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.

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.

ABNORMAL FLOWER AND GRAIN 1 encodes OsMADS6 and determines palea identity and affects rice grain yield and quality

The palea and lemma are floral organ structures unique to grasses;these structures form the hull and directly affect grain size and quality. However, the molecular mechanisms controlling the development of the hull are not well understood. In this study, we characterized the rice(Oryza sativa) abnormal flower and grain1(afg1) mutant, a new allele of OsMADS6. Similar to previously characterized osmads6 alleles, in the afg1 floret, the palea lost its marginal region and acquired the lemma identity. However, in contrast to other osmads6 alleles, the afg1 mutant showed altered grain size and grain quality, with decreased total starch and amylose contents, and increased protein and soluble sugar contents. The analysis of transcriptional activity suggested that AFG1 is a transcriptional activator and may affect grain size by regulating the expression levels of several genes related to cell expansion and proliferation in the afg1 mutant. These results revealed that AFG1 plays an important role in determining palea identity and affecting grain yield and quality in rice.

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.

GBSS-BINDING PROTEIN,encoding a CBM48 domain-containing protein,affects rice quality and yield^∞

The percentage of amylose in the endosperm of rice(Oryza sativa)largely determines grain cooking and eating qualities.Granule-bound starch synthase I(GBSSI)and GBSSII are responsible for amylose biosynthesis in the endosperm and leaf,respectively.Here,we identified OsGBP,a rice GBSS-binding protein that interacted with GBSSI and GBSSII in vitro and in vivo.The total starch and amylose contents in osgbp mutants were significantly lower than those of wild type in leaves and grains,resulting in reduced grain weight and quality.The carbohydrate-binding module 48(CBM48)domain present in the C-terminus of OsGBP is crucial for OsGBP binding to starch.In the osgbp mutant,the extent of GBSSI and GBSSII binding to starch in the leaf and endospermwas significantly lower than wild type.Our data suggest that OsGBP plays an important role in leaf and endosperm starch biosynthesis by mediating the binding of GBSS proteins to developing starch granules.This elucidation of the function of OsGBP enhances our understanding of the molecular basis of starch biosyn-thesis in rice and contributes information that can be potentially used for the genetic improvement of yield and grain quality.

The OsNAC23-Tre6P-SnRK1 a feed-forward loop regulates sugar homeostasis and grain yield in rice

Tre6P(trehalose-6-phosphate)mediates sensing of carbon availability to maintain sugar homeostasis in plants,which underpins crop yield and resilience.However,how Tre6P responds to fluctuations in sugar levels and regulates the utilization of sugars for growth remains to be addressed.Here,we report that the sugar-inducible rice NAC transcription factor OsNAC23 directly represses the transcription of the Tre6P phosphatase gene TPP1 to simultaneously elevate Tre6P and repress trehalose levels,thus facilitating carbon partitioning from source to sink organs.Meanwhile,OsNAC23 and Tre6P suppress the transcription and enzyme activity of SnRK1a,a low-carbon sensor and antagonist of OsNAC23,to prevent the SnRK1a-mediated phosphorylation and degradation of OsNAC23.Thus,OsNAC23,Tre6P,and SnRK1a form a feed-forward loop to sense sugar and maintain sugar homeostasis by transporting sugars to sink organs.Importantly,plants over-expressing OsNAC23 exhibited an elevated photosynthetic rate,sugar transport,and sink organ size,which consistently increased rice yields by 13%–17%in three elite-variety backgrounds and two locations,suggesting that manipulation of OsNAC23 expression has great potential for rice improvement.Collectively,these findings enhance our understanding of Tre6P-mediated sugar signaling and homeostasis,and provide a new strategy for genetic improvement of rice and possibly also other crops.

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.

Alanine aminotransferase(OsAlaAT1)modulates nitrogen utilization,grain yield,and quality in rice

Widespread use of inorganic nitrogen fertilizers has increased yields of many grains, including rice (Oryza sativa L.);however, fertilizers are costly for farmers and cause serious environmental problems, such as soil acidification and water eutrophication (Guo et al., 2010, 2021;Liu et al., 2021). Improving nitrogen use efficiency(NUE) in rice offers a practical way to enhance grain yield and alleviate the environmental problems caused by overuse of nitrogen fertilizer. Identifying and characterizing genes involved in NUE provides crucial information for this aim.

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.