CRISPR/Cas9-mediated mutagenesis of ClBG1 decreased seed size and promoted seed germination in watermelon

Abstract Abscisic acid (ABA) is a critical regulator of seed development and germination. β-glucosidases (BGs) have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA. However, whether BGs are involved in seed development is unclear. In this study, a candidate gene, ClBG1 , in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system. Seed size and weight were significantly reduced in the Clbg1 -mutant watermelon lines, which was mainly attributed to decreased cell number resulting from decreased ABA levels. A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination (DAP), respectively. Cytoskeleton- and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1- mutant lines during seed development. Moreover, the expression of genes in the major signaling pathways of seed size control was also changed. In addition, seed germination was promoted in the Clbg1 -mutant lines due to decreased ABA content. These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-, cell cycle- and signaling-related genes. Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size, a key agricultural trait of significant economic importance.