N⁶-methyladenosine (m⁶A) RNA modification is the most prevalent form of RNA methylation and plays a crucial role in plant development. However, our understanding of m⁶A modification in Masson pine (Pinus massoniana Lamb.) remains limited. In this study, a complete analysis of m⁶A writers, erasers, and readers in Masson pine was performed, and 22 m⁶A regulatory genes were identified in total, including 7 m⁶A writers, 7 m⁶A erases, and 8 readers. Phylogenetic analysis revealed that all m⁶A regulators involved in Masson pine could be classified into three distinct groups based on their domains and motifs. The tissue expression analysis revealed that the m⁶A regulatory gene may exert a significant influence on the development of reproductive organs and leaves in Masson pine. Moreover, the results from stress and hormone expression analysis indicated that the m⁶A regulatory gene in Masson pine might be involved in drought stress response, ABA-signaling-pathway activation, as well as resistance to Monochamus alternatus. This study provided valuable and anticipated insights into the regulatory genes of m⁶A modification and their potential epigenetic regulatory mechanisms in Masson pine.

Pathogenesis-related protein 1 (PR1) plays important roles in plant responses to both biotic and abiotic stresses; however, its role in mediating defense against pine wood nematode in Pinus massoniana remains unclear. In this study, a total of 63 PR1 family members were identified in P. massoniana using bioinformatics approaches and were named PmPR1-1 to PmPR1-63 based on their phylogenetic relationships. Phylogenetic analysis showed that these members were distributed among four of the six subfamilies. Most of the encoded proteins were hydrophilic, with lengths ranging from 131 to 406 amino acids. Their promoter regions contained multiple cis-acting elements associated with phytohormone signaling and stress responses, and some members formed gene clusters on chromosomes 2, 5, and 9. qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis showed that the clustered genes PmPR1-46, PmPR1-55, PmPR1-56, and PmPR1-61 were significantly upregulated in the early stage of pine wood nematode inoculation in both resistant and susceptible P. massoniana plants, with higher expression levels in resistant plants. Transient overexpression of PmPR1-61 increased SOD and PPO activities as well as proline content while decreasing CAT activity. These results suggest that the PmPR1 family may be involved in the defense response of P. massoniana against pine wood nematode. Among them, PmPR1-55, PmPR1-56, and PmPR1-61 represent candidate resistance genes worthy of further investigation and provide valuable gene resources for elucidating resistance mechanisms and supporting molecular breeding in P. massoniana.

Ubiquitins (Ubs) play a crucial role in plant–pathogen interactions, particularly the RPL40 family, which is essential for protein synthesis. While Pinus massoniana is highly susceptible to pine wilt disease (PWD) caused by Bursaphelenchus xylophilus, the defense mechanisms mediated by RPL40s remain poorly understood. Here, we performed a genome-wide identification of the ubiquitin and ubiquitin-like gene family (PmUBQs) in P. massoniana. We identified 30 PmUBQ genes unevenly distributed across 11 chromosomes, which were classified into six subfamilies based on phylogenetic analysis. An analysis of promoter regions indicated that the PmUBQ genes were enriched with cis-acting elements associated with stress responses, hormone regulation, and development. Specifically, two group II members, PmRPL40-1 and PmRPL40-2, located on chromosomes 12 and 11, respectively, were identified and exhibited distinct responses to B. xylophilus infection in resistant and susceptible P. massoniana. PmRPL40-1 was significantly highly expressed in the 15 days post-inoculation, while PmRPL40-2 was downregulated on day 1 and then upregulated. Moreover, both genes showed peak divergence at 15 days post-inoculation; the expression levels of PmRPL40-1 and PmRPL40-2 in resistant P. massoniana were approximately 1.8- and 3.7-fold higher, respectively, than in susceptible P. massoniana. These patterns suggest that PmRPL40s might be involved in the rapid activation of defense responses and late-stage cell repair. Notably, transient overexpression of PmRPL40-1 in P. massoniana led to a significant 1.6-fold increase in the jasmonic acid (JA) content (p < 0.0001). These findings reveal the key PmUBQ genes and suggest that PmRPL40s contribute to PWD resistance potentially through the modulation of JA signaling, offering potential targets for molecular breeding in P. massoniana.