Xiang Cheng

Pinus massoniana Lamb. is an important native economic and ecological tree species in southern China. However, adventitious root regeneration severely restricts the asexual propagation of elite traits. The WOX gene family, a plant-specific transcription factor family, plays critical roles in various aspects of plant growth and development, particularly in root development, including the maintenance of the root apical meristem, root elongation, and the initiation and formation of lateral roots. In this study, a total of 21 WOX family proteins were identified from the genome of P. massoniana and designated as PmWOX1 to PmWOX21 based on their chromosomal locations. These 21 members were unevenly distributed across nine chromosomes, with a clustered distribution observed on chr7. Collinearity analysis suggested that gene duplication and purifying selection may serve as key driving forces in the evolution of WOX genes. Conserved motif analysis revealed divergence among different PmWOX clades, implying distinct functions in growth and development. Cis-element analysis indicated that PmWOX genes may be involved in the regulation of stress responses, hormone signaling, and developmental processes. Expression pattern analysis based on transcriptome data from root tips and shoot tips identified nine PmWOX genes with significantly higher expression in root tips. RT-qPCR further screened PmWOX4, which exhibited the highest expression in root primordia. Functional experiments verified that PmWOX4 genes encoded nuclear-localized proteins with transcriptional activity. The PPI results indicate that PmWOX4 may interact with CLE41/44, LBD, ATHB8 and CLV3/CLV1 to regulate adventitious root formation. Collectively, our findings suggest that PmWOX4 possesses functional potential in adventitious rooting, which could be further exploited to improve the efficiency of asexual propagation in P. massoniana.

The NAC transcription factor superfamily is one of the most prominent plant-specific regulatory gene families, extensively participating in multiple metabolic processes that govern plant growth, tissue development and stress adaptation. Masson pine (Pinus massoniana Lamb.) is a native dominant conifer widely cultivated across South China, whose timber resources possess great exploitation potential in pulp manufacturing and the paper industry. In this study, a total of 98 non-redundant NAC family members were mined at the genome-wide level. Functional validation revealed that PmNAC82, a member belonging to the VND evolutionary subgroup, acts as a core regulatory factor controlling wood formation. Subcellular localization tests confirmed PmNAC82 exclusively resides in the cell nucleus. Heterologous genetic transformation in poplar demonstrated that this gene positively regulates the accumulation of lignin and cellulose. Furthermore, through RT-qPCR, yeast one-hybrid assays, and EMSA, we confirmed that PmNAC82 can bind to the promoters of PtrMYB3, PtrMYB21 and PmCesA7. These findings provide a solid foundation for further investigation into the molecular functions of NAC genes in Masson pine as well as their potential application towards molecular breeding strategies aimed at improving wood quality.

N⁶-methyladenosine (m⁶A) is a widespread post-transcriptional modification of RNA in eukaryotes. The conserved YTH-domain-containing RNA binding protein has been widely reported to serve as a typical m⁶A reader in various species. However, no studies have reported the m⁶A readers in Ginkgo biloba (G. biloba). In this study, a systematic analysis of the m⁶A reader (YTH) gene family was performed on G. biloba, identifying 10 YTH genes in its genome. Phylogenetic analysis of protein-coding sequences revealed that YTH genes from G. biloba could be classified into two subgroups: GbDC1 and GbDC2 in GbDC and GbDF1-8 in GbDF, each with similar motifs and gene structures. In G. biloba, the predicated aromatic cage pocket of the YTH domains in the YTH gene family is uniformly composed of tryptophan residues (WWW). Subcellular localization experiments verified that GbDC1 is indeed localized in the nucleus, while GbDF1 is localized in both the nucleus and the cytoplasm. The expression patterns of the identified m⁶A reader genes showed a wide distribution but were tissue-specific. Most genes were highly expressed in leaves, followed by the stem, while the lowest expression tendency was found in the roots. Cis-regulatory element analysis predicted the possible functions of YTH genes in G. biloba, which were mainly responsive to plant hormones such as ABA and MeJA, as well as stress responses. Furthermore, the expression levels of YTH genes indeed changed significantly after ABA, MeJA, and NaCl treatments, suggesting that they can be affected by these abiotic factors. In addition, the PLAAC prediction results indicate that prion domains exist in GbDF1, GbDF2, GbDF3, GbDF4, GbDF6, GbDF7, GbDF8, and GbDC1, and phase separation is possible. This study provides a foundation for further investigation of the effects of m⁶A methylation on gene expression regulation in G. biloba and other forest trees.

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.

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.

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.

WUSCHEL-related homeobox (WOX) transcription factors (TFs) play a crucial role in regulating plant development and responding to various abiotic stresses. However, the members and functions of WOX proteins in Pinus massoniana remain unclear. In this study, a total of 11 WOX genes were identified, and bioinformatics methods were used for preliminary identification and analysis. The phylogenetic tree revealed that most PmWOXs were distributed in ancient and WUS clades, with only one member found in the intermediate clade. We selected four highly conserved WOX genes within plants for further expression analysis. These genes exhibited expressions across almost all tissues, while PmWOX2, PmWOX3, and PmWOX4 showed high expression levels in the callus, suggesting their potential involvement in specific functions during callus development. Expression patterns under different abiotic stresses indicated that PmWOXs could participate in resisting multiple stresses in P. massoniana. The identification and preliminary analysis of PmWOXs lay the foundation for further research on analyzing the resistance molecular mechanism of P. massoniana to abiotic stresses.

N⁶-methyladenosine (m⁶A) is becoming one of the most important RNA modifications in plant growth and development, including defense, cell differentiation, and secondary metabolism. YT521-B homology (YTH) domain-containing RNA-binding proteins, identified as m⁶A readers in epitranscriptomics, could affect the fate of m⁶A-containing RNA by recognizing and binding the m⁶A site. Therefore, the identification and study of the YTH gene family in Liriodendron chinense (L. chinense) can provide a molecular basis for the study of the role of m⁶A in L. chinense, but studies on the YTH gene in L. chinense have not been reported. We identified nine putative YTH gene models in the L. chinense genome, which can be divided into DF subgroups and DC subgroups. Domain sequence analysis showed that the LcYTH protein had high sequence conservation. A LcYTH aromatic cage bag is composed of tryptophan and tryptophan (WWW). PrLDs were found in the protein results of YTH, suggesting that these genes may be involved in the process of liquid–liquid phase separation. LcYTH genes have different tissue expression patterns, but the expression of LcYTHDF2 is absolutely dominant in all tissues. In addition, the expression of the LcYTH genes is changed in response to ABA and MeJA. In this study, We identified and analyzed the expression pattern of LcYTH genes. Our results laid a foundation for further study of the function of the LcYTH gene and further genetic and functional analyses of m⁶A RNA modification in forest trees.