryza longistaminata and 9311. Three rice seedling heat tolerance QTLs, qTT4, qTT5 and qTT6, were identified. Among them, qTT4 and qTT5 are novel loci, whereas qTT6 expresses the heat shock protein-encoding gene OsHSP74.8 in response to heat stress. The appearance and quality of rice grains under high-temperature stress are also traits worthy of study. Murata et al. (2014) developed CSSLs using the indica rice variety ‘Habataki’ against the background of japonica rice ‘Koshihikari’, and located Apq1, which regulates grain quality under high temperature, between the markers Tak6166-3 and RM21971 on chromosome 7. Miyahara et al. (2022) bred RILs from a cross between the high-temperature-tolerant variety ‘Genkitsukushi’ and the high-temperature-sensitive variety ‘Tsukushiroman’ and mapped two antagonistic QTLs: qWB6, which increased rice back chalkiness, and qWB8, which reduced rice back chalkiness at high temperatures. Coincidentally, qWC6 was shown to be located in the same region as qWB6 through the cross of ‘Chikushi 52’ and ‘Tsukushiroman’ as early as 2015. However, the allele of qWC6 in ‘Genkitsukushi’ could reduce the occurrence of heart chalkiness in rice at high temperatures. Genome-wide association study (GWAS) have also been widely used to locate QTLs. Li et al. (2023b) identified a new locus, qHT7, related to heat tolerance at the seedling stage through a GWAS of 620 rice varieties. An analysis of the transcriptomes of the heat-tolerant variety FACAGRO (F64) and heat-sensitive variety PUILLIPINA KATARI finely located qHT7 to OsVQ30 (LOC_Os07g48710) and LOC_Os07g48710^Hap4, and these loci were revealed to be beneficial for heat resistance. Using 1.2 million SNPs from 173 rice varieties to analyze relative spikelet fertility (RSF) at high temperature and room temperature, Hu et al. (2022) located a new locus, qRSF9.2, where only the gene LOC_Os09g38500 contained a nonsynonymous SNP significantly associated with RSF, and its relative expression level was significantly different between heat-tolerant controls and heat-sensitive controls. The two haplotypes, Hap2 and Hap3, of this gene increased heat tolerance by 7.9% and 11.3%, respectively. These results provide powerful information for qRSF9.2 cloning and breeding of heat-tolerant rice varieties using molecular markers.

Cloning of Heat Tolerance Genes

Wei et al. (2012) and Liu et al. (2016) created F₂ population by crossing the heat-sensitive variety HT13 and heat-tolerant variety HT54, which could tolerate a high temperature of 48 °C for 79 h at the seedling stage. After high-temperature treatment, the segregation ratio of survival to death was 3:1, and the single dominant genetic gene, OsHTAS, encoding a RING-type E3 ubiquitin ligase that enhances the heat tolerance of rice by promoting H₂O₂-induced stomatal closure, was cloned. Park et al. (2020) bred DH strains with the F₁ generation of Cheongcheong/Nagdong hybridization and cloned the heat tolerance gene Osbht, which encodes an Hsps-p23-like calmodulin-binding protein and participates in the heat shock response as a molecular chaperone, at the booting stage. Subsequently, using the same materials, OsSFq3, which contributes to spikelet fertility and grain quality under high temperature conditions, was fine-mapped in the region RM15749-RM15689 on chromosome 3 in the filling stage. It is highly homologous to FLO6 and participates in glycometabolism and maintenance of sugar homeostasis (Park et al. 2021a). Takehara et al. (2018) cloned Sus3 in Apq1, encoding a key enzyme for the conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, catalyzing starch synthesis and reducing chalkiness at high temperatures. Using African cultivated rice CG14 as the donor parent and Asian cultivated rice WYJ as the recipient parent, Li et al. (2015b) cloned the gene TT1 (OgTT1), which encodes a 26 S proteasome α2 subunit, with increased high-temperature tolerance during vegetative and reproductive growth. Compared with its allele OsTT1, OgTT1 can rapidly degrade ubiquitinated proteins in Asian cultivated rice. It significantly eliminates the toxic proteins that accumulate in the cells at high temperatures and thus protects the rice cells. Kan et al. (2021) created a near-isogenic line, NIL-HPS32 using the African rice variety HP21 and the conventional rice variety HJX, in which a new heat tolerance-related gene, TT2, encoding a G protein γ subunit was cloned. Compared with that of wild-type TT2^HJX, the 165th base of the TT2^HPS32 gene is replaced, which causes TT2^HPS32 to terminate prematurely, resulting in a reduction in the intracellular calcium signal under high-temperature stress. It weakens the interaction of SCT1/2-CaM, weakens the increase in the transcriptional activity of CaM toward SCT1/2, and stabilizes the expression of the downstream transcription factor OsWR2, which regulates wax synthesis, thus endowing rice with heat tolerance. TT3.1 and TT3.2, as mentioned above, are two recently cloned genes that antagonize each other and regulate heat tolerance in rice. TT3, which was mapped by Zhang et al. (2022a), is another locus from African rice CG14. Under high-temperature stress at the heading and filling stages, the yield of NIL-TT3^CG14 was approximately twice as high as that of NIL-TT3^WYJ, which included three annotated genes, LOC_Os03g49900 (TT3.1), LOC_Os03g49930 and LOC_Os03g49940 (TT3.2). Knockout of LOC_Os03g49930 had no effect on heat tolerance, whereas the overexpression of TT3.1 or knockout of TT3.2 increased yield by more than 2.5 times. Cao et al. (2022) constructed a series of BC and NIL populations with wild rice HHT3 as the donor parent and heat-sensitive indica rice and japonica rice through years of crossing and backcrossing and cloned the gene HTH5, which encodes a phospholipid phosphate homeostasis protein (PLPHP) that positively regulates heat tolerance at the heading stage. High temperature induces an increase in the PLPHP content, reduces ROS accumulation and stabilizes the seed setting rate. Based on the rice supra/pan-genome and multitissue transcriptome, Lin et al. (2023b) cloned the TTL1 gene, which negatively regulates heat tolerance at the seedling stage and reproductive growth stage, as well as grain size and weight; this gene is located in the nucleus and cell membrane. It functions as a transcriptional activator to cooperate with TT2, TGW6 and other genes to regulate heat tolerance and grain size. Its function-deficient mutation enhances heat tolerance and increases grain size by coordinating cell expansion and proliferation, which is a new valuable target spot for improving the heat tolerance and yield of rice.

miRNAs

An miRNA is a small noncoding single-stranded RNA with a length of approximately 22 nucleotides. It can identify target genes by complementary pairing, cut the target mRNA posttranscriptionally or inhibit the translation of the target mRNA, thus affecting plant growth and development and environmental adaptation. The miRNA-coding gene is located mainly in the intergenic region (IGR) and in the introns of the host gene, which are called mitrons (Carthew and Sontheimer 2009; Berezikov et al. 2007). Mitrons are processed by the intron of the host transcript, while the miRNA gene located in the intergenic region is first transcribed into the primary miRNA (pri-miRNA), which is then catalyzed by RNA polymerase II. Under the action of a heteropolymeric complex formed by ribonuclease III (RNase III) DCL1, the coenzyme factor HYL1 and the zinc finger protein SE, pri-miRNA is further processed into precursor miRNA (pre-miRNA) to form a self-complementary stem‒loop secondary structure. The DCL1 complex can continue to cut it into an miRNA/miRNA* double-stranded structure (miRNA/miRNA* duplex). The double-stranded miRNA is methylated by HEN1, transported out of the nucleus and separated into the cytoplasmic double-stranded miRNA with the help of the nuclear transport protein HASTY. One of the two strands of the miRNA is degraded by a nuclease, and the other strand, known as the guide chain, becomes a mature miRNA, which can bind to the AGO1 protein and be loaded into the RISC to achieve transcriptional cleavage or translational inhibition of the target gene (Carthew and Sontheimer 2009; Berezikov et al. 2007; Dong et al. 2008; Shang et al. 2023). Currently, many miRNAs have been shown to be involved in the responses of plants to biotic and abiotic stresses; therefore, thoroughly studying the upstream and downstream regulatory mechanisms of certain miRNAs is highly important.

With the continuous development of high-throughput sequencing technology, several rice thermal-responsive miRNAs have been identified through reverse genetics, and potential downstream target genes have been predicted. Li et al. (2014) constructed a small RNA library of young panicles of the heat-tolerant variety N22 at high temperature and normal temperature. Ion torrent sequencing and differential expression analysis revealed that the expression of 26 miRNAs was downregulated and that the expression of 21 miRNAs was upregulated under heat stress. Five heat tolerance-related miRNAs, namely, osa-miR162b, osa-miR529a-p5, osa-miR169n, osa-miR171b, and PC-5P-62245-9, which were not reported in other plants, were verified by qRT‒PCR. The expression pattern was consistent with the sequencing results. Moreover, several predicted miRNA targets are also noteworthy; for example, a target gene of osa-miR-2869-5p encodes PI5K, which can phosphorylate PIPs on the plasma membrane, while heat stress can trigger the accumulation of PIP on the plasma membrane of rice leaves, and thus miR2869-PI5K-PIP is very likely to be one of the pathways regulating heat tolerance in rice (Mishkind et al. 2009). Later, Luo et al. (2021) used young panicles of N22 and 9311 as materials, constructed 8 small RNA libraries of two rice varieties at four different heat treatment time points and performed deep sequencing. Using the 9311 library as a control, 90 differentially expressed miRNAs were identified at the booting stage. The results of target gene predictions showed that these differentially expressed miRNA target genes were significantly enriched in the plant hormone signal transduction pathway. The negative regulatory effects of osa-miR166k-5p and osa-miR5496 on the ethylene signaling regulators OsEIN2 and OsEIL1, respectively, and the negative regulatory effects of osa-miR408-3p on the auxin primary response gene OsIAA6 were also verified via a subsequent comparative expression analysis. Miao et al. (2018) constructed an miRNA library of the heat-tolerant indica rice variety HT54 at the seedling stage. A series of miRNAs that respond to high-temperature stress were identified by next-generation sequencing (NGS). Among them, the expression of miR396e and miR396f, which are representative of the miR396 family, was significantly upregulated. The analysis of the degradation group suggested that these genes might target the growth regulation factor OsGRFs to regulate heat tolerance. In addition, the expression of Os-miR397b.2, a member of the MiR397 family, was induced under heat stress, and a predicted gene encoding ascorbate oxidase was confirmed by 5’-RACE to be the target (Jeong et al. 2011). Fan et al. (2024) used XN0437T and XN0437S, which showed significant differences in heat resistance during the grain filling stage, as materials to identify 102 differentially expressed miRNAs at high night temperature. The miRNAs-mRNA target relationship was further validated through degradome sequencing, and enriched in biological processes such as protein folding, hormone regulation, and redox. Some pathways by which miRNAs respond to drought, salinization and low-temperature stress in rice have been characterized, but reports of the roles of miRNAs in heat stress are limited.

Plant Hormones

As chemical messengers, plant hormones are very important for coordinating intracellular and extracellular activities. When plants are subjected to various stresses, they experience changes in the levels of various hormones in tissues to cope with adverse external factors (Khan et al. 2023). The exogenous application of plant hormones can change the ability of plants to defend against stress, which fundamentally affects the synthesis and activity of endogenous hormones and results in bilateral and multilateral hormonal signaling during the stress response process, regulating the expression of stress-responsive genes and hence plant growth and development. Auxin, cytokinin (CK), gibberellin (GA), abscisic acid (ABA) and ethylene (ET) are five classic plant hormones, and much progress has been made in investigating the responses of plant hormones to abiotic stress (Khan et al. 2023; Kosakivska et al. 2021).

Auxin

Heat stress inhibits the expression of most YUCCAs genes and their transcriptional regulators SPL, NGA and TFL in rice florets; significantly decreases the average endogenous IAA content; and represses the auxin receptor genes TIR1/AFB2, auxin response factor ARFs and their target gene HAF, which damage the IAA signaling pathway. All these factors lead to the abnormal growth of pistil pollen tubes, a decrease in pollen viability, and increased spikelet sterility (Sharma et al. 2018; Zhang et al. 2018). OsIAA29 regulates seed development in high temperature through competition with OsIAA21 in the binding to OsARF17, mediating auxin signaling pathway in rice (Chen et al. 2024). Naphthylacetic acid (NAA), an auxin analog, can alleviate the damage caused by high temperature in rice. Exogenous application of NAA at the flowering stage can upregulate the expression of YUC1, YUC9, YUC11 and other genes in the pistil, which ensures the normal development of pollen tubes under heat stress. ROS are also involved in this process through crosstalk between signaling molecules and auxin. Although POD can reduce ROS accumulation, it also degrades auxin. Exogenous NAA can reduce POD activity by inhibiting the expression of POX, while the accumulation of ROS at high temperatures induces an increase in POD activity. Several pathways cooperatively regulate the development of pollen tubes at high temperatures (Zhang et al. 2018; Fig. 3).

Cytokinin

The biosynthesis of cytokinin (CK) involves a variety of enzymes, including isopentenyl transferase (IPT), cytochrome P450 monooxygenase and nucleoside 5-monophosphoribosylhydrolase encoded by LONELY GUY (LOG), and its decomposition is catalyzed mainly by cytokinin oxidase/dehydrogenase. High temperature is not conducive to the transport of cytokinins and inhibits the activity of related synthases, increases the activity of oxidase/dehydrogenase and promotes their degradation. As a result, the abundance of CK in the rice panicles decreased, and the number of spikelets per panicle decreased. The exogenous application of 6-benzylaminopurine could alleviate this harmful effect (Wu et al. 2017). Kinetin (KT) is a type of cytokinin. Exogenous application of a low concentration of KT (10^− 9 M) to rice seedlings can improve the heat tolerance of rice seedlings by increasing the activities of SOD and POD, reducing the level of ROS accumulation, reducing the relative exosmosis rate of electrolysate and MDA contents, and alleviating membrane damage. Moreover, exogenous application of KT induces the expression of the CK-responsive genes OsRR2, OsRR4, and OsRR6; the antioxidant enzyme-encoding genes OsCATB, OsAXP1, and Fe⁺-SOD; the stress defense genes OsSNAC1, OsDREB2A, and OsLEA3; the heat shock protein-encoding genes OsHSP70 and OsHSP90; and the heat shock transcription factor OsHsfA2d (Mei et al. 2023). Another cytokinin derivative, meta-topolin-9-(tetrahydropyran-2-yl) purine (mT9THP), can cooperate with heat acclimation to promote the expression of OsHSP90, OsHsfA2d and other genes and moderately increase the emission of volatile organic compounds at high temperatures. The release of its active form meta-topolin prevents the rapid inactivation of exogenous cytokinins, which is highly beneficial for the heat tolerance of rice (Prerostova et al. 2023; Fig. 3).

Gibberellin

Gibberellin (GA) participates in the regulation of α-amylase activity by ROS under heat stress, which affects the chalkiness of rice during grain filling. The increase in H₂O₂ levels under heat stress upregulates the expression of the GA biosynthesis genes OsGA3ox1 and OsGA20ox1, thus increasing the activity of α-amylase to result in starch degradation and increased grain chalkiness (Suriyasak et al. 2017). The GA/ABA dual-luciferase reporter system designed by Wu and Hong can be used to detect dynamic changes in GA and ABA responses and crosstalk in rice under heat stress. High induction of OsAmy7 expression by GA is closely related to seed germination at high temperatures (Wu and Hong 2021; Fig. 3). Pantoja-Benavides et al. (2021) comprehensively evaluated the effects of several foliar growth regulators on the physiological and biochemical responses of rice under high-temperature stress. The application of gibberellin increased the crop stress index (CSI) and relative tolerance index (RTI), indicating that exogenous gibberellin could protect rice from heat damage to a certain extent at high temperatures.

Abscisic Acid

High temperature increases the content of abscisic acid (ABA) in anthers, leads to the accumulation of ROS in anthers, causes oxidative damage and the early induction of programmed cell death (PCD) in tapetum cells, and impairs pollen fertility. Tapetum PCD is regulated by the interaction between the stress-activated protein kinase SAPK2 and the DEAD-box ATP-dependent RNA helicase eIF4A-1. Knockout of OsSAPK2 blocks ABA signal transduction, resulting in abnormal tapetum degeneration during normal-temperature growth, and cannot alleviate the oxidative stress caused by ABA-mediated ROS accumulation in anthers under heat stress; thus, the pollen viability of sapk2 mutants decreases more significantly at high temperatures than that of wild-type plants (Zhao et al. 2023a). The heat-induced ABA synthesis gene OsNCED1 positively regulates heat tolerance, and overexpression of OsNCED1 significantly activates the expression of antioxidant enzymes, ABA signaling pathways, heat response, and defense related genes (Zhang et al. 2022b). Exogenous ABA pretreatment significantly activates the ABA signaling pathway, upregulates the ABA response genes OsSalT and OsWsi18, and upregulates the expression of ROS-scavenging genes such as OsCATA, OsAPX6, OsCu/Zn-SOD and OsSodCc, which increase the activities of CAT, APX and SOD. ABA pretreatment also increases the activity of starch synthetase under heat stress, increases the starch content and gel consistency, decreases the amylose content, increases the rice filling ability at high temperatures, and improves rice quality (Liu et al. 2023c). Another study using ABA pretreatment showed that the exogenous application of ABA at the rice seedling stage under heat stress reduced the relative electrolyte leakage rate and MDA content; inhibited the expression of the apoptosis-related genes OsKOD1, OsCP1 and OsNAC4; increased the expression of the apoptosis-inhibiting gene OsBI1; reduced cell damage; and reduced seedling leaf wilt by 2.5–28.5% and chlorophyll loss by 12.8–35.1%. Exogenous ABA can also induce the expression of the ABA biosynthesis genes OsNCED3 and OsNCED4 and the heat shock-related genes OsHSP23.7, OsHSP17.7, OsHsf7 and OsHsfA2a, which potentially initiate heat tolerance in rice seedlings (Liu et al. 2022). Spraying ABA at the flowering stage can increase the seed setting rate of rice under heat stress, and its regulatory mechanism may be related to trehalose metabolism and ATP consumption (Zhu et al. 2022; Fig. 3).

Ethylene

The ethylene response factor OsERF115/AP2EREBP110 is strongly induced by high temperature treatment and plays an important role in ethylene signaling transduction. Its overexpression improved the water-saving traits of rice (Park et al. 2021b). Ethylene-mediated signal transduction is also regulated by heat stress combined with treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic (ACC) acid. The induction of the heat shock transcription factors OsHsfA1a, OsHsfA2a,c,d,e, and f and the ACC oxidase genes OsACO1 and OsACO3 and the expression of OsEIN2, OsEIL1 and OsEIL2, which are involved in the ethylene signaling pathway, reduce ion leakage and the MDA content, contribute to water maintenance and significantly reduce intracellular lipid peroxidation. Rice seedlings are endowed with heat tolerance (Wu and Yang 2019). The exogenous application of ethephon optimizes the ethylene level under high-temperature stress and enhances the activity of the enzymatic antioxidant defense system, thus reducing the contents of H₂O₂ and thiobarbituric acid reactive substances (TBARS). In addition to regulating carbohydrate metabolism, the expression of psbA and psbB in photosystem II is upregulated, and the photosynthesis of rice under heat stress is increased (Gautam et al. 2022). Using plant growth-promoting bacteria (PGPB) to address heat stress is a sustainable and environmentally friendly method, and Brevibacterium flax RS16 can produce ACC deaminase. After inoculation with RS16, the expression of ethylene and the concentration of ROS decrease, whereas the expression of glutathione S-transferase (GST) and two sHSPs, HSP16 and HSP26, increase in rice. The restriction of DNA damage at high temperatures effectively promotes photosynthesis, increases biomass accumulation, and positively regulates the high-temperature tolerance of rice (Choi et al. 2021a, b; Fig. 3).

Fig. 3

Plant hormone related genes involved in the regulation of rice heat tolerance, divided into five sectors: auxin, cytokinin, gibberellin, abscisic acid, and ethylene. The yellowish layer in the middle represents endogenous heat-associated genes related to plant hormones. The outer light-green layer represents genes induced by exogenous hormone treatment. Black font genes indicate positive regulation of heat tolerance, while red font genes indicate negative regulation of heat tolerance

Source‒Sink Heat Resistance Theory

The sensitivity of plant photosynthesis to heat stress shows that photosystem II (PSII), the electron transport chain and Rubisco-activating enzyme (RCA) are vulnerable to high-temperature damage, and usually, a decrease in PSII activity is the decisive factor for a decrease in photosynthetic efficiency (Ferguson et al. 2021). The fluidity of the thylakoid membrane increases at high temperatures, and the light-harvesting complex falls off the thylakoid membrane, which leads to the loss of integrity of PSII and affects subsequent electron transfer and ATP synthesis (Hu et al. 2020). Rubisco is a key step in the immobilization of CO₂ in the Calvin cycle, which requires RCA to consume ATP to guide its conformational transition and prevent the inhibition of activity. However, due to the thermal instability of RCA itself, it cannot help Rubisco reach the activation state at high temperatures, and thus it weakens photosynthesis at high temperatures (Hu et al. 2020; Chen et al. 2022a). The respiration rate of plants increases due to heat stress, and a strong heat wave disrupts the ratio of the net carbon balance in the plant, namely, the ratio of respiratory consumption (R) to photosynthetic assimilation (A). An increase in the R/A ratio will transfer assimilated nonstructural carbohydrates to the respiratory metabolic pathway, and this imbalance in the allocation of carbon resources will limit energy allocation from the source to sink. Insufficient accumulation of sinks leads to losses in crop yield and quality (Hu et al. 2020; Posch et al. 2019).

Protecting Photosynthesis

The D1 subunit protein encoded by PsbA can repair the damage to photosystem II caused by heat stress. The supplementation of D1 protein from the nucleus increases the photosynthetic capacity of transgenic rice under heat stress, increases the assimilation rate of net CO₂, and increases biomass accumulation and grain yield (Chen et al. 2020). Following the overexpression of RCA, the content of Rubisco in the oxRCA lines decreases significantly compared with that in the wild type line, but the strain overexpressing both Rubisco and RCA does not significantly decrease the content of Rubisco, and thus it has a higher CO₂ assimilation rate and shoot dry weight at high temperature than the wild type line, indicating that increasing the content of RCA can improve the heat tolerance of rice without reducing the content of Rubisco (Qu et al. 2021). PWL1 regulates premature leaf senescence and heat tolerance by affecting ROS homeostasis and chloroplast function. Compared with the wild type line, the pwl1 mutant presented severe premature senescence, increased sensitivity to high temperature, increased intracellular ROS accumulation, decreased CAT and SOD enzyme activities and chloroplast degradation. The overexpression of PWL1 enhances leaf antiaging ability, stabilizes chloroplast structure and function, and ensures photosynthesis at high temperatures (Xu et al. 2023). The antagonistic effect of TT3.1 on TT3.2 also protects chloroplasts from heat stress, which provides a strategy for accumulation from the source to sink at high temperatures (Zhang et al. 2022a). HSA1 encodes fructokinase-like protein 2 (FLN2), which is located in chloroplasts and regulates chloroplast development. Recessive mutations of hsa1 mutants in FLN2 cause an albino phenotype in rice and significantly delay chloroplast biogenesis under high-temperature stress, which can protect chloroplasts under late heat stress (Qiu et al. 2018b). The intermediate filament gene OsIF is involved in protecting the chloroplast structure under high temperature, and the overexpression of OsIF can stabilize the photosynthetic mechanism and yield under heat stress (Soda et al. 2018). HYR, a transcription factor related to photosynthetic carbon metabolism, activates photosynthesis-related genes, promotes downstream multistage signaling connections, and increases yield stability at high temperatures (Ambavaram et al. 2014). GRY3 is the decisive factor in chlorophyll synthesis, and the methylerythritol 4-phosphate pathway is disrupted in the gry3 mutant. ROS accumulation increases under high temperature, leading to cell death and degradation of photosynthetic proteins (Jiang et al. 2023). High temperature induces HES1 expression, and there are differences in the expression of photosystem related genes in hes1 mutant, indicating that HES1 plays an important role in maintaining chloroplast function (Xia et al. 2021). PGL, encoding chlorophyllide an oxygenase 1 (OsCAO1), impacts leaf senescence. The pgl mutation leads to a decrease in the photosynthetic rate of rice and makes it sensitive to heat stress (Yang et al. 2016). PGL10 encodes PORB: protochlorophyllide oxidoreductase B, which can revert prochlorophyll lactone to chlorophyll lactone. It is necessary for chlorophyll biosynthesis and photosynthetic activity under heat stress (Ahmad et al. 2024). OsNSUN2 is the major mRNA m⁵C methyltransferase in rice. It can regulate mRNA translation, maintain chloroplast function, and enhance rice adaptability to heat stress (Tang et al. 2020). TCM5 plays an important role in chloroplast development and PSII function maintenance in rice under high temperature conditions (Zheng et al. 2016). The missense mutation of TSCD5 leads to defects in chloroplast thermosensitivity (Yang et al. 2023; Fig. 4).

Maintaining the Steady State of ROS

ROS can be byproducts of metabolic pathways such as photosynthesis and respiration or produced by specialized oxidases. When abiotic stress forces the excessive accumulation of ROS, oxidative damage occurs in plant cells, and thus strict control of ROS homeostasis is necessary for heat tolerance in rice (Mittler et al. 2022; You and Chan 2015). Cis-aconitase is an important component of the tricarboxylic acid cycle (TCA) in the respiratory metabolic pathway. The expression of the cis-aconitase gene OsACO1 in rice is upregulated by heat stress, and the W-box element located in its promoter participates in the transcriptional regulation of the heat stress response. An increase in cis-aconitase activity can block the TCA cycle under heat stress, produce protective metabolites and provide energy for ROS scavenging (Li et al. 2015a). The overexpression of the transcription factor WRKY10 accelerates the accumulation of (ROS) in chloroplasts and apoplasts and negatively regulates heat tolerance but also induces expression of Hsfs and HSPs. VQ8 can interact with WRKY10 to inhibit its DNA binding activity and antagonize its negative regulation (Chen et al. 2022b). When the gene OsPDIL1-1 was silenced via RNAi, the activity of NADPH oxidase increased, the accumulation of ROS during anther development increased, and pollen viability and floret fertility were more severely damaged by high temperature, which indicated that OsPDIL1-1 is a positive regulator of ROS homeostasis (Zhao et al. 2023b). The stress-responsive NAC transcription factor SNAC3 can endow rice with heat tolerance, which can regulate the expression of many ROS-scavenging genes to regulate ROS homeostasis. Compared with that in wild type plants, the H₂O₂ content in SNAC-OE plants decreased significantly under heat stress, whereas the opposite trend was detected in SNAC-RNAi plants (Fang et al. 2015). The GDSL lipase gene HTA1 is a negative regulator of heat tolerance in rice. At high temperatures, the expression of antioxidant enzymes in the hta1 mutant increased, the level of ROS decreased, and the degree of membrane damage decreased (Su et al. 2024). OsEDS1 can bind to the catalase OsCATC, increase its activity and increase its ability to scavenge hydrogen peroxide, thus regulating ROS homeostasis (Liao et al. 2023). The pyridoxal phosphate protein encoded by HTH5 helps control the level of ROS at high temperatures and is a valuable genetic resource of heat tolerance in rice (Cao et al. 2022). OsProDH, located in mitochondria, controls H₂O₂ levels through proline metabolism, negatively regulates heat tolerance (Guo et al. 2020). The mutation of PSL50 leads to excessive accumulation of H₂O₂ and increased cell death in rice under heat stress (He et al. 2021). The OsHis1.1-OE lines accumulate more ROS under heat stress than the wild type, negatively regulating heat tolerance (Wan et al. 2023; Fig. 4).

Stomatal Movement and Development

Stomata play an important role in the plant adaptation to adverse environments by controlling water loss via transpiration. Plants continue to evolve stomatal structures to tolerate stress factors and cope with extreme temperatures (Hetherington and Woodward 2003; Wang and Chang 2024). OsHTAS positively regulates the heat tolerance of rice by promoting stomatal closure induced by H₂O₂, while the function of OsMDHAR4 is opposite that of OsHTAS. The heat tolerance of mdhar4 mutants increases, and that of OsMDHAR4-overexpressing lines decreases (Liu et al. 2016, 2018). HST1 encodes a DST transcription factor that negatively regulates heat tolerance through ROS-mediated stomatal movement and heat response gene expression. Under heat stress, the expression of the peroxidase gene in the hst1 mutant is inhibited, the level of ROS increases, the expression of sHSPs, including HSP58, HSP74 and HSP80, is upregulated, stomatal conductance decreases, and water loss decreases (Ding et al. 2023). Compared with the wild type plants, the overexpression of OsEPF1 can significantly reduce stomatal density, maintain stomatal conductance better, and increase the water retention capacity, which can increase the survival of rice plants at high temperatures (Caine et al. 2018). The knockout of OsSTOMAGEN, a positive regulator of stomatal development, and its sibling homologous gene OsEPFL10 decrease stomatal density, and the deletion of OsSTOMAGEN induces a certain level of epistasis on OsEPFL10-mediated stomatal development, all of which affect leaf temperature and water retention and improve agronomic performance to cope with the increase in temperature caused by global climate change (Karavolias et al. 2023; Fig. 4).

Fig. 4

The theoretical model of heat tolerance of rice source-sink from the point of view of photosynthesis, respiration and transpiration. Under heat stress, protect chloroplasts to ensure normal photosynthesis, enhance ROS scavenging ability to protect cells from oxidative damage, and mediate stomatal opening and closing and development to improve rice water retention capacity. The gene in the green box indicates positive regulation, while the gene in the yellow box indicates negative regulation

Breeding by Genomic Selection

Although rice is a thermophilic crop, rice is unable to survive at extreme high temperature. Therefore, the breeding of heat-tolerant rice varieties by introducing heat-resistant loci via conventional breeding and molecular marker-assisted selection is urgently needed. First, screening heat-tolerant lines from public rice germplasm resources is highly important. In the early stage, the International Rice Research Institute used an artificial climate chamber to screen a number of rice germplasm resources with strong heat tolerance. For example, the seed setting rate of N22 and other varieties can reach more than 80% under heat stress (IRRI 1984). Li et al. (2019) evaluated the heat tolerance of some rice germplasms using an artificial climate chamber with a constant temperature of 39 °C during the day, combined with the performance of natural conditions in the field. The selected heat-tolerant germplasms, ZY11, ZY18 and ZY67, promisingly provided donor materials for subsequent heat-tolerant rice breeding. Liu et al. (2019b) evaluated the heat tolerance of varieties from Africa by the cascade temperature method and identified several valuable varieties, such as ‘SDWG005’, ‘SDWG001’, ‘SDBN00’ and ‘SDNR005’. The Rice Institute at the Guangdong Academy of Agricultural Sciences evaluated the heat tolerance of rice resources from 11 countries at the heading and flowering stages and revealed that Ganxiangnuo and N22 presented the greatest heat tolerance (Yang et al. 2012). Understanding the genotype differences between rice varieties with different heat tolerance through omics techniques is particularly promising. The proteomic analysis of anthers from three rice varieties with different heat tolerance, N22, IR64, and Moroberekan, revealed that the protein of heat-resistant variety N22 was enriched in biological processes related to unfolded protein binding and carboxylic acid metabolism under long-term heat stress. In short-term heat stress, N22 anthers were enriched in proteins associated with vitamin E biosynthesis and GTPase activator activity. This laid the foundation for the breeding of flowering heat-resistant varieties through molecular breeding (Kumar et al. 2023a).

Although a few QTLs have been successfully cloned, their application in heat-tolerant breeding is still in progress. With the progress of rice molecular genetics and genomics, compared with traditional breeding methods, molecular marker-assisted selection (MAS) has significantly improved breeding efficiency and has been widely used in the breeding and development of various crops (Collard and Mackill 2008). However, with the wide application and breeding practice of MAS, its limitations are gradually being observed. In general, MAS can identify and mark the genes of interest with SNP molecular markers closely adjacent to the target traits to integrate the major genes or high-effect QTLs controlling the target traits into new varieties (Gregorio et al. 2013). However, many traits of rice, especially heat tolerance, are controlled by many genes with minor effects (Spindel et al. 2015). Because the general MAS only focuses on the utility of some major genes, MAS breeding is far from suitable for the genetic structure of these traits, resulting in the elimination of genes with minor effects early in selection.

Meuwissen et al. (2001) proposed a new alternative to traditional MAS breeding—genome selection (GS). Marker-assisted selection using high-density molecular genetic markers covering the whole genome can shorten the generation gap by early selection. As a new generation of breeding technology developed based on the traditional breeding methods and MAS, GS has achieved a breakthrough in identifying candidate individuals from phenotypic selection to genome selection and has been applied in some crops, such as corn, sugar beet and cassava (Riedelsheimer et al. 2012; Würschum et al. 2013; de Oliveira et al. 2012). To date, although related research on GS in rice breeding populations is lacking, preliminary explorations of the genetic improvement of rice yield have been conducted (Cui et al. 2020b; Xu et al. 2014). Pan et al. (2021) evaluated the heat tolerance of 205 domestic and foreign germplasm resources in combination with high-density SNP markers for GS and preliminarily predicted candidate genes. A total of 130 SNP markers significantly associated with heat tolerance were screened by genome-wide association analysis, and 18 heat-resistant QTLs were identified, of which 6 colocalized with reported heat tolerance-related QTLs. Nevertheless, the high cost of genotyping and sequencing is one of the main factors limiting the promotion of GS (Budhlakoti et al. 2022). The determination and sequencing of individual SNP genotypes are performed using SNP chips. With the increasing popularity of commercial high-density SNP chips and the decline in the price of second-generation sequencing, several high-quality breeding chips, such as RICE6K, RiceSNP50, and RICE90K, have been developed, but the high cost of genotyping is still a limiting factor (Qiu et al. 2018a; Chen et al. 2014a; Yu et al. 2014, 2022). This high cost forces many breeders to limit long-term genotyping and they do not dare to incorporate GS into large-scale application. Moreover, many studies have reduced the input by reducing the size of the reference population or reducing the marker density to reduce the cost, which affect the accuracy of the GEBV to a certain extent, thus underestimating the potential of GS technology in crop breeding and hindering the popularization and application of this technology (Budhlakoti et al. 2022). Therefore, reducing the cost of individual genotyping has always been one of the research hotspots in GS.

Genetic Engineering for Rice Heat Tolerance

With the continuous development of biotechnology, people hope to improve crops productivity in a direction beneficial to humans through genetic engineering. The emergence of the CRISPR-Cas9 system and transgenic technology has supported us reach this conception. For the improvement of rice heat tolerance, we can selectively edit or silence a negative regulatory gene or overexpress a positive regulatory gene, or introduce exogenous heat tolerance genes into rice. Such as, TT2, TT3.2, and TTL1 are new target spots for heat resistance and high yield in rice. Overexpression of receptor-like kinase ERECTA in rice confers thermotolerance that is independent of water loss (Shen et al. 2015). Overexpression of OsRab7 can improve the heat tolerance and yield of transgenic rice (El-Esawi and Alayafi 2019). Overexpression of OsMYB55 can promote plant growth under high temperature and reduce the negative impact of high temperature on grain yield (El-kereamy et al. 2012). Introducing AtHSP101 into rice, transgenic rice lines exhibit excellent growth performance during the recovery period after heat stress (Katiyar-Agarwal et al. 2003). The ectopic overexpression of TaHsfA5 and TaMBF1c significantly enhances tolerance rice to high temperatures (Samtani et al. 2023; Qin et al. 2014), and so on. Nowadays, genetic engineering biotechnologies are gradually being ameliorated. For example, CRISPR-Cpf1 is a newly identified CRISPR-Cas system, which can efficiently generate specific and heritable targeted mutations in rice (Xu et al. 2017). Applying this technology to heat-resistant breeding of rice will be more efficient and convenient.

Prospects

African rice and wild rice are valuable germplasm resources. Making full use of their advantages to improve domesticated rice is highly beneficial for agricultural production (Atwell et al. 2014). Focusing on the design of a new type of heat-tolerant ‘smart’ rice and aggregating multiple heat-tolerant QTLs or genes to improve heat tolerance is necessary to ensure the stability of yield–quality structures.

Additionally, tetraploid rice is a valuable new germplasm with good fertility and yield potential that can not only promote heterosis for heat tolerance but also provide a new haplotype model for many other key genes related to agronomic traits (Yu et al. 2021). This topic is worthy of our attention and research.

The exploitation of C₄ plant genes to improve rice, such as introducing genes related to some enzymes in the photosynthetic pathway encoded by C₄ plants into rice to increase the photosynthetic efficiency of C₄ plants, is prospective (Ermakova et al. 2019). From the point of view of source‒sink, this approach may improve the heat tolerance of rice and how to ultimately achieve this concept in the future is challenging but promising.