Characterization and Functional Analysis of <i>PmCMK</i>: A Gene Involved in Terpenoid Synthesis in <i>Pinus massoniana</i>

Abstract

In Pinus massoniana, the methyl-D-erythritol-4-phosphate (MEP) pathway plays a crucial role in the biosynthesis of terpenoids. The fourth step of this pathway is specifically regulated by 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK). In this study, PmCMK (MW892445.1) was isolated. As a member of the GHMP kinase family, PmCMK exhibits homology with CMK genes across diverse species. The examination of relative expression patterns revealed that PmCMK exhibited higher expression levels in tissues of P. massoniana that are rich in resin. We successfully cloned the PmCMK promoter (1654 bp) and integrated it into a GUS reporter vector. This construct was then transformed into the leaves of tobacco (Nicotiana × sanderae) to assess transient expression patterns. The results demonstrated that the promoter was active not only in the roots, leaves, and stems of the tobacco plants but also exhibited varying expression levels in response to treatments with IAA, SA, MeJA, and PEG6000. This suggested that PmCMK expression was modulated by a variety of signals. It revealed that the expression of PmCMK was affected by different treatments. Further allogeneic expression studies showed that tobacco overexpressing PmCMK exhibited increased levels of chlorophyll and carotene compared to the wild type. This enhancement in content indicates that PmCMK has a significant role in isoprene biosynthesis. These findings provide valuable insights for future research aimed at elucidating the biosynthetic pathways of terpenoids and developing breeding strategies to enhance resin production in P. massoniana.

Keywords

• MEP pathway
• CMK
• <i>Pinus massoniana</i>
• terpenoids