Yufang Xu,Chengcai Chu*,Shanguo Yao*

a State Key Laboratory of Plant Genomics,Institute of Genetics and Developmental Biology,the Innovative Academy for Seed Design,Chinese Academy of Sciences,Beijing 100101,China

b College of Advanced Agricultural Sciences,University of Chinese Academy of Sciences,Beijing 100039,China

c College of Life Science,Henan Agricultural University,Zhengzhou 450002,Henan,China

Keywords:Heat stress Rice Transcriptional regulation Protein homeostasis Thermotolerance breeding

ABSTRACT Heat stress (HS) caused by rapidly warming climate has become a serious threat to global food security.Rice(Oryza sativa L.)is a staple food crop for over half of the world’s population,and its yield and quality are often reduced by HS.There is an urgent need for breeding heat-tolerant rice cultivars.Rice plants show various morphological and physiological symptoms under HS.Precise analysis of the symptoms(phenotyping) is essential for the selection of elite germplasm and the identification of thermotolerance genes.In response to HS,rice plants trigger a cascade of events and activate complex transcriptional regulatory networks.Protein homeostasis under HS is especially important for rice thermotolerance,which is affected by protein quality control,effective elimination of toxic proteins,and translational regulation.Although some agronomic and genetic approaches for improving heat tolerance have been adopted in rice,the molecular mechanisms underlying rice response to HS are still elusive,and success in engineering rice thermotolerance in breeding has been limited.In this review,we summarize HS-caused symptoms in rice and progress in heat-stress sensing and signal cascade research,and propose approaches for improving rice thermotolerance in future.

Contents

1.Introduction .........................................................................................................964

2.Growth and developmental effects of heat stress ...........................................................................964

2.1.Vegetative stage ................................................................................................964

2.2.Reproductive stage ..............................................................................................965

2.3.Grain-filling stage ...............................................................................................966

3.Physiological effects of heat stress .......................................................................................966

3.1.Membrane damage ..............................................................................................966

3.2.Reactive oxygen species accumulation ..............................................................................966

3.3.Photosynthesis damage...........................................................................................966

3.4.Disturbance of carbohydrate metabolism and partitioning ..............................................................967

3.5.Phytohormone imbalance.........................................................................................967

4.Molecular mechanisms of plant responses to heat stress .....................................................................967

4.1.Heat stress sensing ..............................................................................................967

4.2.Heat-induced signal cascades......................................................................................967

4.3.Transcriptional regulatory network of heat stress response..............................................................968

4.4.Protein homeostasis under heat stress ..............................................................................969

5.Approaches for improving thermotolerance................................................................................970

5.1.Agronomic management..........................................................................................970

5.2.Conventional breeding ...........................................................................................970

5.3.Identification of heat-tolerant quantitative trait loci and marker-assisted breeding ..........................................971

5.4.Transgenic approach and genome editing technologies.................................................................972

6.Perspectives .........................................................................................................972

Declaration of competing interest .......................................................................................972

Acknowledgments ....................................................................................................972

References ..........................................................................................................972

1.Introduction

Environmental temperature is one of the most crucial factors governing the seasonal growth and geographic distribution of crops [1].With population growth and industrial development,global warming has become a problem that cannot be ignored[2].The fifth assessment report of the IPCC [3] shows that the increase in global average temperature from 1880 to 2012 was 0.85 °C and projects an increase of 3–5 °C in global mean surface temperature in Southeast Asia by 2100.The data from 23 global climate models also show a high (greater than 90%) probability that temperatures during the growing season in the tropics and subtropics by the end of the 21st century will exceed the most extreme seasonal temperatures recorded from 1900 to 2006,and in many locations of temperate regions the hottest seasons on record will represent the future norm[4].Global warming is coming,and heat waves will be more frequent and longer lasting than we think (Fig.1) [5].

Global warming has become a serious threat to the productivity of agricultural crops worldwide [8].It is estimated that without CO2fertilization,effective adaptation,and genetic improvement,every 1 °C increase in global mean temperature will reduce global yields of wheat by 6.0%,rice by 3.2%,maize by 7.4%,and soybean by 3.1%[9].FAO data show that the relative rates of yield increase for the major cereal crops are declining.However,as population expands,crop production must increase to sustain food security,and it is estimated[10]that a 70%increase in food production will be necessary to meet the demand of an expected population of 9 billion in 2050.

Rice (Oryza sativaL.) is a staple food crop for over half of the world’s population,with a harvest area of 167 million hectares and production of over 782 million tons in 2018(http://www.fao.org/-faostat/zh/#data).Rice provides 76% of the calorific intake of the population of Southeast Asia [11],where is predicted to be the fastest-warming region [3].It is estimated [12] that by 2030,16%of the rice harvested area will be exposed to at least 5 reproductive days of temperatures aboveTcrit(physiologically critical temperatures in the reproductive stage),and this area will increase to 27%by 2050.Rapidly warming climate causes heat stress (HS),commonly defined[13]as an increase in temperature above a threshold level for a certain period that causes irreversible damage to the growth and development of plants.Shi et al.[14]reported that rice yield decreased by 1.5%–9.7%in four planting areas of South China because of post-heading HS during 1981–2010.Lyman et al.[15]reported that every 1°C increase in average temperature during rice growing season reduced paddy yield by 6.2%,total milled rice yield by 7.1%–8.0%,head rice yield by 9.0%–13.8%,and total milling revenue by 8.1%–11.0%.Given the role of rice in world food security and the negative effect of global warming on rice productivity,there is an urgent need to breed thermotolerant rice.Identification of the physiological and biochemical characteristics of rice responsive to HS,the genes and proteins responsible for heat tolerance,and the molecular mechanisms underlying HS response are essential steps for thermotolerance breeding[8,16,17].In this review,we summarize HS-caused morphological and physiological symptoms of rice and progress in elucidating molecular mechanisms underlying rice response to HS,and discuss possible approaches to dissect the regulatory network of HS response and improving rice adaptation to global warming.

2.Growth and developmental effects of heat stress

2.1.Vegetative stage

Fig.1.Trends in global temperature change.(A) Map of the annual mean temperature change (°C) during 2000–2009 relative to 1951–1980.(B) Map of the annual mean temperature change (°C) during 2010–2019 relative to 1951–1980.The data for land surface air temperature are from GHCNv4 (GISS analysis based on global historical climatology network v4),and the data of sea surface temperature are from ERSST_v5(NOAA/NCEI’s extended reconstructed sea surface temperature v5).The number at the top right-hand corner of the map plot is an estimate (°C) of the global mean of the calculated area.Gray areas signify missing data.Ocean data are not used over land nor within 100 km of a reporting land station.The maps were made using the website of GISS Surface Temperature Analysis(https://data.giss.nasa.gov/gistemp/maps/index.html)[6,7].

Fig.2.Morphological and physiological characteristics of rice under heat stress.

Germinability and early seedling growth are major components of seedling vigor.Prolonged temperature elevation reduces seed germination potential and leads to poor germination rate and seedling vigor (Fig.2) [18,19].The optimum growth temperature of rice at the seedling stage is 25–28 °C.Heat stress (42–45 °C) at the seedling stage results in increased water loss,withered and yellow leaves,impaired seedling and root growth,and even death of seedlings [20–22].Rice plant resistance to HS at the seedling stage varies with genetic background.After exposure to HS(45 °C for 72 h),seedlings of thejaponicacultivar Nipponbare are nearly all wilted [21],whereas seedlings of theindicacultivar HT54 can tolerate up to 48 °C for 79 h [23].Plants subjected to HS at the tillering stage show various morphological symptoms,such as leaf wilting,leaf curling and yellowing,and reduced tiller number and biomass[24,25].Rice showed about 35%lower panicle number and 86% lower total yield per plants under HS(40°C day/35°C night)for 15 days than at 28°C[26].The influence of HS on tiller and panicle number is more severe injaponicathan inindicarice[27].Tiller number under HS is often used as a marker for the selection of thermotolerant rice cultivars [28].

2.2.Reproductive stage

Rice plants at the reproductive stage,including the processes of panicle initiation,male and female gametophyte development,anthesis,pollination and fertilization,are more susceptible to HS than at the vegetative stage(Fig.2)[29,30].HS impairs panicle initiation and spikelet development,leads to deformed floral organs,and reduces spikelet number and size[24,25].The spikelet number ofindicacultivar IR64 decreased by 66% following exposure to HS(40°C day/35°C night)at pre-flowering stage for 15 days,in comparison with that under normal growth condition(28°C)[26].Spikelet reduction is caused mainly by the degeneration of spikelets at the tops of panicles [25,31].Inside the floret,anther development and pollen viability are more sensitive to high temperature than the ovule [18].HS during anther development,especially at the pollen mother cell meiosis stage,may result in premature degradation and disintegration of the tapetal cells,impairing the nutritional supply of microspores and the formation of the pollen wall and causing pollen-grain abortion [32–35].Decreases of 78.8% in pollen viability and 48.5% in seed-setting rate were observed [35]when rice plants were subjected to HS (40 °C day/30 °C night) at the pollen mother cell meiosis stage for 10 days in comparison with the values under normal conditions (30 °C day/24 °C night).The early microspore stage following meiosis is the most sensitive to HS,and spikelet fertility is completely lost at this point after HS stress (39 °C day/30 °C night) for 7 days [36].HS during anthesis results in altered anther shape[37],diminished anther dehiscence[38,39],poor pollen viability [36],reduced pollen number on stigma[40,41],reduced pollen swelling,poor germination of pollen grains on the stigma [36,42],inhibited elongation of pollen tubes[41,43],and reduced stigma length [37],all of which severely impair the pollination and fertilization process,thus eventually reducing spikelet fertility [44].After double fertilization,exposure to a short period of HS(39°C for 48 h)can result in abnormal cellularization of early endosperm development,and impair the subsequent establishment of endosperm[45,46].The occurrence of HS during the reproductive stage resulted in an up to 80%reduction in spikelet fertility of rice [25].However,different genetic backgrounds show differing susceptibility to HS.For example,high temperature (38 °C) reduced the number of pollen grains on the stigma in theAustype N22 by 55%and thejaponicatype Moroberekan by 86%,but not in theindicatype IR64[37].And the number of grains on the stigmas of superior spikelets is more sensitive to HS than that on inferior spikelets,owing to their different organ temperatures [40].

2.3.Grain-filling stage

Grain filling is the completion of growth and development in crop plants and involves the transport and synthesis of carbohydrates,proteins,and lipids in seeds [47].It has been reported [48]that the grain-filling rate was increased and the total grain-filling duration was reduced by 21.3%–37.1% for different genotypes following exposure to HS at the grain-filling stage.Heat stress (35°C for 72 h) at early seed development stage impaired endosperm and embryo development[49].HS also results in altered kernel size and reduced grain weight and yield[15,45,48].Following exposure to high temperature(38°C day/30°C night)during grain filling for 20 consecutive days,grain weight was reduced by 24.6%and 39.1%for N22 and IR64,respectively,compared with that under normal conditions(31°C day/23°C night)[48].HS at the grain-filling stage results in poor rice quality,expressed as reduced palatability[50,51],undesirable grain appearance [52,53],and increased grain chalkiness [54].Chalky kernels are the most obvious symptoms caused by HS at the grain-filling stage.Chalkiness is defined as the opaque portion found in the translucent white endosperm,and reduces rice grain quality [47,52].When subjected to HS(33 °C day/27 °C night) during 3–35 days after flowering,90.2% of the kernels ofjaponicacultivar Koshihikari showed combined chalk,including milky-white and white-back kernels[55].The chalkiness rate was increased while brown rice rate,milled rice rate,and head rice rate were decreased with an increase of high temperature and prolonged duration at the early grain-filling stage [56].Several studies [48,53,55–57] suggest that HS triggers nonuniform filling and impairment of starch biosynthesis,resulting in irregular and smaller starch granules and deposition of loosely packed starch granules,thus increasing chalky kernel formation.Amylose content is lower under HS (16.1%) than under normal conditions (19.8%),suggesting that lower activity of amylose synthesis may be involved in chalk formation[55].

3.Physiological effects of heat stress

3.1.Membrane damage

Impairment of growth and development by HS is frequently associated with disturbed physiological and metabolic processes of plant cells.As the primary barrier,with highly ordered structures consisting of lipids and proteins,biomembranes are considered the most heat-sensitive components of the plant cell[24,58].Elevated temperature can impair the structure and function of plasma membranes,alter the ratio of saturated to unsaturated fatty acids,and trigger protein denaturation,leading to increased fluidity and permeability,compromised membrane integrity,and increased leakage of organic and inorganic ions from cells [24,58–60].Elevated temperature also impairs the activities of fatty acid desaturases and consequently the unsaturation degree of fatty acid chains,which influences plant adaptation to HS [58].

3.2.Reactive oxygen species accumulation

Intracellular reactive oxygen species (ROS) levels are dramatically increased under HS [61–63].When rice plants are subjected to HS(38°C day/30°C night)at the meiosis stage,the ROS content in anthers is over three times higher than that under normal temperature(28°C day/22°C night)[62].HS also results in a ROS burst in rice pistils,possibly owing to HS-induced upregulation of a series ofRespiratory Burst Oxidase Homolog(RBOH) genes [40,43].At the same time,HS impairs the activities of antioxidant enzymes,especially superoxide dismutase (SOD) and catalase (CAT)[17,62].Increased expression ofOsANN1provides increased thermotolerance by promoting the activities of CAT and SOD [63].When plants continue to suffer from severe HS,intracellular ROS will accumulate in excess,leading to disturbed ROS homeostasis and oxidative damage such as cell death [58,63],growth retardation [63],grain chalkiness [64],and even seedling death [65] and spikelet sterility [61,62].In particular,excessive ROS will further damage the structure and function of biomembranes by aggravating membrane lipid peroxidation and protein oxidation,leading to an increased content of intracellular malondialdehyde (MDA) that can impair the normal function of proteins and nucleic acids[66,67].Accordingly,electrolyte leakage,ROS level,transcript abundance of antioxidative genes,activities of antioxidant enzymes,and MDA content are frequently used as indicators of membrane and oxidative damage and also reflect the thermotolerance of plants [17,59,67].For example,heat-tolerant rice cultivars such as NERICA-L-44 and Nagina 22 exhibit high membrane stability and lower ROS and MDA contents because of high antioxidant enzyme activities [17,67].

3.3.Photosynthesis damage

Photosynthesis is very sensitive to elevated temperature [68].HS results in destroyed permeability of thylakoid membrane or even thylakoid grana disintegration and triggers a decrease in chlorophyll content,leading to alterations of photochemical reactions with reduction in the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) and photosynthetic rate [17,69,70].Photosystem II (PSII) is the most sensitive component of the photosynthesis apparatus [68].HS-induced oxidative stress causes the dissociation of the oxygen-evolving complex (OEC) in PSII,resulting in inhibition of electron transport from OEC toward the acceptor side of PSII [17,70–72].In vivoamounts of three OEC proteins decreased when rice seedlings were subjected to HS[73].High temperature inhibits the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco),mainly as a consequence of the inactivation of Rubisco activase [74].Transgenic rice plants with increased amounts of Rubisco activase grew better under high temperature and exhibited higher photosynthetic rates than wild-type(WT)plants[75].Similarly,overexpression of thermotolerant Rubisco activase from a wild rice dramatically improved the growth and grain yield of cultivated rice after exposure to HS,suggesting that engineering Rubisco activase may be an effective method for thermotolerance breeding [76].

3.4.Disturbance of carbohydrate metabolism and partitioning

HS disturbs carbohydrate metabolism and photoassimilate partitioning in plants [29,48,77].Abundances of two key enzymes in the glycolytic pathway,phosphoglucose isomerase and phosphofructokinase,were very low,and the abundance of phosphoglycerate mutase was also reduced when rice cells were exposed to high temperature (44 °C),suggesting impaired energy generation in cells under HS[78].When rice plants experience HS during anthesis,the sugar content in anthers is disordered,impairing normal nutrition supply for pollen development [79,80].Susceptible rice cultivars showed high expression of theCarbon Starved Anthers(CSA) gene,whereas tolerant cultivars showed high expression of the sugar transporter geneMST8and cell wall invertase geneINV4in floral organs under HS,suggesting that sugar starvation is a factor in spikelet sterility under HS [81].Similarly,a heattolerant rice cultivar showed higher expression of the sucrose transporter geneOsSUT1than a sensitive cultivar,leading to a higher supply of photoassimilates to filling kernels [82].HS at the filling stage led to down-regulation of a series of genes encoding starch synthesis-related proteins such as granule-bound starch synthase I and branching enzymes,possibly resulting in inhibition of starch accumulation and increased grain chalkiness [83].

3.5.Phytohormone imbalance

HS causes phytohormone imbalance.HS reduced the levels of active cytokinin (CTK),gibberellin (GA),and indole-3-acetic acid(IAA) in rice spikelets and developing kernels,impairing cell proliferation and panicle formation and reducing spikelet number,pollen fertility,and kernel weight[31,84].Under exposure to HS,a marked inhibition of CTK transportation rate and CTK synthesis enzymes,but increased cytokinin oxidase/dehydrogenase (CKX)activity,were observed in heat-susceptible in comparison with heat-tolerant cultivars,possibly accounting for a reduction in panicle CTK abundance [85].Exogenous application of 6-benzylaminopurine (6-BA),a synthetic CTK,alleviates heatinduced damage to spikelet fertility,spikelet number,and kernel weight[31].Similarly,rice plants following exogenous application of brassinosteroids (BR),salicylic acid (SA),and ethylene precursors showed amelioration of heat-triggered oxidative damage and morphological symptoms,suggesting the important roles of these phytohormones in heat stress response (HSR) [35,86,87].In contrast,HS leads to increased levels of abscisic acid (ABA) in anthers and seeds,in turn leading to pollen abortion and inhibition of the germination and seedling establishment [19,84].

4.Molecular mechanisms of plant responses to heat stress

4.1.Heat stress sensing

To identify the complex mechanisms underlying HSR,a key question is how plants rapidly sense HS and then transduce HS signal into intracellular responses [58].The cell wall is the first protective barrier in plants and its structures and properties can be remodeled when plants are subjected to HS,influencing the release of apoplastic Ca2+and increasing the content of free cytosolic Ca2+[88].Pectin methylesterase(PME)is a cell-wall remodeling protein whose expression can be upregulated by HS,leading to an increased level of demethylesterified pectin[88].During nonlethal HS,the PME-mediated demethylesterification of pectin results in lower cell wall pH,promoting endopolygalacturonase (PG) action and contributing to cell-wall loosening and transient release of Ca2+from Ca2+-pectate-enriched structures in the cell wall to cytoplasm by the Ca2+-permeable channels [89].Ca2+/calmodulin(CaM)-dependent signal transduction pathways are then activated.Increased H2O2content in rice can also increase PME activity and pectin demethylesterification,which may further active the downstream regulatory pathway of HSR [90].

The plasma membrane can directly link HS signals to intracellular signaling molecules,and HS-induced changes in membrane structures and properties such as fluidity,permeability,and thickness can affect the functions of membrane proteins,thereby triggering intracellular HS-responsive signaling cascades [58].Ca2+channels located in plasma membranes are known as cyclic nucleotide gated ion channels (CNGCs) and their functions in heat sensing are well documented[91].Transient opening of Ca2+channels induced by membrane fluidity allows extracellular Ca2+to enter the cytoplasm [91].In rice,16 full-length CNGC genes have been identified to date,of which 13 are predicted to be localized in the plasma membrane,and most are responsive to ambient temperature [92].OsCNGC14 and OsCNGC16 are positive modulators in the process of heat-induced increase of cytosolic Ca2+in rice[93].Bothoscngc14andoscngc16mutants display disturbed Ca2+influx to the cytosol and reduced or abolished cytosolic Ca2+-mediated signal transduction in response to HS.The expressions of many genes encoding heat shock transcription factors(HSF) and heat shock proteins (HSP) are subsequently impaired,leading to reduced survival rates,elevated hydrogen peroxide(H2O2) level,and increased cell death [93].OsCNGC13 and OsCNGC9 channel are also Ca2+-permeable and function in cytosolic-Ca2+mediated signaling cascades,but their functions in HSR await further characterization [94,95].Another membraneassociated Ca2+channel,Annexin 1 (ANN1),also contributes to the HS-induced increase in cytosolic Ca2+and plays an important role in thermotolerance [63,96].Some plasma membrane-located phosphoinositide-specific phospholipases C (PLCs) may also be activated by heat-induced change in membrane fluidity [58].PLC3 and PLC9 are reported [97] to be heat sensors and to contribute to the heat-induced increase of cytosolic Ca2+and signal transduction of HSR inArabidopsis.In rice,dysfunction ofOsPLC4impairs the increase of free Ca2+in cytosol and the expression of genes involved in Ca2+sensing,but its function in HSR is still unknown [98].

Elevated ROS produced by the plasma membrane-located protein NADPH oxidase,RBOH,is also considered [58] to be an early event in the response to HS.InArabidopsis,RBOHD is located primarily in the plasma membrane,and abiotic stresses can stimulate RBOHD endocytosis via membrane microdomains,thus regulating its activity [99].Increased cytosolic Ca2+and/or phosphorylation mediated by calcium-dependent protein kinases (CDPKs) can directly activate RBOHD,thus inducing a rapid increase of ROS level [58,100].In rice,nine RBOH proteins have been identified,with all but OsNox6 containing EF-hand Ca2+-binding motifs,suggesting a direct regulatory effect of Ca2+on OsRBOH activity[101].The plasma membrane-located OsRBOHA/OsNox2 is induced by HS,suggesting its function in HSR [102],andOsNox5-9are also upregulated by high temperature but the subcellular localization of OsNox5-9 is not documented yet[101].The roles of these RBOHs in HSR in rice await elucidation.HS also impairs the normal function of chloroplasts and mitochondria and disturbs electron transport during photosynthesis and respiration,thereby promoting the production of ROS and in turn the activation of ROS-mediated signal transduction in the HSR [58,91].

4.2.Heat-induced signal cascades

A transient cytosolic Ca2+influx from the extracellular matrix to the cytosol is considered to be an early event in HSR.Ca2+then acts as a second messenger recognized by calcium sensor proteins,thus rapidly transducing external HS signals inside the cell[58].CaM,a Ca2+-binding protein,is well documented to function in HS signal transduction in plants [103].InArabidopsis,theatcam3mutant showed a markedly reduced survival rate,whereasAtCaM3-overexpressing plants showed increased survival rates compared with the WT following exposure to HS.DNA-binding activity of HSFs and the protein accumulation of HSPs were reduced in theatcam3mutant,suggesting the importance of the CaM3 protein in HS signal transduction [103].CaM3 also interacts with calmodulinbinding protein kinase 3 (CBK3) and protein phosphatase 7 (PP7)[91].CBK3 and PP7 may affect plant thermotolerance by promoting respectively the phosphorylation and dephosphorylation of HSFA1,and dysfunction ofCBK3orPP7led to a thermosensitive phenotype[104,105].Similarly,HS-triggered increase of cytosolic Ca2+together with increased expression ofOsCaM1-1may contribute to HS signal transduction in rice,and the expression of genes encoding CBK3,PP7,HSFs,and HSPs and plant thermotolerance are all increased inOsCaM1-1-overexpressing lines [106].CDPKs can sense Ca2+via their EF-hand domain and transduce Ca2+signal via their protein kinase domain [107].A total of 29 CDPK proteins have been identified in the rice genome,andOsCDPK25/OsCPK25is markedly up-regulated by HS,suggesting its potential functions in thermotolerance [108].CaM-like protein (CML),calcineurin B-like(CBL) protein,and CBL-interacting protein kinases (CIPK) are all important Ca2+sensors and their functions in cold-signal transduction in rice are documented[107],but their functions in HS-signal transduction await discovery.

HS-induced damage to plasma membrane,chloroplasts,and mitochondria results in the overaccumulation of cellular ROS,which is considered as a key second messenger [1,109].When plants were subjected to HS,endogenous H2O2levels were markedly lower in three NADPH oxidase-defective mutants (atrbohB,atrbohD,andatrbohB/D) than in the WT,and the survival rates of these mutants were reduced,confirming the indispensable role of ROS-mediated signal transduction in plant thermotolerance[110].H2O2can directly activate HSFA1a and induce the binding of HSFA1a to heat shock element (HSE) in the promoters of HSP genes [111],and this binding ability is weaker in H2O2-deficient mutants ofatrbohB,atrbohD,andatrbohB/Dthan in the WT under exposure to HS [110].HSFA4a and HSFA8 are also reported [1] to be ROS sensors.ROS also acts as a signaling molecule that contributes to the activation of mitogen-activated protein kinase(MAPK) in HSR.InArabidopsis,H2O2activates the MAPK kinase kinase ANP1,which promotes the phosphorylation of AtMAPK3 and AtMAPK6 [112].Activated MAPK6 can phosphorylate HSFA2 at Thr249,thus contributing to the HS-induced nuclear accumulation of HSFA2[113].Activated MAPK3 and MAPK6 can phosphorylate HSFA4a on Ser309,thus affecting heat tolerance by reducing oxidative damage [114].However,the components in rice HSinduced ROS signaling pathways are still largely unknown.

The integration between Ca2+signaling and the ROS regulatory network in response to HS is well documented.HS induces overproduction of H2O2,resulting in increased rice annexinOsANN1expression[63].The overexpression ofOsANN1can maintain redox homeostasis under HS condition by promoting SOD and CAT activities,thereby increasing the heat tolerance of rice seedlings,and this process may be mediated by the interaction of OsANN1 and OsCDPK,suggesting crosstalk between ROS and Ca2+signals in HSR [63].The production of ROS by RBOHD can be directly promoted by Ca2+or CDPK-mediated phosphorylation[58,100].Application of exogenous H2O2can also induce a sharp increase in cytosolic Ca2+,thus activating Ca2+signaling pathway [115].HSinduced H2O2production can also regulate nitric oxide (NO) accumulation and trigger the NO-dependent HS signal pathway.The HS-induced increase of NO levels is defective in H2O2-deficient mutants ofatrbohB,atrbohD,andatrbohB/D[110],and an elevated internal NO level markedly increased the survival rates of H2O2-deficient mutants after heat treatment.However,an elevated internal H2O2level showed no significant effect on the thermotolerance of an NO-deficientatnoa1mutant,suggesting that NO may function downstream of the H2O2signal[110].NO signal activates CaM3 and subsequently increased the DNA-binding activity of HSFs and HSP accumulation at high temperatures [116].

4.3.Transcriptional regulatory network of heat stress response

HSFs are the most important components in the complex transcriptional regulatory network of HSR in plants,and are responsible for triggering a transcriptional cascade to activate downstream genes encoding HSR-induced transcription factors,ROS scavenging enzymes,metabolic enzymes,and HSPs (Fig.3)[117].Rice has 25 HSFs,which can be classified into three classes:HSFA(1a,2a to 2f,3,4b,4d,5,7,9),HSFB(1,2a to 2c,4a to 4d),and HSFC (1a,1b,2a,2b),and the expressions of 22 of these genes are induced by high temperature[118].Among these HSFs,HSFA1s are considered‘‘master regulators”in the transcriptional network[91].Up-regulation of more than 65% of HS-induced genes inArabidopsis,includingHSFA2,HSFBs,Dehydration-responsive Element Binding Protein 2A(DREB2A),Multiprotein Bridging Factor 1C(MBF1c) and manyHSPs,was abolished in thehsfa1a hsfa1b hsfa1dtriple knockout mutant [1,119].HSFA2is a direct target gene of HSFA1s and plays an essential role in HSR[91].When rice plants are subjected to HS,the transcriptionally active form of OsHSFA2d is induced by alternative splicing [120],increasing the expression of genes such asHSP17.7,HSP18.2,HSP21,HSP83.1andHSP101.Similarly,overexpression ofOsHSFA2einduces the expression of many HSP genes and increases thermotolerance inArabidopsis[121].OsHSFA2c can interact with OsHSFB4b and is involved in the transcriptional regulation ofHSP100by binding specifically to the promoter ofHSP100[122].HSFBsare also reported [91] to be downstream target genes of HSFA1s inArabidopsisand tomato.RiceOsHSFB2bis strongly induced by HS,indicating its important role in thermotolerance [123].

In addition to HSFs,the ERF/AP2 family transcription factor DREB2A is a regulator of thermotolerance and is a direct target of HSFA1 [1].InArabidopsis,DREB2A forms a complex with Nuclear Factor Y,Subunit A2 (NF-YA2),NF-YB3,and DNA Polymerase II Subunit B3-1(DPB3-1)to bind the promoters ofHSFA3and induce its expression [124].Dysfunctions ofDPB3-1,HSFA3,orDREB2Aall cause heat-sensitive phenotypes[124–126].Similarly,interactions between DPB3-1 and OsDREB2B2 (the rice homolog of DREB2A in rice),or OsDPB3-2 (the rice homolog of DPB3-1) and OsDREB2B2,are known [127].The transactivation effect of OsDREB2B2 on the promoter ofHSFA3is markedly increased when the proteins of NF-YA2,NF-YB3,and DPB3-1 are coexpressed,suggesting that the trimer of NF-YA2,NF-YB3,and DPB3-1 shows a highly conserved effect on DREB2A protein in plants [127].DPB3-1-overexpressing rice plants also show increased thermotolerance without a growth and yield penalty,indicating the high breeding potential of DPB3-1-family proteins[127].MBF1c is another direct target of HSFA1.HSFA1b directly regulated the expression ofMBF1cby binding to the heat shock element in theMBF1cpromoter[128],and MBF1c rapidly accumulated and bound to the CTAGA motif in the promoters of its target genesDREB2A,HSFB2a,andHSFB2b[129].Overexpression ofTaMBF1cconferred thermotolerance in both yeast and rice,indicating the conserved function of MBF1c in thermotolerance [130].

Fig.3.The regulatory network involved in heat stress response in plants.The structures and properties of cell wall can be remodeled when plants are subjected to heat stress(HS),promoting the release of apoplastic Ca2+.HS changes membrane fluidity and permeability,affects the activities of membrane proteins,and causes dysfunctions in chloroplasts and mitochondria,resulting in the increase of cytosolic Ca2+,ROS,and NO and the over-accumulation of misfolded or unfolded proteins.Increased cytosolic Ca2+,ROS,and NO act as second messengers and rapidly activate downstream regulatory networks.Heat stress transcription factor HSFA1s are considered to be master regulators whose activities can be regulated by protein–protein interactions and post-translational modifications.Disturbed protein homeostasis under HS induces unfolded-protein response(UPR),and the signal pathways mediated by IRE1-bZIP60 and bZIP17/bZIP28 are two main pathways in UPR.The ubiquitin/26S proteasome system and translational regulation affected by the normal function of RNAs also function in maintaining protein homeostasis under HS.

Many protein–protein interactions and post-translational modifications also act in regulating the activities of HSR-associated transcription factors,thereby participating in HSR (Fig.3) [91].InArabidopsis,interaction of HSP70 and HSP90 negatively regulated HSFA1 activity by repressing the transactivation activity and nuclear localization of HSFA1 [91,131].The rice HSF-binding proteins OsHSBP1 and OsHSBP1 bind to activated HSFs and act as negative regulators in HSR,and their overexpressing plants showed reduced survival rates after heat treatment [132].Protein phosphorylation and sumoylation also contribute to HSR [91].AtCBK3 can phosphorylate AtHSFA1a,and the binding ability of HSFs to the HSE was upregulated inAtCBK3-overexpressing lines[105].Sumoylation reduced the transcriptional activation of AtHSFA2 inArabidopsis,and seedlings overexpressingAtSUMO1showed heat-sensitive phenotypes similar to those of AtHSFA2-knockout seedlings [133].However,overexpression of the rice SUMO E3 ligase geneOsSIZ1increased thermotolerance inArabidopsisand cotton [134,135].Sumoylation stabilized the DREB2A protein under HS,thereby increasing plant thermotolerance[136].

NAC transcription factors are also regulators in HSR.NAC019 activated the signal cascade of HSR by directly binding to the promoters ofHSFA1b,HSFA6b,HSFA7a,andHSFC1[137].Another NAC transcription factor,Jungbrunnen1 (JUB1),was strongly induced by ROS inArabidopsis,and transactivatedDREB2Aexpression by directly binding its promoter[138].As the rice homolog of JUB1,ONAC066 also acts as a positive regulator in oxidative stress,and directly bound to the promoter ofOsDREB2Aand activated its transcription [139].Many HSFA1-independent transcription factors including OsMYB55 [140],OsbZIP46 [141],SNAC3 [65],and OsWRKY11[142],are also necessary for HSR and thermotolerance in rice.

4.4.Protein homeostasis under heat stress

HS disturbs protein homeostasis in the cell,including protein synthesis,folding,quality control,and subcellular localization,leading to the over-accumulation of unfolded or misfolded proteins,which are toxic to plant cells [143].The accumulation of such proteins in the endoplasmic reticulum can initiate the unfoldedprotein response (UPR) [144].There are two main signaling pathways of UPR in plants,one mediated by IRE1 via unconventional splicing of the mRNA ofbZIP60and the other by bZIP17 and bZIP28 transcription factors [145].InArabidopsis,HS can activate the IRE1-,bZIP60-,or bZIP28-mediated UPR [146,147],and the UPR-deficientbzip28bzip60double mutant orbzip28single mutant are both sensitive to HS,suggesting the protective roles of UPR in plant thermotolerance [147,148].Maizebzip60mutants also show heatsensitive phenotypes [149].Similarly,HS can trigger the mRNA splicing ofOsbZIP74/OsbZIP50,the rice homologs ofbZIP60,thus producing the nucleus-localized form and activating the expression of UPR-associated genes includingBiPs,PDIL1-1,Cal-nexin,andSAR1B-like[144,150].The nucleus-localized form of OsNTL3 produced after exposure to HS can regulate the expression ofOsbZIP74,and overexpression ofOsNTL3confers thermotoler-ance in rice seedlings [151].The expression levels of genes encod-ing the homologs of the UPR sensors IRE1,bZIP28,bZIP17,and the UPR marker BiP1 are all upregulated by HS in rice [120],indicating that UPR functions in the reestablishment of cellular protein homeostasis under HS conditions.

HSPs are important molecular chaperones that contribute to the stabilizing of unfolded proteins,and promote the renaturation of aggregated proteins induced by HS [152].HSPs are classified into many classes according to their molecular weight:HSP100,HSP90,HSP70,HSP60,HSP40,and small HSPs with low molecular weights (sHSPs) [152].The expression levels of five small HSPs:HSP26.7,HSP23.2,HSP17.9A,HSP17.4,and HSP16.9A,are upregulated in response to HS,and their expression levels are higher in heat-tolerant than in heat-susceptible rice cultivars,suggesting their use as markers for the selection of high-thermotolerance rice cultivars [153].The purified OsHSP17.4 and OsHSP17.9A proteins showed chaperone activity by preventing the formation of aggregated proteins [154].The protein abundances of HSP101,HSP90,and HSP70 in rice were also significantly increased after heat exposure [155,156],possibly contributing to heat tolerance.

Large amounts of aggregated proteins rapidly accumulate in cells under severe HS,and removal of these toxic proteins is more critical than recovery of their activity[157].The ubiquitin/26S proteasome system is an essential proteolytic complex responsible for degrading proteins conjugated with ubiquitin.Many RING finger ubiquitin E3 ligases,such as OsHIRP1 [158],OsHTAS [21],and OsHCI1 [159],are reported to be indispensable for rice tolerance to HS,and may function in recognizing and ubiquitinating their target proteins for subsequent degradation by the 26S proteasome.OgTT1has been cloned from African rice (O.glaberrima) and encodes a 26S proteasome α2 subunit,and OgTT1 achieves more effective elimination of cytotoxic denatured proteins than OsTT1,thereby protecting cells from heat damage [157].The expression levels ofOsHIRP1[158],OsHTAS[21],OsHCI1[159],andOsTT1[157] are all induced by rising temperature,and overexpression of any of these genes increases thermotolerance in rice.

Protein homeostasis under HS is closely associated with translational regulation,which is related to the normal functions of mRNA,tRNA,and rRNA.A tRNAHisguanylyltransferase,AET1,is essential for global tRNA homeostasis and translational efficiency under HS conditions,and the thermosensitive phenotype of theaet1mutant may be due to the reduced translational efficiency of growth-essential proteins [160].The extensive influence of tRNA thiolation defects on protein homeostasis may cause chronic proteotoxic stress,which may be responsible for the thermosensitivity of plants with dysfunction of the tRNA thiolation protein SLG1[41].Dysfunction of OsNSUN2,an RNA 5-methylcytosine(m5C)methyltransferase in rice,reduces the accumulation of proteins associated with the photosynthesis and detoxification systems under HS and causes severe heat sensitivity[161].rRNA homeostasis is also crucial to translation.The DEAD-box RNA helicase TOGR1 is essential for maintaining normal rRNA homeostasis under HS conditions,and its overexpression can protect rice growth under high temperature [162].

5.Approaches for improving thermotolerance

5.1.Agronomic management

Several agronomic management strategies have been shown[13] to be helpful for alleviating or avoiding heat damage in rice.Proper application of growth regulators such as CTK,SA,BR,and ethylene precursors can alleviate HS-induced damage to rice plants such as pollen abortion,reduced spikelets per panicle and kernel weight,and poor seed-setting rate [31,35,85–87].Plant antioxidants,osmoprotectants,and polyamines have also been reported to be useful for mitigating HS injury.Endogenous ascorbic acid reduces ROS accumulation and maintains leaf function [163].Exogenous application of spermidine increased photosynthetic and antioxidant capacity and partially alleviated HS-caused yield penalty in several rice cultivars[164–166].Glycine betaine or proline can mitigate yield reduction by reducing heat damage to membranes and maintaining the enzymatic function of Rubisco[13].The moderate increase of nitrogen application and combined application of biochar and phosphorus have also been shown[167,168] to alleviate rice yield losses caused by HS at the reproductive stage.Mist spray treatment during the flowering period rapidly reduces temperatures in the rice field,delays leaf senescence,and increases the activities of antioxidant enzymes,thus alleviating HS-caused yield loss [169].

5.2.Conventional breeding

Increasing thermotolerance by conventional breeding is a promising approach for reducing the negative effects of HS on rice yield and quality.Conventional breeding is generally based on thermotolerance-related phenotype selection and is applied in a climatic region similar to that where the crop is to be grown[170].Accurate evaluation of the degree of thermotolerance,selection of elite rice cultivars or breeding lines,and successful transfer of thermotolerance traits into specific cultivars with good agronomic performance are of great importance to conventional breeding.As described above,the heat-caused influences on seedling growth,tiller number,pollen fertility,seed-setting rate,grain chalkiness,and grain yield can be used as indices of the thermotolerance of rice germplasm.However,the typical index is seed-setting rate,assessed by the naked eye,or the ratio of seedsetting rate under HS to that under normal conditions,a direct,simple,and reliable index for conventional breeding for HT.Using this index,a series of heat-tolerant rice materials have been identified and used as donors to develop breeding lines,including N22[22,171],Giza178 [16],HHT4 [172],996 [16],IR2061 [173],and Habataki [42].Another kind of donor,EMF20,is also used to develop cultivars that escape heat at flowering,because of its early-morning flowering trait [174].Some heat-tolerant hybrid rice,such as Guodao 6,show seed-setting stability under HS conditions owing to their heat avoidance adaptability based on erratic floral traits,such as shortened flowering phase and decentralized flowering clock [175].

5.3.Identification of heat-tolerant quantitative trait loci and markerassisted breeding

Because strategies for agronomic management and conventional breeding for thermotolerance are few,an urgent task for breeders is to discover heat-resistance genes or quantitative trait loci (QTL) and apply them to thermotolerance breeding.To date,many QTL responsible for thermotolerance at various developmental stages of seedling,booting,flowering,and grain filling have been identified and validated (Fig.4).OsHTAS,a dominant major QTL on chromosome 9,has been cloned and verified,and confers tolerance to 48 °C temperatures in rice seedlings [21,23].Kilasi et al.[22] detected numerous QTL for seedling growth under HS and identified one QTL,RLHT5.1,for root length under HS with phenotypic contribution up to 20.4%.Many QTL associated with heat tolerance at the reproductive stage have been mapped using the phenotypes of spikelet fertility or seed-setting rate under HS.qHTB1-1has been fine-mapped to a 47.1-kb region nearqRRS1on chromosome 1[176],and explained 13.1%–17.8%of the phenotypic variance observed in several generations [172].Zhu et al.[22,34,177] identified 12 QTL associated with heat tolerance at the booting stage,with one of the major-effect QTL (qHTB3-3)located nearqTL3.4andRLPC3.1.Two major QTL located on chromosome 1 (qHTSF1.1) and chromosome 4 (qHTSF4.1) explained respectively 12.6% and 17.6% of spikelet fertility variation under HS [178],andqHTSF4.1has been further localized to a 1.2 Mb region shared withqTL4.1,SSPF4,andSSPC4[171,177,179].Zhao et al.[42]detected 11 QTL for heat tolerance at anthesis in rice,further verifyingqPSLht4.1at several temperatures.This locus was detected in multiple studies (Fig.4).Many heat-tolerance QTL,includingqSF1,qSF2,qSF3.2,qSSIPSS12.1,qSTIPSS9.1,qSTIY3.1,andqSTIY5.1,have been detected at the flowering stage by a sequencing-assisted approach [180,181].Many QTL contributing to thermotolerance at the filling stage have been identified.One QTL,Appearance quality of brown rice 1(Apq1),has been localized to a 19.4-kb region,and the underlying gene has been cloned and namedsucrose synthase 3(Sus3)[182].Wada et al.[54]identified 10 QTL for white-back and basal-white grains caused by HS,andqWB8has been shown to improve quality of rice grain under HS.The effects ofqWB6,qWB9,andqMW4.1have been verified in multiple environments [52,183].

Fig.4.Identified quantitative trait loci (QTL) associated with heat tolerance in rice.These QTL were identified using heat stress induced-phenotypes such as seedling root length or shoot length,survival rate,spikelet fertility or sterility,flowering time,yield per plant,seed-setting rate,frequency of white-back,basal-white,or milky-white kernels,or kernel weight.Different names for a single locus indicate that the locus has been identified in at least two studies,suggesting that the QTL may have large and stable effects.Red names indicate that these QTL have been cloned and functionally validated by transgenic approaches.

Although many heat-resistance QTL have been detected in rice,identification of the causal genes underlying these QTL remains challenging [170],and only a few causal genes have been successfully cloned and functionally validated to date.TT1is a major QTL for thermotolerance at the seedling stage identified in African rice,and the more thermotolerantTT1CG14allele,as revealed by nearisogenic lines (NILs) and transgenic plants,appears to have great potential for thermotolerance breeding [157].The natural variations ofSLG1confer high-temperature tolerance inindicarice[41].Compared withSLG1Tej-carrying plants,SLG1Ind-carrying plants show strong thermotolerance at both seedling and reproductive stages with similar yield traits,suggesting the high potential of theSLG1Indallele for improving thermotolerance injaponicacultivars [41].QTLOsHTASandSus3,involved in thermotolerance at the seedling and grain filling stages,respectively,have also been cloned and functionally verified [21,23,182].

Identified QTL can be introduced into recipient cultivars using DNA markers linked to the QTL,even if the underlying gene is unknown.Markers RM11633 and RM11642,linked to theqHTB1-1locus,were used to improve rice thermotolerance at the booting stage by marker-assisted selection [172].qMW4.1-introgressed NILs genotyped with the linked marker RM16424 showed higher grain quality than recipient cultivars when exposed to HS[52,54].Identification and validation of thermotolerance QTL with stable effects across different genetic background and environments,and pyramiding of these nonallelic QTL,are targets of thermotolerance breeding [25].

5.4.Transgenic approach and genome editing technologies

Genetic engineering is an efficient and time-saving approach to generating heat-tolerant rice [29,170].The receptor-like kinaseERECTAis the best-known gene used for producing thermotolerant rice.Most leaves and tillers ofERECTA-overexpressing plants remained green and survived after exposure to HS(42 °C day/35 °C night) for 10 days at the reproductive stage,whereas tillers in control lines became dried and withered,and the seed-setting rate ofERECTA-overexpressing plants was 55%–70% higher than that of the control line (～35%) after HS [60].Field trials in multiple locations during a summer heat wave confirmed thatERECTA-overexpressing rice plants showed higher seedsetting rate and yield potential than control plants.Overexpression of rice genes such asOsIF[26],OsMYB55[140],OsANN1[63],SNAC3[65],OsbZIP46CA1[141],SAPK6[141],OsRGB1[184],OsSIZ1[134],OsHIRP1[158],Rca[76],andOsWRKY11[142],was shown to improve plant thermotolerance.Transgenic plants specifically suppressingOsMADS7in endosperm via RNA interference (RNAi)showed improved stability of amylose content under HS [57],and such transgenic materials are a valuable genetic resource for breeding thermotolerant rice at the filling stage.A T-DNA-tagged knockout mutation ofosmdhar4also showed increased tolerance to seedling-stage HS [20].

Despite the promise of transgenic rice for improving thermotolerance,its use and commercialization are still strongly affected by public concerns about unsubstantiated health and environmental safety questions,hindering the application of transgenic technology in practical breeding [185].The emergence of genomeediting technology provides a new opportunity for the application of plant molecular breeding,as it can produce plants harboring only mutations in target genes without expression cassettes[186].Genome editing has been successfully used to engineer plant thermotolerance and identify the molecular mechanism of heat resistance.The roles ofAET1[160],OsNAC006[187],HSA1[188],OsCNGC14[93],andOsCNGC16[93] in rice thermotolerance were validated using the CRISPR-Cas9 system.

6.Perspectives

Artificial domestication inevitably results in a genetic bottleneck and reduced genetic diversity,owing to strong selection for favored traits such as crop yield,and for this reason many stresstolerance traits may have been lost [170].The identification and use of favored natural alleles from wild rice and landraces should accordingly be a preferred approach to stress tolerance breeding.To date,several novel heat-tolerant donors have been identified in rice,including SDWG005 (African landrace) [33],NERICA-L-44(African rice)[67],and FR13A(indicalandrace)[189].But the alleles underlying thermotolerant phenotypes await identification.

Considering the complex molecular mechanism and disturbed homeostasis of proteins and metabolites under HS,a combination of genomics,transcriptomics,proteomics,and metabolomics should be adopted for systemically characterizing regulatory networks involved in response to HS [8,24].By use of omics technologies,several rice genes or proteins closely associated with HSR,such asOsACT[33],OsHSP74.8[189],3,8-divinyl protochlorophyllidea8-vinyl reductase [161],and chaperonin 60[190],have been identified,and their effects on thermotolerance await discovery.

Research results from one plant species should be extended to others.For example,overexpression ofERECTAconfers thermotolerance in rice and tomato [60],andERECTA-like genes are widely distributed in plants [191].It is thus of great interest to identify eliteERECTAalleles with higher expression levels in other crops,and validate their function in thermotolerance [60].Natural variants ofSLG1contribute to heat tolerance ofindicarice by increasing tRNA thiolation [41].Considering the conserved function of tRNAthiolation proteins among different species,a question worthy of study is whether different alleles lead to thermotolerance variation in other crop species.In view of the abundant variation across theSLG1region among wild rice accessions,selecting favorableSLG1alleles from wild rice may contribute to thermotolerance breeding[41].

The interaction of HS with other abiotic stresses,especially drought stress,should be studied,given that drought stress is a secondary effect of high temperature [2].Given that some plant species require microbial associations for stress tolerance and survival [192,193],research into root-zone and endophytic microbes may provide an opportunity for improving heat and/or drought tolerance in crops.

CRediT authorship contribution statement

Yufang Xu:wrote the manuscript and prepared the Figures;Chengcai Chu and Shanguo Yao:designed,supervised,reviewed,and edited the writing.

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 the authors whose work could not be cited in this review owing to space limitations.This work was supported by the National Key Research and Development Program of China(2016YFD0101801),the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24030201),and the State Key Laboratory of Plant Genomics.We thank Yahui Li for his help in drawing the Figures.