Overexpressing GLUTAMINE SYNTHETASE 1;2 maintains carbon and nitrogen balance under high-ammonium conditions and results in increased tolerance to ammonium toxicity in hybrid poplar.

The glutamine synthetase/glutamic acid synthetase (GS/GOGAT) cycle plays important roles in N metabolism, growth, development, and stress resistance in plants. Excess ammonium (NH4+) restricts growth, but GS can help to alleviate its toxicity. In this study, the 84K model clone of hybrid poplar (Populus alba × P. tremula var. glandulosa), which has reduced biomass accumulation and leaf chlorosis under high-NH4+ stress, showed less severe symptoms in transgenic lines overexpressing GLUTAMINE SYNTHETASE 1;2 (GS1;2-OE), and more severe symptoms in RNAi lines (GS1;2-RNAi). Compared with the wild type, the GS1;2-OE lines had increased GS and GOGAT activities and higher contents of free amino acids, soluble proteins, total N, and chlorophyll under high-NH4+ stress, whilst the antioxidant and NH4+ assimilation capacities of the GS1;2-RNAi lines were decreased. The total C content and C/N ratio in roots and leaves of the overexpression lines were higher under stress, and there were increased contents of various amino acids and sugar alcohols, and reduced contents of carbohydrates in the roots. Under high-NH4+ stress, genes related to amino acid biosynthesis, sucrose and starch degradation, galactose metabolism, and the antioxidant system were significantly up-regulated in the roots of the overexpression lines. Thus, overexpression of GS1;2 affected the carbon and amino acid metabolism pathways under high-NH4+ stress to help maintain the balance between C and N metabolism and alleviate the symptoms of toxicity. Modification of the GS/GOGAT cycle by genetic engineering is therefore a potential strategy for improving the NH4+ tolerance of cultivated trees.

Metabolome and transcriptome profiling unveil the mechanisms of light-induced anthocyanin synthesis in rabbiteye blueberry (vaccinium ashei: Reade).

BACKGROUND: Blueberry is one of the most important fruit crops worldwide. Anthocyanin is an important secondary metabolites that affects the appearance and nutritive quality of blueberries. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation induced by light intensity in blueberries. RESULTS: The metabolic analysis revealed that there were 134 significantly changed metabolites in the natural light compared to the control, and flavone, flavonol, and anthocyanins were the most significantly increased. Transcriptome analysis found 6 candidate genes for the anthocyanin synthesis pathway. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the light intensity-treatment increased the accumulation of delphinidin-3-O-arabinoside compared to under the shading-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted that the gene of VcF3'5'H4 may be a candidate gene for anthocyanin accumulation and is highly expressed in light intensity-treated fruit. Through the co-expression analysis of transcription factors and anthocyanin synthesis pathway genes, we found that the VcbHLH004 gene may regulate VcF3'5'H4, and then we transformed VcbHLH004 heterologously into tomato to verify its function. CONCLUSION: These results provide novel insights into light intensity regulation of blueberry anthocyanin accumulation and represent a valuable data set to guide future functional studies and blueberry breeding.

Genome Identification and Expression Profiles in Response to Nitrogen Treatment Analysis of the Class I CCoAOMT Gene Family in Populus.

Lignin is essential for the characteristics and quality of timber. Nitrogen has significant effects on lignin contents in plants. Nitrogen has been found to affect wood quality in plantations and lignin content in plants. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is an important methyltransferase in lignin biosynthesis. However, the classification of woody plant CCoAOMT gene family members and the regulation mechanism of nitrogen are not clear. Bioinformatics methods were used to predict the members, classification, and transcriptional distribution of the CCoAOMT gene family in Populus trichocarpa. The results showed that there were five PtCCoAOMTs identified, and they could be divided into three sub-groups according to their structural and phylogenetic features. The results of tissue expression specificity analysis showed that: PtCCoAOMT1 was highly expressed in roots and internodes; PtCCoAOMT2 was highly expressed in roots, nodes, and internodes, PtCCoAOMT3 was highly expressed in stems; PtCCoAOMT4 was highly expressed in young leaves, and, PtCCoAOMT5 was highly expressed in roots. Different forms and concentrations of nitrogen had varying effects on the expression patterns of genes in different plant tissue types. The results of real-time PCR showed that the expression levels of PtCCoAOMT1 and PtCCoAOMT2 in stems increased significantly under different forms of nitrogen. PtCCoAOMT3 and PtCCoAOMT4 were induced by nitrate nitrogen in upper stems and lower leaves, respectively. PtCCoAOMT4 and PtCCoAOMT5 were induced by different concentrations of nitrate nitrogen in lower stems and roots, respectively. These results could provide valuable information for revealing the differences between functions and expression patterns of the various CCoAOMT gene family members under different forms and concentrations of exogenous nitrogen in poplar.

Genome-wide identification of FRK genes in Populus trichocarpa and their expression under different nitrogen treatments.

Fructokinase (FRK) is the main fructose phosphorylase and plays an important role in catalyzing the irreversible reaction of free fructose phosphorylation. In order to study the regulatory effect of different forms and concentrations of nitrogen on PtFRK genes in Populus trichocarpa, seven genes encoding the hypothetical FRK proteins were identified in Populus trichocarpa genome by bioinformatics method. Phylogenetic analysis revealed that PtFRK family genes can be divided into two subgroups: SI (PtFRK 1, 3, 4, 6) and SII (PtFRK 2, 5, 7). The tissue-specific expression data obtained from PopGenIE indicate that PtFRK2, 3, 4 and 5 are expressed highly in the stem. Quantitative real-time RT-PCR illustrate that PtFRK1-7 showed different expression patterns in different tissues under different concentrations and morphological nitrogen application. Under high nitrate treatment, the expression levels of PtFRK1, 2, 3 and 6 in stem increased significantly, while under low nitrate treatment, only the expression of PtFRK1, 4 in the upper stem and the expression of PtFRK3, 5 in the lower stem increased significantly. In contrast, ammonium tends to inhibit the expression of PtFRKs in lower stems, the expression levels of PtFRK2, 3, 4 and 5 are significantly reduced under ammonium treatment. However, high ammonium had significant effects on PtFRK6 in the apical bud and upper leaves, which were 6 and 8 times of the control, respectively. These results laid the foundation for the study of the PtFRK gene family of poplar and provided a theoretical basis for the molecular mechanism of nitrogen regulating cell wall development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01055-6.

Comparison of the fecal microbiomes of healthy and diarrheic captive wild boar.

Diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) is one of the most common clinical diseases observed in captive wild boars, is usually caused by an imbalance in the gut microbiome, and is responsible for piglets significant mortality. However, little research has been undertaken into the structure and function of the intestinal microbial communities in wild boar with diarrhea influenced by enterotoxigenic E. coli. In this study, fecal samples were collected and 16S-rRNA gene sequencing was used to compare the intestinal microbiome of healthy captive wild boar and wild boar with diarrhea on the same farm. We found that the intestinal microbial diversity of healthy wild boar (HWB) was relatively high, while that of diarrheic wild boar (DWB) was significantly lower. Line Discriminant Analysis Effect Size showed that at the genus level, the abundance of Escherichia-Shigella and Fusobacterium was significantly higher in DWB. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States analysis showed that the expression of genes in pathways including infectious diseases: bacterial, metabolism of amino acids, membrane transport, and signal transduction was significantly higher in DWB. In summary, this study provides a theoretical basis for the design of appropriate means of diarrhea treatment in captive wild boar.

The transcriptional events and their relationship to physiological changes during poplar seed germination and post-germination.

BACKGROUND: Seed germination, the foundation of plant propagation, involves a series of changes at the molecular level. Poplar is a model woody plant, but the molecular events occurring during seed germination in this species are unclear. RESULTS: In this study, we investigated changes in gene transcriptional levels during different germination periods in poplar by high-throughput sequencing technology. Analysis of genes expressed at specific germination stages indicated that these genes are distributed in many metabolic pathways. Enrichment analysis of significantly differentially expressed genes based on hypergeometric testing revealed that multiple pathways, such as pathways related to glycolysis, lipid, amino acid, protein and ATP synthesis metabolism, changed significantly at the transcriptional level during seed germination. A comparison of ΣZ values uncovered a series of transcriptional changes in biological processes related to primary metabolism during poplar seed germination. Among these changes, genes related to CHO metabolism were the first to be activated, with subsequent expression of genes involved in lipid metabolism and then those associated with protein metabolism. The pattern of metabolomic and physiological index changes further verified the sequence of some biological events. CONCLUSIONS: Our study revealed molecular events occurring at the transcriptional level during seed germination and determined their order. These events were further verified by patterns of changes of metabolites and physiological indexes. Our findings lay a foundation for the elucidation of the molecular mechanisms responsible for poplar seed germination.

Comprehensive dissection of transcript and metabolite shifts during seed germination and post-germination stages in poplar.

BACKGROUND: Seed germination, a complex, physiological-morphogenetic process, is a critical stage in the life cycle of plants. Biological changes in germinating seeds have not been investigated in poplar, a model woody plant. RESULTS: In this study, we exploited next-generation sequencing and metabolomics analysis and uncovered a series of significantly different genes and metabolites at various stages of seed germination and post germination. The K-means method was used to identify multiple transcription factors, including AP2/EREBP, DOF, and YABBY, involved in specific seed germination and post-germination stages. A weighted gene coexpression network analysis revealed that cell wall, amino acid metabolism, and transport-related pathways were significantly enriched during stages 3 and 5, with no significant enrichment observed in primary metabolic processes such as glycolysis and the tricarboxylic acid cycle. A metabolomics analysis detected significant changes in intermediate metabolites in these primary metabolic processes, while a targeted correlation network analysis identified the gene family members most relevant to these changing metabolites. CONCLUSIONS: Taken together, our results provide important insights into the molecular networks underlying poplar seed germination and post-germination processes. The targeted correlation network analysis approach developed in this study can be applied to search for key candidate genes in specific biochemical reactions and represents a new strategy for joint multiomics analyses.

Impacts of AlaAT3 transgenic poplar on rhizosphere soil chemical properties, enzyme activity, bacterial community, and metabolites under two nitrogen conditions.

Poplar stands as one of the primary afforestation trees globally. We successfully generated transgenic poplar trees characterized by enhanced biomass under identical nutrient conditions, through the overexpression of the pivotal nitrogen assimilation gene, pxAlaAT3. An environmental risk assessment was conducted for investigate the potential changes in rhizosphere soil associated with these overexpressing lines (OL). The results show that acid phosphatase activity was significantly altered under ammonium in OL compared to the wild-type control (WT), and a similar difference was observed for protease under nitrate. 16SrDNA sequencing indicated no significant divergence in rhizosphere soil microbial community diversity between WT and OL. Metabolomics analysis revealed that the OL caused minimal alterations in the metabolites of the rhizosphere soil, posing no potential harm to the environment. With these findings in mind, we anticipate that overexpressed plants will not adversely impact the surrounding soil environment.

Identification and expression analysis of the PtGATL genes under different nitrogen and carbon dioxide treatments in Populus trichocarpa.

Pectin is one of the most important components of the plant cell wall. Galacturonosyltransferase-like (GATL) is an important enzyme involved in forming pectin in Arabidopsis thaliana. In this study, 12 PtGATL genes were identified and characterized based on the Populus trichocarpa genome using bioinformatics methods. The results showed that the PtGATLs contained four typical motifs, including DXD, LPPF, GLG, and HXXGXXKPW. According to phylogenetic analysis, PtGATLs were divided into six groups. Chromosome distribution and genome synteny analysis showed that there were 11 segmental-duplicated gene pairs with repeated fragments on chromosomes 2, 5, 7, 8, 10, and 14. Tissue-specific expression profiles indicated that these PtGATLs had different expression patterns. The transcription level of PtGATLs was regulated by different carbon dioxide and nitrogen concentrations. In conclusion, the identification and analysis of PtGATL genes in poplar provide important information on the gene function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03129-y.

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.

Bioinformatics analysis of PAE family in Populus trichocarpa and responsiveness to carbon and nitrogen treatment.

Plant Pectin acetylesterase (PAE) belongs to family CE13 of carbohydrate esterases in the CAZy database. The ability of PAE to regulate the degree of acetylation of pectin, an important polysaccharide in the cell wall, affects the structure of plant cell wall. In this study, ten PtPAE genes were identified and characterized in Populus trichocarpa genome using bioinformatics methods, and the physiochemical properties such as molecular weight, isoelectric points, and hydrophilicity, as well as the secondary and tertiary structure of the protein were predicted. According to phylogenetic analysis, ten PtPAEs can be divided into three evolutionary clades, each of which had similar gene structure and motifs. Tissue-specific expression profiles indicated that the PtPAEs had different expression patterns. Real-time quantitative PCR (RT-qPCR) analysis showed that transcription level of PtPAEs was regulated by different CO2 and nitrogen concentrations. These results provide important information for the study of the phylogenetic relationship and function of PtPAEs in Populus trichocarpa. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02918-1.

Genome-wide analysis of UGDH genes in Populus trichocarpa and responsiveness to nitrogen treatment.

Plant UDP-glucose 6-dehydrogenase (UGDH) is an important enzyme for the formation of hemicellulose and pectin. Previous studies on UGDH have primarily focused on the biosynthesis of cell wall polysaccharides, while few studies have focused on their regulation by exogenous nitrogen. In the present study, four genes encoding PtUGDH proteins were analyzed by bioinformatics methods. And, the expression profiles of PtUGDH genes under different nitrogen treatments were evaluated with qRT-PCR. The results showed that PtUGDHs have conserved NAD coenzyme binding motif GAGYVGG and the catalytic motif GFGGSCFQKDIL. According to the phylogenetic analysis, PtUGDHs were divided into two subgroups. PtUGDH3 and PtUGDH4 were closely related to AtUGDH1 (important for normal development of Arabidopsis cell wall structure). Chromosomal distribution and genome synteny analysis revealed four segmental-duplicated gene pairs on chr4, 8, 10 and 17. Tissue-specific data from PlantGenIE showed that PtUGDH3 and PtUGDH4 were highly expressed in stems. The qRT-PCR detection showed that the expression of PtUGDH3 in the lower stem and PtUGDH2 of upper leaves were significantly increased induced by low ammonium or nitrate condition. This comprehensive analysis of the UGDH family in poplar provides new insights into their regulation by nitrogen, and would increase our understanding of the roles of UGDHs in hemicellulose and pectin biosynthesis in the cell wall and during poplar development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02697-9.

Differential expression of copper-zinc superoxide dismutase gene of Polygonum sibiricum leaves, stems and underground stems, subjected to high-salt stress.

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5' untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3' untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1-16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO(3). The different mRNA levels' expression of PS-CuZnSOD show the gene's different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.

Analysis of the energy source at the early stage of poplar seed germination: verification of Perl's pathway.

Adenosine triphosphate (ATP) is produced at the early stage of seed germination and provides the energy for metabolism. The source of ATP in seeds may be Perl's pathway, but this has not yet been confirmed. In this study, using germinating seeds of poplar as the experimental materials, the transcript levels of genes related to Perl's pathway were determined by real-time PCR. The activities of enzymes in Perl's pathway were also determined. The results were verified by comparison with RNA-Seq and metabolomics data. The results showed that there were high transcript levels of some genes encoding malate dehydrogenase (MDH), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and pyruvate kinase (PK) at the early stage of germination (0.75 h). The enzymes MDH, PEPCK, PK, PDC, and ADH showed peaks in activity at around 0.75 h and 6 h during germination. The oxaloacetate concentration was high in poplar seeds at the early stage of germination. This study provides experimental data showing that Perl's pathway participates in supplying energy during the early stages of poplar seed germination, and lays the foundation for further studies on the complex metabolic processes that function during seed germination.

Genome-wide analysis of the RGP gene family in Populus trichocarpa and their expression under nitrogen treatment.

Reversible glycosylation polypeptide (RGP) is a type of plant-specific protein, primarily involved in the biosynthesis of cell wall polysaccharides, which in turn changes the shape of the cell walls and affects the wood properties of plants. Poplar is a major industrial timber species, and the RGP gene has not been studied. This study uses bioinformatics methods to predict physical and chemical characters such as molecular weight, isoelectric point, and hydrophilicity; and fluorescent quantitative method to determine the effect of different forms of nitrogen on the transcription level of the gene family. The results showed that there are six RGP homologous genes in the Populus trichocarpa genome, which were distributed on the six chromosomes of P. trichocarpa. The family members have a simple gene structure and contain four exons and introns. Phylogenetic tree analysis showed that RGP genes all belong to Class I in P. trichocarpa. Tissue-specific expression analysis showed that PtRGP1 and PtRGP2 were highly expressed in the stems, PtRGP4 and PtRGP5 were highly expressed in the upper leaves, PtRGR3 and PtRGR6 were expressed in stems and internodes, but the relative expression is not high. Quantitative real-time RT-PCR (qRT-PCR) analyses revealed that PtRGP3 and 6 were up-regulated in the upper stem in response to the low ammonium and high nitrate treatments. The influence of nitrogen on the expression of PtRGP3 and 6 genes may affect the formation of the plant secondary cell wall. This study lays a foundation for further study on the function of RGP genes in P. trichocarpa.

Genome-wide identification of BXL genes in Populus trichocarpa and their expression under different nitrogen treatments.

ß-d-xylosidase (BXL) hydrolyzes xylobiose and xylo-oligosaccharides into xylose monomers, and is a rate-limiting enzyme in the degradation of hemicellulose in the cell wall. In this study, ten genes encoding putative BXL proteins were identified in the Populus trichocarpa genome by bioinformatics methods. In the phylogenetic analysis, the PtBXLs formed two subfamilies. PtBXL8 and PtBXL9 were closely related to AtBXL1, an important enzyme in the normal development of the Arabidopsis cell wall structure. Chromosomal distribution and genome synteny analyses revealed two tandem-duplicated gene pairs PtBXL3/4 and PtBXL6/7 on chromosomes II and V, respectively, and six segmental-duplicated gene pairs on chromosomes II, V, VIII, X, and XIV among the PtBXL gene family. Tissue-specific expression data from PlantGenIE indicated that PtBXL2, 4, 5, and 10 were highly expressed in stems. Quantitative real-time RT-PCR analyses revealed that PtBXL4, 5, and 9 were up-regulated in the upper stem in response to the low and high ammonium and nitrate treatments. The influence of nitrogen on the expression of PtBXL4, 5, and 9 genes may affect the formation of the plant secondary cell wall. This comprehensive analysis of the BXL family in poplar provides new insights into their regulation by nitrogen and increases our understanding of the roles of BXLs in hemicellulose metabolism in the secondary cell wall and during plant development.

Functional characterization and expression patterns of PnATX genes under different abiotic stress treatments in Populus.

The copper chaperone ATX1 has been investigated previously in the herbaceous plants Arabidopsis and rice. However, the molecular mechanisms of ATX1 underlying copper transport and functional characteristics in the woody plant Populus are poorly understood. In this study, PnATX1 and PnATX2 of Populus simonii × P. nigra were identified and characterized. Sequence analysis showed that PnATXs contained the metal-binding motif MXCXXC in the N-terminus and a lysine-rich region. Phylogenetic analysis of ATX protein sequences revealed that PnATXs were clustered in the same group as AtATX1. PnATX proteins were localized in the cytoplasm and nucleus. Tissue-specific expression analysis showed that PnATX1 and PnATX2 were expressed in all analyzed tissues and, in particular, expressed to a higher relative expression level in young leaves. Quantitative real-time PCR analysis indicated that each PnATX gene was differentially expressed in different tissues under treatments with copper, zinc, iron, jasmonate and salicylic acid (SA). The copper-response element GTAC, methyl jasmonate and salicylic acid responsiveness elements and other cis-acting elements were identified in the PnATX1 and PnATX2 promoters. Expression of ß-glucuronidase driven by the PnATX1 promoter was observed in the apical meristem of 7-day-old Arabidopsis transgenic seedlings, and the signal strength was not influenced by deficient or excessive copper conditions. Both PnATX1 and PnATX2 functionally rescued the defective phenotypes of yeast atx1Δ and sod1Δ strains. Under copper excess and deficiency conditions, transgenic Arabidopsis atx1 mutants harboring 35S::PnATX constructs exhibited root length and fresh weight similar to those of the wild type and higher than those of Arabidopsis atx1 mutants. Superoxide dismutase activity decreased in transgenic lines compared with that of atx1 mutants, whereas peroxidase and catalase activities increased significantly under excess copper. The results provide a basis for elucidating the role of Populus PnATX genes in copper homeostasis.

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 (Populussimonii× 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.

[Cloning of PsPIP1 gene from Polygonum sibiricum Laxm. and analy-sis of its expression in response to NaHCO3].

Gene PsPIP1 (GenBank accession No. EU626398) containing a complete ORF was obtained using rapid amplification of cDNA ends (RACE) from the cDNA library of Polygonum sibiricum Laxm. leaves. The length of cDNA was 1 004 bp, which encoded a peptide of 285 amino acid residues. Based on other kinds of plant aquaporin amino acid sequences, the phylogenetic evolution, and tertiary structure of protein comparison, this gene was classified into aquaporin subfamily. Expression analysis by Real-time PCR showed that PsPIP1 gene was expressed in leaves, stems, and underground stems. The expression level of PsPIP1 gene was higher in leaves than in underground stems and was the lowest in stems. The expression pattern of PsPIP1 gene induced by NaHCO3 stress and de-stressing also varied remarkably.

[Cloning and expression of cysteine synthase gene from Polygonum sibiricum Laxm.].

Cysteine synthase is a key enzyme for restricting plant cysteine synthesis. Cysteine synthase gene, designated PcCSase1 (GenBank accession no. EU597481), was successfully isolated from Polygonum sibiricum Laxm. by RACE technique. This gene was 1 260 bp in full-length and encoded a peptide of 382 amino acids. Based on bioinformatic analysis, PcCSase1 is a cytoplasm cysteine synthesis and contains a 16 amino-terminal (N-terminal) signal peptide, which led the PcCSase1 to go to the cytoplasm. The results obtained through homologous sequence analysis indicated that PcCSase1 mature protein was highly conserved in plants, which shared approximate 90% in the amino acid sequence. Expression analysis by RT-PCR showed that PcCSase1 gene was expressed in leaf, stem and root with the largest expression in leaf. Under 3% NaHCO3 stress, the largest expression of PcCSase1 gene was detected in leaf, stem and root at the second day following stress. PcCSase1 gene was inserted into pYES2 and transformed into yeast cells (Saccharomyces cerevisiae). The contents of the glutathione in the recombinant yeast and the cysteine in the medium were increased. INVSc1-pYES2-PcCSase1 was more tolerant to salt treatment than INVSc1-pYES2 and the former survival rate was higher than that of the later under the stress of 10% NaHCO3 and 5 mol/L NaCl. These results proved that PcCSase1 gene may confer high salt-tolerance.