Abstract
NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in fruit ripening and quality. The watermelon genome encodes 80 NAC genes, and 21 of these NAC genes are highly expressed in both the flesh and vascular tissues. Among these genes, ClNAC68 expression was significantly higher in flesh than in rind. However, the intrinsic regulatory mechanism of ClNAC68 in fruit ripening and quality is still unknown. In this study, we found that ClNAC68 is a transcriptional repressor and that the repression domain is located in the C-terminus. Knockout of ClNAC68 by the CRISPR-Cas9 system decreased the soluble solid content and sucrose accumulation in mutant flesh. Development was delayed, germination was inhibited, and the IAA content was significantly decreased in mutant seeds. Transcriptome analysis showed that the invertase gene ClINV was the only gene involved in sucrose metabolism that was upregulated in mutant flesh, and expression of the indole-3-acetic acid-amido synthetase gene ClGH3.6 in the IAA signaling pathway was also induced in mutant seeds. EMSA and dual-luciferase assays showed that ClNAC68 directly bound to the promoters of ClINV and ClGH3.6 to repress their expression. These results indicated that ClNAC68 positively regulated sugar and IAA accumulation by repressing ClINV and ClGH3.6. Our findings provide new insights into the regulatory mechanisms by which NAC transcription factors affect fruit quality and seed development.
Highlights
Fruit ripening is a complex process, and multiple metabolic changes, including changes that affect sugar, organic acids, and pigments, occur during fruit ripening and postharvest storage[1]
differentially expressed genes (DEGs) involved in ABA biosynthesis, and we found that the expression of the key ABA biosynthesis gene Cla97C07G137260, encoding 9-cis-epoxycarotenoid dioxygenase (NCED), was significantly lower in the flesh of clnac[68] mutant fruits than in that of WT fruits at 26 days after pollination (DAP) (Figure S5A)
Knockout of ClNAC68 resulted in reduced sugar accumulation, and the total contents of sugar, sucrose, fructose, and glucose were significantly lower in clnac[68] mutant fruits, especially at 26 DAP (Fig. 2)
Summary
Fruit ripening is a complex process, and multiple metabolic changes, including changes that affect sugar, organic acids, and pigments, occur during fruit ripening and postharvest storage[1]. The network of underlying molecular mechanisms that directly regulate fruit ripening and determine fruit quality has been revealed; this network includes hormones and signaling pathways, transcriptional regulators, and other regulatory elements[2]. Numerous studies have shown that enzymes involved in sugar metabolism, including sucrose synthase (Sus), sucrose phosphate synthase (SPS), and invertases (INVs), play important roles in regulating sucrose accumulation in fruit[4]. Among these enzymes, Sus or INVs catalyze the hydrolysis of sucrose into fructose and glucose, and the activity of these two enzymes shows a negative correlation with sugar content[5]. HpWRKY3 was found to activate the expression of HpINV2 and HpSuSy1 to
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