Abstract

4-Coumarate:CoA ligase(4CL, EC6.2.1.12), located at the end of the phenylpropanoid metabolic pathway, regulates the metabolic direction of phenylpropanoid derivatives and plays a pivotal role in the biosynthesis of flavonoids, lignin, and other secondary metabolites. In order to understand the molecular characteristics and potential biological functions of the4CLgene family in the pomegranate, a bioinformatics analysis was carried out on the identified4CLs. In this study, 12Pg4CLswere identified in the pomegranate genome, which contained two conserved amino acid domains: AMP-binding domain Box I (SSGTTGLPKGV) and Box II (GEICIRG). During the identification, it was found thatPg4CL2was missing Box II. The gene cloning and sequencing verified that this partial amino acid deletion was caused by genome sequencing and splicing errors, and the gene cloning results corrected thePg4CL2sequence information in the ‘Taishanhong’ genome. According to the phylogenetic tree,Pg4CLswere divided into three subfamilies, and each subfamily had 1, 1, and 10 members, respectively. Analysis of cis-acting elements found that all the upstream sequences ofPg4CLscontained at least one phytohormone response element. An RNA-seq and protein interaction network analysis suggested thatPg4CL5was highly expressed in different tissues and may participate in lignin synthesis of pomegranate. The expression ofPg4CLin developing pomegranate fruits was analyzed by quantitative real-time PCR (qRT-PCR), and the expression level ofPg4CL2demonstrated a decreasing trend, similar to the trend of flavonoid content, indicatingPg4CL2may involve in flavonoid synthesis and pigment accumulation.Pg4CL3,Pg4CL7,Pg4CL8, andPg4CL10were almost not expressed or lowly expressed, the expression level ofPg4CL4was higher in the later stage of fruit development, suggesting thatPg4CL4played a crucial role in fruit ripening. The expression levels of4CLgenes were significantly different in various fruit development stages. The res