OsSPL5 promotes rice outcrossing efficiency by G‐protein pathway

The yield of rice F1 hybrid seed production is influenced by parental line traits, including stigma exsertion rate (SER), which impacts seed pricing and utilization (Marathi and Jena, 2015). SER is highly susceptible to environmental fluctuations, phenotypic and complex genetic factors (Miyata et al., 2007). Although over 40 QTLs related to SER have been identified, none have been molecularly characterized due to differences in genetic background and small additive effects (Zhu et al., 2023). We have demonstrated a positive correlation between stigma size and SER previously (Tan et al., 2023). Then we previously located the stigma size gene SER1 within a 470 kb interval on chromosome 2 based on SSSL-42, a Single Segment Substitution Line with Huajingxian74 (HJX74) as the recipient parent (Tan et al., 2021). In this study, homozygous recombinant lines derived from the crossing of SSSL-42 and HJX74 allowed the region of SER1 to be narrowed down to a 29.48 kb stretch flanked by markers QY18 and LST2 (Figure 1a, Table S1). The line R4, with the shortest substitution segment, was identified as a near-isogenic line for SER1 (NIL-SER1), while the HJX74 was referred to as NIL-ser1 (Figure 1a). The NILs did not differ from one another in many agronomic traits, but the significant difference in SER and stigma size were detected between NILs (Figures 1b–e and S1). There are three candidate genes (OsSPL5, OsCH240 and OsSm-F) detected related to the mapped interval. Variant analysis revealed that OsSPL5 harbours two nucleotide polymorphisms in the third exon in the mapped interval, resulting in amino acid substitutions (Figure S2). The transcriptome assays of the stigma revealed no significant differences in the gene expression among these three genes between the NILs (Figure S3). To investigate the candidate gene for SER1, we obtained over-expression lines and knockout lines for the three candidate genes. Either the gene-edited lines in the NIL-SER1 background or over-expression lines in the NIL-ser1 background, the transgenic lines of OsCH240 and OsSm-F exhibited no phenotypic changes in stigma size (Figure S4). However, the over-expression of OsSPL5 resulted in enlarged stigmas, whereas the knockout of OsSPL5 led to smaller stigmas (Figure 1g–j). Furthermore, the stigma exertion rate changed accordingly in different transgenic lines of OsSPL5 (Figure 1e,g,h). Thus, the candidate gene for SER1 is OsSPL5. The further RT-qPCR assay detected no variation in transcriptional levels across different tissues (Figure S5). Furthermore, the stigma size dramatically decreased in the gene-edited lines, KO-SER1-3rd exon (Figure S6). It strongly suggests that the sequence variation located in the third exon of OsSPL5 is the primary cause of the phenotypic differences between NILs. Scanning electron microscopy (SEM) indicated a significant increase in the epidermal cell length of both stigma brush (SB) and no-brush parts (SNB) in NIL-SER1 (Figures 1f and S7). Anatomical observations revealed that stigma size differences between NILs gradually increased as the spikelet developed (Figure S8). These results indicated that OsSPL5 regulates stigma size in rice by affecting cell size, which subsequently impacts SER. OsSPL5 is an important member of the SPL family, and subcellular localization indicates that it is primarily located in the nucleus (Figure S9). Transcriptional activation assays elucidated that the activation domain of OsSPL5 is located in the N-terminal region (Figure S10). Transcriptome analysis identified 3331 and 759 significant different expression genes in NIL-SER1 vs NIL-ser1 and NIL-ser1 vs KO-SER1, respectively (Table S2). Single transcription factor differentially regulate downstream target genes based on their functional strength in diverse allelic backgrounds, while gene knockout typically results in loss of function. A total of 379 genes exhibited increased expression in NIL-SER1 compared to NIL-ser1, while exhibiting decreased expression in KO-SER1 compared to NIL-SER1 (Figure 1k and Table S2). The Cut&Tag-Seq assays with GFP-SER1 fusion-transformed protoplasts revealed 2153 promoter- (−1000 to 0) related peaks associated with 1852 genes (Figure 1l; Table S3). Subsequent association analysis pinpointed 27 candidate downstream target genes of SER1, which displayed upregulation in NIL-SER1 and down regulation in KO-SER1 (Figure 1k; Table S4). Notably, DEP1, a gene that encodes the γ subunit of the heterotrimeric G-protein and is known as a crucial factor for spikelet and flower development in rice (Huang et al., 2009, 2022), was identified as one of the key candidate genes. The enrichment of SBP binding motifs (GTAC) was observed in its promoter, overlapping with the Cut&Tag peak summit (Figure 1k,l). Alphafold3 docking illustrated a strong binding interaction between the DEP1 promoter motif and SER1 (Figure S11). The combination of data from Cut&Tag-Seq and further assays of DAP-Seq illustrated a ~230 bp binding window of SER1 in the DEP1 promoter (Figure S12). The further promoter-LUC assay confirmed that SER1 binding to the DEP1 promoter enhanced downstream gene expression (Figure 1m). This finding was reinforced by Y1H assays, indicating a positive regulatory relationship between SER1 and DEP1 (Figure 1n). The investigation of relationship between DEP1 alleles and stigma size elucidated that the NIL-dep1-ser1 shows an elevation in stigma width compared to NIL-ser1 (NIL-DEP1-ser1) (Figure 1o,p). Furthermore, DEP1 knockout lines derived from NIL-SER1 showed a significant reduction in stigma size (Figure 1q,r). Additionally, the over-expression of SER1 resulted in shorter panicles, while KO-SER1 exhibited elongated panicles (Figures S13 and S14), consistent with known DEP1 functions. These findings suggest that SER1 exerts a positive regulatory effect on DEP1, thereby modulating stigma and panicle development in rice through the G-protein signalling pathway. To explore the natural variation of the SER1 gene, a total of 2042 Oryza accessions displaying extensive genetic diversity were analysed (Yao et al., 2019). SNP analysis of OsSPL5 indicated that SER1 and ser1 are the two predominant haplotypes. These two haplotypes are widespread in wild rice, but in cultivated rice, nearly all varieties of the indica subspecies carry ser1, while the japonica subspecies predominantly carry SER1. This indicates near complete differentiation between indica and japonica rice at this locus (Figure 1s; Table S5). The nucleotide diversity (π) of the SER1 gene is extremely low within the two cultivated rice subspecies (Figure 1t). This suggests significant potential for SER1 in indica rice breeding programs. The downstream DEP1 gene also shows strong inter-subspecific differentiation, suggesting co-selection during domestication (Figure S15; Table S5). We further introgressed the SER1 gene into lines of P132-16A and P132-16B, which is an indica male sterile line and its corresponding restorer line derived from HJX74 with a ser1 genetic background (Figure 1u–w). The restorer line, P132-16B-SER1, exhibited a heritably higher SER compared to both HJX74 and P132-16B (Figure 1x,y). The generated P132-16A-SER1 lines, which maintained pollen sterility (Figure 1w), showed a higher seed-setting rate than P132-16A in outcrossing rates analysis (Figure 1z). These results indicate the potential for effective application of SER1 in rice breeding programs aimed at improving hybrid seed production. In this study, we identified SER1 (synonymous with OsSPL5) as a pivotal regulator of stigma exertion rate and stigma size in rice. Previous research has demonstrated that the SPL family influences inflorescence morphology (Wang and Zhang, 2017). The potential interaction between SER1 and DEP1 suggests an intricate regulatory network. The differential expression of genes, such as OsRAC3, OsBMY4 and OsGASR2, further suggests extensive genetic interactions (Table S4). Previous studies have elucidated that stigma exsertion is crucial for hybrid seed production and significantly impacts rice domestication. The transition from the high SER and outcrossing behaviour of wild rice to the low SER and predominantly self-pollinating behaviour in cultivated rice has been reported (Zhu et al., 2023). Haplotype analysis revealed higher nucleotide diversity for SER1 in wild rice, suggesting additional functional roles and highlighting the evolutionary significance of SER in rice domestication. This work supported by Biological Breeding-National Science and Technology Major Project (2023ZD04069), the National Natural Science Foundation of China (32401881, 32201841, 91435207), the Key Research and Development Program of Guangdong Province (2022B0202060002), the major science and technology research projects of Guangdong Laboratory for Lingnan Modern Agriculture (NT2021001), and the China Postdoctoral Science Foundation (2021M701265, 2022M721213). The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Figures S1-S15 Supplementary Figures. Tables S1-S5 Supplementary Tables. 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.