OsVP1 activates Sdr4 expression to control rice seed dormancy via the ABA signaling pathway

Pre-harvest sprouting(PHS)is a disadvantageous trait in cereal production worldwide,causing large economic losses each year.Its regulation mechanism is still unclear.We generated the Oryza sativa Viviparous1(OsVP1)mutant using gene editing technique,which shows increased PHS compared with that of the wild type Nipponbare.OsVP1 is localized mainly in the nucleus and expressed in various tissues and organs.Expression of Seed dormancy 4(Sdr4),a key gene controlling PHS,was sharply reduced in OsVP1 mutants.OsVP1 bound to the specific motif CACCTG in the promoter of Sdr4 and activated its expression in rice protoplasts.Overexpression of Sdr4 reduced the high seed germination rate of OsVP1 mutant cr-osvp1-1,showing that Sdr4 acts as a downstream target of OsVP1.Both OsVP1 and Sdr4 loss-of-function mutants were insensitive to exogenous ABA and employed the ABA signaling pathway in regulating seed dormancy.These findings shed light on the control of seed dormancy aimed at preventing PHS in rice.

Integrated transcriptome, small RNA, and degradome analysis to elucidate the regulation of rice seedling mesocotyl development during the passage from darkness to light

The mesocotyl,a structure located between the basal part of the seminal root and the coleoptile node of seedlings,contributes to pushing the shoot tip through the soil surface,a function that is essential for the uniform emergence of direct-seeded rice.Its elongation is inhibited by light and induced in darkness.This investigation of an indica rice(P25)with vigorous mesocotyl elongation was aimed at identifying the"omics"basis of its lightinduced growth inhibition.A transcriptomic comparison between mesocotyl tissues that had developed in the dark and then been exposed to light identified many differentially expressed genes(DEGs)and differentially abundant micro RNAs(mi RNAs).Degradome sequencing analysis revealed 27 negative mi RNA-target pairs.A co-expression regulatory network was constructed based on the mi RNAs,their corresponding targets,and DEGs with a common Gene Ontology term.It suggested that auxin and light,probably antagonistically,affect mesocotyl elongation by regulating polyamine oxidase activity.

An orchestrated ethylene-gibberellin signaling cascade contributes to mesocotyl elongation and emergence of rice direct seeding

A mechanized direct seeding of rice with less labor and water usage,has been widely adopted.However,this approach requires varieties that exhibit uniform seedling emergence.Mesocotyl elongation(ME)offers the main drive of fast emergence of rice seedlings from soils;nevertheless,its genetic basis remains unknown.Here,we identify a major rice quantitative trait locus Mesocotyl Elongation1(qME1),an allele of the Green Revolution gene Semi-Dwarf1(SD1),encoding GA20-oxidase for gibberellin(GA)biosynthesis.ME1 expression is strongly induced by soil depth and ethylene.When rice grains are direct-seeded in soils,the ethylene core signaling factor OsEIL1 directly promotes ME1 transcription,accelerating bioactive GA biosynthesis.The GAs further degrade the DELLA protein SLENDER RICE 1(SLR1),alleviating its inhibition of rice PHYTOCHROME-INTERACTING FACTOR-LIKE13(OsPIL13)to activate the downstream expansion gene OsEXPA4 and ultimately promote rice seedling ME and emergence.The ancient traits of long mesocotyl and strong emergence ability in wild rice and landrace were gradually lost in company with the Green Revolution dwarf breeding process,and an elite ME1-R allele(D349H)is found in some modern Geng varieties(long mesocotyl lengths)in northern China,which can be used in the direct seeding and dwarf breeding of Geng varieties.Furthermore,the ectopic and high expression of ME1 driven by mesocotyl-specific promoters resulted in rice plants that could be direct-seeded without obvious plant architecture or yield penalties.Collectively,we reveal the molecular mechanism of rice ME,and provide useful information for breeding new Green Revolution varieties with long mesocotyl suitable for direct-seeding practice.

OsLESV and OsESV1 promote transitory and storage starch biosynthesis to determine rice grain quality and yield

Transitory starch is an important carbon source in leaves,and its biosynthesis and metabolism are closely related to grain quality and yield.The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice(Oryza sativa)quality and yield remain unclear.Here,we show that OsLESV and OsESV1,the rice orthologs of AtLESV and AtESV1,are associated with transitory starch biosynthesis in rice.The total starch and amylose contents in leaves and endosperms are significantly reduced,and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants.Furthermore,we found that OsLESV and OsESV1 bind to starch,and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain.Importantly,OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis,granule-bound starch synthase I(GBSSI),GBSSII,and pyruvate orthophosphote dikiase(PPDKB),to maintain their protein stability and activity.OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules.Overexpression of GBSSI,GBSSII,and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants.Thus,we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice.These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield,providing useful information for the genetic improvement of rice grain quality and yield.

Tiller Bud Formation Regulators MOC1 and MOC3 Cooperatively Promote Tiller Bud Outgrowth by Activating F0N1 Expression in Rice

Tillering in rice is one of the most important agronomic traits.Rice tiller development can be divided into two main processes: the formation of the axillary bud and its subsequent outgrowth.Several genes critical for bud formation in rice have been identified by genetic studies;however,their molecular functions and relationships are still largely unknown.Here,we report that MONOCULM 1 (MOC1) and MONOCULM 3/ TILLERS ABSENT 1/STERILE AND REDUCED TILLERING 1 (MOC3/TAB1/SRT1),two vital regulators for tiller formation in rice,physically interact to regulate tiller bud outgrowth through upregulating the expression of FLORAL ORGAN NUMBER 1 (FON1),the homolog of CLAVATA1 in rice.We found that M0C3 is able to directly bind the promoter ofFONI and subsequently activate FON1 expression.MOC1 functions as a coactivator of MOC3,whereas it could not directly bind the FON1 promoter,and further activated FON1 expression in the presence of MOC3.Accordingly,FON1 is highly expressed at axillary meristems and shows remarkably decreased expression levels in mod and moc3 mutants.Loss-of-function mutants of FON1 exhibit normal bud formation but defective bud outgrowth and reduced tiller number.Collectively,these results shed light on a joint transcriptional regulatory mechanim by MOC1 and MOC3,and establish a new framework for the control of tiller bud formation and outgrowth.

GRAIN INCOMPLETE FILLING 2 regulates grain filling and starch synthesis during rice caryopsis development

Rice grain filling determines grain weight, final yield and grain quality. Here, a rice defective grain filling mutant, gif2, was identified. Grains ofgif2 showed a slower filling rate and a significant lower final grain weight and yield compared to wild-type. The starch content in gilt2 was noticeably decreased and its physicochemical properties were also altered. Moreover, gif2 endosperm cells showed obvious defects in compound granule formation. Posi- tional cloning identified GIF2 to encode an ADP-glucose pyrophosphorylase （AGP） large subunit, AGPL2; consequently, AGP enzyme activity in gif2 endosperms was remarkably decreased. GIF2 is mainly expressed in developing grains and the coded protein localizes in the cytosol. Yeast two hybrid assay showed that GIF2 interacted with AGP small subunits OsAGPS% OsAGPS2a and OsAGPS2b. Transcript levels for granule-bound starch synthase, starch synthase, starch branching enzyme and starch debranching enzyme were distinctly elevated in gif2 grains. In addition, the level of nucleotide diversity of the GIF2 locus was extremely low in both cultivated and wild rice. All of these results suggest that GIF2 plays important roles in the regulation of grain filling and starch biosynthesis during caryopsis development, and that it has been preserved during selection throughout domestication of modern rice.

MONOCULM 3,an Ortholog of WUSCHEL in Rice,Is Required for Tiller Bud Formation

WUSCHEL （WUS） plays an essential role for the maintenance of meristem activity in dicots, but its function is still elusive in monocots. We isolated a new monoculm mutant, monoculm 3 （moc3）, in which a point mutation causes the premature termination of rice O. sativa WUS （OsWUS）. Morphological observation revealed that the formation of tiller buds was disrupted in moc3. MOC3 was localized in the nuclear and could interact with TOPLESS-RELATED PROTEINS （TPRs）. The expression of MOC3 was induced by cytokinins and defection of MOC3 affected the expression of several two-component cytokinin response regulators, OsRRs and ORRs. Our results suggest that MOC3 is required for the formation of axillary buds and has a complex relationship with cytokinins.

Targeted mutagenesis of POLYAMINE OXIDASE 5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield

Under conditions of labor or resource scarcity,direct seeding,rather than transplantation,is the preferred mode of rice(Oryza sativa)cultivation.This approach requires varieties that exhibit uniform seedling emergence.Mesocotyl elongation(ME),the main driver of rapid emergence of rice seedlings from soil,is enhanced by darkness and inhibited by light.Plant polyamine oxidases(PAOs)oxidize polyamines(PAs)and release H2O2,Here,we established that OsPAO5 expression in rice seedlings is increased in the presence of light and inhibited by darkness.To determine its role in ME,we created OsPAO5 mutants using CRISPR/Cas9.Compared with the wild type,pao5 mutants had longer mesocotyls,released less H2O2,and synthesized more ethylene.The mutant seedlings emerged at a higher and more uniform rate,indicating their potential for use in direct seeding.Nucleotide polymorphism analysis revealed that an SNP(PAO5-578G/A)located 578 bp upstream of the OsPAO5 start codon alters its expression,and was selected during rice mesocotyl domestication.The PAO5-578G genotype conferring a long mesocotyl mainly exists in wild rice,most Aus accessions,and some Geng(Japonica)accessions.Intriguingly,knocking out OsPAO5 can remarkably increase the grain weight,grain number,and yield potential.In summary,we developed a novel strategy to obtain elite rice with higher emergence vigor and yield potential,which can be conveniently and widely used to breed varieties of direct-seeding rice.

OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice

Starch and storage proteins are the main components of rice(Oryza sativa L.)grains.Despite their importance,the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive.Here,we identified a rice opaque endosperm mutant,opaque3(o3),that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type.The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells.OPAQUE3(O3)encodes a transmembrane basic leucine zipper(bZIP)transcription factor(OsbZIP60)and is localized in the endoplasmic reticulum(ER)and the nucleus,but it is localized mostly in the nucleus under ER stress.We demonstrated that O3 could activate the expression of several starch synthesis-related genes(GBSSI,AGPL2,SBEI,and ISA2)and storage protein synthesis-related genes(OsGluA2,Prol14,and Glb1).O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1,two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells.High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants.We also revealed that OsbZIP50 can assist O3 in response to ER stress,especially under high-temperature conditions.We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

CDE4 encodes a pentatricopeptide repeat protein involved in chloroplast RNA splicing and affects chloroplast development under low-temperature conditions in rice

Pentatricopeptide repeat(PPR)proteins play important roles in the post-transcriptional modification of organellar RNAs in plants.However,the function of most PPR proteins remains unknown.Here,we characterized the rice(Oryza sativa L.)chlorophyll deficient 4(cde4)mutant which exhibits an albino phenotype during early leaf development,with decreased chlorophyll contents and abnormal chloroplasts at low-temperature(20℃).Positional cloning revealed that CDE4 encodes a P-type PPR protein localized in chloroplasts.In the cde4 mutant,plastid-encoded polymerase(PEP)-dependent transcript levels were significantly reduced,but transcript levels of nuclear-encoded genes were increased compared to wild-type plants at 20℃.CDE4 directly binds to the transcripts of the chloroplast genes rpl2,ndhA,and ndhB.Intron splicing of these transcripts was defective in the cde4 mutant at 20℃,but was normal at 32℃.Moreover,CDE4 interacts with the guanylate kinase VIRESCENT 2(V2);overexpression of V2 enhanced CDE4 protein stability,thereby rescuing the cde4 phenotype at 20℃.Our results suggest that CDE4 participates in plastid RNA splicing and plays an important role in rice chloroplast development under lowtemperature conditions.