Search

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.

Populus alba × P. glandulosa clone 84 K, derived from South Korea, is widely cultivated in China and used as a model in the molecular research of woody plants because of high gene transformation efficiency. Here, we combined 63-fold coverage Illumina short reads and 126-fold coverage PacBio long reads to assemble the genome. Due to the high heterozygosity level at 2.1% estimated by k-mer analysis, we exploited TrioCanu for genome assembly. The PacBio clean subreads of P. alba × P. glandulosa were separated into two parts according to the similarities, compared with the parental genomes of P. alba and P. glandulosa. The two parts of the subreads were assembled to two sets of subgenomes comprising subgenome A (405.31 Mb, from P. alba) and subgenome G (376.05 Mb, from P. glandulosa) with the contig N50 size of 5.43 Mb and 2.15 Mb, respectively. A high-quality P. alba × P. glandulosa genome assembly was obtained. The genome size was 781.36 Mb with the contig N50 size of 3.66 Mb and the longest contig was 19.47 Mb. In addition, a total of 176.95 Mb (43.7%), 152.37 Mb (40.5%) of repetitive elements were identified and a total of 38,701 and 38,449 protein-coding genes were predicted in subgenomes A and G, respectively. For functional annotation, 96.98% of subgenome A and 96.96% of subgenome G genes were annotated with public databases. This de novo assembled genome will facilitate systematic and comprehensive study, such as multi-omics analysis, in the model tree P. alba × P. glandulosa.

High-throughput single-cell RNA sequencing (scRNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba × Populus glandulosa. The scRNA-seq profiled 9,798 cells, which were grouped into 12 clusters. Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations, we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type (cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.

Pollen tube growth and penetration in female tissues are essential for the transfer of sperm to the embryo sac during plant pollination. Despite its importance during pollination, little is known about the mechanisms that mediate pollen tube growth in female tissues. In this study, we identified an Arabidopsis thaliana pollen/pollen tube-specific gene, SKU5-SIMILAR 13 (SKS13), which was critical for the growth of pollen tubes in the transmitting tract. The SKS13 protein was distributed throughout the cytoplasm and pollen tube walls at the apical region. In comparison with wild-type pollen tubes, those of the sks13 mutants burst more frequently when grown in vitro. Additionally, the growth of sks13 pollen tubes was retarded in the transmitting tract, thereby resulting in decreased male fertility. The accumulation of pectin and cellulose in the cell wall of sks13 pollen tubes was altered, and the content of jasmonic acid (JA) in sks13 pollen was reduced. The pollen tubes treated with an inhibitor of JA biosynthesis grew much more slowly and had an altered distribution of pectin, which is similar to the pollen tube phenotypes of the SKS13 mutation. Our results suggest that SKS13 is essential for pollen tube growth in the transmitting tract by mediating the biosynthesis of JA that modifies the components of pollen tube cell walls.

Cellulose synthase-like D (CSLD) family was characterized for their expression and functions in Populus trichocarpa. Ten members, PtrCslD1-10, were identified in the P. trichocarpa genome, and they belong to 4 clades by phylogenetic tree analysis. qRT-PCR and promoter:GUS assays in Arabidopsis and P. trichocarpa displayed divergent expression patterns of these 10 PtrCSLD genes in root hairs, root tips, leaves, vascular tissues, xylem and flowers. Among PtrCslD2, PtrCslD4, PtrCslD5, PtrCslD6, and PtrCslD8 that all exhibited expression in root hairs, only PtrCslD5 could restore the root hairless phenotype of the atcsld3 mutant, demonstrating that PtrCslD5 is the functional ortholog of AtCslD3 for root hair formation. Our results suggest more possible functions for other PtrCslD genes in poplar.

MiRNA transcriptome analysis of different tissues in poplar and larch suggests variant roles of miRNAs in regulating wood formation between two kinds of phyla. Poplar and larch belong to two different phyla. Both are ecological woody species and major resources for wood-related industrial applications. However, wood properties are different between these two species and the molecular basis is largely unknown. In this study, we performed high-throughput sequencing of microRNAs (miRNAs) in the three tissues, xylem, phloem and leaf of Populus alba × Populus glandulosa and Larix kaempferi. Differentially expressed miRNA (DEmiRNA) analysis identified 85 xylem-specific miRNAs in P. alba × P. glandulosa and 158 xylem-specific miRNAs in L. kaempferi. Among 36 common miRNAs, 12 were conserved between the two species. GO and KEGG analyses of the miRNA target genes showed similar metabolism in two species. Through KEGG and BLASTN, we predicted target genes of xylem differentially expressed (DEmiRNA) in the wood formation-related pathways and located DEmiRNAs in these pathways. A network was built for wood formation-related DEmiRNAs, their target genes and orthologous genes in Arabidopsis thaliana. Comparison of DEmiRNA and target gene annotation between P. alba × P. glandulosa and L. kaempferi suggested the different functions of DEmiRNAs and divergent mechanism in wood formation between two species, providing knowledge to understand wood formation mechanism in gymnosperm and angiosperm woody plants.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.