Background: As an important epigenetic mark, 5-methylcytosine (5mC) methylation is involved in many DNAdependent biological processes and plays a role during development and differentiation of multicellular organisms. However, there is still a lack of knowledge about the dynamic aspects and the roles of global 5mC methylation in wood formation in tree trunks. In this study, we not only scrutinized single-base resolution methylomes of primary stems (PS), transitional stems (TS), and secondary stems (SS) of Populus trichocarpa using a high-throughput bisulfite sequencing technique, but also analyzed the effects of 5mC methylation on the expression of genes involved in wood formation. Results: The overall average percentages of CG, CHG, and CHH methylation in poplar stems were~53.6%,~37.7%, and 8.5%, respectively, and the differences of 5mC in genome-wide CG/CHG/CHH contexts among PS, TS, and SS were statistically significant (p < 0.05). The evident differences in CG, CHG, and CHH methylation contexts among 2 kb proximal promoters, gene bodies, and 2 kb downstream regions were observed among PS, TS, and SS. Further analysis revealed a perceptible global correlation between 5mC methylation levels of gene bodies and transcript levels but failed to reveal a correlation between 5mC methylation levels of proximal promoter regions and transcript levels. We identified 653 and 858 DMGs and 4978 and 4780 DEGs in PS vs TS and TS vs SS comparisons, respectively. Only 113 genes of 653 DMGs and 4978 DEGs, and 114 genes of 858 DMGs and 4780 DEG were common. Counterparts of some of these common genes in other species, including Arabidopsis thaliana, are known to be involved in secondary cell wall biosynthesis and hormone signaling. This indicates that methylation may directly modulate wood formation genes and indirectly attune hormone signaling genes, which in turn impact wood formation. Conclusions: DNA methylation only marginally affects pathway genes or regulators involved in wood formation, suggesting that further studies of wood formation should lean towards the indirect effects of methylation. The information and data we provide here will be instrumental for understanding the roles of methylation in wood formation in tree species.

In plants, GATA transcription factors (TFs) have been reported to play vital roles in to a wide range of biological processes. To date, there is still no report about the involvement and functions of woody plant GATA TFs in wood formation. In this study, we described the functional characterization of a Populus trichocarpa GATA TF, PtrGATA12, which encodes a nuclear-localized transcriptional activator predominantly expressing in developing xylem tissues. Overexpression of PtrGATA12 not only inhibited growths of most phenotypic traits and biomass accumulation, but also altered the expressions of some master TFs and pathway genes involved in secondary cell wall (SCW) and programmed cell death, leading to alternated SCW components and breaking forces of stems of transgenic lines. The significant changes occurred in the contents of hemicellulose and lignin and SCW thicknesses of fiber and vessel that increased by 13.5 and 10.8%, and 20.83 and 11.83%, respectively. Furthermore, PtrGATA12 bound directly to the promoters of a battery of TFs and pathway genes and activated them; the binding sites include two cis-acting elements that were specifically enriched in their promoter regions. Taken together, our results suggest PtrGATA12, as a higher hierarchical TF on the top of PtrWND6A, PtrWND6B, PtrMYB152, and PtrMYB21, exert a coordinated regulation of SCW components biosynthesis pathways through directly and indirectly controlling master TFs, middle-level TFs, and further downstream pathway genes of the currently known hierarchical transcription network that governs SCW formation.

We applied miRNA expression profiling method to Populus trichocarpa stems of the three developmental stages, primary stem (PS), transitional stem (TS), and secondary stem (SS), to investigate miRNA species and their regulation on lignocellulosic synthesis and related processes. We obtained 892, 872, and 882 known miRNAs and 1,727, 1,723, and 1,597 novel miRNAs, from PS, TS, and SS, respectively. Comparisons of these miRNA species among different developmental stages led to the identification of 114, 306, and 152 differentially expressed miRNAs (DE-miRNAs), which had 921, 2,639, and 2,042 candidate target genes (CTGs) in the three respective stages of the same order. Corelation analysis revealed 47, 439, and 71 DE-miRNA-CTG pairs of high negative correlation in PS, TS and SS, respectively. Through biological process analysis, we finally identified 34, 6, and 76 miRNA-CTG pairs from PS, TS, and SS, respectively, and the miRNA target genes in these pairs regulate or participate lignocellulosic biosynthesis related biological processes: cell division and differentiation, cell wall modification, secondary cell wall biosynthesis, lignification, and programmed cell death processes. This is the first report on an integrated analysis of genome-wide mRNA and miRNA profilings during multiple phases of poplar stem development. Our analysis results imply that individual miRNAs modulate secondary growth and lignocellulosic biosynthesis through regulating transcription factors and lignocellulosic biosynthetic pathway genes, resulting in more dynamic promotion, suppression, or regulatory circuits. This study advanced our understanding of many individual miRNAs and their essential, diversified roles in dynamic regulation of secondary growth in woody tree species.

Background: MicroRNAs (miRNAs) are small, non-coding RNAs that have important regulatory functions in plant growth and development. However, the miRNAs that are involved in different developmental stages of tree stems have not been systemically characterized. In this study, we applied miRNA expression profiling method to the Populus trichocarpa trunks of the three distinct developmental stages defined as the primary stem (PS), transitional stem (TS), and secondary stem (SS) to investigate the miRNA species, their dynamic regulation and functions during the transitions of wood formation in different developmental stages at the genome-wide scale by Solexa sequencing.Results: We obtained 892, 872, and 882 known miRNAs and 1,727, 1,723, and 1,597 novel miRNAs, from PS, TS, and SS, respectively. And identified 114, 306, and 152 differentially expressed miRNAs (DE-miRNAs) with 921, 2,639, and 2,042 candidate target genes (CTGs), which formed 158, 855, and 297 DE-miRNA-CTG pairs in PS vs TS, PS vs SS, and TS vs SS , respectively. Among these, 47, 439, and 71 DE-miRNA-CTG pairs showed a significant negative correlation, respectively. Finally, we identified 39, 9, and 92 miRNA-CTG pairs involved in PS, TS, and SS, respectively. These DE-miRNA-CTG pairs in poplar or whose counterparts in other plant species are known to be transcriptional factors or structural genes involved in cell division and differentiation, cell wall modification, secondary cell wall (SCW) biosynthesis, lignification, and programmed cell death processes of wood formation. Moreover, qRT–PCR analysis confirmed that the results of small RNA-seq were robust and reliable and most miRNA-CTG pairs exhibited an inverse correlation.Conclusions: This is the first report on an integrated analysis of genome-wide mRNA and miRNA profiling of diverse phases of wood formation in poplar trunks. We showed that even though miRNAs involved in diverse developmental phases were not in a considerable number, their roles in the regulatory network that govern wood formation during different developmental stages cannot be negligible or underestimated. The information and data obtained in this paper significantly advanced our understanding of these miRNAs and their essential, dynamic and diversified roles as well as functions in diverse phases of wood formation in tree species.