N⁶-methyladenosine (m⁶A) is one of the most abundant chemical modifications on mRNA in eukaryotes. RNA-binding proteins containing the YT521-B (YTH) domain play crucial roles in post-transcriptional regulation of plant growth, development, and stress response by reading the m⁶A mark. However, the YTH domain-containing RNA-binding protein family has not been studied in a valuable and medicinal tree such as Cinnamomum camphora (C. camphora) yet. In this study, we identified 10 YTH genes in C. camphora, located on eight out of 12 chromosomes. Phylogenetic analysis revealed that these genes can be classified into two major classes, YTHDF (CcDF) and YTHDC (CcDC). Closely related CcYTHs within the same class exhibited a similar distribution of conserved motifs and domain organization, suggesting functional similarities among these closely related CcYTHs. All CcYTH proteins possessed a highly conserved YTH domain, with CcDC1A containing an additional CCCH domain. The liquid–liquid phase separation (LLPS) predictions indicate that CcDC1A, CcDF1A, CcDF1C, CcDF3C, CcDF4C, and CcDF5C may undergo phase transitions. Quantitative expression analysis revealed that tissue-specific expression was observed fo CcYTHs. Notably, there were two genes, CcDF1A and CcDF5C; both exhibited significantly higher expression levels in various tissues than other genes, indicating that the m⁶A-YTH regulatory network in C. camphora might be quite distinct from that in most plants such as Arabidopsis thaliana (A. thaliana) with only one abundant YTH protein. According to the analysis of the up-stream cis-regulatory elements of these YTH genes, these genes could be closely related to stress, hormones, and development. The following stress response experiments further verified that their expression levels indeed changed under both PEG and NaCl treatments. These findings not only provide a foundation for future functional analysis of CcYTHs in C. camphora, but also provide insights into the functions of epigenetic mark m⁶A in forest trees.

Stress-associated proteins (SAPs), belonging to the A20/AN1 zinc finger protein family, are key regulators in plant stress responses. Despite their importance, studies on the SAP gene family in Pinus massoniana are still relatively scarce. This study aimed to systematically identify and characterize SAP genes in P. massoniana and to explore their potential roles in stress response mechanisms. A total of 17 PmSAP genes were identified from P. massoniana. Phylogenetic analysis revealed that these genes group into five distinct clades, and 10 conserved motifs were identified. Using transcriptome data and qRT-PCR, we analyzed their expression patterns and employed yeast systems to validate their transcriptional activities. The responses of PmSAP gene family members to different stress treatments showed significant differences. For example, PmSAP8 and PmSAP12 responded strongly to ABA, MeJA, and H₂O₂ treatments, while PmSAP3 and PmSAP5 showed significant upregulation under ETH and NaCl stress. Yeast experiments indicated that PmSAP6/8/12 were transcriptional activators, and PmSAP3 and PmSAP5 were transcriptional suppressors. The identification and preliminary analysis of PmSAP genes provided a theoretical foundation for understanding stress resistance mechanisms in P. massoniana.

Terpenoids, abundant and structurally diverse secondary metabolites in plants, especially in conifer species, play crucial roles in the plant defense mechanism and plant growth and development. In Pinus massoniana, terpenoids’ biosynthesis relies on both the mevalonate (MVA) pathway and the 2-methyl-D-erythritol-4-phosphate (MEP) pathway, with 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) catalyzing the sixth step of the MEP pathway. In this study, we cloned and conducted bioinformatics analysis of the PmHDS gene from P. massoniana. The results showed that PmHDS shares homology with HDS proteins from other species. Analysis of tissue expression patterns indicated that PmHDS exhibits the highest expression level in xylem tissue, followed by stems, with significantly lowest expression in the apical meristem. Treatment with NaCl, abscisic acid (ABA), ethylene (ETH), methyl jasmonate (MeJA), and salicylic acid (SA) upregulated the expression of PmHDS. Furthermore, we successfully cloned the PmHDS promoter (about 2220 bp) and integrated it into a GUS reporter vector, which resulted in GUS activity being observed in various tissues of Arabidopsis thaliana. Overexpression of the PmHDS gene in A. thaliana significantly increased the content of carotenoids, chlorophylls a and b, and related enzyme activities, as well as the levels of terpenoid derivatives such as cytokinin (CTK), gibberellic acid (GA), and ABA, thereby enhancing the resistance to those abiotic stresses. These findings suggest that PmHDS plays an important role in the terpenoid synthesis pathway. This study provides a theoretical basis for understanding the biosynthesis of terpenoids and lays a foundation for future research on the regulation of terpene synthesis and resistance in molecular breeding.

Transcription factors refer to types of proteins that perform significant functions in the process of gene expression regulation. The ethylene insensitive 3/ethylene insensitive 3-like (EIN3/EIL) family, functioning as significant transcription factors regulating ethylene, plays a critical role in the growth and development of plants and participates in the plant’s response to diverse environmental stresses. Pinus massoniana is an excellent native tree with high economic and ecological value. However, the study of EIN3/EIL genes in gymnosperms, for instance, P. massoniana, is still relatively limited. In this research, four putative EIN3/EIL genes were identified in the transcriptome of P. massoniana. Bioinformatics analysis showed that PmEIL genes contain a highly conserved EIN3 domain and other structural features of acidic, proline-rich and glutamine-rich sites. The molecular evolution tree analysis demonstrated that the EIN3/EIL family was partitioned into three categories (A, B, and C), and the number, type, and distribution of conserved motifs grouped in one category were similar. The results of qRT-PCR indicated that the expression levels of PmEIL genes were markedly elevated in needles compared to other tissues. Through the analysis of expression patterns of the PmEIL genes under various stress treatments, it was found that the PmEIL genes could participate in plant hormone stimulation induction, osmosis, drought and other response processes. In addition, PmEIL is a nuclear localization protein. PmEIL1, PmEIL3, and PmEIL4 are transcriptional activators, while PmEIL2 is a transcriptional suppressor. This research provides a basis for further elucidating the function of EIN3/EIL transcription factors in growth, development and stress response of P. massoniana.