Functional Research on Three Presumed Asparagine Synthetase Family Members in Poplar

Asparagine synthetase (AS), a key enzyme in plant nitrogen metabolism, plays an important role in plant nitrogen assimilation and distribution. Asparagine (Asn), the product of asparagine synthetase, is one of the main compounds responsible for organic nitrogen transport and storage in plants. In this study, we performed complementation experiments using an Asn-deficient Escherichia coli strain to demonstrate that three putative asparagine synthetase family members in poplar (Populus simonii × P. nigra) function in Asn synthesis. Quantitative real-time PCR revealed that the three members had high expression levels in different tissues of poplar and were regulated by exogenous nitrogen. PnAS1 and PnAS2 were also affected by diurnal rhythm. Long-term dark treatment resulted in a significant increase in PnAS1 and PnAS3 expression levels. Under long-term light conditions, however, PnAS2 expression decreased significantly in the intermediate region of leaves. Exogenous application of ammonium nitrogen, glutamine, and a glutamine synthetase inhibitor revealed that PnAS3 was more sensitive to exogenous glutamine, while PnAS1 and PnAS2 were more susceptible to exogenous ammonium nitrogen. Our results suggest that the various members of the PnAS gene family have distinct roles in different tissues and are regulated in different ways

Identification and Characterization of the APX Gene Family and Its Expression Pattern under Phytohormone Treatment and Abiotic Stress in Populus trichocarpa

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

Genome-wide identification and expression profile analysis of CCH gene family in Populus

Copper plays key roles in plant physiological activities. To maintain copper cellular homeostasis, copper chaperones have important functions in binding and transporting copper to target proteins. Detailed characterization and function analysis of a copper chaperone, CCH, is presently limited to Arabidopsis. This study reports the identification of 21 genes encoding putative CCH proteins in Populus trichocarpa. Besides sharing the conserved metal-binding motif MXCXXC and forming a βαββαβ secondary structure at the N-terminal, all the PtCCHs possessed the plant-exclusive extended C-terminal. Based on their gene structure, conserved motifs, and phylogenetic analysis, the PtCCHs were divided into three subgroups. Our analysis indicated that whole-genome duplication and tandem duplication events likely contributed to expansion of the CCH gene family in Populus. Tissue-specific data from PlantGenIE revealed that PtCCH genes had broad expression patterns in different tissues. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that PnCCH genes of P. simonii × P. nigra also had different tissue-specific expression traits, as well as different inducible-expression patterns in response to copper stresses (excessive and deficiency). In summary, our study of CCH genes in the Populus genome provides a comprehensive analysis of this gene family, and lays an important foundation for further investigation of their roles in copper homeostasis of poplar

A Weighted Mean Value Analysis to Identify Biological Pathway Activity Changes during Poplar Seed Germination

Poplar (Populus × xiaohei T. S. Hwang et Liang) is an excellent model plant, with a known genome sequence, for studying woody plant developmental processes, such as seed germination. Here, we report the transcriptional profiling of poplar seeds at five germination stages using RNA-Seq technology. We focused on identifying biological pathway activity changes during seed germination and transcription factors that play important roles in different stages. Among the 16 significantly changing clusters obtained using the STEM method, transcription was significantly enriched in five different clusters, 8, 21, 25, 27, and 35. The oxidative phosphorylation-related genes were only enriched in cluster 9, and expression patterns decreased in 6 and 24 HAI, while ubiquitin-dependent protein catabolic processes were only enriched in cluster 16, and expression patterns increased in 6 HAI. A weighted mean method analysis determined that most primary metabolism-associated categories, such as major carbohydrate metabolism, glycolysis, oxidative pentose phosphate, tricarboxylic acid cycle, lipid metabolism, nucleotide metabolism, amino acid metabolism, and protein metabolism, were elevated between 6 and 48 h after imbibition (HAI). ATP synthesis and C1 metabolism had highly active expression patterns between 0.75 and 48 HAI. The photosynthesis category-associated genes that were identified appeared highly active at 144 HAI. The homogenization of transcription factors in each cluster revealed that the HAP2, C3H zinc finger family, and C2C2(Zn) GATA transcription factors were present in relatively high numbers in cluster 8, while HAP5, Zn-finger (CCHC), FHA, and E2F/DP transcription factor families, as well as SNF7, were present in high numbers in cluster 25. Thus, we identified a series of biological pathway activity changes that occur, and transcription factors that are active, during poplar seed germination. Moreover, this study provides an integrated view of transcriptional regulation that can reveal the molecular events occurring during seed germination

Non-Targeted Metabolomics Reveals Patterns of Metabolic Changes during Poplar Seed Germination

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