In the methyl-D-erythritol-4-phosphate (MEP) pathway, 1-deoxy-D-xylose-5-phosphate synthase (DXS) is considered the key enzyme for the biosynthesis of terpenoids. In this study, PmDXS (MK970590) was isolated from Pinus massoniana. Bioinformatics analysis revealed homology of MK970590 with DXS proteins from other species. Relative expression analysis suggested that PmDXS expression was higher in roots than in other plant parts, and the treatment of P. massoniana seedlings with mechanical injury via 15% polyethylene glycol 6000, 10 mM H₂O₂, 50 μM ethephon (ETH), 10 mM methyl jasmonate (MeJA), and 1 mM salicylic acid (SA) resulted in an increased expression of PmDXS. pET28a-PmDXS was expressed in Escherichia coli TransB (DE3) cells, and stress analysis showed that the recombinant protein was involved in resistance to NaCl and drought stresses. The subcellular localization of PmDXS was in the chloroplast. We also cloned a full-length 1024 bp PmDXS promoter. GUS expression was observed in Nicotiana benthamiana roots, stems, and leaves. PmDXS overexpression significantly increased carotenoid, chlorophyll a, and chlorophyll b contents and DXS enzyme activity, suggesting that DXS is important in isoprenoid biosynthesis. This study provides a theoretical basis for molecular breeding for terpene synthesis regulation and resistance.

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

Basic leucine zipper (bZIP) transcription factors (TFs) are ubiquitous in eukaryotes. Members of this family play significant roles in the regulation of plant growth, signal transduction, and various stresses. To date, bZIP TFs have been extensively studied in various plants, but there is little information about them in Pinus massoniana Lamb. In this study, 55 bZIP TFs were identified based on data from four different P. massoniana transcriptomes, and a systematic analysis was performed. According to the phylogenetic results, P. massoniana bZIP TFs were divided into 11 groups. Each bZIP protein contained a highly conserved bZIP domain, and the numbers and types of motifs were similar in the same group. The PmbZIPs were nuclear localization proteins. Based on the pine wood nematode inoculation transcriptome, the transcriptional profiles revealed that 25 PmbZIP genes could respond to pine wood nematodes at different levels. Genes PmbZIP3, PmbZIP4, PmbZIP8, PmbZIP20, and PmbZIP23 were selected to be upregulated in the process of inoculation with pine wood nematodes. These five genes had different expression levels in different tissues and were responsive to the related treatment conditions. Transcriptional activity analysis showed that PmbZIP3 and PmbZIP8 were transcriptional activators; PmbZIP4, PmbZIP20 and PmbZIP23 were transcriptional repressors. These findings provide preliminary information on PmbZIP TFs, which is helpful for further study of other physiological functions of bZIP TFs in P. massoniana.