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.

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.

CCCH-type zinc finger proteins play an important role in multiple biotic and abiotic stresses. More and more reports about CCCH functions in plant development and stress responses have appeared over the past few years, focusing especially on tandem CCCH zinc finger proteins (TZFs). However, this has not been reported in Pinaceae. In this study, we identified 46 CCCH proteins, including 6 plant TZF members in Pinus massoniana, and performed bioinformatic analysis. According to RT-PCR analysis, we revealed the expression patterns of five RR-TZF genes under different abiotic stresses and hormone treatments. Meanwhile, tissue-specific expression analysis suggested that all genes were mainly expressed in needles. Additionally, RR-TZF genes showed transcriptional activation activity in yeast. The results in this study will be beneficial in improving the stress resistance of P. massoniana and facilitating further studies on the biological and molecular functions of CCCH zinc finger proteins.

Pinus massoniana is a species used in afforestation and has high economic, ecological, and therapeutic significance. P. massoniana experiences a variety of biotic and abiotic stresses, and thus presents a suitable model for studying how woody plants respond to such stress. Numerous families of transcription factors are involved in the research of stress resistance, with the GRAS family playing a significant role in plant development and stress response. Though GRASs have been well explored in various plant species, much research remains to be undertaken on the GRAS family in P. massoniana. In this study, 21 PmGRASs were identified in the P. massoniana transcriptome. P. massoniana and Arabidopsis thaliana phylogenetic analyses revealed that the PmGRAS family can be separated into nine subfamilies. The results of qRT-PCR and transcriptome analyses under various stress and hormone treatments reveal that PmGRASs, particularly PmGRAS9, PmGRAS10 and PmGRAS17, may be crucial for stress resistance. The majority of PmGRASs were significantly expressed in needles and may function at multiple locales and developmental stages, according to tissue-specific expression analyses. Furthermore, the DELLA subfamily members PmGRAS9 and PmGRAS17 were nuclear localization proteins, while PmGRAS9 demonstrated transcriptional activation activity in yeast. The results of this study will help explore the relevant factors regulating the development of P. massoniana, improve stress resistance and lay the foundation for further identification of the biological functions of PmGRASs.