A CYP78As-small grain4-coat protein complex â ¡ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

E3 ligase DECREASED GRAIN SIZE 1 promotes degradation of a G-protein subunit and positively regulates grain size in rice.

Grain size is one of the most important traits determining crop yield. However, the mechanism controlling grain size remains unclear. Here, we confirmed the E3 ligase activity of DECREASED GRAIN SIZE 1 (DGS1) in positive regulation of grain size in rice (Oryza sativa) suggested in a previous study. Rice G-protein subunit gamma 2 (RGG2), which negatively regulates grain size, was identified as an interacting protein of DGS1. Biochemical analysis suggested that DGS1 specifically interacts with canonical Gγ subunits (rice G-protein subunit gamma 1 [RGG1] and rice G-protein subunit gamma 2 [RGG2]) rather than non-canonical Gγ subunits (DENSE AND ERECT PANICLE 1 [DEP1], rice G-protein gamma subunit type C 2 [GCC2], GRAIN SIZE 3 [GS3]). We also identified the necessary domains for interaction between DGS1 and RGG2. As an E3 ligase, DGS1 ubiquitinated and degraded RGG2 via a proteasome pathway in several experiments. DGS1 also ubiquitinated RGG2 by its K140, K145 and S147 residues. Thus, this work identified a substrate of the E3 ligase DGS1 and elucidated the post transcriptional regulatory mechanism of the G-protein signalling pathway in the control of grain size.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

Young Leaf White Stripe encodes a P-type PPR protein required for chloroplast development.

Pentatricopeptide repeat (PPR) proteins function in post-transcriptional regulation of organellar gene expression. Although several PPR proteins are known to function in chloroplast development in rice (Oryza sativa), the detailed molecular functions of many PPR proteins remain unclear. Here, we characterized a rice young leaf white stripe (ylws) mutant, which has defective chloroplast development during early seedling growth. Map-based cloning revealed that YLWS encodes a novel P-type chloroplast-targeted PPR protein with 11 PPR motifs. Further expression analyses showed that many nuclear- and plastid-encoded genes in the ylws mutant were significantly changed at the RNA and protein levels. The ylws mutant was impaired in chloroplast ribosome biogenesis and chloroplast development under low-temperature conditions. The ylws mutation causes defects in the splicing of atpF, ndhA, rpl2, and rps12, and editing of ndhA, ndhB, and rps14 transcripts. YLWS directly binds to specific sites in the atpF, ndhA, and rpl2 pre-mRNAs. Our results suggest that YLWS participates in chloroplast RNA group II intron splicing and plays an important role in chloroplast development during early leaf development.

Improving pre-harvest sprouting resistance in rice by editing OsABA8ox using CRISPR/Cas9.

KEY MESSAGE: Knock out OsABA8ox helps improve pre-harvest spouting resistance and do not affect rice yield. Pre-harvest sprouting(PHS) is a phenomenon that the seeds of crops germinate preharvest, which reduces the yield and quality of rice. Abscisic acid(ABA) is one of the phytohormones that promotes seed dormancy. ABA8' hydroxylase is the main enzyme that can catabolism ABA in plant. There are three genes that encode ABA8' hydroxylase in rice, named OsABA8ox1, OsABA8ox2 and OsABA8ox3. In this study, we use CRISPR/Cas9 gene editing technology to target these three genes in Ningjing6 and find that the knockout transgenic lines are all significantly strengthen in seed dormancy and have no effect on the yield. By a series of quantitative experiments, we consider that after knock out OsABA8ox, the high endogenous ABA level will influence the ABA signal which suppress the substantial and energy metabolism in the seeds, and finally led to higher dormancy.

Ribonuclease H-like gene SMALL GRAIN2 regulates grain size in rice through brassinosteroid signaling pathway.

Grain size is a key agronomic trait that determines the yield in plants. Regulation of grain size by brassinosteroids (BRs) in rice has been widely reported. However, the relationship between the BR signaling pathway and grain size still requires further study. Here, we isolated a rice mutant, named small grain2 (sg2), which displayed smaller grain and a semi-dwarf phenotype. The decreased grain size was caused by repressed cell expansion in spikelet hulls of the sg2 mutant. Using map-based cloning combined with a MutMap approach, we cloned SG2, which encodes a plant-specific protein with a ribonuclease H-like domain. SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2 (GSK2) in response to BR signaling, and its mutation causes insensitivity to exogenous BR treatment. Genetical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2. We further found that BRs enhance the accumulation of SG2 in the nucleus, and subcellular distribution of SG2 is regulated by GSK2 kinase activity. In addition, Oryza sativa OVATE family protein 19 (OsOFP19), a negative regulator of grain shape, interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size. Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regulates grain size by interacting with OsOFP19.

Small grain and semi-dwarf 3, a WRKY transcription factor, negatively regulates plant height and grain size by stabilizing SLR1 expression in rice.

KEY MESSAGE: OsWRKY36 represses plant height and grain size by inhibiting gibberellin signaling. Plant height and grain size are important agronomic traits affecting yield in cereals, including rice. Gibberellins (GAs) are plant hormones that promote plant growth and developmental processions such as stem elongation and grain size. WRKYs are transcription factors that regulate stress tolerance and plant development including height and grain size. However, the relationship between GA signaling and WRKY genes is still poorly understood. Here, we characterized a small grain and semi-dwarf 3 (sgsd3) mutant in rice cv. Hwayoung (WT). A T-DNA insertion in the 5'-UTR of OsWRKY36 induced overexpression of OsWRKY36 in the sgsd3 mutant, likely leading to the mutant phenotype. This was confirmed by the finding that overexpression of OsWRKY36 caused a similar small grain and semi-dwarf phenotype to the sgsd3 mutant whereas knock down and knock out caused larger grain phenotypes. The sgsd3 mutant was also hyposensitive to GA and accumulated higher mRNA and protein levels of SLR1 (a GA signaling DELLA-like inhibitor) compared with the WT. Further assays showed that OsWRKY36 enhanced SLR1 transcription by directly binding to its promoter. In addition, we found that OsWRKY36 can protect SLR1 from GA-mediated degradation. We thus identified a new GA signaling repressor OsWRKY36 that represses GA signaling through stabilizing the expression of SLR1.

GW5-Like, a homolog of GW5, negatively regulates grain width, weight and salt resistance in rice.

Grain size is an important determinant of yield potential in crops. We previously demonstrated that natural mutations in the regulatory sequences of qSW5/GW5 confer grain width diversity in rice. However, the biological function of a GW5 homolog, named GW5-Like (GW5L), remains unknown. In this study, we report on GW5L knockout mutants in Kitaake, a japonica cultivar (cv.) considered to have a weak gw5 variant allele that confers shorter and wider grains. GW5L is evenly expressed in various tissues, and its protein product is localized to the plasma membrane. Biochemical assays verified that GW5L functions in a similar fashion to GW5. It positively regulates brassinosteroid (BR) signaling through repression of the phosphorylation activity of GSK2. Genetic data show that GW5L overexpression in either Kitaake or a GW5 knockout line, Kasaorf3 (indica cv. Kasalath background), causes more slender, longer grains relative to the wild-type. We also show that GW5L could confer salt stress resistance through an association with calmodulin protein OsCaM1-1. These findings identify GW5L as a negative regulator of both grain size and salt stress tolerance, and provide a potential target for breeders to improve grain yield and salt stress resistance in rice.

WEAK SEED DORMANCY 1, an aminotransferase protein, regulates seed dormancy in rice through the GA and ABA pathways.

Seed dormancy is a critical trait that enhances plant survival by preventing seed germination at the wrong time or under unsuitable conditions. Lack of seed dormancy in rice can lead to pre-harvest sprouting on mother plants leading to reduced yield and seed quality. Although some genes have been identified, knowledge of regulation of seed dormancy is limited. Here, we characterized a weak seed dormancy mutant named weak seed dormancy 1 (wsd1) that showed a higher seed germination percentage than the wild-type following the harvest ripeness. We cloned the WSD1 encoding an aminotransferase protein using a MutMap approach. WSD1 was stably expressed after imbibition and its protein was localized in the endoplasm reticulum. A widely targeted metabolomics assay and amino acid analysis showed that WSD1 had a role in regulating homeostasis of amino acids. PAC treatment and RNA-seq analysis showed that WSD1 regulates seed dormancy by involvement in the GA biosynthesis pathway. GA1 content and expression of GA biosynthesis-related genes were increased in the wsd1 mutant compared with the wild-type. The wsd1 mutant had reduced sensitivity to ABA. Our overall results indicated that WSD1 regulates seed dormancy by balancing the ABA and GA pathways.

OsDOG1L-3 regulates seed dormancy through the abscisic acid pathway in rice.

Seed dormancy is closely related to pre-harvest sprouting resistance. Both plant hormone abscisic acid (ABA) and DELAY OF GERMINATION 1 (DOG1) protein are key regulators of seed dormancy. Their relationship is well reported in Arabidopsis, but little is known in rice. Here, we show that a quantitative trait locus, qSd-1-1 contributes significantly to seed dormancy differences between the strongly dormant indica variety N22 and non-dormant japonica variety Nanjing35. It encodes a DOG1-like protein named OsDOG1L-3 with homology to Arabidopsis DOG1. There were evident promoter and expression differences in OsDOG1L-3 between N22 and Nanjing35, and overexpression or introduction of the N22 OsDOG1L-3 allele in Nanjing35 enhanced its seed dormancy. OsDOG1L-3 expression was positively correlated with seed dormancy and induced by ABA. OsbZIP75 and OsbZIP78 bound directly with the promoter of OsDOG1L-3 to induce its expression. Overexpression of OsbZIP75 increased OsDOG1L-3 protein abundance and promoted seed dormancy. OsDOG1L-3 upregulated expression of ABA-related genes and increased ABA content. We propose that the N22 OsDOG1L-3 allele is a candidate gene for the seed dormancy in QTL qSd-1-1, and that it participates in the ABA pathway to establish seed dormancy in rice.

WSL9 Encodes an HNH Endonuclease Domain-Containing Protein that Is Essential for Early Chloroplast Development in Rice.

BACKGROUND: The plant chloroplast is essential for photosynthesis and other cellular processes, but an understanding of the biological mechanisms of plant chloroplast development are incomplete. RESULTS: A new temperature-sensitive white stripe leaf 9(wsl9) rice mutant is described. The mutant develops white stripes during early leaf development, but becomes green after the three-leaf stage under field conditions. The wsl9 mutant was albinic when grown at low temperature. Gene mapping of the WSL9 locus, together with complementation tests indicated that WSL9 encodes a novel protein with an HNH domain. WSL9 was expressed in various tissues. Under low temperature, the wsl9 mutation caused defects in splicing of rpl2, but increased the editing efficiency of rpoB. Expression levels of plastid genome-encoded genes, which are transcribed by plastid-coded RNA polymerase (PEP), chloroplast development genes and photosynthesis-related genes were altered in the wsl9 mutant. CONCLUSION: WSL9 encodes an HNH endonuclease domain-containing protein that is essential for early chloroplast development. Our study provides opportunities for further research on regulatory mechanisms of chloroplast development in rice.

WRKY Transcription Factor OsWRKY29 Represses Seed Dormancy in Rice by Weakening Abscisic Acid Response.

For efficient plant reproduction, seed dormancy delays seed germination until the environment is suitable for the next generation growth and development. The phytohormone abscisic acid (ABA) plays important role in the induction and maintenance of seed dormancy. Previous studies have identified that WRKY transcription factors can regulate ABA signaling pathway. Here, we identified an Oswrky29 mutant with enhanced dormancy in a screen of T-DNA insertion population. OsWRKY29 is a member of WRKY transcription factor family which located in the nuclear. The genetic analyses showed that both knockout and RNAi lines of OsWRKY29 had enhanced seed dormancy whereas its overexpression lines displayed reduced seed dormancy. When treated with ABA, OsWRKY29 knockout and RNAi lines showed greater sensitivity than its overexpression lines. In addition, the expression levels of ABA positive response factors OsVP1 and OsABF1 were higher in the OsWRKY29 mutants but were lower in its overexpression lines. Further assays showed that OsWRKY29 could bind to the promoters of OsABF1 and OsVP1 to inhibit their expression. In summary, we identified a new ABA signaling repressor OsWRKY29 that represses seed dormancy by directly downregulating the expression of OsABF1 and OsVP1.

OsLUGL is involved in the regulating auxin level and OsARFs expression in rice (Oryza sativa L.).

Specification of floral organ identity is critical for floral morphology and inflorescence architecture. Floral organ identity in plants is controlled by floral homeotic A/B/C/D/E-class genes. Although multiple genes regulate floral organogenesis, our understanding of the regulatory network remains fragmentary. Here, we characterized a rice floral organ gene KAIKOUXIAO (KKX), mutation of which produces an uncharacteristic open hull, abnormal seed and semi-sterility. KKX encodes a putative LEUNIG-like (LUGL) transcriptional regulator OsLUGL. OsLUGL is preferentially expressed in young panicles and its protein can interact with OsSEU, which functions were reported as an adaptor for LEUNIG. OsLUGL-OsSEU functions together as a transcriptional co-regulatory complex to control organ identity. SEP3 (such as OsMADS8) and AP1 (such as OsMADS18) serve as the DNA-binding partner of OsLUGL-OsSEU complex. Further studies indicated that OsMADS8 and OsMADS18 could bind to the promoter of OsGH3-8. The altered expression of OsGH3-8 might cause the increased auxin level and the decreased expression of OsARFs. Overall, our results demonstrate a possible pathway whereby OsLUGL-OsSEU-OsAP1-OsSEP3 complex as a transcriptional co-regulator by targeting the promoter of OsGH3-8, then affecting auxin level, OsARFs expression and thereby influencing floral development. These findings provide a valuable insight into the molecular functions of OsLUGL in rice floral development.

Overexpression of OsbHLH107, a member of the basic helix-loop-helix transcription factor family, enhances grain size in rice (Oryza sativa L.).

BACKGROUND: Grain size, which is determined by grain length, grain width, and grain thickness, is an important determinant for grain yield in rice. Identification and characterization of new genes that are associated with grain size will be helpful for the improvement of grain yield in rice. RESULTS: We characterized the grain size mutant, larger grain size 1 (lgs1), derived from rice activation-tagged T-DNA insertion lines. Histological analysis showed that increased cell numbers in the longitudinal direction of spikelet hulls was responsible for the grain mutant phenotype in lgs1. Quantitative real-time PCR (qRT-PCR) analysis further showed that the expression levels of genes associated with the cell cycle in the young panicles of the lgs1 were higher than those in the wild type (WT), which might result in the increased cell numbers in lgs1 spikelet hulls. Insertion site analysis together with transgenic experiments confirmed that the lgs1 phenotype was caused by enhanced expression of truncated OsbHLH107, corresponding to the nucleotide (nt) 331-846 region (i.e., the transcriptional activation region of OsbHLH107) of the OsbHLH107 coding sequence (CDS). OsbHLH107 is a nucleus-localized bHLH transcription factor, which can form a homodimer with itself. Phylogenetic analysis showed that OsbHLH107 belonged to the same subfamily as OsPILs. OsPIL13 (OsPIL1) and OsPIL16 (APG) were reported to regulate grain size in rice. By transgenic experiments, we found that OsPIL11 could also regulate grain size. CONCLUSION: We concluded that OsbHLH107 and its homologs are important regulators of grain size development and might be useful for grain yield improvement in rice.