Cito Liu, Bigng Mo, Dingyng Yun, Chengi Chu, Meijun Dun,*

a College of Agriculture, Hunan Agricultural University, Changsha 410128, Hunan, China

b State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, Hunan, China

c 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

Keywords:Salt stress Rice (Oryza sativa L.)Salt tolerance genes Physiological response Salt signal transduction

ABSTRACT Crop yield loss due to soil salinization is an increasing threat to agriculture worldwide.Salt stress drastically affects the growth,development,and grain productivity of rice(Oryza sativa L.),and the improvement of rice tolerance to salt stress is a desirable approach for meeting increasing food demand.The main contributors to salt toxicity at a global scale are Na+and Cl-ions,which affect up to 50%of irrigated soils.Plant responses to salt stress occur at the organismic, cellular, and molecular levels and are pleiotropic,involving (1) maintenance of ionic homeostasis, (2) osmotic adjustment, (3) ROS scavenging, and (4)nutritional balance.In this review, we discuss recent research progress on these four aspects of plant physiological response, with particular attention to hormonal and gene expression regulation and salt tolerance signaling pathways in rice.The information summarized here will be useful for accelerating the breeding of salt-tolerant rice.

1.Introduction

Soil salinity is an environmental factor that has severe negative effects on seed germination,crop growth,and productivity[1],and it is becoming a serious problem worldwide [1,2].Of the 230 million hectares of farmland currently in use around the world,20%is affected by salt,and this percentage increases every year as a result of inappropriate crop irrigation practices, overfertilization, and excessive plowing, as well as natural causes such as salt intrusion into coastal zones resulting from rising sea levels[2-4].The threats of salinity are more obvious in arid and semiarid regions where limited rainfall, high evapotranspiration, and extreme temperatures coupled with poor water and soil management are the main contributing factors [5,6].In China, there are as many as 100 million hectares of saline farmland [7].Globally, salinity is a significant abiotic stress that affects one-quarter to one-third of the crop productivity of on agricultural soils [8].As the world population increases,more food is needed to meet the rising demand,and the use of saline land to make up for the resulting food shortage is becoming more urgent [9].Scientists are attempting to increase food production by 70%in order to avoid the risk of food shortages for 9.3 billion people by 2050 [10].Rice, an important cereal crop that feeds half of the world’s population, is sensitive to salt stress[11,12].Improvement of rice salt stress tolerance is therefore of paramount importance for global food security.

Combating salt stress to increase rice production requires a thorough understanding of physiology, biochemistry, metabolism,and gene expression under salt stress conditions.In this review,we systematically summarize the current understanding of salt stress effects on rice, the mechanisms that underlie rice salt stress tolerance, and salt stress signaling in rice.This information may be helpful for developing salt-tolerant rice varieties.

2.Effects of salt stress on rice

Plant morphological changes under high salt conditions include impaired root system establishment, leaf rolling, chlorosis, fewer tillers per plant,reduced biomass,shorter plant height,lower thousand grain weight,fewer spikelets per panicle,and more sterile florets, ultimately leading to reductions in harvest index and grain yield [13-16].

Salt stress affects plant physiology and biochemistry at all developmental stages from germination to senescence [6,17,18].Salt stress has both osmotic and ionic or ion-toxicity effects on plants, ultimately causing oxidative stress and nutrient depletion in plant cells[6,19].Continuous salt stress reduces plant cell turgor pressure, which in turn reduces cell growth, and plants must osmotically adjust to maintain cell expansion and growth [20-22].Osmotic stress (caused by lower water potential of the external solution) is rapidly sensed by the plant soon after exposure to saline conditions and leads to plant water and solute deficits[9,21-23].Osmotic stress also results in rapid stomatal closure, which reduces the plant’s ability to assimilate CO2and inhibits photosynthesis [24].Ionic stress causes sodium (Na+) and chlorine (Cl-)accumulation in plant cells, eventually resulting in premature leaf senescence and even plant death [1,25,26].The most common explanation for Na+toxicity is that it has an inhibitory effect on enzyme activities, negatively affecting metabolism, including the Calvin cycle and other pathways [27,28].Excess Na+in the cytoplasm also interferes with the uptake and transport of potassium(K+) and other macro- and micronutrients, including nitrogen (N),phosphorus (P), potassium (K+), calcium (Ca2+) and zinc (Zn2+)[13,29-32].In addition to osmotic and ionic stresses, salt stress causes the cellular accumulation of reactive oxygen species(ROS), which can severely damage cellular structures and macromolecules such as DNA, lipids, and enzymes [33,34].

Under salt stress, plants must adjust the physiological and biochemical processes involved in the regulation of ion and osmotic homeostasis, oxidative stress, and nutritional balance [1,25].

3.Mechanisms underlying rice salt stress tolerance

3.1.Regulation of ionic balance

Salt stress is commonly caused by high concentrations of Na+and Cl-in the soil [9,35].Na+and K+are imported into the cell using the same suite of transporters,and the two cations compete with each other [36].K+is important for the catalytic activities of many central enzymes, and excess Na+competes with K+for uptake across the plasma membranes of plant cells [37].K+is also necessary for osmoregulation and protein synthesis, the preservation of cell turgor, and optimal photosynthetic activity [6,38,39].The maintenance of cellular Na+/K+homeostasis is therefore a crucial factor that determines the plant’s ability to survive during salt stress [1,25].

Mechanisms to reduce cytoplasmic Na+include restriction of Na+uptake, increase of Na+efflux, and compartmentalization of Na+in the vacuole [1,40].The rice plasma membrane Na+/H+antiporter (OsSOS1) excludes Na+from the shoot, promoting a lower cellular Na+/K+ratio and increasing salt tolerance[41,42].The vacuolar Na+/H+antiporters OsNHX1, OsNHX2, OsNHX3, OsNHX4,OsNHX5, and OsARP/OsCTP play important roles in the vacuolar compartmentalization of Na+and K+that accumulate in the cytoplasm and thereby determine rice salt tolerance[43-45].Members of the rice high-affinity K+transporter (HKT) family, OsHKT1;1,OsHKT1;4/OsHKT7, and SKC1/OsHKT1;5/OsHKT8, help to reduce Na+accumulation in leaves during salt stress [46-48].OsHKT1;1,OsHKT2;1, OsHKT2;3/OsHKT3, OsKAT1, OsKAT2, OsHAK5, and OsHAK21/qSE3 transport Na+or both Na+and K+,helping to maintain Na+/K+homeostasis in the cytoplasm and regulate rice response to salt stress [46,49-58].The vacuolar H+-translocating inorganic pyrophosphatase OsVP1 pumps H+from the cytosol into the vacuole,increasing the electrochemical potential gradient of H+between the cytoplasm and vacuole, thereby promoting the exchange of Na+/H+and improving rice tolerance to salt stress[43].Researchers have demonstrated that Cl-induces deficiencies in key macronutrients (e.g., N and S) because the uptake ofandis mediated by the same (non-selective) anion transporters as Cl-[23,59].OsCLC1,OsCLC-1, andOsCLC-2encode rice chloride channel proteins that promote plant growth under ionic stress and improve salt stress tolerance [60,61] (Table 1; Fig.1).

A number of genes influence rice salt tolerance by regulating the expression ofHKT,NHX, andCLCgenes.These include transcription factors such as OsMYBc, OsbZIP71, OsNF-YC13, and the sucrose nonfermenting 1-related protein kinase2 (SnRK2) SAPK4,which directly activate the expression of K+/Na+transporters,thereby improving the K+/Na+ratio and positively regulating salt tolerance [46,62-64].The myristoylated Ca2+-binding protein OsSOS3/OsCBL4 phosphorylates OsSOS2/OsCIPK24, thereby activating the Na+/H+antiporter OsSOS1 and promoting rice tolerance to salt stress[41,66].The mitogen-activated protein kinase(MAPK)OsMAPK33 plays a negative role in salt tolerance by promoting higher sodium uptake into cells and thereby lowering the K+/Na+ratio[65](Table 1;Fig.1).The results above suggest that the modulation of Na+/K+homeostasis under salt stress may provide an effective way to improve salt tolerance in rice.

Table 1 Proteins that function in Na+/K+ homeostasis during salt stress.

Fig.1.Genes involved in the regulation of salt tolerance in rice,including Na+/K+ion homeostasis(upper right),osmotic adjustment(upper left),ROS scavenging(lower left),and nutritional balance (lower right).The middle yellow layer contains functional genes whose encoded proteins directly protect membranes and macromolecules under stress conditions.The outer gray layer contains regulatory genes whose products protect plants from adversity by regulating the expression of functional genes under stress conditions.

3.2.Osmotic adjustment

Salt stress also causes osmotic stress and promotes the biosynthesis and accumulation of compatible osmolytes such as sugar,proline, glycine betaine, polyamines, and proteins from the late embryogenesis abundant (LEA) superfamily.These osmolytes play a dominant role in osmotic adjustment under salt stress by reducing cell osmotic potential and stabilizing proteins and cellular structures[1,23].The proline synthesis genesOsP5CS1andOsP5CRincrease proline accumulation and improve rice tolerance to salt stress [67].OsTPS1,OsTPS2,OsTPS4,OsTPS5,OsTPS8, andOsTPS9,which encode trehalose-6-phosphate synthase (TPS), enhance rice tolerance to cold,salt,and drought stresses by increasing trehalose and proline contents under abiotic stress [68].OsGMST1, which encodes a monosaccharide transporter, increases monosaccharide accumulation and improves salt tolerance in plants [69].The Sugars Will Eventually be Exported Transporters (SWEETs) OsSWEET13 and OsSWEET15 regulate the transport and distribution of sucrose and maintain sugar homeostasis in rice under drought and salinity stresses [70].Rice glycine betaine is synthesized by the choline monooxygenase OsCMO and the betaine aldehyde dehydrogenase OsBADH1, which enhance rice tolerance to salt stress by promoting glycine betaine accumulation [71,72].TheLEAgenesOsLEA3-2,OsLEA4,OsLEA5, andOsEm1significantly improve plant tolerance to salt and osmotic stresses [73-76].

The salt-regulated geneOsSALP1encodes a small plant-specific membrane protein that improves salt tolerance by increasingOsP5CSexpression and free proline content under salt stress [77].The transcription factors OsZFP252, OsZFP179, OsZFP182, OsNFYC13, OsCOIN, and OsNAC5 increase the amount of free proline and soluble sugars, upregulate the expression of stress tolerant genes,and enhance rice tolerance to salt stress[63,78-82](Table 2;Fig.1).

3.3.ROS scavenging

Plant exposure to salt stress can upregulate the production of ROS such as1O2(singlet oxygen), O2-(superoxide radical), H2O2(hydrogen peroxide), and OH-(hydroxyl radical) [9,34].Although low ROS concentrations can function as a signal to activate salt stress responses,excess ROS accumulation causes phytotoxic reactions including DNA mutation,protein degradation,and the peroxidation of carbohydrates and lipids [1,34].Plants use enzymatic and nonenzymatic antioxidants to mitigate ROS stress [34,83,84].Enzymatic scavengers include nicotinamide adenine dinucleotide phosphate oxidases (NOXs, also called respiratory burst oxidase homologs [Rbohs]), superoxide dismutase (SOD), ascorbate peroxidase(APX),catalase(CAT),glutaredoxin(GRX),glutathione peroxidase (GR), glutathione S-transferase (GST), and glutathione peroxidases (GPXs) [1,85-90].Nonenzymatic scavengers include ascorbic acid (ASH), alkaloids, carotenoids, flavonoids, glutathione(GSH), phenolic compounds, and tocopherol [83,91,92].OsRbohAandOsRbohIare induced by salt treatment,whereasOsRbohB,OsRbohC,OsRbohE, andOsNox6are repressed [93].OsMn-SOD1andOsCu/Zn-SODoverexpression lines show lower accumulation of O2-and H2O2under salt stress [94,95].OsAPX2,OsAPX7,OsAPx8,OsAPXa, andOsAPXbincrease APX activity,lower H2O2and malondialdehyde (MDA) levels, decrease oxidative stress damage, and enhance rice tolerance to salt stress [96-99].OsGST4is induced by heavy metals,hypoxia,and salt stress in rice[100].Glutathione responsive rice glyoxalase II(OsGLYII-2)functions in salinity adaptation by maintaining better photosynthetic efficiency and increasing the antioxidant pool[101].OsGR2,OsGR3,andOsGRX8increase GSH content and enhance tolerance to various abiotic stresses,including salinity, osmotic, and oxidative stress [99,102-106].The cytosolic dihydroorotate dehydrogenase geneOsDHODH1improves rice tolerance to salt and osmotic stresses [107].Rice GDP-mannose pyrophosphorylase OsVTC1-1 and dehydroascorbate reductase (OsDHAR) play critical roles in plant salt tolerance by promoting the ASH scavenging of excess ROS[108,109].OsGSA1encodes a UDP-glucosyltransferase that causes plants to accumulate flavonoid glycosides, which protect rice against abiotic stress[110] (Table 3; Fig.1).

Table 2 Proteins and metabolites that function in osmotic adjustment during salt stress.

A number of genes improve the salt tolerance of rice by regulating genes involved in ROS biosynthesis and scavenging pathways.The calcium-dependent protein kinases OsCPK4 and OsCPK12 and the receptor-like kinase OsSIK1 promote tolerance to salt stress by reducing the accumulation of ROS [111-113].The transcription factors OsZFP179, OsZFP182, OsZFP213, OsHBP1b/Osb-ZIP3, OsMADS25, OsMyb2, and OsMyb6 positively regulate salt tolerance by increasing ROS-scavenging ability [79,80,114-118],whereas the zinc-finger proteins DST and DCA1 negatively affect rice salt tolerance by regulating the transcription of ROSscavenging genes [119,120](Table 3;Fig.1).All these studies suggest that enhancing ROS-scavenging ability can efficiently increase the salt tolerance of rice.

3.4.Nutrient imbalance

Salt stress causes plant nutritional deficiencies due to reduced transport efficiency and decreased uptake of nutrients such as N,P, K, Ca, Zn, and magnesium (Mg) [6,13,29,121].Mg2+transport by OsMGT1 in the mature root zone synchronously enhances OsHKT1;5 activity, which restricts Na+accumulation in the shoots and improves salt tolerance [122].The rice aminotransferase OsAMTR1 interacts with stress associated protein 1 (OsSAP1) and regulates abiotic stress responses [123].The cytokinin type-B response regulator (RR) OsRR22 regulates Zn acquisition by directly modulating the expression of Zn-regulated transporter genes and the sensitivity to salt stress [124,125].OsMADS27regulates root development in a-dependent manner and modulates salt tolerance in rice [126] (Table 4 ; Fig.1).

Table 3 Proteins and metabolites that function in ROS homeostasis during salt stress.

Table 4 Proteins that function in nutritional adjustment during salt stress.

3.5.Developmental adjustment

Plant response to salinity is the collective outcome of intricate communications among various processes linked to plant morphology, biochemistry, and physiology, as well as the inhibition of growth and photosynthesis and the reduction of grain yield[1,127,128].Roots take up water and nutrients and have a key role in plant growth,development,and survival.The root system is the first tissue to perceive salt stress [129].Transgenic rice lines that overexpressedOsAHL1, OsHAL3, andOsMADS25had greater root volume under saline conditions and exhibited enhanced salt avoidance [116,130,131].Transgenic lines that overexpressedOsiSAP8andOsCFM2had higher chlorophyll contents and photosynthetic rates than wild-type plants under abiotic stress [132,133].

4.Salt stress signaling in rice

As sessile organisms, plants must cope with abiotic stresses such as soil salinity[19].To adapt to salt stress,plants have developed various strategies to integrate exogenous salinity stress signals with endogenous developmental cues to optimize the balance between growth and stress response [134].Na+influx occurs through voltage-insensitive monovalent cation channels(VICs) [135,136].These channels are regulated using bivalent cations such as Ca2+to maintain ion homeostasis [31].Early salt signaling responses are induced, including Ca2+signaling, and these early Na+-induced signals reduce Na+import [14].Ca2+acts as a second messenger, and its levels are rapidly elevated when plants encounter abiotic stresses [31].An initial Ca2+-dependent signaling network is involved in salt stress responses and includes both Ca2+transport and downstream targets such as calmodulin(CaM), CMLs (CaM-like proteins), CDPK/CPK (calcium-dependent protein kinase), CBLs (calcineurin B-like proteins), and CIPKs(CBL-interacting protein kinases) [31,137].

4.1.The SOS signaling pathway

Plants use a calcium-dependent protein kinase pathway known as the salt overly sensitive (SOS) pathway for salt stress signaling and the development of Na+tolerance[138,139].The SOS pathway was the first abiotic stress signaling pathway to be characterized in plants[139].It is a major mechanism for the exclusion of Na+from the cytosol,and its elucidation was a milestone in our understanding of how plants deal with salt load[140,141].In this pathway,the calcium-binding protein OsSOS3/CBL4 senses the cytosolic calcium signal elicited by salt stress, then interacts with and activates a sucrose non-fermenting-1-related protein kinase-3 (SnRK3),OsSOS2/OsCIPK24.Activated OsSOS2/OsCIPK24 phosphorylates and activates OsSOS1, a Na+/H+antiporter at the plasma membrane, which in turn controls Na+homeostasis and improves rice tolerance to salt stress [41] (Fig.2).

Other Ca2+signal response proteins also play a role in salt stress signaling.The calmodulin protein OsMSR2, OsCam1-1, OsCCD1,OsCam1, and the CML protein OsCBL8 act as Ca2+sensors and improve rice tolerance to salt stress[142-146].The CDPK/CPK proteins OsCPK4, OsCPK7, and OsCPK12 help to lower Na+accumulation and maintain osmotic balance under salt stress conditions[112,147,148].

4.2.Hormonal regulation during salt stress

Downstream of the early salt signaling phase, phytohormone levels change, and the salt-induced signaling cascade ultimately result in adaptive responses [14].Phytohormones are crucial endogenous chemical signals that coordinate plant growth and development under both optimal conditions and environmental challenge [14].Response and adaptation to salt stress require the integration and coordination of multiple phytohormones, including abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA), gibberellic acid (GA), cytokinin (CK), and salicylic acid (SA), which regulate normal growth and mediate responses to abiotic stress[14,149-151].

4.2.1.Abscisic acid

Fig.2.Known salt stress signaling pathways in rice.(1) The most classic pathway is the SOS signaling pathway (left), which consists of OsSOS3/OsCBL4, OsSOS2/OsCIPK24,and OsSOS1.It is important for sensing salt-induced Ca2+signals and regulating ion homeostasis by removing excess Na+from cells or compartmentalizing it in the vacuole.(2)In the ABA-dependent pathway(middle),salt stress induces ABA accumulation and promotes ABA binding to PYR/PYL ABA receptors such as OsPYR/RCAR5,leading to the inactivation of core ABA-signaling components (PP2Cs such as PP108).The PYL-ABA-PP2C complex forms, releasing SnRK2s such as SAPK4 from association with and inhibition by PP2Cs.The released SnRK2s are activated through autophosphorylation and in turn activate the expression of many downstream effectors.(3) In one typical ABA-independent pathway(right),ethylene regulates stress responses.OsSIT1 mediates salt sensitivity by activating MPK3/6,thereby promoting ethylene biosynthesis and ROS accumulation.The ethylene-response genes MHZ6/OsEIL1 and OsEIL2 negatively regulate salt tolerance by controlling OsHKT2;1 expression and Na+uptake in rice roots.

ABA is the central regulator of abiotic stress tolerance in plants[151-153].It has a major role in the salt stress response,regulating stomatal closure, ion homeostasis, salt stress-responsive gene expression, and metabolic changes [14].Salt stress causes an increase in ABA accumulation, and exogenous ABA may alleviate the deleterious effects of salt stress[154,155].ABA signal transduction requires three core components:PYR/PYL/RCAR ABA receptors(PYLs), type 2C protein phosphatases (PP2Cs), and class III SNF-1-related protein kinase 2 s (SnRK2s) [156].Under salt stress conditions, ABA binds to PYR/PYL/RCAR receptors, which then interact with PP2Cs and inhibit their activity, thus releasing SnRK2s from repression [157-159].SnRK2s phosphorylate various ABAresponsive element (ABRE)-binding protein/ABRE-binding factor(AREB/ABF)transcription factors,which further regulate ROS scavenging, ion homeostasis, and stomatal closure in response to salt stress [14,160-162].The ABA biosynthesis genesOsNCED3andOsNCED5, encoding 9-cis-epoxycarotenoid dioxygenases, increase ABA levels and enhance salt stress tolerance [163,164].The rice ABA receptor geneOsPYL/RCAR5is a positive regulator of the ABA signal transduction pathway in abiotic stress tolerance [165,166](Fig.2).The PP2C genes (such asOsPP108) negatively regulate ABA signaling but positively regulate abiotic stress signaling[167] (Table 5; Fig.2).The transcription factors OsABF1/OsbZIP12,OsABF2/OsbZIP46,OsBZ8/OsbZIP05,OsbZIP71,OsWRKY45,SNAC1,OsNAC2, ONAC022, OsMADS25, OsMYB48-1, OsMYB91, and OsRHP1 play positive roles in ABA-mediated salt tolerance of rice[62,116,168-179], whereas OsABI5/OsbZIP10, ZFP185, and Oshox22 play negative roles [180-183].OsbZIP23 directly targets the ABA synthesis geneOsNCED4and the ABA signaling component geneOsPP2C49, which act as positive and negative regulators,respectively, and it can therefore feedback-regulate ABA signaling and biosynthesis in response to abiotic stress [184] (Table 5).

Table 5 Phytohormones that mediate salt stress responses.

4.2.2.Ethylene

Etiolated seedlings treated with ethylene exhibit a double response,i.e.the promotion of coleoptile growth and the inhibition of root elongation[213].The function of ethylene in rice salt tolerance has been studied extensively.Promotion of ethylene biosynthesis and signal transduction can enhance plant salinity tolerance, whereas their inhibition increases sensitivity to salinity inArabidopsisand soybean [214,215].However, in tomato, the direct ethylene precursor ACC causes Na+accumulation and oxidative stress in leaves and promotes leaf senescence under salinity stress[216-218].Ethylene treatment of Nipponbare rice seedlings increased their sensitivity to salinity, whereas treatment with 1-MCP(a blocker of ethylene perception)enhanced salinity tolerance[185,219].Salt Intolerance 1(OsSIT1),a lectin receptor-like kinase,is an ethylene sensor that positively mediates salt sensitivity by activating MITOGEN-ACTIVATED PROTEIN KINASE3/6 (MPK3/6),thereby promoting ethylene biosynthesis, ROS accumulation, and sensitivity to salt stress [187] (Table 5; Fig.2).The ethylene response factors(AP2/ERFs),such as OsAP23,OsERF922,OsEREBP1,and OsEREBP2,as well as the ethylene signaling component genesMHZ6/OsEIL1andOsEIL2, are positively regulated by ethylene signaling and negatively affect salt tolerance in rice [185,188-191](Table 5; Fig.2).The DOF transcription factor OsDOF15 and the ethylene-responsive element binding protein (EREBP) transcription factor OsBIERF3 positively regulate primary root elongation under salt stress by restricting ethylene biosynthesis [192-194](Table 5).

4.2.3.Gibberellic acid

GA is an important hormone that regulates plant growth and has been linked to the regulation of growth under abiotic stress[220].The GA catabolic pathway genesgibberellin 2-oxidase 5(OsGA2ox5) andOsCYP71D8Lreduced GA accumulation and enhanced plant salt tolerance by retarding growth [195,221], suggesting a negative role for GA in rice salt tolerance.The rice homolog of theArabidopsisDELLA geneSlender rice1,OsSLR1, encodes a GA signaling component that regulates mesocotyl and root growth,specifically in the dark and under salt stress[196](Table 5).Therefore, rice growth inhibition under salt stress may be an active adaptation mechanism whereby rice reduces GA levels or GA signaling in response to changing environmental conditions [134].

4.2.4.Auxin

Auxin plays an important role in root system growth in response to local soil conditions [134,222].Rice Big Grain 1(RBG1) regulates cell division, auxin accumulation, and tolerance to drought,salt,and heat stresses[197].The two rice auxin receptor genesOsTIR1andOsAFB2are targeted by OsmiR393, downregulated the expression ofOsAUX1(an auxin transporter) andOsTB1(a tillering inhibitor), repressing auxin signaling and leading to more tillers, earlier flowering, and reduced salt and drought tolerance [198].The NAC transcription factor OsNAC2 decreases auxin biosynthesis,content,and gene responses while increasing cytokinin (CK) biosynthesis gene expression and CK content, thereby integrating the auxin and CK pathways to modulate rice root development under normal and saline conditions [175,199] (Table 5).

4.2.5.Cytokinins

CKs function in the control of plant adaptation to environmental stress.InArabidopsis, plants with reduced levels of various CKs exhibit enhanced salt tolerance [223], whereas exogenous CK application increases salt tolerance inSolanum melongena[224].The cytokinin oxidase OsCKX2, an enzyme of the CK degradation pathway, negatively regulates salt stress tolerance by controlling CK levels [200].The CK response regulatorsOsRR9,OsRR10, andOsRR22inhibit the response to CK signaling by a negative feedback mechanism and improve rice tolerance to salt stress[124,125,201].OsAGO2increases salt tolerance by activating the expression ofOsBG3and altering CK distribution [202,203].Two rice authentic histidine phosphotransferases, OsAHP1 and OsAHP2, and the NAC transcription factor OsNAC2 mediate CK signaling and enhance plant tolerance to salt stress [199,204] (Table 5).

4.2.6.Brassinosteroids

Brassinosteroids (BRs) are plant steroid hormones that play essential roles in plant growth, development, and abiotic stress responses [225].Exogenous application of BR enhances salt tolerance in rice [226].GW5-Like (GW5L) positively regulates BR signaling by repressing the phosphorylation activity of GSK2 and confers salt stress sensitivity [205,207].The rice microRNAosamiR1848targets the obtusifoliol 14α-demethylase geneOsCYP51G3and mediates the biosynthesis of phytosterols and BR during development and in response to stress [206] (Table 5).

4.2.7.Jasmonic acid

Jasmonic acid (JA) is required for the inhibition of root elongation and the activation of antioxidant enzymes upon exposure to high salinity[227].Endogenous JA accumulates in rice roots under salt stress,and exogenous JA improves salt stress tolerance in both rice and wheat [228,229].Overexpression of the Cyt P450 family geneOsCYP94C2b, encoding a JA-catabolizing enzyme, reduces JA content and improves performance at high salt concentrations[208].The plant-specific TIFY transcription factors OsJAZ8 and OsJAZ9 act as negative regulators of jasmonic signaling but positively modulate salt stress tolerance in rice [209,210] (Table 5).

4.2.8.Salicylic acid

Salicylic acid (SA) plays an important role in plant salt tolerance.Exogenous SA application enhances the antioxidant system,increases the synthesis of osmolytes,and promotes photosynthesis and nitrogen fixation under salt stress [230-232].The pathogenesis-related protein RSOsPR10 (root specific rice PR10)is regulated antagonistically by JA/ethylene and SA signaling and improves plant tolerance to salt stress [211,212] (Table 5).

4.3.Conclusions

Over the course of evolution, plants have developed the capacity to detoxify the excessive Na+levels that accompany high salt concentrations and to utilize multiple strategies for the alleviation of salt stress damage[31].Plant salt responses caused by hyperosmotic tension are closely linked to Ca2+channels and Ca2+signaling[31,233].(1) The most typical Ca2+signaling pathway is the SOS pathway,in which OsSOS3/OsCBL4 senses the cytosolic Ca2+signal elicited by salt stress and phosphorylates OsSOS2/OsCIPK24,which activates OsSOS1, a plasma membrane Na+/H+antiporter that regulates ion homeostasis by transporting excess Na+out of cells or compartmentalizing it in the vacuole [41] (Fig.2).(2) Phytohormones mediate the response to various environmental stresses,including salt stress, and thus regulate plant growth adaptation[9,134].ABA is the hormone usually associated with major plant responses to stress [153].In the ABA-dependent pathway, the PYL-ABA-PP2C complex forms, releasing SnRK2s such as SAPK4 to phosphorylate many downstream effectors(Fig.2).(3)Ethylene is also a widely known abiotic stress signal.For example, the receptor-like kinase OsSIT1 mediates salt sensitivity by activating MPK3/6, which promotes ethylene biosynthesis and ROS accumulation[187](Fig.2).OsEIL1 and OsEIL2 are ethylene response genes that repress the expression of K+transporters[185](Fig.2).In brief,plants have developed various signaling cascades for sustaining ion homeostasis and cell turgor pressure, avoiding cellular oxidative damage or nutritional imbalances, and optimizing the balance between growth and stress responses under salt stress.

5.Future perspectives

Ongoing soil salinity threatens rice production and future food security, and breeding and cultivation of salt-tolerant varieties is the most effective means for overcoming this environmental challenge[23].Many salt response genes have been cloned and identified in the past three decades, and mechanisms of rice salt stress tolerance have been characterized.Two major approaches have been used to improve crop salt tolerance:(1)exploitation of natural genetic variation between tolerant and sensitive varieties, and(2) generation of transgenic plants with novel genes or altered expression levels of existing genes[234].However,the application of this fundamental knowledge to improve salt stress tolerance of crops in the field is a slow and challenging process.

Because salt tolerance is a complex quantitative trait,only a few haplotypes of genes or loci have been reported that could be used for molecular marker-assisted breeding.For example, the T67K and P140A mutations may destabilize the transmembrane domain or alter the phosphorylation probability, respectively, of OsHKT1;5/SKC1, thereby influencing rice tolerance to salt stress[235-237].The K24E mutation in the SalT protein appears to alter its interactions with other proteins,affecting its function and influencing salinity tolerance [235,238].TheSaltolquantitative trait locus (QTL) contributes to salinity tolerance and has been fine mapped on chromosome 1 using a Pokkali/IR29 recombinant inbred line (RIL) population, and the simple sequence repeat(SSR) markers RM493 and RM3412b have been used effectively in marker-assisted breeding [239-243].

However, marker-assisted breeding has two problems:first,QTL mapping results based on specific parents are not usually universal and cannot readily be applied to breeding populations.Second,salt tolerance is controlled by multiple minor genes,and these genes or loci cannot be used effectively to improve quantitative traits.Genome selection(GS)using SNPs or alleles of the whole rice genome can be used to predict genomic estimated breeding value(GEBV) of individuals in rice populations based on allele effects using a genetic relationship matrix.GS is time-efficient and can combine pedigree and genotype information, making it superior to other methods of plant breeding [244,245].

Improvements in crop salt stress tolerance will become more feasible with the aid of gene editing technologies and advances in the efficient genetic transformation of different species [246].Genes that confer salt sensitivity, such asDST,OsRR22, OsEIL1,OsEIN2, andOsSIT1, could be knocked out to improve rice salt tolerance [120,125,185,187].TheOsbZIP23promoter has a 35-nucleotide deletion in the 5′-UTR in the drought-sensitive IR20 cultivar, and the deleted sequence is associated with decreasedOsbZIP23expression and reduced tolerance to abiotic stress relative toOryza rufipogon[247].We can also use genome editing technology for knock-in of the deleted sequence in cultivated rice.

An understanding of the molecular mechanisms of rice salt tolerance can now be combined with the development of molecular salt tolerance markers,the complete sequencing of plant genomes,and the widespread use of microarray analysis and genome editing technology.These powerful techniques offer advantages and provide solutions to the complex and intriguing trait of plant salt resistance.

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.

CRediT authorship contribution statement

Citao Liuwrote the manuscript.Bigang Mao, Dingyang Yuan,Chengcai Chu, and Meijuan Duanrevised and edited the manuscript.

Acknowledgments

This work was supported by the Research Initiation Fund of Hunan Agricultural University (20154/5407419002), the Open Research Fund of the State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center (2020KF05), the Hunan Science and Technology Major Project (2018NK1010), and the Hunan Science and Technology Talents Support Project(2019TJ-Q08).The authors would like to thank TopEdit(www.topeditsci.com)for its linguistic assistance during the preparation of this manuscript.