RAL6 encodes a seed allergenic protein that positively regulates grain weight and seed germination

The rice albumin (RAG) gene family belongs to the Tryp_alpha_amyl family. RAG2, specifically expressed in 14-21 DAP (days after pollination) seeds, regulates grain yield and quality. In this study, we identified another RAG family gene, RAL6, which exhibits specific expression in developing seeds, particularly in 7, 10, and 15 DAP seeds. Employing the CRISPR/Cas9 system, we analyzed functions of RAL6 and found that the ral6 lines (ral6-1, ral6-2, ral6-3, and ral6-4) displayed thinner seeds with significantly decreased 1000-grain weight and grain thickness compared to ZH11. Additionally, the cell width of spikelet cells, total protein and glutelin contents were significantly reduced in ral6. The germination assay and 1% TTC staining revealed a significant decrease in seed vigor among the ral6 lines. The alpha-amylase activity in ral6 mutant seeds was also markedly lower than in ZH11 seeds after 2 days of imbibition. Furthermore, co-expression analysis and GO annotation showed that co-expressed genes were involved in immune response, oligopeptide transport, and the glucan biosynthetic process. Collectively, our findings suggest that RAL6 plays a coordinating role in regulating grain weight and seed germination in rice.