Characterization of 4-coumarate CoA ligase (4CL) gene family and functional study of Sm4CL2/3/7/9 in Salvia miltiorrhiza

Abstract

The 4-coumarate CoA ligase (4CL, EC6.2.1.12) enzyme is pivotal for the final step of three shared stages in the general phenylpropane metabolic pathway, which plays a critical role in the biosynthesis of a diverse array of metabolites (e.g., phenolic acids, flavonoids, lignin, anthocyanins, and coumarins). Ten 4CL genes have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), the majority of these genes have not yet been characterized. In this study, the expression profiles of the Sm4CLs gene family were analyzed by qRT-PCR. Among the ten members, Sm4CL7 had the highest expression across various tissues, while Sm4CL5 showed the lowest transcription levels. Notably, Sm4CL2/3/4/6/7/9/10 were significantly responsive to methyl jasmonate (MeJA) treatments. Ten 4CLs were divided into four groups via phylogenetic analysis, while Sm4CL2/3/7/9 that represented each group were selected for further investigation. The GUS staining results of proSm4CL2/3/7/9::GUS transgenic Arabidopsis thaliana were strongly consistent with the qRT-PCR results, and all four Sm4CLs were located in the cytoplasm. Compared with the control, transgenic S. miltiorrhiza plants that overexpressed Sm4CL2/3/7/9 possessed reduced contents of p-coumaric acid and caffeic acid, albeit higher levels of rosmarinic acid and salvianolic acid. In conclusion, this research expands our understanding of the Sm4CL gene family and offers insights into enhancing the quality of S. miltiorrhiza and the production of secondary metabolites through the targeted manipulation of 4CL enzymes.