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.

The basic helix–loop–helix (bHLH) family members are involved in plant growth and development, physiological metabolism, and various stress response processes. Pinus massoniana is a major turpentine-producing and wood-producing tree in seasonally dry areas of southern China. Its economic and ecological values are well known. The forestry industry holds it in exceptionally high regard. Drought severely limits the growth and productivity of P. massoniana, and the functional role of PmbHLH58 in drought stress is not clear. Therefore, PmbHLH58 was cloned from P. massoniana and its bioinformation was analyzed. Subcellular mapping of the gene was performed. The biological function of PmbHLH58 overexpression in Populus davidiana × P. bolleana was studied. The results show that the drought tolerance of PmbHLH58-overexpressed poplar was significantly improved, which may be due to the increase in water use efficiency and reactive oxygen species (ROS) accumulation under drought stress. In an ethylene-responsive manner, PmERF71 interacted with the PmbHLH58 protein, which was found by yeast two-hybridization. We further demonstrated that the drought-induced PmbHLH58 transcription factor increased the expression of key enzyme genes in ABA receptor family genes in PmbHLH58-overexpressing poplar lines (OE). These findings provide new insights into transcriptional regulation mechanisms related to drought stress and will promote the progression of the genetic improvement and plantation development of P. massonsiana.

MYB transcription factors (TFs) have been shown to be important regulators of plant growth and development, and the R2R3−MYB family plays an important role in the regulation of plant primary and secondary metabolism, growth, and development. In this study, the PmMYB45 gene sequence was successfully cloned from Pinus massoniana, with an open reading frame of 1476 bp, encoding 491 amino acids. The results of subcellular localization and transcriptional self-activation showed that the gene was localized in the nucleus and was self-activating. qPCR showed that the highest expression of PmMYB45 was found in stems. PmMYB45 can promote lignin synthesis when overexpressed in tobacco. These results suggest that PmMYB45 promotes lignin synthesis by regulating the lignin biosynthesis pathway. These findings improve our understanding of the mechanism of lignin biosynthesis in P. massoniana and provide a basis for molecular breeding functions. It also provides insights into the role of the MYB transcription factor family in lignin accumulation.