The normalization of data by choosing suitable reference genes is fundamental for obtaining accurate and reliable results in quantitative real-time polymerase chain reaction (qPCR) analyses. In this study, the expression stability of 12 candidate reference genes of Pinus massoniana under different abiotic stresses was evaluated using four statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The results indicate that the following genes could be used as reference genes under different treatments: Actin 2 (ACT2) and F-box family gene (F-box) for salinity treatment, cyclophilin (CYP) and alpha-tubulin (TUA) for ABA treatment, actin 7 (ACT7) and CYP for drought treatment, actin 1 (ACT1) and ACT7 for cold treatment, ACT1 and CYP for heat treatment, and TUA and ACT2 for the “Total” group. To validate the suitability of the selected reference genes in this study, the Short-Root protein (SHR), Alpha-pinene synthase (APS), and Pyrabactin resistance-like protein (PYL) gene expression patterns were analyzed. The expression patterns had significant biases when the most unstable reference genes were used for normalization, compared with when the optimum reference gene or gene combinations were used for normalization. These results will be beneficial for further studies on gene transcription in early-stage, unlignified seedlings of P. massoniana.

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.

In vascular plants, the importance of R2R3-myeloblastosis (R2R3-MYB) transcription factors (TFs) in the formation of secondary cell walls (SCWs) has long been a controversial topic due to the lack of empirical evidence of an association between TFs and downstream target genes. Here, we found that the transcription factor PmMYB7, which belongs to the R2R3-MYB subfamily, is involved in lignin biosynthesis in Pinus massoniana. PmMYB7 was highly expressed in lignified tissues and upon abiotic stress. As a bait carrier, the PmMYB7 protein had no toxicity or autoactivation in the nucleus. Forty-seven proteins were screened from the P. massoniana yeast library. These proteins were predicted to be mainly involved in resistance, abiotic stress, cell wall biosynthesis, and cell development. We found that the PmMYB7 protein interacted with caffeoyl CoA 3-O-methyltransferase-2 (PmCCoAOMT2)—which is involved in lignin biosynthesis—but not with beta-1, 2-xylosyltransferase (PmXYXT1) yeast two-hybrid (Y2H) studies. Our in vivo coimmunoprecipitation (Co-IP) assay further showed that the PmMYB7 and PmCCoAOMT2 proteins could interact. Therefore, we concluded that PmMYB7 is an upstream TF that can interact with PmCCoAOMT2 in plant cells. These findings lay a foundation for further research on the function of PmMYB7, lignin biosynthesis and molecular breeding in P. massoniana.