Molecular mechanisms underlying seed enlargement mediated by seed coat development in autotetraploid castor bean

Polyploids, particularly synthetic lines, often produce larger seeds than their diploid progenitors. However, little is known regarding the role of seed coat development in the larger seeds formation in polyploid plants. Here, we investigated the cellular and molecular basis by which seed coat development mediates seed enlargement in the oilseed crop castor bean (Ricinus communis L.) following homologous chromosome doubling. We found that seeds of autotetraploid castor bean, including seed length, width, thickness and hundred-seed weight, were significantly greater than those of diploids. Histological analysis further revealed that the enlarged seeds were associated with increased seed coat cell area. At the transcriptome level, we showed that genome doubling led to stage-specific and non-linear transcriptional changes. A total of 6043 non-redundant differentially expressed genes (DEGs) were identified between tetraploid and diploid seed coats. Among them, several key genes related to cell expansion and cell wall loosening (e.g. RcEXPA1/5/9/12/15 and RcEXLA1, RcEXTs, RcELP, RcCESA4/7/8, RcXYL1, and RcXTH15/27/30) were significantly up-regulated in tetraploids, consistent with enhanced seed coat expansion. In addition, lignin biosynthesis-related genes (RcPAL1, RcCOMT1, Rc4CL2, RcCSE, RcC4H, RcCCRs, RcCADs, RcCCoAOMT1, and RcHCT) were significantly down-regulated in tetraploids, which may facilitate seed coat cell enlargement. The stage-specific DEGs in the indole-3-acetic acid (IAA), gibberellin, and brassinosteroid signaling pathways (RcIAA3/13/16/26/27, RcRGL2, and RcBSK6) may contribute to regulating seed coat cell development. Importantly, co-expression analysis together with yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase reporter (DLR) assays showed that RcPDF2 bound to two L1-box elements in the RcEXLA1 promoter and increased its promoter activity, suggesting that RcPDF2 contributes to seed size determination by modulating seed coat cell expansion. Collectively, our study provides important insights into the underlying molecular basis of gene dosage effects on seed size formation in autopolyploid plants, thereby laying a solid foundation for enhancing yield in castor bean through polyploid breeding.