Yi Kan ,Hong-Xuan Lin ,c,*

a National Key Laboratory of Plant Molecular Genetics,CAS Centre for Excellence in Molecular Plant Sciences and Collaborative Innovation Center of Genetics&Development,Shanghai Institute of Plant Physiology and Ecology,Chinese Academy of Sciences,Shanghai 200032,China

b University of the Chinese Academy of Sciences,Beijing 100049,China

c School of Life Science and Technology,ShanghaiTech University,Shanghai 201210,China

Keywords:Rice Thermotolerance Thermal response Thermosensitivity Molecular regulation

ABSTRACT Global warming threatens food security.Rice(Oryza sativa L.),a vital food crop,is vulnerable to heat stress,especially at the reproductive stage.Here we summarize putative mechanisms of hightemperature perception(via RNA secondary structure,the phyB gene,and phase separation)and response(membrane fluidity,heat shock factors,heat shock proteins,and ROS(reactive oxygen species)scavenging)in plants.We describe how rice responds to heat stress at different cell-component levels(membrane,endoplasmic reticulum,chloroplasts,and mitochondria)and functional levels(denatured protein elimination,ROS scavenging,stabilization of DNA and RNA,translation,and metabolic flux changes).We list temperature-sensitive genetic male sterility loci available for use in rice hybrid breeding and explain the regulatory mechanisms associated with some of them.Breeding thermotolerant rice species without yield penalties via natural alleles mining and transgenic editing should be the focus of future work.

1.Introduction

Climate change,mainly in the form of global warming,has been projected since the 1980s as an unprecedented and enormous problem threatening future human societies[1].Agriculture is one of the sectors most vulnerable to global warming.Temperature elevation can lead to severe crop yield losses and reduced grain quality,and is predicted to imperil food security.

Rice(Oryza sativa L.)is the most important crop in Asia,providing a major part of the dietary energy for more than 4 billion people.Each 1°C elevation in global average temperature reduces global yield of rice by 3.2%[2].Rice is sensitive to heat stress,especially in the heading and filling stages,when it causes severe yield reduction[3,4].Heat stress in these stages reduces pollen viability[5],resulting in low grain setting rate,poor milling quality,and reduced final yield,as well as changes in grain quality such as gel consistency and chalkiness[6].

To adapt to extreme temperatures,rice has employed various protective mechanisms to cope with damage from heat stress,including cell component-and biochemically-mediated thermal regulation.Identifying molecular regulatory mechanisms involved in response to high temperature and developing genetic control via gene introgression or genome editing may allow the breeding of rice adapted to target temperatures without a yield penalty.Thermosensitivity is widely used in breeding temperature-sensitive genetic male sterility(TGMS)lines applied in hybrid breeding[7].

2.How plants perceive elevated temperature

RNA secondary structure is considered a temperature sensor,especially in prokaryotes.In bacteria,the translation of heat shock transcripts is inhibited,wherein a secondary structure traps ribosome binding site(RBS);whereas,high temperature impairs the structure and releases RBS for normal translation[8](Fig.1).The translation of PIF7 is rapidly induced via the formation of an RNA hairpin in its 5′untranslated region under high temperature environment[9].phyB(phytochrome B),once thought to be a photoreceptor,plays major thermosensory roles during darkness.phyB perceives temperature via reversion from the active Pfr state to the inactive Pr state,and binds to promoters of genes responsive to temperature,wherein phyB may recruit or modulate other regulators in Arabidopsis[10,11].ELF3(EARLY FLOWERING 3),a member of the evening complex,was recently shown to perceive temperature by phase separation,namely that ELF3 is able to adopt an active soluble form under normal conditions and rapidly formsvisible bright speckles at elevated temperatures in a predicted prion domain-dependent manner,shifting ELF3 to an inactive state and releasing the inhibition of downstream target genes[12](Fig.1).These results support that there are multiple thermosensors functioning in various regulatory pathways to transduce increasing temperature into downstream molecular responses.

Fig.1.Schematic model for thermal sensing and response inplants.

Elevated temperature affects many processes.High temperature alters membrane fluidity,resulting in lipid composition changes,membrane lipid saturation level changes,and even the generation of ROS.In this way,membrane-localized calcium channels may be directly or indirectly activated,followed by elevation of the cytoplasmic calcium level,which activates or represses the activity of Ca2+/CaM-related kinases,phosphatases,and even transcription factors.To buffer the heat-triggered decrease of saturation level,FADs are downregulated to increase saturation levels of membrane lipids.RNA secondary structure is also influenced by high temperature via the spliceosome or another pathway.The alteration changes the activity and expression of HSF-associated genes,in turn altering the heat shock response.Changes in high-temperature-triggered protein state,phase separation,and chromatin may also affect transcription activity of downstream target genes,participating in the regulation of thermomorphogenesis.There may be some receptor-like kinases(RLKs)that have not been identified in plants.These RLKs would perceive high temperature via multiple mechanisms,such as those involving extracellular residues or other unknown molecules.

All thermosensors are identified under warm-or relatively high-temperature conditions,which is different from heat stress,and these thermosensors are always involved in thermomorphogenesis but not in resistance to heat stress.To date no crop thermosensors have been identified.We propose that putative heatstress sensors should have at least one of the following characteristics:(1)fast perceiving temperature following a protein status change(such as in activity or interaction)and/or(2)fast perceiving temperature by rapidly mediating Ca2+-signaling induction.For example,putative heat-stress sensors can function as Ca2+channels that directly regulate Ca2+signaling in response to extracellular stimuli,like OSCA1(reduced hyperosmolality-induced[Ca2+]iincrease 1)for osmotic sensing in Arabidopsis[13];as regulators affecting membrane lipids composition which associates with the activity of Ca2+channels,like the glucuronosyl transferase MOCA1 for GIPC(glycosyl inositol phosphorylceramide)sphingolipids for salt perception in Arabidopsis[14];or as RLKs(receptor-like kinases)inducing Ca2+influxes upon sensing extracellular stimuli,like HPCA1(hydrogen-peroxide-induced Ca2+increases)/CARD1(cannot respond to DMBQ 1)in H2O2and quinone perception in Arabidopsis[15,16].All sensors of abiotic stress that have been well characterized are associated with stress-triggered cytosolic Ca2+elevation.Accordingly,in searching for mutants unresponsive to heat stress via calcium monitoring assays,such as aequorin,scanning ion-selective electrode technique,and YC3.6(Yellow cameleon 3.6)[17],may be an efficient way to identify heat-stress sensors.

3.How plants respond to heat stress

Mild increases in average temperatures can result in altered morphogenesis[18],biorhythms[19],and immunity responses[20],but do not cause extensive cell damage or heat-shock responses.In contrast,heat stress always causes deleterious changes,including membrane damage,ROS burst,cytotoxic denatured proteins[7],and even plant death.Plants employ various thermal responses to cope with heat stress.

Ca2+signal induction is the most rapid response in plants.A maximal concentration of cytosolic Ca2+occurs within the first 10 to 15 min upon an abrupt increase in temperature from 24 to 36°C in the moss protonemata,which eventually subsides to a baseline level with ongoing heat shock response[21].CNGCs(Cyclic nucleotide-gated channels)are nonspecific cation channels that regulate Ca2+fluxes under various abiotic and biotic stresses[22].AtCNGC2 in Arabidopsis mediates Ca2+influx into leaf cells and loss of function of AtCNGC2 contributes to enhanced tolerance to heat stress at seedling stage,wherein a moderate higher accumulation of heat response proteins was observed compared with wild-type plants.[23,24].Interestingly,dysfunction in AtCNGC2 also leads to thermosensitivity at the reproductive stage,in contrast to its function at the seedling stage[24].Changes in Ca2+channels activity may be the primary responses enabling plants to transduce extracellular stimulation into Ca2+signal.

Heat stress also changes membrane fluidity,followed by changes in lipid composition and membrane saturation level.Reducing membrane saturation is thus a required strategic response for plants to adapt to a high-temperature environment.FAD(fatty acid desaturase)is destabilized by high temperatures,contributing to an increased saturation level that confers thermotolerance in plants[25](Fig.1).Reductions in the expression levels of FAD-associated genes are detected in heat-tolerant soybean genotypes under heat stress conditions[26].Transcription profiling analysis shows that ZmFAD2.1&2.3 and ZmSLD1&3 expression levels are detectably down-regulated in maize under heat stress[27].Thus,FADs play essential roles in heat stress response.Heat-triggered membrane fluidity changes might activate PM(plasma membrane)-bound phospholipases and kinases,leading to rapidly increasing PA(phosphatidic acid)and PIP2(phosphatidylinositol diphosphate),which are essential in stress-signal transduction[25].Heat-triggered Ca2+influx is also regulated by overall membrane-lipid saturation level.High saturation of membrane-associated lipids attenuates heat-triggered Ca2+elevation and heat-shock response[28].Thus,plants employ various pathways to deal with changes in membranes,both by downregulating FADs to elevate membrane saturation levels and by releasing signal molecules to downstream pathways.

Heat stress leads to accumulation of unfolded,misfolded,and denatured proteins;and plants employ multiple methods to generate enough HSPs(heat shock proteins)or other HSR(heat shock response)genes to cope with this disruption of protein homeostasis.Heat-triggered Ca2+signal is rapidly amplified by CaM(calmodulin)and transduced to downstream proteins.AtCBK3 and AtPP7 function as a CaM-binding protein kinase and phosphatase,and AtCBK3 has been demonstrated to regulate the activity of HsfA1s(HEAT STRESS TRANSCRIPTION FACTOR A1),which function as‘‘master regulators”to activate HSR and improve resistance to heat stress[29,30](Fig.1).The cyclin-dependent kinase CDC2a represses HsfA1 binding activity[31].HSPs function as chaperones to renature or degrade misfolded proteins in plants[32].Overexpressing HSP100 confers thermotolerance in Arabidopsis[33]and OsHSP101 contributes to thermotolerance in rice[34].Moreover,the activity and nuclear localization of HsfA1 are inhibited under normal conditions in an HSP70 and HSP90-dependent manner,whereas,HSP70/HSP90-HsfA1 interactions are dissociated under heat stress,contributing to the activation of HsfA1[35,36].In addition,HsfAs may self-regulate in transcription.The heat-activated splicing process occurs in HsfA2-III mRNA through a cryptic 5’splice site in the intron and generates a S-HsfA2(small truncated isoform),and S-HsfA2 can bind to the HsfA2 promoter to activate its own transcription[37,38].SUMO1 is reported[39,40]to interact with HsfA2 and reduce its transcriptional activity.HsfA1-induced DREB2A is also known to participate in heat-tolerance regulation,and its degradation under unstressed conditions is mediated by DRIP1/2(DREB2A-INTERACTING PROTEIN 1/2),RCD1(RADICALINDUCED CELLDEATH 1),and CK1(CASEIN KINASE 1)[41-43].

Heat stress causes disruption in ROS homeostasis,contributing to ROS burst and oxidative damage[44].PSI and PSII(photosystems I and II)in chloroplasts and the electron transport chain of mitochondria are major sites for the production of ROS.Numerous antioxidant regulatory pathways have been reported to protect cells against oxidant stress,including enzymatic(SOD,CAT,APX,GR,etc.)and non-enzymatic molecules(ASH,GSH,etc.)[45](Fig.1).The wheat genotype WH730 showed increased thermotolerance and sustained grain yield under heat stress as a consequence of elevated ROS scavenging activity[46].

4.Thermal response and tolerance in rice

4.1.Thermal response of the plasma membrane

The primary response to heat stress occurs on membranes,typically followed by changes in membrane fluidity and activation of channels or receptors[47,48].Transient Ca2+signal and signaling molecules are induced,and extracellular stimuli are subsequently transduced into intracellular physiological responses[49-51].OsCNGC14 and OsCNGC16 were recently determined[52]to regulate thermotolerance by altering the heat-triggered elevation of cytosolic Ca2+(Fig.2;Table 1).Several membrane-located receptors have also been identified as playing essential roles in rice thermotolerance(Fig.2;Table 1).For example,ERECTA overexpression increases thermotolerance independent of water loss and after minimal membrane disruption[53].25L1 and 25L2,functioning as leucine-rich repeat receptor-like kinases, regulate temperature-associated growth in rice[54].But whether these receptors are activated by sensing membrane fluidity or ligands is still unknown.

OsCNGC14 and OsCNGC16 respond to heat stress by triggering calcium signals.Leucine-repeat receptor-like kinases such as ERECTA and Hwi1 regulate thermal response in rice by an unknown transduction pathway.The ER-localized OsbZIP74 moves to the nucleus under heat stress and activates the expression of OsNTL3.In turn,membrane-localized OsNTL3 also respond to heat by moving to the nucleus and directly regulating the expression of OsZIP74.In chloroplasts,the de novo synthesis of D1 subunit is required for repairing the destroyed PSII,and PGL and the OsTRXz-OsFLN1/2 complex protect chloroplasts against heatinduced damage.Mitochondria-localized EG1 protects the organelle and sustains floral robustness under heat stress.OsANN1,DST,SNAC3,OsHTAS and the above mentioned chloroplast genes cooperate to maintain ROS homeostasis.In case of disrupted protein homeostasis,OsHSPs stabilize,renature,and help to degrade unfolded proteins.TT1 and OsHATS protect cells against cytotoxic denatured proteins via the 26S proteasome system.For RNA homeostasis,TOGR1,OsNSNU2,SLG1 and AET1 protect the processing,modification,and stability of RNA under heat stress.For DNA homeostasis,LS1 maintains genome stability against DNA damage.AET1 plays an essential role in mRNA translation.GSA1 controls the accumulation of flavonoid glycosides to protect rice against heat damage.

4.2.Thermal response in the endoplasmic reticulum

Heat stress also disrupts the ER(endoplasmic reticulum),especially by causing the accumulation of misfolded proteins,which activates the unfolded-protein response[55].Under ER stress conditions,OsIRE1 splices out the double stem-loop structure,which encodes hydrophobic amino acids,in ER membrane-associated OsbZIP74(also known as OsbZIP50)mRNA,and this splicing process is also induced by heat stress[56].The location of spliced OsbZIP74 is shifted from the ER to the nucleus.OsNTL3,a plasma membraneassociated NAC transcription factor,is required for thermotolerance and relocates from the plasma membrane to the nucleus in response to heat stress.OsNTL3 binds directly to the OsbZIP74 promoter and regulates its transcription in response to heat stress.In turn,OsNTL3 is also up-regulated by heat stress in an OsbZIP74-dependent manner[57](Fig.2;Table 1).OsbZIP17(also known as OsbZIP39)and OsbZIP16(also known as OsbZIP60)have also been shown [57,58]to regulate downstream transcriptional responses.

4.3.Thermal response in chloroplasts

Chloroplasts are vulnerable to damage from heat stress.D1,the core subunit of PSII,is susceptible to light and heat stress.To avoid the accumulation of damage,PSII plants employ de novo synthesis of proteins,among which the D1 subunit protein encoded by a chloroplast gene psbA plays an essential role in the process(Fig.2;Table 1).A heat-responsive promoter-driven D1 proteinincreased thermotolerance and led to a large boost in aboveground biomass(20.6%-22.9%)and grain yield per plant(8.1%-21.0%)compared with wild-type plants in rice[59].Chloroplasts have also been shown to be essential for activation of cellular heat stress signaling.Disruption of PGL(pale green leaf,encoding chlorophyllide a oxygenase 1)leads to thermosensitivity in rice[60](Fig.2).WLP2,a putative pfkB-type carbohydrate kinase,belongs to a set of plastid-encoded RNA polymerase associated proteins and maintains redox balance via a TRX-FLN regulatory pathway by interacting with thioredoxin OsTRXz to protect chloroplast development against heat stress in rice[61](Fig.2).HSA1/OsFLN2 also contributes to guaranteeing normal chloroplast development in the early stages and protects chloroplasts against heat stress at later stages in rice[62](Fig.2;Table 1).Loss of OsNSUN2 function leads to a vulnerable photosystem with reduced photosynthesis efficiency and ROS accumulation upon heat treatment[63].

Fig.2.OsCNGC14 and OsCNGC16 respond to heat stress by triggering calcium signals.Leucine-repeat receptor-like kinases such as ERECTA and Hwi1 regulate thermal response in rice by an unknown transduction pathway.The ER-localized OsbZIP74 moves to the nucleus under heat stress and activates the expression of OsNTL3.In turn,membrane-localized OsNTL3 also respond to heat by moving to the nucleus and directly regulating the expression of OsZIP74.In chloroplasts,the de novo synthesis of D1 subunit is required for repairing the destroyed PSII,and PGL and the OsTRXz-OsFLN1/2 complex protect chloroplasts against heat-induced damage.Mitochondria-localized EG1 protects the organelle and sustains floral robustness under heat stress.OsANN1,DST,SNAC3,OsHTAS and the above mentioned chloroplast genes cooperate to maintain ROS homeostasis.In case of disrupted protein homeostasis,OsHSPs stabilize,renature,and help to degrade unfolded proteins.TT1 and OsHATS protect cells against cytotoxic denatured proteins via the 26S proteasome system.For RNA homeostasis,TOGR1,OsNSUN2,SLG1 and AET1 protect the processing,modification,and stability of RNA under heat stress;aaRS,aminoacyl tRNA synthetases.For DNA homeostasis,LS1 maintains genome stability against DNA damage.AET1 plays an essential role in mRNA translation.GSA1 controls the accumulation of flavonoid glycosides to protect rice against heat damage.

4.4.Thermal response in mitochondria

Stabilization of the mitochondrial genome is associated with thermotolerance in plants.Simultaneous disruption of two genes,MSH1(MutS Homolog1)and RECA3,both of which are involved in regulating plant mitochondrial recombination,associated with thermotolerance in Arabidopsis[64].Mitochondrial lipid metabolism also associates with thermotolerance in rice.A mitochondria-localized lipase,EG1(EXTRA GLUME1),protects the normal expression pattern of some floral identity genes under heat stress,suggesting that mitochondria function as a buffer to promote floral robustness against temperature fluctuation[65](Fig.2;Table 1).

4.5.Thermal response to disruption of protein homeostasis

Besides membrane integrity and fluidity,another major heatinduced injury is the disruption in protein homeostasis,which leads to cell death and cytotoxicity.A set of molecular chaperones called HSPs can stabilize,renature,or degrade these unfolded proteins.Overexpressing OsHSP1 increases thermotolerance in Arabidopsis[95]and OsHSP101 modulates long-term acquired thermotolerance in rice by acting in a positive feedback loop with HSA32[34].Compared with recovering denatured proteins by HSPs,proteasome-mediated degradation is more effective when toxic proteins are accumulated within a short period.TT1(THERMOTOLERANCE1),anα2 subunit of the 26S proteasome,efficiently eliminates cytotoxic denatured proteins involved in ubiquitination,maintains protein homeostasis under heat stress[3](Fig.2;Table 1).

4.6.Thermal response to cope with ROS burst induced by heat stress

Heat-induced H2O2is always produced in chloroplasts and mitochondria,and not only acts as a messenger molecule for early signal transduction,but also causes cell damage during later periods,even triggering programmed cell death[66].OsANN1,acalcium-binding annexin,enhances heat stress tolerance by modulating antioxidant accumulation under heat stress[67](Fig.2;Table 1).DST(drought and salt tolerance),a C2H2 zinc finger transcription factor,determines thermotolerance by regulating the expression of genes determining H2O2-homeostasis,such as those encoding scavenging enzymes[7,68](Fig.2).OsHTAs,encoding a RING finger ubiquitin E3 ligase,improves thermotolerance by mediating hydrogen peroxide accumulation to alter stomatal aperture in an ABA-dependent and DST-mediated pathway,and also participate in unfolded-protein elimination by interacting with components of the ubiquitin/26S proteasome system[69](Fig.2;Table 1).OsSNAC3,a NAC transcription factor,directly activates many genes encoding ROS scavengers to sustain ROS homeostasis and confer rice thermotolerance[70](Fig.2;Table 1).Cu/Zn-SODa,a superoxide dismutase,is abundantly expressed in rice anthers and displays a thermosensitive response to heat stress.Heatstress exposure represses the expression,especially at the meiosis stage,leading to decreased pollen viability and low grain setting rate in rice[71].LS1(local lesion 1)indirectly blocks DNA damage-induced ROS accumulation under high light and temperature stress[72].

Table 1QTL mapping and gene cloning related to heat stresses in rice.

4.7.Thermal response in metabolic flux

GSA1,encoding a UDP-glucosyltransferase,is essential for both grain size and heat-stress tolerance.GSA1 redirects metabolic flux from the pathways involved in lignin biosynthesis to flavonoid biosynthesis pathways and controls the accumulation of flavonoid glycosides and anthocyanin,which reduce damage and protect plants against heat stress[73](Fig.2;Table 1).

4.8.Thermal response to maintain nucleic acid stabilization and translation

High temperatures cause extensive damage to DNA replication,transcription,and translation.LS1,encoding a subunit of the RNase H2 complex,maintains genome stability,subsequently preventing DNA damage,chloroplast degradation,and cell apoptosis under high light and temperature environments[72](Fig.2;Table 1).A nucleolar-located DEAD-box RNA helicase,TOGR1,transcription of which is in a high-temperature inducible state,guarantees pre-rRNA structure stability by promoting its helicase activity to confer rice tolerance against heat stress[74](Fig.2;Table 1).OsNSUN2,encoding an m5C(RNA 5-methylcytosine)methyltransferase in rice,is required for heat-triggered m5C modification of mRNAs involved in detoxification and photosynthesis pathways,following by elevated protein synthesis[63](Fig.2;Table 1).SLG1,encoding tRNA 2-thiolation protein 2,positively regulates thermotolerance in rice by maintaining normal thiolated tRNA levels under heat stress[75](Fig.2;Table 1).AET1,encoding a tRNAHisguanylyltransferase,protects pre-tRNAHismodification under hightemperature conditions and translationally regulates auxin signaling in response to high temperature via interaction with RACK1A and eIF3h[76](Fig.2;Table 1).

5.Roles of thermosensitivity in fertility in rice hybrid breeding

Identifying heat-tolerant rice genotypes using genes conferring thermotolerance is a promising way to cope with food insecurity caused by global warming.However,because rice is selfpollinating,agricultural production should employ some thermosensitive mutations in hybrid breeding combined with heterosis.

During the past decades,the three-line system[comprising CMS(cytoplasmic male sterility),maintainer,and restorer lines]has contributed to increasing crop productivity,but breeding using this system is time-consuming and labor-intensive[77].Two-line hybrids have been generated using EGMS(environment-sensitive genetic male sterility),including PGMS(photoperiod-sensitive GMS)and TGMS(temperature-sensitive GMS).TGMS plants are fully male-sterile at high temperatures,but male-fertile at low temperatures.rTGMS(reverse TGMS)has also been developed and displays the opposite characteristics[7].

Numerous TGMS and rTGMS loci have been identified in various cultivars.tms6 was derived from a Korean-origin japonica cultivar,sokcho-MS,which is only fertile at temperatures ranging from 25to 27°C[78].However,G20S,a variety with an opposite phenotype to TGMS,is fertile only at temperature higher than 29.5°C,a character controlled by a single recessive gene named tms6(t)[79].A non-pollen type TGMS line,XianS,originating from indica rice cultivar Xianhuazhan,displays sterility at temperatures higher than 27°C.The candidate gene that contributes to the phenotype is localized close to the OsNAC6 gene,a previously identified gene associated with tms5 in AnnongS-1.However,no sequence variation has been identified between the TGMS lines and wild-type plants[80].

TMS5(thermosensitive male sterility 5)encodes an evolutionarily conserved RNase Z protein,known to process 3′ends of tRNA,that has been shown to process mRNA of UbL40(ubiquitin fusion ribosomal protein L40)genes into fragments.Overaccumulation of UbL40mRNA in tms5 mutant leads to dysfunction of pollen production and causes male sterility[81].OsbHLH138,a bHLH-type transcription factor,was recently shown[82]to activate the expression of TMS5 via forming a helix-loop-helix structure and directly binding to the promoter of TMS5.Thus,OsbHLH138 expression level can be altered in order to regulate TMS5 expression and the accumulation of UbL40 mRNA to determine male sterility in rice.Another transcription factor,OsGATA10,has been reported[83]to target UbL40,and loss of OsGATA10 function leads to a low level of UbL40 transcripts and increased male fertility.

p/tms12-1(a photo-or thermo-sensitive genic male sterility locus on chromosome 12)responds variably in a rice subspeciesdependent manner and confers PGMS in japonica Nongken 58S(NK58S)and TGMS in indica Peiai 64S(PA64S,originating from NK58S).P/TMS12-1 encodes a long noncoding RNA termed longday-specific male-fertility-associated RNA,which produces a 21-nucleotide small RNA.A substitution(C to G)occurs in p/tms12-producing small RNA and causes PGMS and TGMS in japonica and indica species[84].

UGPase(UDP-glucose pyrophosphorylase),which is required for callose deposition,plays an essential role in male sterility.There are two homologous UGPase genes,Ugp1 and Ugp2.RNA interference of Ugp1 causes suppression of Ugp1 and Ugp2,combined with various developmental dysfunctions.Ugp1-cosuppressing plants generate unprocessed intron-containing primary transcripts,an operation that is sensitive to high temperature owing to the splice efficiency of Ugp1 mRNA,and shows TGMS[85].Two motifs,TTTCT and TTTC,characterized in the promoter regions of Ugp2,are known[86]to be pollen-specific cis elements.

tms2 is reported[87]to harbor a 70-kb deletion in chromosome 7 and confers TGMS owing to disrupted sphingolipid homeostasis.tms3(t),located on chromosome 6,contributes to rice TGMS phenotypes[88].tms9-1 was identified in the conventional TGMS line HengnongS-1,and the rice MALE STERILITY1 homolog OsMS1,a PHD finger transcription factor,was identified[89]as the candidate gene for tms9-1;however,osms1 in Zh8015 variety showed no similar phenotypes with TGMS[90].TMS10 and TMS10L(homologous TMS10-like),which encode two leucine-rich repeat receptorlike kinases,redundantly maintain male fertility,especially under high temperature.Natural alleles or artificial genome editing of TMS10 cause male sterility in either japonica or indica species,indicating that TMS10 and TMS10L can buffer environmental changes to guarantee the switch between postmeiotic tapetal development and pollen development[91].

6.Prospects

6.1.Thermotolerant germplasm in rice

African rice(O.glaberrima),a typical tropical crop,has been domesticated and exhibits outstanding thermotolerance,thus providing a valuable gene resource for breeding thermotolerant crops.Among O.glaberrima cultivars,CG14,IRGC102635,IRGC102580,IRGC100127,IRGC102370,IRGC102239,and ACC 102,265 have been reported[3]to exhibit strong thermotolerance after longterm heat treatment(45°C for 72 h).Some japonica cultivars grown in Taiwan of China also exhibited high thermotolerance,including TK14,HC56,TT30,TNG70,and TK8[92].Introgression of SLGIndallele from ZF802(indica variety)confers thermotolerance on the thermosensitive temperate japonica cultivar KY131,indicating that indica cultivars can also be treated as thermotolerant germplasm for breeding[75].

6.2.Approaches to improving rice thermotolerance without yield penalty

Resistance to stress is always accompanied by compromised crop yield.The trade off between thermotolerance and normal development should be addressed in breeding research.For improvement of rice thermotolerance,functional molecules encoded by QTL have a particularly important role,such as TT1,the first and only known crop thermotolerance QTL that has been identified.TT1 offers the possibility of directly breeding thermally resistant species without yield penalty via molecular DNA-markerassisted introgression,as well as of identifying regulatory networks controlling major traits[3].Transgenic approaches also provide an opportunity for pleiotropic trait improvement.Overexpression of TOGR1 not only protects rice against heat damage but also contributes to increasing 1000-grain weight and grain number per panicle[74].Upregulation of the chloroplast D1 subunit driven by a heat-responsive promoter increased the survival of Arabidopsis,tobacco,and rice under heat stress,as well as increasing biomass and plant yield via elevated net CO2assimilation[59].

6.3.Combination of thermotolerance regulators to improve tolerance to heat stress in rice

Disruption in protein homeostasis is mostly deleterious for cells[7].TT1 is able to eliminate unfolded and cytotoxic proteins[3],whereas HSPs function to stabilize and renature unfolded proteins[32].Accordingly,we can introduce the O.glaberrima allele of TT1 into HSPs overexpression lines,thereby possibly generating a more thermotolerant rice cultivar.OsANN1 and SNAC1 confer tolerance to heat stress on rice by positively regulating the expression of H2O2-scavenging enzymes[67,70].However,H2O2,as a signal molecule,also induces stomatal closure,restricting water loss by transpiration during stress[68,93].High water loss also causes thermosensitivity.Given the role of ERECTA in transpiration efficiency[53],we propose a combination of increased ROS scavenging and restriction of water loss as a strategy for improving rice tolerance to heat stress.

6.4.Additional components of the adaptability-independent pathway remain to be identified

A plant pathway centered on HSF-HSP regulation and ROS scavenging has been established,in which plants employ various measures to cope with heat-triggered homeostasis disturbance,such as renaturing or eliminating unfolded proteins and scavenging ROS to protect cells from damage[7].However,in addition to adaptation,preserving normal function is still an indispensable strategy for plants to avoid overreaction or endure extreme environmental stress without high thermal response-induced energy consumption.In Arabidopsis,knockout of cMCU diminishes response to osmotic stress in calcium level and confers resistance to longterm osmotic stress[94].We thus have reason to believe that riceregulatory mechanisms that reduce thermal response and thereby confer thermotolerance await discovery.

CRediT authorship contribution statement

Hong-Xuan Linconceived and supervised the project,Yi Kan and Hong-Xuan Linwrote the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We apologize to colleagues whose work could not be cited owing to space limitations.This work was supported by the National Natural Science Foundation of China (31630052,31788103),Chinese Academy of Sciences(XDB27010104,QYZDYSSW-SMC023,159231KYSB20200008),the National Key Research and Development Program of China(2016YFD0100604),and the Shanghai Science and Technology Development(18JC1415000).