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.

To explore the molecular mechanism of the response of Masson pine (Pinus massoniana), the main coniferous tree in southern China, to high CO₂ stress, transcriptome sequencing was carried out to analyze the genome-wide responses of annual seedlings under different durations (0 h, 6 h, 12 h and 24 h) of high CO₂ stress. The results showed that a total of 3080/1908, 3110/2115 and 2684/1483 genes were up-/down-regulated after 6 h, 12 h and 24 h of treatment, respectively, compared with control check group (CK, 0 h). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most of these differentially expressed genes (DEGs) were enriched in energy metabolism, carbohydrate synthesis, cell wall precursor synthesis and hormone regulation pathways. For energy metabolism, the expression of most genes involved in photosynthesis (including the light reaction and Calvin cycle) was generally inhibited, while the expression of genes related glycolysis, the tricarboxylic acid (TCA) cycle and PPP pathway was up-regulated. In addition, the increase in the CO₂ concentration induced the up-regulation of gene expression in the sucrose synthesis pathway. Among all starch synthesis genes, GBSS (granule-bound starch synthase) had the highest expression level. On the other hand, during the synthesis of hemicellulose and pectin (cell wall precursor substances), the expression levels of GMD (GDP-mannose 4,6-dehydratase), MGP (Mannose-1-phosphate guanylyl transferase) and RHM (Rhamnose biosynthetic enzyme) were the highest, suggesting that the synthesis of the raw materials hemicellulose and pectin in Masson pine under stress were mainly supplied by GDP-Man, GDP-Fuc and UDP-Rha. Finally, stress inhibited gene expression in the ABA (Abscisic Acid) synthesis pathway and induced gene expression in the GA (Gibberellin), SA (Salicylic acid), BR(Brassinolide) and MeJA (Methyl Jasmonate) pathways. Stomatal switches were regulated by hormonal interactions. This experiment elaborated on the response and molecular mechanism of Masson pine to CO₂ stress and aided in screening carbon sequestration genes for the corresponding molecular research of Masson pine in the future.

Transcription factors (TFs) are a class of proteins that play an important regulatory role in controlling the expression of plant target genes by interacting with downstream regulatory genes. The lateral organ boundary (LOB) structural domain (LBD) genes are a family of genes encoding plant-specific transcription factors that play important roles in regulating plant growth and development, nutrient metabolism, and environmental stresses. However, the LBD gene family has not been systematically identified in Pinus massoniana, one of the most important conifers in southern China. Therefore, in this study, we combined cell biology and bioinformatics approaches to identify the LBD gene family of P. massoniana by systematic gene structure and functional evolutionary analysis. We obtained 47 LBD gene family members, and all PmLBD members can be divided into two subfamilies, (Class I and Class II). By treating the plants with abiotic stress and growth hormone, etc., under qPCR-based analysis, we found that the expression of PmLBD genes was regulated by growth hormone and abiotic stress treatments, and thus this gene family in growth and development may be actively involved in plant growth and development and responses to adversity stress, etc. By subcellular localization analysis, PmLBD is a nuclear protein, and two of the genes, PmLBD44 and PmLBD45, were selected for functional characterization; secondly, yeast self-activation analysis showed that PmLBD44, PmLBD45, PmLBD46 and PmLBD47 had no self-activating activity. This study lays the foundation for an in-depth study of the role of the LBD gene family in other physiological activities of P. massoniana.

The basic helix-loop-helix (bHLH) protein transcription factor family is the most widely distributed transcription factor family in eukaryotes. Members of this family play important roles in secondary metabolic biosynthesis, signal transduction, and plant resistance. Research on the bHLH family in animals is more extensive than that in plants, and members of the family in plants are classified according to the classification criteria for those in animals. To date, no research on the bHLH gene family in Pinus massoniana (Masson pine) has been reported. In this study, we identified 88 bHLH genes from four transcriptomes of Masson pine and performed bioinformatics analysis. These genes were divided into 10 groups in total. RT-PCR analysis revealed that the expression levels of the six genes increased under abiotic stress and hormone treatments. These findings will facilitate further studies on the functions of bHLH transcription factors.

The methylerythritol phosphate (MEP) pathway provides the universal basic blocks for the biosynthesis of terpenoids and plays a critical role in the growth and development of higher plants. Pinus massoniana is the most valuable oleoresin producer tree with an extensive terrestrial range. It has the potential to produce more oleoresin with commercial value, while being resistant to pine wood nematode (PWN) disease. For this study, eleven MEP pathway associated enzyme-encoding genes and ten promoters were isolated from P. massoniana. Three PmDXS and two PmHDR existed as multi-copy genes, whereas the other six genes existed as single copies. All eleven of these MEP enzymes exhibited chloroplast localization with transient expression. Most of the MEP genes showed higher expression in the needles, while PmDXS2, PmDXS3, and PmHDR1 had high expression in the roots. The expressions of a few MEP genes could be induced under exogenous elicitor conditions. The functional complementation in a dxs-mutant Escherichia coli strain showed the DXS enzymatic activities of the three PmDXSs. High throughput TAIL PCR was employed to obtain the upstream sequences of the genes encoding for enzymes in the MEP pathway, whereby abundant light responsive cis-elements and transcription factor (TF) binding sites were identified within the ten promoters. This study provides a theoretical basis for research on the functionality and transcriptional regulation of MEP enzymes, as well as a potential strategy for high-resin generation and improved genetic resistance in P. massoniana.

Cellulose synthase (CESA) is a key enzyme in the synthesis of cellulose, which plays an important role in cell wall construction and plant growth and development. In this study, seven CesA genes of P. massoniana were identified by searching the transcriptome data. Bioinformatics analysis showed that the putative CESA proteins were composed of 984–1101 amino acids, each containing the typical motifs of CESA proteins. Phylogenetic analysis showed that Transcript4609, Tran-script2643 and Transcript1263 were clustered into three groups with proteins related to regulating secondary wall synthesis, while Transcript691, Transcript1283, Transcript418 and Transcript556 were categorized into three clades with those associated with the formation of the primary cell walls. RT-qPCR analysis showed that the CesA genes were differentially expressed in different tissues, and most of the genes were induced by different abiotic stress and hormones. Transcript4609, Tran-script2643 and Transcript1263 were mainly expressed in the xylem and could respond to drought and salt stress induced by ABA, MeJA, ETH and SA hormones, indicating that these three CesA genes may play an important role in the response to abiotic stress in P. massoniana. This study revealed the possible biochemical and physiological functions of the CesA gene in P. massoniana, which can provide a basis for further exploration of the function of the CesA gene in cell wall formation and the response to external stress.