Kai Gao

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.

This study conducted a comprehensive analysis of the SsuWOX gene family in Schima superba, elucidating its role in plant growth and stress response mechanisms. The genome contains 15 WOX genes primarily encoding nuclear proteins unevenly distributed across 18 chromosomes. Phylogenetic classification grouped these genes into three distinct subfamilies, with members in each subfamily showing conserved gene structures. Interaction network analysis and cis-regulatory element characterization revealed that SsuWOX gene expression is influenced by hormones and various abiotic stresses. Tissue-specific expression profiles showed six genes exhibiting spatial specificity with significant expression level variations across developmental stages. Notably, SsuWOX1 overexpression in callus tissue significantly elevated CLAVATA3 (CLV3) expression levels. CLV3, a crucial small peptide signaling molecule, primarily regulates stem cell maintenance and differentiation in the shoot apical meristem (SAM). Transgenic callus cells displayed bud-like cell characteristics, including increased cell density and organized spatial arrangement. These findings establish a foundation for functional characterization of SsuWOX1 and provide insights into its regulatory mechanisms in plant development.

Pine wilt disease has caused great economic loss and become an ecological threat since it was introduced into East Asia. In China, Pinus massoniana Lamb. is highly susceptible. The pathogenic of this disease is linked to the invasion of P. massoniana by the pine wood nematode (PWN, Bursaphelenchus xylophilus), leading to various physiological activities. However, the migration pathway of PWN and the defense mechanisms of P. massoniana tissue structure following invasion remain unclear. This knowledge is vital for understanding the pathogenesis of pine wood nematode disease. To address this issue, we analyzed the tissue structure damage, horizontal and vertical migration pathways, and histochemical reactions of P. massoniana after PWN inoculation. The results are as follows: susceptible P. massoniana exhibited more tissue structure damage compared to highly resistant P. massoniana. PWN reproduced and migrated by feeding on and damaging cells. In susceptible P. massoniana, PWN propagated and migrated throughout the entire plant. Highly resistant P. massoniana displayed limited horizontal and vertical migration of PWN, making it challenging for PWNs to move from cambium to xylem. After P. massoniana was damaged by PWNs, a protein cross-linking phenomenon appeared rapidly, with highly resistant P. massoniana exhibiting less protein cross-linking than the susceptible variety. Lignin synthesis is a crucial factor in the tissue defense of P. massoniana. Protein crosslinking provides time for lignin synthesis and is an vital component of tissue defense.

Pinus massoniana Lamb. is a crucial timber and resin conifer in China, but its plantation industry is threatened by outbreaks of pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (pinewood nematode; PWN). However, as of yet, there is no comprehensive analysis of NBS-LRR genes in P. massoniana involved in its defense against PWN. In this study, 507 NBS genes were identified in the transcriptome of resistant and susceptible P. masoniana inoculated with the PWN. The phylogenetic analysis and expression profiles of resistant and susceptible P. massoniana revealed that the up-regulated PmNBS-LRR97 gene was involved in conferring resistance to PWN. The results of real-time quantitative PCR (qRT-PCR) showed that PmNBS-LRR97 was significantly up-regulated after PWN infection, especially in the stems. Subcellular localization indicated that PmNBS-LRR97 located to the cell membrane. PmNBS-LRR97 significantly activated the expression of reactive oxygen species (ROS)-related genes in P. massoniana. In addition, the overexpression of PmNBS-LRR97 was capable of promoting the production of ROS, aiding in plant growth and development. In summary, PmNBS-LRR97 participates in the defense response to PWN and plays an active role in conferring resistance in P. massoniana. This finding provides new insight into the regulatory mechanism of the R gene in P. massoniana.

RT-qPCR is considered a rapid and reliable technique for analyzing gene expression. This technique is commonly used to analyze the expression of various genes at diverse transcriptional levels in different samples. However, few studies have characterized ornamental Koelreuteria species for reliable reference genes. In this study, eight reference genes were evaluated as controls in RT-qPCR with SYBR green to quantify gene expression in different Koelreuteria paniculata samples. All selected reference genes showed a broad range of C_t values in all samples, which was supportive of their variable expression. Our results showed significant variation in the stable expression of K. paniculata genes. Sample data, analyzed using geNorm, NormFinder, and BestKeeper, showed that phospholipase (PLA2) and β-actin (ACT) were the most suitable and statistically reliable reference genes, whereas ribosomal protein L13 (RPL13) and elongation factor 1-α (EF1α) were less stable and unsuitable for use as internal controls. To compare gene expression levels, two or more reference genes should be used for data normalization. Thus, the stability and expression of both PLA2 and ACT were believed to provide better normalization and quantification of the transcript levels for gene expression studies in K. paniculata.

Flowering is an important link in the life process of angiosperms, and it is also an important sign of the transformation of plants from vegetative to reproductive growth. Although the flowering regulation network of Arabidopsis is well-understood, there has been little research on the molecular mechanisms of perennial woody plant flower development regulation. Populus tomentosa is a unique Chinese poplar species with fast growth, strong ecological adaptability, and a long lifecycle. However, it has a long juvenile phase, which seriously affects its breeding process. Nuclear factor-Y (NF-Y) is an important type of transcription factor involved in the regulation of plant flowering. However, there are few reports on PtoNF-Y gene flowering regulation, and the members of the PtNF-YC subfamily are unknown. In this study, four key genes were cloned and analyzed for sequence characteristics, gene structure, genetic evolution, expression patterns, and subcellular localization. The plant expression vector was further constructed, and transgenic Arabidopsis and P. tomentosa plants were obtained through genetic transformation and a series of molecular tests. The flowering time and other growth characteristics were analyzed. Finally, the expression level of flowering genes was detected by quantitative PCR, the interaction between PtoNF-YC and PtoCOL proteins was measured using the yeast two-hybrid system to further explain the flowering regulation mechanism, and the molecular mechanisms by which PtNF-YC6 and PtNF-YC8 regulate poplar flowering were discussed. These results lay the foundation for elucidating the molecular regulation mechanism of PtoNF-YC in flowering and furthering the molecular design and breeding of poplar, while providing a reference for other flowering woody plants.