Rice OsGA2ox9 regulates seed GA metabolism and dormancy

Seed development is important for grain yield and quality and finely controlled by complex regulatory networks involving various factors. Phytohormone gibberellin (GA) plays an essential role in late embryogenesis of Arabidopsis and GA metabolism and signalling-related genes are preferentially expressed in rice embryo. Considering the effect in promoting seed germination, how to ensure that GA will not promote germination of early developing seeds, that is, coordinates the embryo development regulation and germination inhibition, is largely unknown. C20 GA 2-oxidase (GA2ox) hydroxylates C20-GA precursors to render them being converted to active GAs, thus reducing bioactive GA levels and regulating rice growth (Lo et al., 2008). Investigation of expression pattern of GA-synthesis/metabolism-related genes showed that OsGA2ox9 is transcribed in various tissues including root, leaf, pollen and seed, and relatively highly expressed in ovary and early developing seed (1 day after pollination) during seed development (Figure S1A). Promoter-reporter (GUS) fusion study further revealed the OsGA2ox9 transcription in spikelet and young seeds, particularly the embryo surrounding region (ESR) at early developmental stages (Figures 1a, S1B). ESR contains several cell layers around young embryo and functions in exchanging signals between embryo and endosperm (Dai et al., 2021). Distinct expression at ESR suggests a role of OsGA2ox9 in early seed development. Loss-of-function mutant was generated by CRISPR-Cas9 technology and six independent homozygous lines were obtained (Figure 1b). Measurement revealed the increased levels of GA precursors (GA53, GA19 and GA20) and bioactive GA (GA1 and GA7) in OsGA2ox9-Cas9 developing seeds (Figure 1c). Compared to ZH11, OsGA2ox9-Cas9 plants presented no visible abnormality during vegetative growth except for less seed setting, longer panicles, and more branches of panicles (Figure S2), while overexpression of OsGA2ox9 driven by native promoter (OsGA2ox9-OE) resulted in extremely dwarf and delayed heading time (Figure S3A). Analysis showed the altered germination rate under OsGA2ox9 deficiency or overexpression (Figure 1d). Interestingly, dry OsGA2ox9-Cas9 grains showed advanced embryo germination (Figure 1e, left) and paddy field-growing plants presented obvious preharvest sprouting (PHS) under successive rainy weather (Figure 1e, right). Consistently, increased grain dormancy of OsGA2ox9-OE was observed (Figure S3B), which can be rescued by exogenous GA3 (Figure 1f), confirming the OsGA2ox9 effect in regulating seed dormancy through altering GA metabolism. Dormancy of seed is with ecological importance of seed spreading. During breeding, varieties with rapid germination, fast growth and high yield are selected, leading to loss of seed dormancy, which causes seed germination on panicle when encountering high temperature and rainy weather in harvest season (the preharvest sprout, PHS). Abscisic acid (ABA) and GA antagonistically regulate seed development and dormancy, and ABA- and GA-mediated PHS has been observed in cereals rice, wheat and maize (Skubacz and Daszkowska-Golec, 2017; Xu et al., 2022), however, how GA functions in PHS remains elusive. GA stimulates the expression of α-amylase encoding genes and whose abnormally high expression after wheat ripens will result in PHS (Simsek et al., 2014). A preliminary agar plate assay showed a much higher α-amylase activity of OsGA2ox9-Cas9 seeds, compared to ZH11 (Figure 1g, left). Consistently, transcription analysis confirmed the significantly increased expression of α-amylase encoding genes OsAmy (1B, 1C, 3A, 3B, 3C and 3E) in OsGA2ox9-Cas9 seeds (Figure 1g, right). The promoter activities of OsAmy1C, 3A, 3B and 3E were detected in the endosperm adjacent to scutellum (Nakata et al., 2017), which closely related to OsGA2ox9 expression, suggesting that OsGA2ox9 might regulate the transcription of α-amylase encoding genes through GA signalling. As α-amylases hydrolyse starch granules into soluble sugars, measurement indeed showed the increased contents of glucose and sucrose in OsGA2ox9-Cas9 seeds (Figure 1h). Sugar of rice endosperm is sufficient to suppress the expression of ABA signalling genes and result in PHS (Du et al., 2018), suggesting that sugar may suppress seed dormancy through modulating ABA signalling. Indeed, developing OsGA2ox9-Cas9 panicles showed reduced dormancy (higher germination) under ABA (Figure 1i), and transcriptions of OsABI3 and OsABI5, key positive regulators controlling seed dormancy, were significantly decreased (Figure 1j), indicating that GA-promoted starch hydrolysis led to reduced ABA signalling. It is thus proposed that OsGA2ox9 maintains the relatively low-level bioactive GAs in early developing grains under normal conditions by hydroxylating GA precursors. As a result, transcriptions and activities of α-amylases are suppressed, resulting in low concentrations of soluble sugars and hence released inhibition on ABA signalling, to ensure the normal seed dormancy and inhibited advanced germination (Figure 1k). OsGA2ox9 prevents GA in embryo from entering endosperm to inhibit GA-soluble sugar-ABA signalling, ensuring that early seeds (embryo-maturation stage) will not germinate in advance. Our finding is consistent with the recent study showing that disruption of ABA biosynthesis gene OsNCED3 impairs GA signalling pathway, leading to increased bioactive GAs in seeds and enhanced PHS in rice. Among the altered GA metabolism genes, OsGA2ox9 was significantly downregulated in Osnced3 mutant (Chen et al., 2022). A series of PHS-related QTLs or genes in cereals, while few GA-related phs mutants, have been identified (Skubacz and Daszkowska-Golec, 2017). We demonstrated the role of OsGA2ox9 in determining seed dormancy by regulating contents of soluble sugars, providing direct evidence and revealing the significance of GA metabolism in PHS. Over-accumulated sugars and reduced ABA signalling of OsGA2ox9-Cas9 developing grains confirmed that endosperm sugar involves in seed dormancy determination, which elucidates the GA-soluble sugar-ABA regulation in cereal PHS and sets up a novel link of GA-ABA signalling in regulating seed dormancy. By collecting single nucleotide polymorphisms (SNPs) across OsGA2ox9 mRNA region from 3K rice genome database, 14 major SNPs were identified, of which nine SNPs located at open reading frame region (Figure S4A). Further analysis showed that OsGA2ox9 could be divided into 14 different haplotypes (Hap1-14) (Table S2) and Hap1-4 were major groups (Hap4 was reference type, Nipponbare) (Figure S4B). Based on the germination rate (GR) data (Zhao et al., 2022), Hap1 and Hap3 had significant decrease in GR (Figure 1l) and one common nonsynonymous SNP at nucleotide position 155 (adenosine to guanosine) and two synonymous SNPs at nucleotide positions 3818 and 4146 were characterized, all three SNPs are associated with reduced GR (Figure S4C). There is no significant difference of Hap2 compared to Hap4, probably owing to SNP 3872 in Hap2, which is correlated with increased GR (Figure S4C). Interestingly, Hap1 and Hap3 are the main group of indica rice (47.19% of indica rice belongs to Hap1), whether and how SNPs contribute to seed germination and dormancy, particularly the differentiation of japonica and indica rice, worth further investigating and may help on relevant breeding. Besides rice, PHS is also a serious problem in wheat and maize. Phylogenetic analysis revealed the presence of OsGA2ox9 orthologs in cereal species particularly maize and wheat (Figure S5), indicating a possibly conserved regulatory mechanism of PHS in monocots and suggesting OsGA2ox9 a useful genetic resource for cultivating PHS-resistant varieties. A seed-specific expression of OsGA2ox9 may help to remove the adverse effects by OsGA2ox9 overexpression in vegetative tissues, and generation of beneficial alleles by SNP substitute can be applied for improving PHS resistance. This work was supported by the National Natural Science Foundation of China (NSFC, 31771741). The authors declare no conflict of interest. H.X designed the project and wrote the paper; M.X and S.C collected the materials, performed the experiments and drafted the manuscript; S.C, M.X and X.Z analysed the data; and X.Z conducted the evolutionary correlation analysis. Figure S1 Rice OsGA2ox9 is transcribed in various tissues. Figure S2 Characterization of OsGA2ox9-Cas9 and OsGA2ox9 overexpression lines. Figure S3 OsGA2ox9 regulates rice growth and seed dormancy. Figure S4 Haplotype analysis of OsGA2ox9 across open reading frame region in 3,000 rice accessions (3K rice genome). Figure S5 Phylogenetic tree of OsGA2ox9. Table S1 Primers used in this study. Table S2 SNPs combination of 14 haplotypes. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.