Glutathione S-transferases (GSTs) play a crucial role in responding to abiotic stress and are an important target for research on plant stress tolerance mechanisms. Populus euphratica is a promising candidate species for investigating the abiotic tolerance mechanisms in woody plants. In our previous study, PeGSTU58 was identified as being associated with seed salinity tolerance. In the present study, PeGSTU58 was cloned from P. euphratica and functionally characterized. PeGSTU58 encodes a Tau class GST and is located in both the cytoplasm and nucleus. Transgenic Arabidopsis overexpressing PeGSTU58 displayed enhanced tolerance to salt and drought stress. Under salt and drought stress, the transgenic plants exhibited significantly higher activities of antioxidant enzymes, including SOD, POD, CAT, and GST, compared to the wild-type (WT) plants. Additionally, the expression levels of several stress-responsive genes, including DREB2A, COR47, RD22, CYP8D11, and SOD1 were upregulated in PeGSTU58 overexpression lines compared to those in WT Arabidopsis under salt and drought stress conditions. Furthermore, yeast one-hybrid assays and luciferase analysis showed that PebHLH35 can directly bind to the promoter region of PeGSTU58 and activate its expression. These results indicated that PeGSTU58 was involved in salt and drought stress tolerances by maintaining ROS homeostasis, and its expression was positively regulated by PebHLH35.

MicroRNA319 (miR319) has been implicated in leaf development in a number of plant species. Here we study the roles of miR319a and its regulated network in leaf development in poplars. Over-expression of miR319a in Populus alba × Populus glandulosa caused dwarf statures, narrow leaf blades and serrated leaf margins. The vascular bundles and bundle sheaths in transgenic leaves had more layers of cells than those in the leaves of control plants, indicating enhanced lignification in these cells. Among the 93 putative targets of miR319a predicted with the psRNATarget tool, only three genes, TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR), were differentially expressed in the leaves of MIR319a-over-expression transgenic lines. With the RNA-seq data sets from multiple MIR319a over-expression transgenic lines, we built a three-layered gene regulatory network mediated by miR319a using Top-down graphic Gaussian model (GGM) algorithm that is capable of capturing causal relationships from transcriptomic data. The results support that miR319a primarily regulates the lignin biosynthesis, leaf development and differentiation as well as photosynthesis via miR319-MEE35/TCP4, miR319-TCP2 and miR319-TCP2-1 regulatory modules.

Enormous distinctions of the stem structure and cell types between gymnosperms and angiosperms tree species are expected to cause quite different wood physical and mechanical attributes, however, the molecular mechanisms underlying the differing wood morphology are still unclear. In this study, we compared the transcriptomes obtained by RNA-Seq betweenPopulus alba×P. glandulosaclone 84K, andLarix kaempferi(Lamb.) Carr trees. Available genome resource served as reference forP. alba×P. glandulosaand the Iso-Seq results of a three-tissues mixture (xylem, phloem, and leaf) were used as the reference forL. kaempferito compare the xylem-specifically expressed genes and their alternative splicing model. Through screening, we obtained 13,907 xylem-specifically expressed genes (5,954 up-regulated, 7,953 down-regulated) in the xylem ofP. alba×P. glandulosa, and 2,596 xylem-specifically expressed genes (1,648 up-regulated, 948 down-regulated) in the xylem ofL. kaempferi. From the GO and KEGG analyses, some genes associated with two wood formation-related pathways, namely those for phenylpropanoid biosynthesis, and starch and sucrose metabolism, were successfully screened. Then the distributions and gene expression models betweenP. alba×P. glandulosaandL. kaempferiin those pathways were compared, which suggested differential wood formation processes between the angiosperm and gymnosperm trees. Furthermore, a Weight Gene Co-expression Network Analysis (WGCNA) for total xylem-specifically expressed genes in two species was conducted, from which wood formation-related modules were selected to build a co-expression network for the two tree species. The genes within this co-expression network showed different co-expression relationships between the angiosperm and gymnosperm woody species. Comparing the alternative splicing events for wood formation-related genes suggests a different post-transcriptional regulation process exists between the angiosperm and gymnosperm trees. Our research thus provides the foundation for the in-depth investigation of different wood formation mechanisms of angiosperm and gymnosperm species.

The BTB (Broad-complex, tramtrack, and bric-a-brac) gene family, characterized by a highly conserved BTB domain, is implicated in a spectrum of biological processes, encompassing growth and development, as well as stress responses. Characterization and functional studies of BTB genes in poplar are still limited, especially regarding their response to hormones and biotic/abiotic stresses. In this study, we conducted an HMMER search in conjunction with BLASTp and identified 95 BTB gene models in Populus trichocarpa. Through domain motif and phylogenetic relationship analyses, these proteins were classified into eight families, NPH3, TAZ, Ankyrin, only BTB, BACK, Armadillo, TPR, and MATH. Collinearity analysis of poplar BTB genes with homologs in six other species elucidated evolutionary relationships and functional conservations. RNA-seq analysis of five tissues of poplar identified BTB genes as playing a pivotal role during developmental processes. Comprehensive RT-qPCR analysis of 11 BTB genes across leaves, roots, and xylem tissues revealed their responsive expression patterns under diverse hormonal and biotic/abiotic stress conditions, with varying degrees of regulation observed in the results. This study marks the first in-depth exploration of the BTB gene family in poplar, providing insights into the potential roles of BTB genes in hormonal regulation and response to stress.