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.

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.

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.

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.

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.