Mutagenesis of <i>SlNAC4</i> by CRISPR/Cas9 alters gene expression and softening of ripening tomato fruit
Ying Gao, Yiping Zhang, Zhongqi Fan, Jing Yuan, Jianye Chen, Donald Grierson, Yang Rui, Daqi Fu
Abstract
Softening is one of the key fruit quality traits, which results from the selective expression of cell wall metabolism genes during ripening. The identification of transcription factors (TFs) that regulate fruit softening is an important field in order to understand and control fruit softening. In tomato, NAC (NAM, ATAF, and CUC) TFs members have been demonstrated to be involved in fruit ripening regulation, including NAC-NOR (non-ripening), NOR-like1, SlNAC4, SlNAC1. Here, we generated <i>slnac4</i> mutant knockout(CR<i>-SlNAC4</i>)tomato plant by a clustered regularly interspaced short palindromic repeats genomic targeting system (CRISPR/Cas9) and <i>SlNAC4</i> overexpressing (OE<i>-SlNAC4</i>) plant. In addition to confirming the previously reported results that SlNAC4 positively regulates fruit ripening, we found that SlNAC4 has a strong effect on tomato fruit softening. Compared with the control fruit, fruit softening was inhibited in <i>slnac4</i> fruit and conversely was accelerated in OE-<i>SlNAC4</i> tomato fruit. Through RNA-sequencing analysis, we found that expression levels of <i>SlEXP1</i> (expansin) and <i>SlCEL2</i> (endo-β-1,4 glucanase) genes involved in cell wall metabolic were significantly different in WT (wild type)/<i>slnac4</i> and WT/OE-<i>SlNAC4</i> fruit. Further study showed that these genes contained a NAC TF binding domain in their promoter regions. In vitro electrophoretic mobility shift assays (EMSA) and dual-luciferase reporter assays (DLR) demonstrated that these two genes were the direct targets of SlNAC4 binding and transactivation. The results enriched the function of <i>SlNAC4</i> and provided a new dimension in understanding the regulation of tomato fruit softening.