Pinus massoniana Lamb, an economically important conifer tree, is widely distributed in China. WRKY transcription factors (TFs) play important roles in plant growth and development, biological and abiotic stress. Nevertheless, there is little information about the WRKY genes in P. massoniana. By searching for conserved WRKY motifs in transcriptomic RNA sequencing data for P. massoniana, 31 sequences were identified as WRKY TFs. Then, phylogenetic and conserved motif analyses of the WRKY family in P. massoniana, Pinus taeda and Arabidopsis thaliana were used to classify WRKY genes. The expression patterns of six PmWRKY genes from different groups were determined using real-time quantitative PCR for 2-year-old P. massoniana seedings grown in their natural environment and challenged by phytohormones (salicylic acid, methyl jasmonate, or ethephon), abiotic stress (H₂O₂) and mechanical damage stress. As a result, the 31 PmWRKY genes identified were divided into three major groups and several subgroups based on structural and phylogenetic features. PmWRKY genes are regulated in response to abiotic stress and phytohormone treatment and may participate in signaling to improve plant stress resistance. Some PmWRKY genes behaved as predicted based on their homology with A. thaliana WRKY genes, but others showed divergent behavior. This systematic analysis lays the foundation for further identification of WRKY gene functions to aid further exploration of the functions and regulatory mechanisms of PmWRKY genes in biological and abiotic stress 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.

N⁶-methyladenosine (m⁶A) is essential for RNA metabolism in cells. The YTH domain, conserved in the kingdom of Eukaryotes, acts as an m⁶A reader that binds m⁶A-containing RNA. In plants, the YTH domain is involved in plant hormone signaling, stress response regulation, RNA stability, translation, and differentiation. However, little is known about the YTH genes in tea-oil tree, which can produce edible oil with high nutritional value. This study aims to identify and characterize the YTH domains within the tea-oil tree (Camellia chekiangoleosa Hu) genome to predict their potential role in development and stress regulation. In this study, 10 members of the YTH family containing the YTH domain named CchYTH1-10 were identified from C. chekiangoleosa. Through analysis of their physical and chemical properties and prediction of subcellular localization, it is known that most family members are located in the nucleus and may have liquid–liquid phase separation. Analysis of cis-acting elements in the CchYTH promoter region revealed that these genes could be closely related to abiotic stress and hormones. The results of expression profiling show that the CchYTH genes were differentially expressed in different tissues, and their expression levels change under drought stress. Overall, these findings could provide a foundation for future research regarding CchYTHs in C. chekiangoleosa and enrich the world in terms of epigenetic mark m⁶A in forest trees.

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.