Shuangshuang Liu,Ziwen Li,Suowei Wu,*,Xiangyuan Wan,*

a Zhongzhi International Institute of Agricultural Biosciences,Shunde Graduate School,Research Center of Biology and Agriculture,University of Science and Technology Beijing,Beijing 100096,China

b Beijing Engineering Laboratory of Main Crop Bio-Tech Breeding,Beijing International Science and Technology Cooperation Base of Bio-Tech Breeding,Beijing Solidwill Sci-Tech Co.Ltd.,Beijing 100192,China

Keywords:

A B S T R A C T Sugar metabolism plays an essential role in plant male reproduction.Defects in sugar metabolism during anther and pollen development often result in genic male sterility(GMS).In this review,we summarize the recent progresses of the sugar metabolism-related GMS genes and their roles during plant anther and pollen development,including callose wall and primexine formation,intine development,pollen maturation and starch accumulation,anther dehiscence,and pollen germination and tube growth.We predict 112 putative sugar metabolic GMS genes in maize based on bioinformatics and RNA-seq analyses,and most of them have peak expression patterns during middle or late anther developmental stages.Finally,we outline the potential applications of sugar metabolic GMS genes in crop hybrid breeding and seed production.This review will deepen our understanding on sugar metabolic pathways in controlling pollen development and male fertility in plants.

Contents

1. Introduction........................................................................................................1224

2. Overview of sugar metabolism and its related GMS genes in plants...........................................................1224

3. Sugar metabolic GMS genes and their essential roles in plant anther and pollen development.....................................1225

3.1. Callose wall and primexine formation..............................................................................1227

3.2. Pollen intine development.......................................................................................1227

3.3. Pollen maturation and starch accumulation.........................................................................1227

3.4. Anther dehiscence,pollen germination and tube growth...............................................................1229

3.5. The specific roles of sugar metabolism and its related genes at different anther and pollen development stages.................1229

4. The coordinated regulation of sugar metabolic GMS genes contributing to plant male fertility.....................................1229

4.1. Transcriptional regulation........................................................................................1229

4.2. Posttranscriptional and other regulations...........................................................................1230

5. Prediction of putative sugar metabolism-related GMS genes in maize.........................................................1231

5.1. Maize orthologues of the cloned sugar metabolic GMS genes in other plants..............................................1231

5.2.De novoprediction of maize sugar metabolic genes based on bioinformatics analysis.......................................1231

6. Potential applications of sugar metabolic GMS genes in crop hybrid breeding and seed production.................................1231

6.1. Environment-sensitive male-sterility systems........................................................................1232

6.2. Genetic stable male-sterility systems..............................................................................1233

7. Conclusions and perspectives..........................................................................................1233

CRediT authorship contribution statement.................................................................................1234

Declaration of competing interest........................................................................................1234

Acknowledgments...................................................................................................1234

Appendix A.Supplementary data.......................................................................................1234

References.........................................................................................................1234

1.Introduction

In plants,sugars including sucrose,glucose,fructose,trehalose and their derivatives,such as callose,cellulose,hemicellulose,pectin and starch,mainly serve as the basic source of energy and structural constituents of plant cells[1,2].During the active vegetative growth and development,plants rely on the reserved energy consisted of carbohydrates(mainly sucrose)produced by photosynthesis in source organs(e.g.,mature leaves)and their transport from the source organs through the phloem to the sink organs(e.g.,anthers,pollen grains and seeds)for their utilization[1].

Pollen wall is the complex multiple-layer outer surface of pollen,which is essential for plant male reproduction through rendering male gametophytes resistant to various biotic and abiotic stresses.Pollen wall development occurs after meiosis when the callose surrounding microspores is degraded by callases(e.g.,β-1,3-glucanase)secreted from anther tapetum,the innermost layer of anther somatic wall enclosing microspores[2].Tapetal cells are responsible for exporting all necessary nutrients,such as sugars and lipids,into the locular fluid through the apoplast to fuel the early development of microspores[3].When programmed cell death(PCD)of tapetal cells takes place after meiosis,young microspores form primexine,a microfibrillar matrix mainly consisting of cellulose that acts as a template for the deposition and assembly of sporopollenin precursors.At the stage of bicellular pollen,pollen wall contains three layers:the outer exine,the inner intine and tryphine.Among them,the major components of intine consist of pectin,cellulose,hemicellulose,hydrolytic enzymes and hydrophobic proteins[2].During the late stages of pollen development,pollen initiates starch accumulation,which is crucial for pollen maturation and viability,and thus provides the reserve of energy for pollen germination and pollen tube growth[4].

Male sterility resulted from insufficient sugar supply for pollen development under various abiotic stresses have been known for many years[5].This type of male sterility is associated with grain yield losses in cereal crops such as rice(Oryza sativa,Os),maize(Zea mays,Zm)and wheat(Triticum aestivum,Ta)due to the reduced fertilization of spikelets and thus reducing the grain number per plant[6].Accordingly,the artificial modification of sugar flux in anthers to impair pollen development has been utilized as a strategy to induce complete male sterility of female lines used to produce hybrid seeds and enhance the heterosis utilization efficiency and productivity in crops[7,8].In addition,as to the stressinduced male sterility associated with sugar metabolism,other reviews are available for references[9–12].

In this review,we focus on the progress and perspective of sugar metabolism-related genic male sterility(GMS)genes and their roles in plant male fertility.Based on the cloned sugar metabolic GMS genes in plants,we propose a regulation network of sugar metabolism underlying anther and pollen development as well as pollen tube growth.Furthermore,we predict 112 putative sugar metabolic GMS genes in maize based on bioinformatics and RNA-seq analyses by using four sets of maize anther transcriptome data.Finally,we outline the potential strategies utilizing sugar metabolism-related genes in plant breeding and hybrid seed production,and discuss the current challenges and future research directions regarding sugar metabolism in plant pollen development and male fertility.

2.Overview of sugar metabolism and its related GMS genes in plants

Sugar metabolism,including sugar biosynthesis,degradation and transport,as well as its regulation,plays an essential role in plant male reproduction.In the reproductive stage of flowering plants,sugars are transported to anthers to support the normal development of microspores[13],including callose wall and primexine development,intine formation,starch accumulation and pollen maturation,pollen germination and pollen tube growth(Fig.1A)[2].Accordingly,any disturbance in anther sugar metabolism could impair pollen development and eventually lead to male sterility[4,14,15],and defects in sugar metabolic genes required for anther/pollen development often result in GMS phenotype[2].Therefore,sugars have a significant impact on male reproduction,and play a crucial role in pollen formation in plants.

To date,at least 62 sugar metabolism-related GMS genes have been cloned and characterized in different plant species,mainly participating in the biosynthesis or degradation(33 genes),transport(15 genes)and regulation(14 genes)of sugars during anther and pollen development.Notably,most of them are identified inArabidopsis thaliana(32 genes)and rice(24 genes),while only one in maize and five in other plants(Fig.1A and B;Table 1).Among them,seven types of sugar metabolic GMS genes are found to be shared in two or more plant species(Fig.1B),indicating that there are conserved sugar metabolic pathways underlying male reproduction existing in different plants.In addition,some orthologous GMS genes have similar or diverse expression patterns in different plants.For example,CALLOSE SYNTHASE 5(AtCalS5)[16]andGLUCAN SYNTHASE-LIKE 5(OsGSL5)[17],AtA6[18]andGLUCANASE 1(OsG1)[19]have the similar expression patterns,whileMALE STERILITY 8(ZmMs8)[20]andUNEVEN PATTERN OF EXINE 1(UPEX1/AtKNS4)[21]display the different expression patterns(Table S1),suggesting that these orthologues probably have both conserved and divergent functions in anther and pollen development during plant evolution.Thus,more sugar metabolic GMS genes need to be further discovered and elucidated deeply in different plants,especially in maize.

Among all the reported sugar metabolic GMS genes,25 genes are investigated on their protein subcellular localizations(Fig.1C;Table S1).Firstly,nine sugar biosynthesis-related GMS genes encoding proteins are found to be localized in several organelles,e.g.,the cytoplasm-localized REVERSIBLY GLYCOSYLATED POLYPEPTIDES 1(AtRGP1)and AtRGP2[22]and HEXOKINASE10(OsHXK10)[23],Golgi-localized GLYCOSYLTRANSFERASE 1(OsGT1)[24],transmembrane proteins AtCalS5[16]and NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION(AtNPU)[25],and plastid-localized PHOSPHOGLUCOMUTASE(OspPGM),ADPGLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT 4(OsAGPL4)[26]and HEXOKINASE 5(OsHXK5)[27](Fig.1C).These results indicate that sugar biosynthesis may take place in various organelles of plant anthers and pollen grains.Secondly,most(8/9)of the sugar transport-related GMS genes encoding proteins are localized mainly in plasm membrane and endoplasmic reticulum(ER),while two(GLUCOSE-6-PHOSPHATE TRANSPORTER1,AtGPT1 and OsGPT1)are localized in plastid[28,29]and only one(NUCLEOTIDE SUGAR TRANSPORTER 3,AtUTr3)in Golgi[30](Fig.1C),suggesting that the sugar transport in plant anthers and pollen mainly occurs in plasm membrane and ER,as well as less in plastids and Golgi.Finally,most(6/7)of the sugar metabolic regulators are localized in nucleus,including CYCLIN-DEPENDENT KINASE G1(AtCDKG1)[31],MALE STERILITY 35(AtMs35/AtMYB26)[32,33],CARBON STARVED ANTHER(OsCSA)[34–36],OsCSA2[37],OsSPX1[38]and DEFECTIVE MICROSPORE DEVELOPMENT 1(OsDMD1)[39],whereas CALLOSE DEFECTIVE MICROSPORE 1(AtCDM1)is found in specific cytoplasmic foci[40].The protein subcellular localization information of sugar metabolic GMS genes are largely consistent with their functions in anther and pollen development,mainly including callose wall and primexine formation,pollen intine development,and pollen maturation and germination(Fig.1A and D).Nevertheless,the detailed molecular mechanism of sugar metabolism and transport during anther and pollen development is largely unclear,which need to be further investigated.

Table 1(continued)

Table 1The sugar metabolism-related GMS genes and their roles in pollen development and male fertility in plants.

Fig.1.Sugar metabolism-related genic male sterility genes and their roles in plant pollen development.(A)The cloned sugar metabolic GMS genes involved in four pollen development processes during various anther development stages in plants.Stages from premeiosis to tricellular pollen and anther dehiscence are defined as Shi et al.[2]and Wan et al.[8].At,Arabidopsis;Os,rice;Zm,maize;Le,tomato;Ph,petunia hybrida;Cs,cucumber;Gh,cotton.(B)Venn diagram of the cloned sugar metabolic GMS genes among Arabidopsis(32 genes),rice(24),maize(1)and other plant species(5).(C)The protein subcellular localizations of the 25 cloned sugar metabolic GMS genes in plants.(D)The essential roles of sugar metabolism and its related GMS genes in callose wall and primexine formation,pollen intine development and pollen maturation and germination.WT,wild type;MT,mutant.

3.Sugar metabolic GMS genes and their essential roles in plant anther and pollen development

Sucrose is generally synthesizedviaphotosynthesis in source organs(e.g.,mature leaves),but our experimental data also shows that chloroplasts in anther endothecium(EN)cells display active photosynthetic ability to enable starch metabolism and carbohydrate supply during the late anther developmental stages in maize[41].Sucrose is symplasmically transported through the terminal phloem to anther outer wall layers(e.g.,EN and middle layer,ML)and apoplasmically transferred across MLviavarious sugar transporters to anther tapetum(Fig.2A).In anther tapetum,carbohydrates are stored as transitory starch,which is later converted into sucrose and its derivatives for anther development and pollen formation,including callose wall and primexine formation,intineformation,starch accumulation and pollen maturation,anther dehiscence,pollen germination and tube growth(Fig.2B and C).

3.1.Callose wall and primexine formation

Callose,a polysaccharide mainly comprised of β-1,3-glucan,plays a vital role in plant microsporogenesis.A temporary callose layer is formed between the primary cell wall and the plasma membrane(PM),and serves to separate microspore mother cells(MMCs)prior to meiosis in anther locules[16].The release of individual microspores requires the coordinated synthesis and degradation of callose[17].Thus,the timing of callose wall formation and degradation is critical for the normal microspore development.

Callose is synthesized from UDP-glucose,which binds directly to the catalytic subunit of callose synthase(CalS)or glucan synthase-like(GSL).InArabidopsisand rice,AtCalS5,OsGSL5[16,17],andAtGSL1andAtGSL5with functional redundance[42],are required for callose wall formation during microsporogenesis,and their single-or double-mutants often display defective callose synthesis and exine patterning,thus leading to male sterility(Fig.2B and 2C).UDP-glucose pyrophosphorylase(UGPase)is an important enzyme in the metabolism of UDP-glucose,a precursor for the synthesis of cell wall carbohydrate components,such as cellulose and callose.Theatugp1/atugp2double mutant ofAtUGP1andAtUGP2,displayed normal PMC development,but without callose deposition around microspores[43].Similarly,OsUGP1is required for rice callose deposition during PMC meiosis,and bridges the apoplastic unloading pathway and pollen development[44].Callose wall appears to participate in the formation of primexine by providing a mold to construct pollen exine during microsporogenesis,and its degradation by β-1,3-glucanases facilitates the release of microspores from the tetrad.Defects ofArabidopsis AtA6and riceOsG1genes,which encode β-1,3-glucanases,cause defective callose degradation and reduced male fertility[18,19](Fig.2B).InArabidopsis,QUARTET 1(AtQRT1),encoding a pectin methylesterase,regulates the separation of tetrads[45].WhileAtQRT3,encoding a polygalacturonase,can degrade cell wall of PMCs during microspore development[46].

Primexine determines pollen wall patterning,while the undulation of microspore PM guides probacula formation along the primexine.InArabidopsis,mutation ofAtNPU,encoding a PM protein,causes complete absence of primexine deposition and PM undulation[25].Arabidopsis AtKNS4and maizeZmMs8genes,encoding a type II arabinogalactan glycosyltransferase,are required for primexine and/or callose degradation during pollen development[20,21].The mutant ofatkns4displays an abnormality of the primexine matrix,and thezmms8mutant meiocytes are covered by excess callose during meiosis stage[47].The mutant ofArabidopsisRUPTURED POLLEN GRAIN 1(AtRPG1),a sugar transporter,exhibits defective primexine formation[48].AtRPG1 shares a redundant function with AtRPG2 in regulating expression ofAtCalS5during pollen development[49](Fig.2C).

3.2.Pollen intine development

As the innermost layer of pollen wall underlying the exine,pollen intine consists of pectin,cellulose,hemicellulose and other components[2].Therefore,sugar metabolism and its related GMS genes are essential for pollen intine development.InArabidopsis,three unique types of components in primary cell-wall cellulose synthase(CESA)complexes,AtCESA1,AtCESA3 and AtCESA6,are necessary for pollen wall development,since their mutations result in aberrant pollen morphology and male sterility[50].Furthermore,some important enzymes/proteins involved in sugar nucleotide metabolism are critical for male gametophyte development.The mutation of UDP-sugar pyrophosphorylase(atusp)results in lack of a pectocellulosic intine and degeneration of cytoplasm in pollen,likely due to defects in the sugar nucleotide supply[51].Reversibly glycosylated polypeptides(RGPs)have been implicated in polysaccharide biosynthesis.Arabidopsis AtRGP1andAtRGP2are crucial for male gametophyte development,and their doublemutant pollen has irregular organization with poorly defined intine,maybe resulting from an inability to convert sugar nucleotides into either wall polysaccharides or other carbohydrates[22](Fig.2C).In rice,OsGT1encoding a Golgi-localized glycosyltransferase[24],COLLAPSED ABNORMAL POLLEN 1(OsCAP1)encoding an arabinokinase-like protein[52],andUDP-ARABINOPYRANOSE MUTASE 3(OsUAM3)encoding an UDP-arabinopyranose mutase[53],are required for the initial development of pollen intine(Fig.2C).Their mutants display the reduced pollen intine formation and male sterility[24,52,53].

3.3.Pollen maturation and starch accumulation

Starch accumulation in microspores is crucial for pollen maturation and viability.Pollen grain is a heterotrophic organ,and pollen starch accumulation is mainly contributed by gametophyte,as well as intine development[2,26].Several sugar metabolic GMS genes involved in pollen starch accumulation have been identified inArabidopsisand rice,and the molecular mechanism of starch accumulation has been explored preliminarily(Fig.2C).For example,down-regulation of riceINVERTASE 4(OsINV4),encoding an anther-specific cell wall invertase,causes a disruption in hexose production and starch formation in pollen grains[54].Silencing ofOsUGP2,a member of rice UGPase family,blocks starch accumulation in pollen,and results in male sterility[55].Deficiencies of riceOsHXK5,OspPGMorOsAGPL4lead to significantly reduced pollen starch synthesis and thus cause male sterility[26,27].These findings suggest the essential roles ofOsINV4,OsHXK5,OspPGMandOsAGPL4in pollen starch biosynthesis and pollen maturation.

Fig.2.The proposed regulation network of sugar metabolism and its related GMS genes during plant anther and pollen development.(A)The simple model of sugar transport across anther wall layers.Sucrose is synthesized in source organs and symplasmically transported from the terminal phloem to anther endothecium(EN)and/or middle layer(ML),and apoplasmically transferred across anther tapetum(TA)via various sugar transporters,such as AtSUC1,OsSUT1,OsMST7/8,AtRPG1/2 and OsSWEET11.In anther tapetum,carbohydrates are stored as transitory starch,which is later converted into sucrose.(B)In anther tapetum,the partial transcriptional and posttranscriptional regulation pathway for callose synthesis and dissolution and their roles in callose wall and primexine formation.(C)The proposed regulation network of sugar metabolism in plant pollen grains.Pollen grains uptake sugars,sucrose and hexose via the apoplast.Sucrose is cleaved into hexose by invertase(e.g.,OsINV4)and phosphorylated by hexokinase(e.g.,OsHXK5)to produce glucose-6-phosphate(G6P).The HXK step is essential for both starch biosynthesis and degradation.During pollen germination,starch degradation occurs very quickly after water imbibition.Glucose(Glu)and maltose(Mal)produced from starch degradation must be phosphorylated to be used for energy generation and building blocks for intine formation,pollen germination and tube growth.The roles of the cloned sugar metabolic GMS genes are shown.G1P,glucose-1-phosphate;ADP-Glu,ADP-glucose;UDP-Glu,UDP-glucose;L-Ara,L-arabinose;L-Ara-1P,L-arabinose-phosphate;UDP-L-Ara,UDP-L-arabinose;TCA cycle,tricarboxylic acid cycle.

Similarly,several sugar transport-related GMS genes involved in pollen maturation and starch accumulation have been cloned and characterized inArabidopsisand rice.For example,nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into ER,and are essential for pollen development inArabidopsis.Their disruption causes pollen lethality without starch accumulation[30].Cold stress suppresses expression of the monosaccharide transporter genesOsMST7andOsMST8,and induces pollen sterility by depletion of starch formation in rice pollen grains[56].RiceOsSWEET11encoding a sucrose transporter,plays a key role in pollen development,and its mutant displays smaller anthers and produces mostly abortive pollen[57].Ara-bidopsis AtGPT1and riceOsGPT1,encoding the plastidial glucose-6-phosphate/phosphate transporters,are required for uptake of cytosolic glucose 6-phosphate into non-green plastids(Fig.2C),which is essential for pollen maturation and starch biosynthesis[28,29].However,little is known about the biochemical mechanism of these genes involved in pollen development.

3.4.Anther dehiscence,pollen germination and tube growth

Anther dehiscence results in mature pollen release and is largely dependent on the secondary wall thickening of anther EN,which is primary due to the deposition of cellulose and lignin during pollen maturation[32].In mature pollen,sugars derived from starch are utilized as an energy source to support pollen germination,and pollen tube formation requires sugars(e.g.,callose)as the major components of cell wall and plugs[58].Thus,starch and sugar metabolism are required for anther dehiscence and pollen germination and tube growth.For example,disruption ofOsSPS1encoding a sucrose phosphate synthase,the rate-limiting enzyme in sucrose synthesis,shows defective pollen germination and male sterility in rice[59],while RNAi-mediated suppression ofOsHXK10expression leads to non-dehiscent anther and reduction of pollen germination in rice[23].The over-accumulation of callose through over-expression ofCALLOSE SYNTHASE 3(AtCalS3)by a sperm cellspecific promoter can induce severe defects in the apical transport of sperm cells into pollen tube and thus partial male sterility[60](Fig.2C).

Several sugar transport-related GMS genes are reported to be required for anther dehiscence and pollen function(Fig.2A and C).For example,partial silencing ofPhNEC1gene affects anther dehiscence inPetunia hybrida,resulting in poor pollen quality and impaired pollen release[61].Disruptions of the homologous genesArabidopsis SUCROSE CARRIER 1(AtSUC1)[62],riceSUCROSE TRANSPORTER 1(OsSUT1)[63]and cucumberCsSUT1[64]display similar phenotypes with impaired pollen germination and male sterility.In tomato,inhibition ofLeSUT2impairs pollen tube growth and prevents pollination[65].However,the detailed mechanism of these genes participate in pollen development and function remains largely unknown.

3.5.The specific roles of sugar metabolism and its related genes at different anther and pollen development stages

Based on the reported results described above,we propose a work model of sugar metabolism and its related GMS genes during different stages of plant anther and pollen development(Fig.2).(1)During premeiotic stages,sucrose is symplasmically transported from the resource organsviathe terminal phloem across the anther outer layers(e.g.,EN and ML)and then is apoplastically transferred in anther tapetum by sucrose transporters(e.g.,AtSUC1,OsSUT1,LeSUT1/2 and CsSUT1).(2)In the developing tapetum,carbohydrates are stored as transitory starch which is later converted to sucrose,and the sucrose may be hydrolyzed to glucose and fructose by OsINV4.Sucrose and hexoses efflux into the loculeviaSWEETs(e.g.,AtRPG1/2 and OsSWEET11)(Fig.2A).(3)At the tetrad stage,callose is synthesized by callose synthases(e.g.,AtCalS5,AtGSL1/5 and OsGSL5)in anther tapetum and timely degraded by callases(e.g.,AtA6 and OsG1)secreted from anther tapetum,which is essential for callose wall and primexine formation of microspores as well as microspore release(Fig.2B).(4)During the later stages of anther/pollen development,microspores take up sucrose and hexosesviavarious sugar transporters,such as sucrose transporters(e.g.,AtSUC1,OsSUT1,LeSUT1/2 and CsSUT1)and monosaccharide transporters(e.g.,OsMST7/8)(Fig.2C).(5)In the developing pollen grains,sucrose is cleaved into hexose by invertases(e.g.,OsINV4)and phosphorylated by hexokinase(e.g.,OsHXK4)to produce glucose-6-phosphate(G6P).This phosphorylated step is essential for both starch biosynthesis and degradation.Then,G6P is transported across the amyloplast membrane by GPTs(e.g.,AtGPT1 and OsGPT1),and the starch synthesis is mediated by the plastidic enzymes OspPGM and OsALP(e.g.,OsAGPL4)(Fig.2C).(6)During pollen intine development,G6P is the major precursor for intine formation,with several sequential reactions mediated by the enzymes/proteins,such as OsUGP2,OsGT1,OsCAP1,AtUSP and AtRGP1/2,and the metabolites are likely transportedviasugar transporters.(7)During anther dehiscence and pollen germination,glucose and maltose produced from starch must be phosphorylatedviahexokinases(e.g.,OsHXK5 and OsHXK10)to be used for energy generation and building blocks(e.g.,callose produced by AtCalS5,AtCalS3,AtGSL1/5 and OsGSL5)for pollen germination and tube growth(Fig.2C).Notably,this proposed network is mainly based on the related reports form rice andArabidopsis,it should be proved by more experimental data from other plants(e.g.,maize)in the future.

4.The coordinated regulation of sugar metabolic GMS genes contributing to plant male fertility

Besides the sugar metabolism and transport-related GMS genes mentioned above,there are also many GMS genes involved in anther and pollen development by the coordinated regulation of sugar metabolism and transport in plant anthers,including transcriptional regulation,posttranscriptional and other regulations.

4.1.Transcriptional regulation

To date,many transcription factors(TFs)have been reported to participate in anther and pollen development by regulating sugar metabolic GMS genes in plants(Fig.1A).During premeiotic to tetrad stages,ArabidopsisAUXIN RESPONSE FACTOR17(AtARF17)is essential for primexine formation and pollen development through regulation ofAtCalS5expression[66](Fig.2B).ArabidopsisCALLOSE DEFECTIVE MICROSPORE1(AtCDM1),a tandem CCCH-type zinc finger protein,is required for male fertility through regulating callose metabolism during microsporogenesis.Its mutation affects expression of callose synthetic genes(AtCalS5andAtGSL5/AtCalS12)and callase-related genes(AtA6andAtMYB80/AtMYB103),as well as three putative β-1,3-glucanase genes(At3g24330,At3g55780andAt3g61810)[40].Meanwhile,AtMYB103 plays an important role in callose dissolution and exine formation by regulating the β-1,3-glucanase geneAtA6inArabidopsis[67](Fig.2B).RiceOsDMD1encodes a nuclear protein with transcriptional activation activity,and is required for callose degradation and primexine formation in microspores by regulating a β-1,3-glucanase geneOsG4and several polysaccharide transport-related genes,such asOsSWEET5,OsSTP14andOsGONST1[39].

As to pollen maturation and starch accumulation,three TFs have been reported to play essential roles in rice anther and pollen development.Two MYB TFs,Carbon Starved Anther(OsCSA)and OsCSA2,are required for rice anther and pollen development under short-day(SD)and long-day(LD)conditions,respectively.OsCSA and OsCSA2 contribute to long-distance sugar partitioning for pollen maturationviapositively modulating expression of several sugar metabolism-and transport-related genes[36,37].Knockdown ofOsBZR1(encoding a BR-signaling TF)expression causes defective pollen maturation with less starch accumulation.Logically,OsBZR1 directly promotesCSAexpression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen development[34].

ArabidopsisNAC TFs AtNST1 and AtNST2 regulate secondary wall thickenings in anther EN and are required for anther dehiscence[68].ArabidopsisAtMYB26 protein is localized specifically to anther EN nuclei and directly regulatesAtNST1 andAtNST2expression,which in turn controls the process of anther EN secondary thickening and subsequent anther dehiscence[32,33](Fig.1A).

4.2.Posttranscriptional and other regulations

Increasing evidence shows that many posttranscriptional and other regulators besides transcription factors are also involved in pollen development and male sterility[69,70]by regulating sugar metabolic genes.For example,miR160 directly regulates expression ofAtARF17,and thus indirectly regulates expression ofAtCalS5andAtRPG1,to control callose biosynthesis and primexine deposition.AtTTP(TRISTERAPROLINE)is involved in miR160 maturation and thus form a regulatory pathway with the module of AtTTPmiR160-AtARF17-AtCalS5[66,71](Fig.2B).

Fig.3.Predicting 71 maize sugar metabolic genes orthologous to the cloned GMS genes in other plants based on bioinformatics and RNA-seq analyses during maize anther development.Hierarchical clustering of 63 differentially expressed(A)and eight low or differentially expressed(B)sugar metabolic genes based on anther RNA-seq data of maize inbred lines W23,B73,Oh43,and Zheng58 in our laboratory.Notes:Anther-specific expression information is retrieved from B73 RNA-seq data in MaizeGDB website(www.maizegdb.org).

Furthermore,other regulators also play important roles in controlling sugar metabolism and its related genes required for anther and pollen development.ArabidopsisAtCDKG1,a member of the cyclin-dependent protein kinase family,regulatesAtCalS5premRNA splicing and thus affects callose wall and primexine formation[31].In rice,OsLecRK5,a plasma membrane-localized lectin receptor-like kinase,phosphorylates OsUGP1 to regulate callose biosynthesis during pollen development[72].In a similar study,OsAP1/OsLecRK5 directly interacts with and phosphorylates OsUGP2 to increase its enzymatic activity for pollen starch accumulation[73](Fig.2C).Down-regulated expression ofOsSPX1,a rice SPX domain gene,significantly affects expression of genes involved in carbohydrate metabolism and sugar transport,thus disrupting rice normal anther and pollen development and leading to semi-male sterility[38].In cotton,a pollen-specific SKS-like protein(GhPSP231),suppresses GhWRKY15-mediated transcriptional repression ofGhCalS4/8,thus activating callose biosynthesis to promote pollen maturation[74].These findings indicate that the regulation of sugar metabolism during plant anther/pollen development is a relatively complex process,which need to be explored deeply in the future.

5.Prediction of putative sugar metabolism-related GMS genes in maize

Compared withArabidopsisand rice,only one sugar metabolic GMS gene(ZmMs8)has been identified in maize,which greatly limits our understanding on the roles of sugar metabolism in maize male fertility.To discover more sugar metabolic GMS genes in maize,the following two strategies are used to predict maize putative sugar metabolism-related GMS genes based on bioinformatics and maize anther RNA-seq analyses.

5.1.Maize orthologues of the cloned sugar metabolic GMS genes in other plants

Through bioinformatic analysis of the orthologous relationship across different plant species by using the BLAST in GRAMENE website(http://www.gramene.org/),we identify 71 independent maize orthologs of sugar metabolism-related GMS genes inArabidopsis,rice and other plants including maizeZmMs8gene(Tables 1 and S1).Based on the temporal-specific expression patterns from four sets of maize anther RNA-seq data covering at least 10 developmental stages in maize inbred lines W23[75],B73[69],Oh43[76]and Zheng58[77]in our laboratory(Table S2),the 71 maize orthologs can be classified into four clusters(Fig.3).

The cluster I includes 16 differentially expressed genes(DEGs)with the expression peaks at early development stages(S2-S5)of maize anthers.These genes are orthologous to seven cloned GMS genes,including three sugar biosynthetic genes(AtCalS3,AtGSL1andAtA6)and four sugar regulatory genes(OsDMD1,OsSPX1,OsBZR1andAtARF17).Nevertheless,all of them are not antherspecific expression in maize,based on the B73 RNA-seq data including 68 distinct samples over 23 tissues in MaizeGDB website(www.maizegdb.org)(Fig.3A–I).

The cluster II contains 19 DEGs with the expression peaks at middle development stages(S6-S9)of maize anthers.These genes are orthologous to 12 cloned GMS genes,including six sugar regulatory genes(OsCSA,AtCDM1,AtARF17,AtMYB103,OsCSA2andOsAP1),five sugar biosynthetic genes(OsUGP1,OsGT1,AtKNS4,OsHXK10andAtQRT1)and one sugar transporter genes(AtGPT1).Among them,four maize orthologous genes(AtCDM1,OsHXK10,AtMYB103andOsAP1)are anther-specific expressed(Fig.3A-II),implying that they are closely associated with male fertility and pollen development in maize.

The cluster III includes 28 DEGs with the expression peaks at late development stages(S10–S13)of maize anthers.These genes are orthologous to 13 cloned sugar biosynthetic GMS genes,five sugar transporter genes,and three sugar regulatory genes inArabidopsisand rice.Interestingly,11 of them show anther-specific expression patterns(Fig.3A-III),indicating that these genes may be important GMS candidates in maize,which need further confirmed by using reverse genetic approaches such as the CRISPR/Cas9 technology.

The cluster IV contains eight genes with low expression levels during the whole anther stages based on W23 RNA-seq data.However,six of them are also DEGs in RNA-seq data from B73,Oh43 and/or Zheng 58 lines,including maize orthologues ofAtUTr1,AtCalS3,OsSUT1andAtARF17.All of them are not anther-specific expression in maize(Fig.3B).

Notably,the anther stage expression patterns of these predicted genes will give some clues to explore their roles in anther and pollen development.All the 15 anther-specific DEGs display their peak expressions at the middle or late anther developmental stages,indicating that these putative sugar metabolic GMS genes may play more important roles in primexine formation,intine development,pollen starch accumulation,pollen maturation and germination.Consistent with this finding,Datta et al.[78]reported that a large number of sugar biosynthetic or transporter genes show altered expression patterns between male-sterile and-fertile maize anthers,and closely associated with starch biosynthesis during pollen maturation.

Most recently,one of the 15 anther-specific expressed genes,Zm00001d025664(ZmMYB84)has been confirmed to be required for maize male fertility by using the CRISPR/Cas9 mutagenesis in our laboratory[79].Therefore,based on the important information obtained here,it is feasible to quickly identify more sugar metabolic GMS genes in maize by using the CRISPR/Cas9 mutagenesis,and this work is ongoing in our laboratory.

Furthermore,to determine the precise chromosomal locations of these predicted putative GMS genes in maize,a physical map is constructed by using the updated V4 version of maize reference genome(Fig.4).This physical map will be helpful for gene mapping and map-based cloning of maize novel sugar metabolic GMS genes in the future.

5.2.De novo prediction of maize sugar metabolic genes based on bioinformatics analysis

To discover more sugar metabolism-related GMS genes in maize,bioinformatics analysis is carried out based on maize W23 anther RNA-seq data covering ten sequential anther stages[75].Among the 639 predicted sugar metabolic genes,593 genes showed stage-differential expression patterns in W23 anther transcriptomes(Table S3).These genes mainly participate in eight pathways,including starch and sucrose metabolism,sugar transport,glycolysis and gluconeogenesis,photosynthesis,antenna proteins,carbon fixation,citrate cycle,and pentose phosphate.Notably,there are 42 novel genes with anther-specific or high expressions in maize anthers(Fig.5),including 18 starch and sucrose metabolic genes,11 sugar transport-related genes,five glycolysis-related genes,three citrate cycle-related genes,and five carbon fixation-related genes.The 42 novel genes are important sugar metabolic GMS candidates in maize.More importantly,these potential maize GMS genes will deepen our understanding on sugar metabolic pathways in controlling pollen development and male fertility since their gene functions are unclear in plant male reproduction.

6.Potential applications of sugar metabolic GMS genes in crop hybrid breeding and seed production

Fig.4.The precise chromosomal locations of the 71 predicted sugar metabolic genes in maize genome.Notes:The red font indicates the two cloned GMS genes(ZmMs8 and ZmMYB84)in maize,and the black font indicates the 69 putative sugar metabolic GMS genes in maize.

Cloning and characterization of sugar metabolic GMS genes not only greatly contribute to our understanding on the molecular mechanism of anther and pollen development in plants,but also provide an important basis to develop novel GMS lines.Several attempts have been made to utilize sugar metabolic pathway and its related GMS genes in constructing genetic engineering malesterility systems in crops.

6.1.Environment-sensitive male-sterility systems

Rice OsCSA and OsCSA2 regulates sugar partitioning from photosynthetic tissues(sources)to anthers(sinks)to promote pollen maturation.The mutants ofOsCSAandOsCSA2display male sterility under SD and LD conditions,while they can recover male fertility under LD and SD conditions,respectively[35,37].Cosuppression by transforming the antisense construct of riceOsUGP1(Ugp1-AS),which is essential for pollen callose deposition,results in a new type of thermosensitive genic male sterility(TGMS)phenotype[44].Therefore,mutants ofOsCSAandOsCSA2can be used to create new photoperiod-sensitive genic male sterility(PGMS)lines,OsUGP1-cosuppressing plants have a potential application in developing TGMS lines,and both two types of male sterility are useful for two-line hybrid rice seed production.Notably,based on the reverse genetic strategies,e.g.,CRISPR/Cas9 and RNA inference technologies,it is reasonable to create more PGMS or TGMS lines by knockout or knockdown of the orthologous genes ofOsCSA,OsCSA2andOsUGP1in other crops,such as maize,sorghum,wheat and barley,ultimately improving efficiencies of heterosis utilization and hybrid seed production in these crops.

Fig.5.De novo prediction of 42 maize putative sugar metabolism-related GMS genes based on bioinformatics analysis of anther RNA-seq data from four maize lines.Note:Hierarchical clustering of 42 maize putative sugar metabolism-related GMS genes based on anther RNA-seq data of four maize inbred lines(W23,B73,Oh43,and Zheng58).The 42 genes belong to five pathways,including starch metabolism,sugar transport,glycolysis,carbon fixation and citrate cycle(Table S3).All these genes are anther-specific or-highly expressed based on the information retrieved from B73 RNA-seq data in MaizeGDB website(www.maizegdb.org).

6.2.Genetic stable male-sterility systems

Anther-specific antisense repression ofNin88,encoding an extracellular invertase isoenzyme,under control of its native promoter in tobacco results in complete male sterility[14].Thus,disturbance of the carbohydrate supply in anthers provides a highly efficient biotechnological method to engineer male sterility for practical applications in crops.

Similarly,the pollen-specific expression ofZmAAgene(encoding an α-amylase)is successfully used for the maize Seed Production Technology(SPT)and Multi-Control Sterility(MCS)systems[4,15].Pollen grains with the transgenes exhibit starch depletion resulting from expression ofZmAAgene in pollen and are unable to germinate,which is the most important point for the nontransgenic seed production from the SPT and MCS systems.More recently,a one-step transformation strategy for creating the GMS line and its maintainer has been successfully developed in maize by using the CRISPR/Cas9 mutagenesis and SPT/MCS-like construct ofZmMs26gene[80],which can be easily extended to the predicted sugar metabolic GMS genes and used to create novel GMS lines and their maintainer lines in other crops.Collectively,these biotechnology-based male-sterility systems have important application values for hybrid breeding and seed production in maize and other crops.

7.Conclusions and perspectives

Sugar metabolism plays pivotal roles in anther and pollen development,and thus affects male fertility and grain yield in crops.Here,we summarize the recent progresses of sugar metabolic GMS genes and their roles in controlling plant anther/pollen development.Sugars are mainly generated from the resource organs and transported into the sink organs functioning as both the reserved energy and essential cell compounds to promote callose wall and primexine formation,intine development,pollen maturation and starch accumulation,and pollen germination and tube growth(Fig.1A).Thus,any disruption of sugar metabolic genes required for anther and pollen development often leads to GMS in plants.

To date,most of the cloned sugar metabolic GMS genes are identified inArabidopsisand rice,while relatively fewer genes are reported in maize(only one)and other plants(five genes)(Fig.1B),which greatly limits our understanding on the molecular mechanism of sugar metabolism involved in anther and pollen development in maize and other plants(Fig.2).Here,we predict 71 maize orthologues of sugar metabolic GMS genes in other plants,analyze their anther stage differential expression patterns,and determine their precise chromosome localizations in maize genome(Figs.3 and 4).Moreover,42 putative novel sugar metabolic GMS genes are predicted in maize by using four sets of anther RNA-seq data(Fig.5;Table S3).After completing functional confirmation of these predicted putative sugar metabolic GMS genes by using the CRISPR/Cas9 mutagenesis,more sugar metabolic GMS genes will greatly facilitate our deep understanding on the molecular mechanism of plant male sterility and promote their utilizations for hybrid breeding and seed production in crops.Furthermore,starch biosynthesis during pollen maturation is associated with the altered expression patterns of many sugarmetabolic genes in an S-type cytoplasmic male-sterility(S-CMS)maize line when compared with its corresponding male-fertility line[78,81],indicating that sugar metabolism is related to pollen development in both GMS and CMS plants,although the detailed mechanism remains to be explored.Notably,genetic engineering improvement of sugar metabolic pathway can increase grain yield and starch quality in maize[82,83].Therefore,investigation of both functional mechanism and practical application values of sugar metabolism-related genes to enhance maize grain yield should be given priority considerations in future work.

CRediT authorship contribution statement

Shuangshuang Liu:Resources,Investigation,Validation,Visualization.Ziwen Li:Methodology,Software,Data curation,Formal analysis.Suowei Wu:Conceptualization,Investigation,Resources,Funding acquisition,Writing-original draft,Writing-review &editing.Xiangyuan Wan:Conceptualization,Funding acquisition,Supervision,Writing-review & editing.All authors have read and approved to the final version of the manuscript.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This research was supported by the National Key Research and Development Program of China (2018YFD0100806,2017YFD0101201 and 2017YFD0102001),the National Natural Science Foundation of China(31871702,31971958 and 31771875),the Fundamental Research Funds for the Central Universities of China(06500136),and the Beijing Science & Technology Plan Program(Z191100004019005).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.08.003.