Gengmi Li ,Jiuyou Tng ,Jikui Zheng *,Chengci Chu ,c,*

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 Key Laboratory of Southwest Rice Biology and Genetic Breeding,Ministry of Agriculture/Luzhou Branch of National Rice Improvement Center,Rice and Sorghum Research Institute,Sichuan Academy of Agricultural Sciences,Deyang 618000,Sichuan,China

c College of Advanced Agricultural Sciences,University of Chinese Academy of Sciences,Beijing 100049,China

Keywords:Rice Yield improvement Nitrogen use efficiency Photosynthetic efficiency Molecular breeding

ABSTRACT Rice grain yield is determined by three major‘‘visible”morphological traits:grain weight,grain number per panicle,and effective tiller number,which are affected by a series of‘‘invisible”physiological factors including nutrient use efficiency and photosynthetic efficiency.In the past few decades,substantial progress has been made on elucidating the molecular mechanisms underlying grain yield formation,laying a solid foundation for improving rice yield by molecular breeding.This review outlines our current understanding of the three morphological yield-determining components and summarizes major progress in decoding physiological traits such as nutrient use efficiency and photosynthetic efficiency.It also discusses the integration of current knowledge about yield formation and crop improvement strategies including genome editing with conventional and molecular breeding.

1.Introduction

Rice is a staple food for more than half the world’s population.However,with decreases in arable land,increasing crop yield to feed the increasing population has become more challenging.It is projected[1]that rice production will need to be doubled by 2050 relative to 2010,requiring an annual yield increase of more than 2.4%to meet the growing demand.

Productivity of cereal crops is influenced by three major agronomic traits:grain weight,grain number per panicle,and effective tiller number per plant.Grain number per panicle is determined by the number of primary and secondary branches,whereas grain weight is determined by grain size(length,width,and thickness)and also the rate of grain filling[2].Effective tiller number per plant is determined by the number of tiller buds and their ability to grow[3].Although these three components do not exert a simple additive effect on rice yield formation,they can be observed directly in conventional breeding.However,physiological traits such as nutrient use efficiency and photosynthetic efficiency,are‘‘invisible”and cannot be simply measured,making it difficult to formulate a reliable and systematic breeding method.In recent years,the progress of molecular biology has not only deepened our understanding of the genetic mechanism of complex rice agronomic traits,but also promoted the transformation of rice breeding from conventional breeding to more efficient molecular breeding.This review summarizes recent progress in the molecular dissection of three yield-determining traits:grain weight,grain number per panicle,and effective tiller number per plant.It also describes major research progress in understanding nitrogen use efficiency and photosynthetic efficiency of plants,two physiological traits that strongly influence crop yield.

2.Control of grain weight

2.1.Phytohormone signaling and homeostasis

Brassinosteroids(BRs)and auxin are two phytohormones that regulate growth,development,and stress responses in plants.They regulate seed growth by shaping maternal tissues and affect endosperm development,influencing grain size.

2.1.1.BRs

The genes encoding the BR biosynthetic enzymes,such as BRD1[4,5],BRD2[6],D2[7],and D11[8],usually have short grains.Therole of these genes in controlling grain length is attributed to their effect on cell size in the lemma and glume via promotion of cell expansion in spikelet hulls[9-11].

Several quantitative trait loci(QTL)controlling seed size have been identified as being associated with the BR signaling pathway,including GRAIN SIZE5(GS5)[12],GRAIN WIDTH5(GW5)/QTL for SEED SIZE5(qSW5)[13,14],GRAIN LENGTH 3.1(GL3.1)/qGL3/qGL3-1/PROTEIN PHOSPHATASE KELCH FAMILY SERINE(OsPPKL1)[15-17],and GRAIN SIZE2(GS2)/GRAIN LENGTH(GL2)/PANICLE TRAITS 2(PT2)/GRAIN LENGTH AND WIDTH2(GLW2)/LARGE GRAIN SIZE 1(LGS1)(Table 1)[18-20].GS5,encoding a putative serine carboxypeptidase,regulates grain width by promoting cell division.GS5 interacts with OsBAK1-7,which promotes BR signaling and results in larger grains[21].GW5 encodes a calmodulin-binding protein[14],which interacts with SHAGGY-LIKE KINASE(GSK2)and regulates its kinase activity.GSK2 functions as a core negative regulator of BR signaling,suggesting that GW5 regulates seed size by affecting BR signaling[22].GL3.1 encodes a protein phosphatase with two Kelch-like repeat domains[15].It has been shown[23]that GL3.1 regulates grain length by working with OsGSK3 to modulate BR signaling.A rare mutation in the AVLDT conserved motif of the second Kelch domain of OsPPKL1 causes extra-large grain and consequently higher rice yield[17].GS2/GL2,encoding the Growth-Regulating Factor 4(OsGRF4)is regulated by OsmiR396 and is also inhibited by GSK2,promoting cell expansion to influence grain size [18-20,24-26].Overexpression of GS2/GL2 increases BR response,suggesting that GS2/GL2 participates in the BR pathway(Fig.1)[27].

Mutant-based and homology-based cloning has revealed multiple genes that participate in BR signaling(Table 1).Mutations in these genes produce short grains,a phenotype similar to those of typical BR-deficient mutants.GSK2,encoding GSK3/SHAGGY-like kinase,participates in multiple signaling pathways to control grain size[22].GSK2 can directly inhibit OsGRF4 activity to regulate grain size.GS9 encodes a transcriptional activator that can be repressed by OVATE FAMILY PROTEIN(OsOFP8).GSK2 phosphorylates OsOFP8 to attenuate the repression of GS9,producing slender grains[28,29].A recent study showed that GSK2 can phosphorylate MEI2-like protein 4(OML4),encoded by the LARGE1 gene,to control grain size and grain number per panicle(Fig.1)[30].

BR signaling genes,such as BRASSINOSTEROID INSENSITIVE1(OsBRI1)[31],BRI1-ASSOCIATED RECEPTOR KINASE(OsBAK1)[32],and DLT/GS6[33,34],regulate not only grain size but grain number.OsBRI1 encodes LRR-RLK which acts as a BR receptor(Table 1).OsBAK1 encodes an OsBRI1-associated kinase which acts as a coreceptor.OsBRI1 and OsBAK1 work synergistically to regulate the BR signaling pathway.DWARF AND LOW-TILLERING(DLT)/GS6,which encodes a member of the plant-specific GRAS family,has been demonstrated as a BR positive regulator in the BR signaling pathway and also can be phosphorylated by GSK2.DLT directly interacts with SMOS1 to regulate BR signaling and associated functions[33,35].Overexpression of a microRNA(OsmiR397)positively regulates the number and size of rice grains,leading to grain yield increases of up to 25%[36].A very recent study showed that OsTCP19,encoding a TEOSINTE BRANCHED 1,CYCLOIDEA AND PROLIFERATING CELL FACTOR(TCP)family transcription factor,targets DLT to modulate tillering response to nitrogen[37].OsmiR397 targets OsLAC,a gene that encodes a laccase-like protein that participates in BR response.Overexpression of OsLAC produces small grains and also reduces grain number[36].BR was reported to participate in the modulation of grain size and number by regulating upstream processes in the guanine nucleotide-binding protein(G-protein)signaling pathway(Fig.1)[38].

2.1.2.Auxin

THOUSAND GRAIN WEIGHT(TGW6),a major QTL for thousandgrain weight,encodes an indole-3-acetic acid(IAA)-glucose hydrolase to produce free IAA[39].A 1-bp frameshift deletion in TGW6results in increased cell number to produce longer grain.BIG GRAIN1(BG1),encoding a membrane-localized protein involved in auxin transport,controls grain size by increasing cell proliferation and cell expansion.Overexpression of BG1 markedly increases grain size,suggesting a role of auxin in the regulation of grain size[40].TGW3,a major QTL encoding SHAGGY-like kinase 41(OsSK41)/OsGSK5,negatively regulates grain size by phosphorylating OsARF4,whereas OsARF4 negatively regulates grain length via the auxin signaling pathway(Table 1 and Fig.1)[41-43].

Table 1The major components involved in controlling rice sink capacity.

Fig.1.A simplified representation of pathways controlling rice grain yield.Rice grain yield is determined by grain weight,grain number per panicle,and effective tiller number,all of which are regulated by complex networks.Absorption and utilization of nutrients as well as photosynthetic effciiency are major physiological factors determining rice grain yield.MAPK,mitogen-activated protein kinase;N,nitrogen;P,phosphorus;K,potassium.

2.2.Ubiquitin-proteasome pathway

Ubiqutination is a post-translational mo dification of proteins that is involved in diverse life processes.To date,at least two deubiquitinating enzymes and two E3 ubiquitin ligases have been found to be involved in the regulation of grain size(Table 1).OsOTUB1/WIDE AND THICK GRAIN(WTG1),encoding a deubiquitinating enzyme,is a major QTL controlling grain size.Overexpression of OsOTUB1/WTG1 leads to long and light grain and lower grain number per panicle,while mutation of OsOTUB1/WTG1 results in wide and heavy grains and greater grain number per panicle[44,45].OsOTUB1/WTG1 controls grain size mainly by affecting cell expansion in the spikelet hull and controls grain number per panicle mainly by affecting meristem activity and panicle branches[44,45].Moreover,LARGE GRAIN1(LG1)encodes ubiquitin-specific protease 15(OsUBP15),which has de-ubiquitination activity.The gene was first identified in a dominant large grain mutant lg1-D that displays greater width and weight of grain[46].Notably,LG1 may be related to GW2,a major grain size QTL that encodes an E3 ubiquitin ligase.The gene regulates both width and weight of rice grains[47].Disruption of GW2 promoted cell proliferation,resulting in wider spikelet hulls,and also accelerated grain filling rate,resulting in increased grain weight and yield[47].Additionally,TAIHU DWARF1(TUD1)which encodes another U-box E3 ubiquitin ligase which directly interacts with D1/RGA1 to regulate plant height and grain size[48].TUD1 and D1 act together in regulating the BR signaling pathway to produce short grains by reducing cell division(Fig.1).

2.3.G-Protein signaling pathway

GS3,encoding a non-canonical Gγ,was the first major QTL shown to regulate grain size(Table 1)[49,50].Four different GS3 alleles have been characterized,one each in Zhenshan 97,Nipponbare,Minghui 63,and Chuan 7,respectively[50,51].A null allele(C-A)of GS3 from Minghui 63 has been widely used in breeding,which leads to longer grain owing to the fewer spikelet hull cell.Overall,GS3 negatively regulates grain size[50-52].

DENSE AND ERECT PANICLE1(DEP1),another major QTL encoding a non-canonical Gγ,exerts multiple effects on panicle morphology,grain size,grain number per panicle,and nitrogen-use efficiency(Table 1)[49,53,54].Overexpression of DEP1 results in large grains and knockout of DEP1 results in small grains,suggesting that DEP1 expression is positively correlated with grain size[55].

GS3 binds competitively to Gβ(RGB1),producing the opposite effect to DEP1 and another non-canonical Gγ(GGC2),to regulate grain size[55].In addition to GS3,DEP1 and GGC2,RGG1 and RGG2 are two additional non-canonical Gγs that negatively regulate grain length[56-59].The non-canonical Gγs harbor an N-terminal with aγ-like domain and a C-terminal with variable cysteine-rich domains.The functional specificity(negative or positive regulation)of the Gγs is dependent on the cysteine-rich C-terminal tail length.Given their long C-terminal tails,DEP1 and GGC2 positively regulate grain length,in contrast to short tailed GS3,which negatively regulates grain length(Fig.1)[55].

qLGY3 is a QTL controlling grain length that encodes a MADS-domain transcription factor,OsMADS1(Table 1)[57].By directly interacting with GS3 and DEP1 in the nucleus,OsMADS1 affects the function of G-proteinβγdimers[55,57].GS3 and DEP1 also directly interact with the conserved keratin-like domain of MADS transcription factors,which function as cofactors to increaseOsMADS1 transcriptional activity to restrict grain growth[57].OsLG3b,an alternatively spliced transcript of OsMADS1,has undergone artificial selection leading to long grains[60],whereas OsMADS1lgy3leads to long and slender grains[57].These findings suggest that OsMADS1 is negatively regulated grain size.However,further research is needed to identify the respective positive and negative regulatory effects of DEP1 and GS3 on grain size via promotion of OsMADS1 transcription(Fig.1).

OsRac1 gene encodes a GTPase,a member of the Rho family.It positively regulates grain size and yield by promoting cell proliferation in the spikelet hull and enhancing cell metabolic processes[61].OsRac1 can interact with OsMAPK6 to control grain size by altering the level of OsMAPK6.

2.4.Mitogen-activated protein kinase(MAPK)signaling pathway

Mutant-based cloning revealed OsMKKK10[62],OsMKK4[63],and OsMAPK6[64]as the key components of a MAPK signaling pathway involved in the regulation of rice grain size(Table 1).They negatively regulate grain size by modulation of cell proliferation in the spikelet hulls.Interestingly,even though overexpression of OsMKKK1,OsMKK4 or OsMAPK6 increases grain size,but reduces grain number[62].

OsER1 encodes ERECTA1,which negatively regulates grain number per panicle and performs a receptor-like protein kinase function upstream of the OsMKKK10-OsMKK4-OsMPK6 signaling pathway[65].The oser1 mutant has small grain but higher grain number per panicle,suggesting that ERECTA1 performs similar functions to OsMKKK10,OsMKK4,and OsMPK6.OsWRKY53,a downstream transcription factor for the MAPK signaling pathway,acts as a regulator of BR signaling[66],and also a positive regulator of grain size.It can interact with and be phosphorylated by OsMAPK6(Fig.1)[67].

GRAIN SIZE AND NUMBER1(GSN1),encoding the MAPK phosphatase OsMKP1,was isolated from a rice mutant with large but few grains per panicle.It was found that GSN1 mediates these changes by disrupting cell proliferation and differentiation[68].OsMKP1 directly interacts with and inhibits the dephosphorylation of OsMPK6.The phenotype of GSN1-overexpression plants is similar to that of the osmapk6 mutant,suggesting that OsMKP1 negatively regulates the OsMKKK10-OsMKK4-OsMPK6 signaling cascade(Fig.1).

2.5.Transcription factors

Transcription factors(TFs)are proteins that modulate multiple essential functions[69].Many TF families,including WRKY,bHLH,SPL,and AP2,participate in the modulation of grain size(Table 1).

2.5.1.SQUAMOSA promoter binding protein-like(SPL)family

OsSPL13,a positive regulator for grain size via promotion of cell expansion in the spikelet hull,is encoded by a major QTL,GRAIN LENGTH AND WIDTH7(GLW7)[70].The tandem repeat sequence of CCATTC in the 5′UTR of tropical japonica rice elevates the expression of GLW7,leading to large grains,while two copies of the CCATTC sequence in the 5′UTR reduce the expression of GLW7,resulting in small grains.OsSPL13 can bind the promoter of SMALL AND ROUND SEED5(SRS5)to promote its transcription.The mutant srs5 produces short grains and overexpression of SRS5 produces long grains(Fig.1)[71].

IDEAL PLANT ARCHITECTURE1(IPA1)/OsSPL14 interacts with OsOTUB1 and also the E2 ubiquitin-conjugating protein OsUBC13[45,72],with the physical interaction of OsSPL14 and OsOTUB1 limiting the K63-linked ubiquitination(K63Ub)of OsSPL14 to control grain size by regulating cell expansion[45].The expression of IPA1 is also regulated by the miRNA OsmiR156[73].Elevated expression of IPA1 results in increased grain weight and grain number per panicle.Furthermore,OsSPL14 can also regulate grain size,grain weight,and number per panicle by binding the promoter of DEP1 and promoting its transcription in the panicle(Fig.1)[74].IPA1 interacting protein(IPI1)encodes a RING-finger E3 ubiquitin ligase that degrades IPA1 in panicles to produce more secondary branches,thereby increasing grain number per panicle[75].

OsSPL16 positively regulates grain size by promoting cell division and grain filling and is encoded by a grain-width QTL,GW8[76].A 10-bp deletion in the GW8 promoter of Basmati rice represses its expression,resulting in longer and higher-quality grains.OsSPL16 can bind directly to the promoter of GW7/GL7,a major QTL influencing grain length by regulating longitudinal cell elongation,by repressing its expression(Fig.1)[77,78].Copy number variation of GL7 increases grain length and improves grain appearance[78].Manipulation of the OsSPL16-GW7 module represents a new strategy for simultaneously improving rice yield and grain quality[77].

2.5.2.Basic helix-loop-helix(bHLH)family

Awn-1(An-1),a major QTL,encodes a typical bHLH TF to regulate awn development and grain length[79].The wild allele An-1 in Guangluai 4 produces long awns and longer grains,but significantly reduces grain number per panicle.In contrast,the An-1 RNAi lines produce short awns and short grains but increased grain number.These observations suggest that An-1 is a positive regulator of awn and grain length but a negative regulator of grain number per panicle(Fig.1).The An-1 locus is a major target of artificial selection for the loss of awns.

POSITIVE REGULATOR OF GRAIN LENGTH 1(PGL1)and ANTAGONIST OF PGL1(APG1),encoding an antagonistic pair of bHLH proteins,are involved in the regulation of rice grain length by controlling cell elongation in the lemma and palea[80].APG1 is a negative regulator of grain length whose function is inhibited by PGL1.Elevated expression of PGL1 or reduced expression of APG1 results in increased BL sensitivity,suggesting that PGL1 and APG1 control grain length by mediating BR signaling.

2.5.3.APETALA2-type(AP2)transcription factors

SMALL ORGAN SIZE1(SMOS1),encoding an auxin-dependent transcription factor with an imperfect AP2 domain,positively regulates grain size[81].SMOS1 can directly regulate the expression of PHOSPHATE-INDUCED PROTEIN1(OsPHI-1),which is involved in cell expansion.It has also been shown that SMOS1 interacts with DLT to participate in the BR signaling pathway to regulate grain size and grain number[35].

SUPPRESSION OF SHATTERING1(SSH1),encoding a plant-specific APETALA2-like transcription factor,negatively regulates grain size by positively regulating the expression of qSH1 and SH5,resulting in the elongation of glume longitudinal cells[82].

2.5.4.Other transcription factors

In addition to the above types of transcription factors,there are many other types involved in regulation of grain size(Fig.1).For example,GW6a encodes a GNAT-like protein that possesses intrinsic histone acetyltransferase activity(OsglHAT1).Elevated GW6a expression increased grain weight and yield by increasing cell number and accelerating grain filling[83].

GS9 encodes a transcriptional activator containing a conserved domain with unknown function[28],and the gs9 null mutant produces slender grains by altering cell division.

GL6/SHORT GRAIN6(SG6),encoding a plant-specifci AT-rich zinc-binding TF,positively regulates rice grain length but negatively regulates spikelet number[84].A recent study showed that GL6/SG6 interacts with the core cell cycle machinery DP proteinand many regulators of cell division,promoting the expression of many DNA replication and cell cycle-related genes,indicating that GL6/SG6 controls grain size by promoting cell proliferation in young panicles and grains[85].

GL4,a Myb-like gene cloned from African cultivated rice,positively modulates grain length by regulating longitudinal cell elongation of outer and inner glumes[86].

2.6.Secreted peptides

GRAIN LENGTH AND AWN DEVELOPMENT1(GAD1),encoding a secreted signaling peptide in the EPIDERMAL PATTERNING FACTOR-LIKE(EPFL)family,regulates awn length,grain length,and grain number[87].gad1 mutant leads to short grain and awn length by reducing cell number and increases grain number by reducing the expression of DST and Gn1a to increase the level of cytokinin[88],a phenotype similar to the loss-of-function phenotype of the An-1 mutant.

2.7.Unknown functional protein

DEP2,encoding a novel plant-specific protein with uncharacterized domains,is highly expressed in young panicles.DEP2 affects mainly the elongation of the rachis and primary and secondary branches[89].Further detailed analysis suggested that DEP2 is essential for panicle growth and elongation,in which polar auxin transport may be involved[89].

3.Control of grain number

3.1.Phytohormones

3.1.1.Cytokinin(CK)

Gn1a is a major QTL controlling grain number in rice.It encodes a cytokinin oxidase/dehydrogenase(OsCKX2)that degrades the phytohormone cytokinin to control grain number(Table 1)[90].Reduced expression of Gn1a increases the activity of panicle meristem and panicle branching and thus increases grain number without affecting grain size.LARGE PANICLE(LP),a Kelch repeatcontaining F-box protein,and DROUGHT AND SALT TOLERANCE(DST),a transcription factor for zinc finger,can regulate Gn1a expression to regulate grain size [91,92].VIL2 encodes a chromatin-interacting factor and can decrease the expression of Gn1a by interacting directly with its promoter region.Overexpression of VIL2 increases grain number by increasing panicle branching[93].Rice cultivars with dep1 have higher grain number,suggesting that DEP1 also regulates panicle branching[53].IPA1 can regulate grain number by directly binding the DEP1 promoter to regulate its expression[74].Two allelic mutants of LP achieve greatly increased panicle size by producing more inflorescence branches as well as more grains.Furthermore,Gn1a was found to be downregulated in the lp mutants,suggesting that LP modulates CK level to regulate grain number[91].DST negatively regulates the expression of Gn1a to control grain size without affecting panicle size(Fig.1)[92].Thus,Gn1a and DST may be ideal targets for yield-improving breeding.

An-2,a QTL,catalyzes the final step of cytokinin synthesis(Table 1)[94,95].Loss of function of An-2 reduces cytokinin content,reducing awn length and increasing both tiller and grain number by limiting cell division.

Interestingly,PURINE PERMEASE7(OsPUP7)and OsPUP4/BIG GRAIN3(BG3),two PUP-type cytokinin transporters,work together to form a cytokinin cell-to-cell transport system(Table 1)[96,97].Elevated expression of OsPUP4/BG3 results in increased grain size but decreased grain number[97].A recent study showed that ARGONAUTE2(AGO2)could activate the expression of BG3 by altering its histone methylation level to control grain size and grain number[98].

A putative zeatin O-glucosyltransferase,encoded by cZOG1,is involved in cytokinin storage.Knockdown of cZOG1 results in increased grain number per panicle induced by greater panicle branching[99].Short Panicle 3(SP3),encoding a DNA-binding transcriptional activator with one finger(Dof),regulates the expression of genes associated with cytokinin catabolism and biosynthesis[100].The decreased cytokinin content in the sp3 mutant reduces grain number by producing fewer panicle branches(Table 1).

STRESS_TOLERANCE AND GRAIN_LENGTH(OsSGL)encodes a putative DUF1645-family protein and participates in rice stress tolerance,grain length,and grain number(Table 1)[101].Overexpression of OsSGL altered certain developmental processes to increase grain number and panicle length by increasing cell size and cell number.Enhanced OsSGL expression modifies the expression level of several genes associated with CK signaling,such as ATYPE RESPONSE REGULATOR GENE1(OsRR1)and OsRR4,suggesting that OsSGL may be an upstream modulator of cytokinin signaling.

3.1.2.Other phytohormones

Grain Number per Panicle1(GNP1)encodes GA20ox1 which participates in GA biosynthesis[102].Elevated expression of GNP1 increases expression of GA2oxs,rice GA catabolism genes,and reduces GA1and GA3accumulation in the apical regions of inflorescence meristems,leading to increased activity of cytokinin owing to a knotted1-like homeobox(KNOX)-mediated transcriptional feedback loop.The highly active cytokinin results in increased grain number and grain yield in rice.

PLANT ARCHITECTURE AND YIELD 1(PAY1),encoding a protein containing the peptidase S64 domain,affects the transport activity of polar auxin and alters the endogenous distribution of IAA[103].Elevated expression of PAY1 produces more panicle branches,particularly secondary branches,to significantly increase grain number(Table 1).

OsmiR167 mediates auxin signaling by modulating the expression of certain auxin-response factor(ARF)genes.Overexpression of miR167 severely repressed the expression of OsARF6,OsARF12,OsARF17,and OsARF25,resulting in decreased panicle length,grain number per panicle,tiller number,and sensitivity to gravity(Table 1)[104,105].

NUMBER OF GRAINS 1(NOG1)encodes an enoyl-CoA isomerase/hydratase that increases grain number per panicle without alteration of panicle number per plant(Table 1)[106].Jasmonic acid treatment reduces NOG1 expression and reduces grain number by suppressing panicle branching.

3.2.Transcription factors

FRIZZY PANICLE(FZP)/CONTROL OF SECONDARY BRANCH1(COS1)/SMALL GRAIN AND DENSE PANICLE7(SGDP7)/QTL for SECONDARY RACHIS BRANCHING on CHROMOSOME7(qSrn7),encoding an AP2/ERF domain-containing TF,was isolated by transposon tagging[107].An 18-bp fragment 5.3 kb upstream of FZP can be bound by OsBZR1[108].OsBZR1-mediated repression of FZP leads to increased grain number(Table 1 and Fig.1).

SUPERNUMERARY BRACT(SNB),encoding an APETALA2-like TF containing two AP2 domains,regulates the transition to floral meristem and floral organ development from the spikelet meristem[109].SNB and OsIDS1,another AP2 family TF,were necessary for branch meristem,inflorescence meristem,and spikelet meristem identity to control grain number by panicle branching(Table 1)[110].A recent study showed that an allele of SNB,SSH1,negatively regulated grain size by modulating cell elongation[82].

4.Control of tiller number

4.1.Transcription factors

MONOCULM 1(MOC1)encodes plant-specific GRAS-family nuclear proteins that act as a vital regulator for controlling the formation of tiller buds[111].moc1 is a loss-of-function rice mutant that has only one culm.MOC3/ORTHOLOG of WUSCHEL(OsWUS)encodes a WOX family protein that acts as a transcriptional suppressor participating in the initiation of axillary meristem development[112,113].A point mutation causes the premature termination of MOC3,preventing the formation of tiller buds to produce a monoculm,similar to the phenotype of moc1.FLORAL ORGAN NUMBER1(FON1)encodes a leucine-rich repeat(LRR)receptor-like kinase that is involved in shoot floral meristem,apical meristem,and inflorescence meristem formation[114,115].MOC3 can directly bind the promoter of FON1 and activate its expression.MOC1 also acts as a MOC3 co-activator for modulating the expression of FON1 in the presence of MOC3[116].fon1 produces normal bud formation but was defective in bud outgrowth,leading to reduced tiller number.These findings imply that there is a interconected transcriptional regulatory mechanism controlled by MOC1,MOC3,and FON1,with a direct relationship between the formation and outgrowth of tiller buds(Table 1;Fig.1).

TILLER ENHANCER(TE)/TILLERING AND DWARF 1(TAD1)encodes an anaphase-promoting complex(APC/C)co-activator that contains a multi-subunit E3 ubiquitin ligase.TAD1 interacts with MOC1 and OsAPC10[117].Its loss-of-function mutant displays drastically increased tiller number(Table 1).The APC/CTEcomplex in the ubiquitin-26S proteasome pathway mediated the degradation of MOC1,repressing axillary meristem initiation and formation and thereby reducing rice tillering(Fig.1)[118,119].

LAX PANICLE 1(LAX1)encodes a putative bHLH TF that interacts with LAX2,a nuclear protein.LAX1 and LAX2 work together to control tiller bud initiation by regulating axillary meristem formation[120,121].

NITROGENMEDIATED TILLER GROWTH RESPONSE 5(NGR5)encodes a rice APETALA2-domain transcription factor that can interact with the polycomb repressive complex 2(PRC2)(Table 1).Histone H3 lysine 27 trimethylation(H3K27me3)modification of target branching inhibitory genes,such as D14 and IPA1,is repressed by the interaction of NGR5 and PRC2.Meanwhile,NGR5 can also interact with GIBBERELLIN INSENSITIVE DWARF1(GID1),a gibberellin receptor.Their interactions are competitively inhibited by DELLA accumulation,resulting in increased stability of NGR5.These findings suggest that NGR5 can increase tiller number but does not change the semi-dwarfsim feature in rice[122].

4.2.Strigolactones(SLs)biosynthesis and signaling pathway

Strigolactones are a group of terpenoid lactones,carotenoidderived hormones,which suppress the outgrowth of tiller buds to repress rice tillering[123-126].SL biosynthesis is initiated by isomerase DWARF27(D27),which changes all-trans-β-carotene to 9-cis-β-carotene[127].In rice,D17/carotenoid cleavage dioxygenase 7(CCD7),D27,and D10/CCD8 are three important catalyzing enzymes that unceasingly transform carotene into carlactone(CL)[128-130].However,CL does not exert a SL activity.The cytochrome P450 enzyme MAX1 in Arabidopsis transforms carlactone into carlactonoic acid [131].Rice contains two genes,Os01g0700900 and Os01g0701400,homologous to Arabidopsis MAX1.As a carlactone oxidase,Os01g0700900 transforms carlactone into 4-deoxyorobanchol,followed by Os01g0701400-mediated catalysis from 4-deoxyorobanchol to orobanchol[132,133].Zaxinone Synthase(ZAS)belongs to the CCD subfamily of plants and has been shown to produce zaxinone,a key regulator of rice,which can reduce the content of SLs by its influence on the transcription of D17,D27,D10,and Os01g0700900(Table 1)[134].A recent study showed that chloroplasticζ-carotene isomerase(ZISO),encoded by T20,is involved in carotenoid biosynthesis as well as the biosynthesis of their metabolites,abscisic acid(ABA)and SL(Table 1)[135].The SL analog rac-GR24 elevates the expression of T20 to increase the biosynthesis of all-trans-β-carotene biosynthesis,and stimulates expression of 9-CIS EPOXYCAROTENOID DIOXYGENASE(OsNCED1)through HOMEODOMAIN-LEUCINE ZIPPER TRANSCRIPTION FACTOR(OsHOX12)to promote ABA biosynthesis.In addition,ABA can repress the expression of D10 and D27 to inhibit SL biosynthesis,suggesting that ABA participates in the regulation of tillering in rice(Table 1).

SL signal transduction depends on hormone-activated proteolysis.An F-box protein component of an SCF E3 ubiquitin ligase complex marks specific protein substrates for polyubiquitination as well as degradation by the 26S proteasome[125,136].In rice,D14,encoding anα/β-hydrolase superfamily protein,acts as both a receptor of SL signaling and an SL hydrolytic enzyme,leading to SCFD3-mediated repressor D53 polyubiquitination and its subsequent breakdown[137-141].IPA1(reviewed in section 2.5)can bind the promoter of TEOSINTE BRANCHED1(TB1)/FINE CULM1(FC1),which encodes a TF with a TCP domain,to regulate its expression[74,142].OsMADS57,a MADS-box family transcriptional factor,can directly bind the D14 promoter,suppressing D14 expression to regulate the outgrowth of axillary buds[143].TB1/FC1 can interact with OsMADS57 to reduce the OsMADS57 inhibition of D14 transcription,suggesting that TB1/FC1 is a downstream response factor of SL signaling inhibiting the outgrowth of axillary buds(Table 1 and Fig.1)[144].

5.Nutrient acquisition and photosynthesis

Nitrogen,phosphorus,and potassium are the three most essential macronutrients for plants.The consumption of N,P,and K fertilizers in rice cultivation is estimated at 15%,13%,and 11%,respectively,of all fertilizers[145].In the plant,photosynthesis provides the metabolic energy for development,and the efficiency of photosynthesis ultimately affects rice yield.Therefore,improvement of nutrient utilization and photosynthetic efficiency,as well as elucidation of their molecular mechanisms,are essential for improving rice yield.

5.1.Nit rogen uptake and transport

Ammonium and nitrate are the two major inorganic nitrogen forms in plants[146].The Peptide Transporter(PTR)family(NPF)/Nitrate Transporter 1(NRT1)and NRT2 family are the two most studied nitrate transporter families in rice(Fig.1)[145].OsNRT1 encodes a low-affinity nitrate transporter which participates in root absorption of nitrate[147].Under high-N conditions,rice increases OsNRT1 expression to increase N uptake.OsNPF4.1 encodes a putative peptide transporter(PTR)family protein that shows no detectable nitrate transporter activity[148].Overexpression of OsNPF7.3 improves rice growth but reduces the efficiency of nitrogen use under high ammonium concentrations[149].OsNPF8.20 is localized in the plasma membrane,and overexpression of OsNPF8.20 increases ammonium uptake to improve grain yield[150].OsNPF2.4 encodes a low-affinity NPF family transporter that is dependent on low pH.Knockout of OsNPF2.4 reduces the absorption of low-affinity nitrate in roots and decreases the potassium concentration in the root,xylem sap,sheath,and culm[151].Similar to OsNPF2.4,OsNPF2.2 also encodes a low-affinity nitrate transporter that is pH dependent and is localized in the plasmamembrane[152].The expression of OsNPF2.2 in parenchyma cells around the xylem is nitrate-induced.The loss-of-function mutant of OsNPF2.2 maintains high nitrate levels in roots but only low levels in shoots,suggesting that OsNPF2.2 is involved in root-toshoot transportation of nitrate.NRT1.1B/OsNPF6.5 encodes a nitrate transporter with dual-affinity,and a one-SNP difference between japonica and indica results in large differences in nitrogen use efficiency between the two subspecies of Asian cultivated rice[153].Expression of NRT1.1B-indica gene in the japonica cultivar enhances nitrate uptake as well as root-to-shoot transportation.A recent study showed that NRT1.1B interacts with SPX4,a signaling repressor for phosphate.Nitrate enhances the interaction of NRT1.1B and SPX4,leading to the degradation of SPX4 as a result of ubiquitination by NBIP1,an E3 ubiquitin ligase,suggesting that OsNRT1.1B acts as an important factor regulating N-P balance[154].

OsNAR2.1 acts as a dual component transporter with OsNRT2.2,OsNRT2.1,and OsNRT2.3a,and is essential for high-affinity uptake of nitrate[155].The OsNRT2.3 gene has two alternative splicing transcripts:OsNRT2.3a and OsNRT2.3b.OsNAR2.1 cooperates with OsNRT2.2,OsNRT2.1,and OsNRT2.3a to regulate nitrate uptake.Intriguingly,OsNRT2.4 and OsNRT2.3b regulate nitrate absorption independent of OsNAR2.1[156].OsNRT2.1,OsNAR2.1,OsNRT2.3a and OsNRT2.2 are up-regulated in the presence of nitrate and downregulated by ammonium at high temperature,while OsNRT2.4 and OsNRT2.3b are not sensitive to these factors.

Ammonium is an important inorganic form of N required for plant growth,especially for rice growing in paddy field.Genes of the HIGH-AFFINITY AMMONIUM TRANSPORTER1(AMT1)family,including OsAMT1;1,OsAMT1;2,and OsAMT1;3,encode putative high-affinity NH+4transporters(Fig.1)[157].OsAMT1;1 is induced by ammonium and is expressed mainly in root epidermal,stele,and mesophyll cells.Downregulation of OsAMT1;1 reduces ammonium uptake and suppresses the growth of shoots and roots under both high-and low-ammonium conditions[158].Ammonium in roots reduces the expression of OsAMT1;2[157].The expression of OsAMT1.3 is strongly induced under low-N conditions in the lateral root emission and root tip zone,and overexpression of OsAMT1.3 promotes the expression of OsAMT1.2[159].A study shows that expression of the OsAMT1 is dependent on the concentration of glutamine,a product of ammonium assimilation[160].OsAMT2;1 was expressed in both shoot and root,whereas the expression of OsAMT3;1 was relatively weak[161].

5.2.Phosphorus uptake and transport

In rice,there are 26 Pi transporters(PHT/PT),classified into four families:PHT1,PHT2,PHT3,and PHT4[162].Among these transporter families,PHT1 is responsible for Pi uptake from the soil.The PHT1 family contains 13 genes,10 of which have been functionally identified[163].qRT-PCR and promoter analysis using reporter gene demonstrated that OsPT1,OsPT4,and OsPT8 are highly expressed in roots,root-shoot junctions,and leaves,and participate in Pi uptake in roots[164-166].OsPT2 is localized in the stele lateral and primary root,whereas OsPT6 is localized in both cortical and epidermal cells of younger lateral and primary roots and both OsPT2 and OsPT6 genes were induced significantly under Pi deficiency conditions[167].Like that of OsPT2 and OsPT6,the expression of OsPT3,OsPT9,and OsPT10 is also induced by Pi deficiency,especially in the root epidermis,root hairs,and lateral roots(Fig.1)[168,169].PHOSPHATE RESPONSE1(OsPHR1),OsPHR2,and OsPHR3 encode MYB domain-containing transcriptional regulators,which are involved in transcriptional activation of most Pi starvation-induced genes(Fig.1)[170].OsPHR2 is a core phosphate regulator regulating many Pi starvation-responsive genes.SPX domain-containing proteins,such as OsSPX1,OsSPX2,OsSPX4,and OsSPX6,can interact with OsPHR2 through their SPX domains,inhibiting its regulation of the PHT1 family genes[171-173].The Phosphate Transporter Traffic Facilitator 1(OsPHF1)regulates the localization of both high-and low-affinity Pi transporters on the plasma membrane,thus determining Pi uptake and translocation(Fig.1)[174].Under Pi-sufficient conditions,phosphate transporters can be phosphorylated by the CK2α3/β3 holoenzyme,blocking the interactions between them and OsPHF1 and resulting in fewer phosphate transporters on the plasma membrane to take up Pi[175].

5.3.Potassium uptake and transport

K+enters the root apoplast and diffuses toward inner cell layers,a process that can be interrupted by the Casparian strip[176,177].To cross this impermeable barrier,the entry of K+must be mediated by membrane transport systems,channels,transporters,and co-transporters.OsHAK1,encoding a high-affinity potassium transporter,is up-regulated by K+deficiency in the root and shoot apical meristem,the epidermis and steles of the root,and the vascular bundles of the shoot[178].Loss of function of OsHAK1 reduces the K+absorption rate,while overexpression of OsHAK1 increases K+uptake and the K+/Na+ratio.OsHAK5,another HAK potassium transporter family gene,is expressed largely in root epidermis and stele,vascular tissues,and mesophyll cells[179].Knockout of OsHAK5 reduces root K+acquisition and transport of K+from roots to aerial parts,while overexpression of OsHAK5 increases K+uptake and increases the K+/Na+ratio.OsAKT1 encodes a shaker K+channel transporter which is responsible for K+uptake[180].Its loss-of-function mutant reduces K+uptake,resulting in a low-K+-sensitive phenotype.Overexpression of OsHAK5 increases the level of K+in the root,leading to improvement of rice osmotic and drought stress tolerance[181].OsHAK1,OsHAK5,and OsAKT1 participate in K+uptake under low and high K+conditions and in translocation of K+from the root to the shoot(Fig.1).

5.4.Photosynthesis

Rice uses the C3photosynthetic pathway,as do wheat,tomato,soybean,and many other crops.The ribulose-1,5-bisphosphate carboxylase-oxygenase(Rubisco)capacity,capacity to regenerate ribulose 1,5-bisphosphate(RuBP),and the diffusion rate of CO2to the site of carboxylation are three main limitations of the C3photosynthetic pathway.Accordingly,increasing the amount of Rubisco,activating Rubisco,increasing the content of the cytochrome b6/f complex,modification of the activity of the RuBP regeneration enzymes,and promoting the diffusion of CO2into the chloroplast stroma might be ways to improve photosynthesis and rice productivity.

Rice has four genes(OsRBCS2/3/4/5)encoding the small subunit of Rubisco(Fig.1)[182].Overexpression of the OsRBCS gene increases Rubisco production and biomass of transgenic plants by 32%and 15%under low and normal levels of CO2,respectively[183].However,suppression of individual OsRBCS2/3/5 genes decreases Rubisco content and increases biomass under elevated CO2conditions[184].Rubisco can be inhibited by certain sugar phosphates,requiring reactivation by Rubisco activase for removing the inhibitors[185].A study showed that overexpression of the maize Rubisco activase gene increased Rubisco activation but decreased Rubisco content[186].The Calvin cycle of RuBP regeneration involves 12 reactions,in which sedoheptulose 1,7-bisphosphatase(SBPase)is the rate-limiting enzyme.Overexpression of the SBPase gene in transgenic plants,such as wheat,tomato,and Arabidopsis,enhances leaf photosynthesis,resulting in increased biomass[187-189].Keeping the stomata open is one of the ways to promote the diffusion of CO2into the chloroplast stroma.A loss-of-function of OsSLAC1,which encodes a nitrate-selective anion channel protein,results in the constitutive opening of stomata,leading to enhanced photosynthesis and decreased drought tolerance[190].OsSAPK8 phosphorylates and activates OsSLAC1 to control the stroma(Fig.1)[191].Mesophyll resistance(rm)is one of the critical factors limiting leaf photosynthesis.A study indicated that variations in rmwere affected by four biochemical factors:photorespiration and respiration,non-uniform photosynthetic status across the leaf,bicarbonate leakage on the chloroplast envelope,and hydration activity in the cytosol and stroma.The authors constructed three-dimensional cell and leaf anatomy to show the hydration of CO2and its diffusion processes from the intercellular air space to the stroma[192].

In C3plants,glycolate can be formed by the oxidation of Rubisco to RuBP.Although photorespiration is required for the metabolism of toxic glycolate,it can reduce yield by 20 to 50%in C3crops.A recent study demonstrated three pathways that can inhibit glycolate export into the native pathway,two of which increased the biomass of tobacco by 13%and 18%,respectively[193].

A new bioengineering technique called the GOC bypass was developed recently,which significantly boosts photosynthesis in rice,leading to a grain yield increase of up to 27%[194].Three genes,OsGLO3,OsOXO3,and OsCATC,establish the bypass and are introduced into rice chloroplasts via a multi-gene assembly and transformation system.Photosynthetic efficiency,biomass,yield,and nitrogen use efficiency of GOC rice plants were greatly improved by the increased ability to concentrate CO2[194].To solve the problem of unstable grain yield in GOC rice plants,a synthetic photorespiratory shortcut(the GCGT bypass)was designed,which consisted of a rice glycolate oxidase gene and three Escherichia coli genes encoding catalase,glyoxylate carboligase,and tartronic semi-aldehyde reductase,respectively[195].The biomass production and grain yield of the GCGT rice plants increase 28%owing to the recovery of 75%of the carbon from glycolate metabolism into the Calvin cycle.

6.Conclusion and perspectives

Rice grain yield is influenced physiologically by sink-source interaction,which is determined by sink capacity,source strength,and the flow of photoassimilates.Green tissues such as mature leaves are major carbon source as well as a major organic nitrogen source but an inorganic nitrogen sink.In contrast,underground tissues such as roots are inorganic nitrogen source as well as carbon sink[196].The biosynthesis of Rubisco,the key rate-limiting enzymes of photosynthesis,consumes the largest fraction of nitrogen in rice leaves uptaking from the roots[197].The improvement of photosynthetic efficiency and nitrogen use efficiency can enhance source activity,and ultimately grain filling.The connection of the sink and the source is the flow,such as phloem sieve tubes and the transport system.Rice grains are a sink for both carbon and inorganic and organic nitrogen,obtaining them from green tissues and roots.The sink capacity is determined by three component traits:panicle number/effective tiller number,grain number per panicle,and grain size or weight.Grain weight is determined by the filling and the volume of seeds influenced by growth of the spikelet hull.Grain number per panicle is determined by two components:(1)the duration of panicle differentiation and(2)the rate of spikelet differentiation.Rice tillering consists of two distinct steps:axillary meristem formation and outgrowth of axillary buds.The driving forces are photosynthesis and nitrogen acquisition,which provide sufficient carbohydratesand organic nitrogen to the sink.Further increasing rice yield potential needs to optimization of source-sink interaction,for instance by overexpression of NRT1.1B/OsNPF6.5 enhance the source strength for nitrogen and thus increase tiller number.Increased tiller number improves sink capacity and change the source strength for carbon owing to the altered plant architecture.There are similar in the relationship between grain weight and number.And high grain yield at the individual level does not mean high field yield at the population level(Fig.2).For this reason,the application of one gene or pyramiding several genes is difficult to greatly increase rice yield.The flow of assimilates is the ability to transport the photoassimilates and other nutrients to fill the sink.In the past,many yield-related genes have been selected by visible phenotypes or agronomic traits.However,physiological traits,such as nutrient use efficiency and photosynthetic efficiency,cannot be observed directly,limiting the development of rice yield breeding.The molecular understanding underlying these physiological traits will be great help for further improvement of rice yield.With the cloning and functional characterization of several key genes,such as NRT1.1B and OsNR2,related to physiological traits,it is now possible to use marker-assisted selection,transgenic approaches,and genome editing systems to tune these invisible physiological traits.Rice breeding will move from conventional breeding to breeding by molecular design.

CRediT authorship contribution statement

Gengmi Li:Funding acquisition,Writing-original draft.Jiuyou Tang:Writing-review&editing.Jiakui Zheng:Project administration.Chengcai Chu:Project administration,Writing-review&editing.

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

This work was supported by the National Natural Science Foundation of China(31901520),and Top Talent Foundation of Sichuan Academy of Agricultural Sciences(2020BJRC008).