Ascorbate peroxidase (APX) is a member of class I of the heme-containing peroxidase family. The enzyme plays important roles in scavenging reactive oxygen species for protection against oxidative damage and maintaining normal plant growth and development, as well as in biotic stress responses. In this study, we identified 11 APX genes in the Populus trichocarpa genome using bioinformatic methods. Phylogenetic analysis revealed that the PtrAPX proteins were classifiable into three clades and the members of each clade shared similar gene structures and motifs. The PtrAPX genes were distributed on six chromosomes and four segmental-duplicated gene pairs were identified. Promoter cis-elements analysis showed that the majority of PtrAPX genes contained a variety of phytohormone- and abiotic stress-related cis-elements. Tissue-specific expression profiles indicated that the PtrAPX genes primarily function in roots and leaves. Real-time quantitative PCR (RT-qPCR) analysis indicated that PtrAPX transcription was induced in response to drought, salinity, high ammonium concentration, and exogenous abscisic acid treatment. These results provide important information on the phylogenetic relationships and functions of the APX gene family in P. trichocarpa.

Secondary development is a key biological characteristic of woody plants and the basis of wood formation. Exogenous nitrogen can affect the secondary growth of poplar, and some regulatory mechanisms have been found in the secondary xylem. However, the effect of nitrogen on cambium has not been reported. Herein, we investigated the effects of different nitrogen concentrations on cambium development using combined transcriptome and metabolome analysis. The results show that, compared with 1 mM NH₄NO₃ (M), the layers of hybrid poplar cambium cells decreased under the 0.15 mM NH₄NO₃ (L) and 0.3 mM NH₄NO₃ (LM) treatments. However, there was no difference in the layers of hybrid poplar cambium cells under the 3 mM NH₄NO₃ (HM) and 5 mM NH₄NO₃ (H) treatments. Totals of 2365, 824, 649 and 398 DEGs were identified in the M versus (vs.) L, M vs. LM, M vs. HM and M vs. H groups, respectively. Expression profile analysis of the DEGs showed that exogenous nitrogen affected the gene expression involved in plant hormone signal transduction, phenylpropanoid biosynthesis, the starch and sucrose metabolism pathway and the ubiquitin-mediated proteolysis pathway. In M vs. L, M vs. LM, M vs. HM and M vs. H, differential metabolites were enriched in flavonoids, lignans, coumarins and saccharides. The combined analysis of the transcriptome and metabolome showed that some genes and metabolites in plant hormone signal transduction, phenylpropanoid biosynthesis and starch and sucrose metabolism pathways may be involved in nitrogen regulation in cambium development, whose functions need to be verified. In this study, from the point of view that nitrogen influences cambium development to regulate wood formation, the network analysis of the transcriptome and metabolomics of cambium under different nitrogen supply levels was studied for the first time, revealing the potential regulatory and metabolic mechanisms involved in this process and providing new insights into the effects of nitrogen on wood development.

Summary: Invertase (EC.3.2.1.26), a key enzyme in sucrose breakdown, is crucial for cellulose synthesis. However, the function of the vacuolar invertase (VINV) in woody plants remains unclear. In this study, transgenic lines of Populus trichocarpa Torr. and Gray were generated to investigate the role of PtrVINV2 in wood formation and under high salinity stress. Compared to wild‐type (WT), VINV activity in the developing xylem of knockout lines was reduced, resulting in a decrease in lignin content and an increase in hemicellulose content, while cellulose content remained unaffected. These changes in structural carbohydrate content were accompanied by reductions in xylem width and fibre cell wall thickness. The overexpression lines of the developing xylem exhibited opposite trends. Transcriptome analyses of developing xylem indicated that the expression level of PtrVINV2 affects the expression of genes involved in hemicellulose and lignin biosynthesis pathways, such as AXS, UAMs, HCT, COMT, CAD and peroxidases, while CesA expression remained unaffected. WGCNA analysis revealed that Potri.001G219100, Potri.009G106600 and Potri.002G081000 serve as ‘hub’ transcription factor genes within the structural/non‐structural carbohydrate modules of PtrVINV2 transgenic lines, potentially involved in plant salt tolerance. Additionally, under 200 mmol/L NaCl treatment, the knockout lines exhibited increased salt sensitivity compared to WT. This increased sensitivity was accompanied by elevated activities of SOD, CAT and MDA, as well as higher sucrose content and reduced contents of glucose and fructose. The findings indicate that although PtrVINV2 is not essential for cellulose synthesis, it enhances salt tolerance in poplar and presents a promising candidate gene for breeding salt‐tolerant poplar.

Exogenous nitrogen and carbon can affect plant cell walls, which are composed of structural carbon. Sucrose synthase (SUS), invertase (INV), hexokinase (HXK), phosphoglucomutase (PGM), and UDP-glucose pyrophosphorylase (UGP) are the key enzymes of sucrose metabolism involved in cell wall synthesis. To understand whether these genes are regulated by carbon and nitrogen to participate in structural carbon biosynthesis, we performed genome-wide identification, analyzed their expression patterns under different carbon and nitrogen treatments, and conducted preliminary functional verification. Different concentrations of nitrogen and carbon were applied to poplar (Populus trichocarpa Torr. and Gray), which caused changes in cellulose, lignin, and hemicellulose contents. In poplar, 6 SUSs, 20 INVs, 6 HXKs, 4 PGMs, and 2 UGPs were identified. Moreover, the physicochemical properties, collinearity, and tissue specificity were analyzed. The correlation analysis showed that the expression levels of PtrSUS3/5, PtrNINV1/2/3/5/12, PtrCWINV3, PtrVINV2, PtrHXK5/6, PtrPGM1/2, and PtrUGP1 were positively correlated with the cellulose content. Meanwhile, the knockout of PtrNINV12 significantly reduced the cellulose content. This study could lay the foundation for revealing the functions of SUSs, INVs, HXKs, PGMs, and UGPs, which affected structural carbon synthesis regulated by nitrogen and carbon, proving that PtrNINV12 is involved in cell wall synthesis.

Research Highlights: This study was the first to use metabolomics techniques to investigate seed germination in poplar, a model woody plant. Our results lay a foundation for uncovering changes in metabolite levels during woody plant seed germination and for understanding the underlying mechanism. Background and Objectives: Poplar is a model woody plant. Because poplar can be easily propagated asexually, the molecular mechanism of poplar seed germination has not been well studied. However, long-term asexual reproduction of poplar leads to seedlings with weak resistance, high vulnerability to degradation, and reduced growth potential. Materials and Methods: The non-targeted metabolomics technique was used to analyze changing trends in metabolite contents during the poplar seed germination process. Results: We found that the number of differential metabolites increased as seed germination progressed. Metabolic pathway analysis of differential metabolites revealed that galactose metabolism and alanine, aspartate, and glutamate metabolism were significantly enriched during all germination periods. MapMan-based visual analysis of metabolic pathways of differential metabolites indicated that glutamine, glutamic acid, phenylalanine, arginine, and asparagine contents increased with germination time, with most metabolites related to glucose metabolism following similar trends. Contents of most metabolites related to the tricarboxylic acid cycle exhibited a fluctuating pattern. Conclusions: This study has revealed the major changes taking place in primary metabolite contents during poplar seed germination and has laid the foundation for elucidation of the molecular mechanism of poplar seed germination.