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.

Pinus massoniana is a major fast-growing timber tree species planted in arid areas of south China, which has a certain drought-resistant ability. However, severe drought and long-term water shortage limit its normal growth and development. Therefore, in this study, physiological indices, and the transcriptome sequencing and cloning of AP2/ERF transcription factor of P. massonsiana were determined to clarify its molecular mechanism of drought stress. The results showed that stomatal conductance (Gs) content was significantly decreased, and superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and abscisic acid (ABA) content were significantly increased under drought stress. Transcriptomic analysis revealed that compared to the control, 9, 3550, and 4142 unigenes with differential expression were identified by comparing plants subjected to light, moderate or severe drought. AP2/ERF with high expression was screened out for cloning. To investigate the biological functions of ERF1, it was over-expressed in wild-type Populus davdianaand × P. bolleana via the leaf disc method. Under drought stress, compared to wild-type plants, ERF1 over-expressing poplar lines (OE) maintained a higher photosynthetic rate and growth, while the transpiration rate and stomatal conductance significantly decreased and water use efficiency was improved, indicating that drought tolerance was enhanced. This study provides an insight into the molecular mechanism of drought stress adaptation in P. massoniana.

Wood formation originates in the biosynthesis of lignin and further leads to secondary cell wall (SCW) biosynthesis in woody plants. Masson pine (Pinus massoniana Lamb) is an economically important industrial timber tree, and its wood yield affects the stable development of the paper industry. However, the regulatory mechanisms of SCW formation in Masson pine are still unclear. In this study, we characterized PmMYB4, which is a Pinus massoniana MYB gene involved in SCW biosynthesis. The open reading frame (ORF) of PmMYB4 was 1473 bp, which encoded a 490 aa protein and contained two distinctive R2 and R3 MYB domains. It was shown to be a transcription factor, with the highest expression in semi-lignified stems. We overexpressed PmMYB4 in tobacco. The results indicated that PmMYB4 overexpression increased lignin deposition, SCW thickness, and the expression of genes involved in SCW formation. Further analysis indicated that PmMYB4 bound to AC-box motifs and might directly activate the promoters of genes (PmPAL and PmCCoAOMT) involved in SCW biosynthesis. In addition, PmMYB4-OE(over expression) transgenic lines had higher lignin and cellulose contents and gene expression than control plants, indicating that PmMYB4 regulates SCW mainly by targeting lignin biosynthetic genes. In summary, this study illustrated the MYB-induced SCW mechanism in Masson pine and will facilitate enhanced lignin and cellulose synthesis in genetically engineered trees.

One of the largest families of transcription factors in plants, the MYB transcription factors family (Myeloblastosis, MYB TF), plays a vital role in regulating plant biochemical and physiological processes. The role of MYB TF in coping with stresses, such as drought, salt and cold, has been reported. Unfortunately, a comprehensive identification of R2R3-MYB TF in Masson pine (Pinus massoniana) has not been achieved. In this study, a total of 49 sequences were identified as R2R3-MYB TF. The structure, function and phylogenetic relationships of the conserved structural domains of Masson pine R2R3-MYB TF and Populus trichocarpa Torr. & A.Gray ex Hook. TFs were compared using bioinformatics tools. The results showed that Masson pine R2R3-MYB TF was divided into 24 groups, mainly located in the nucleus, and mostly lacking signal peptides and transmembrane structural domains with multiple phosphorylation sites. The drought stress-responsive R2R3-MYB gene, PmMYB4, was selected from the drought stress transcriptome based on analysis of the expression pattern and tissue specificity of PmMYB4 gene under abiotic stress using qPCR. The results showed that PmMYB4 can be involved in drought stress treatment through ABA signaling, as well as in multiple stress responses such as salt stress, and there were significant differences in the expression of PmMYB4 in the eight tissues. These results provide a reference scheme for the functional identification of R2R3-MYB transcription factors, which may be involved in plant responses to multiple stresses such as drought, and enrich our understanding of the functions of R2R3-MYB transcription factors in plants.

Phenylpropanoids are crucial for the growth and development of plants and their interaction with the environment. As key transcriptional regulators of plant growth and development, MYB-like transcription factors play a vital role in the biosynthesis of phenylpropanoid metabolites. In this study, we functionally characterized PmMYB6, a Pinus massoniana gene that encodes an R2R3-MYB transcription factor. It was confirmed by qPCR that PmMYB6 was highly expressed in the flowers, xylem, and phloem of P. massoniana. By overexpressing PmMYB6 in tobacco and poplar, we found that transgenic plants had enlarged xylem, increased content of lignin and flavonoids, and up-regulated expression of several enzyme genes of the phenylpropane metabolism pathway to different degrees. The above research results indicate that PmMYB6 is involved in the metabolic flux distribution of different branches of the phenylpropane metabolic pathway, and the results may provide clues for the regulation of metabolic fluxes between flavonoids and the lignin biosynthesis pathways of P. massoniana, as well as provide a basis for the molecular breeding of P. massoniana.

To investigate the clinical effects and security of YiSuiShengXueGranule (YSSXG) on treating 156 patients with beta-thalassemia major. YSSXG was given orally to 156 patients with beta-thalassemia in GuangXi Autonomous Region (the high incidence area of beta-thalassemia in China) for 3 months as one therapeutic course, 3 times a day, 10 g each time (for children, the dose should be reduced properly according to their body weight and age), and no blood transfusion used during the course. Clinical symptoms and levels of hemoglobin (Hb), red blood cell (RBC), reticulocyte (Ret) and hemoglobin F (HbF) were observed before and after treatment, and side-effects were observed during the course. A 3-6 months follow up study was performed after withdrawal of YSSXG. And systemic gene analysis was conducted with PCR, SSCP-PCR, RT-PCR and DNA sequences analysis and mRNA differently expression technique, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation. Levels of Hb, RBC, Ret and HbF obviously elevated, and clinical symptoms markedly ameliorated in patients after treated with YSSXG from the 1st to 3rd month (all p<0.01). Dynamical observation showed that the improvement of symptoms kept accordance with the elevation of hemorrheological indexes. The treatment was effective in 145 patients and ineffective in 11, and the total effective rate was 92.9%, without any adverse reaction founded. Follow-up studies showed the therapeutic effect could sustain for 3 to 4 months after drug-withdrawal. The molecular mechanism study showed: YSSXG did not change the genetic mutation type, but could obviously increase gamma/(beta+gamma) globin ratio, both gamma-globin mRNA and GM-CSF mRNA expression were significantly enhanced so as to induce HbF synthesis increasing after treated with YSSXG. YSSXG had obvious effects in treating beta-thalassemia by unlocking gamma-gene, increasing the gamma-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has provided a new path for the treatment of beta-thalassemia with Traditional Chinese Medicine.