Molecular characterization of XVSAP1, a stress-responsive gene from the resurrection plant Xerophyta viscosa Baker

The strategy of 'complementation by functional sufficiency' was used to isolate a cDNA designated XVSAP1 from a cDNA library constructed from dehydrated Xerophyta viscosa Baker leaves. Analysis of the cDNA sequence indicated a highly hydrophobic protein with six transmembrane regions. Southern blot analysis revealed that there are at least two copies of XVSAP1 in X. viscosa. The deduced amino acid sequence showed 49% identity to WCOR413, a low-temperature-regulated protein from wheat. The protein also showed between 25% to 56% identity to WCOR413-like proteins from Arabidopsis thaliana. Expression of XVSAP1 in Escherichia coli (srl::Tn10) conferred osmotic stress tolerance when the cells were grown in 1 M sorbitol. Analysis of gene expression using semi-quantitative RT-PCR indicated that XVSAP1 is induced by dehydration, salt stress (100 mM), both low (4 degrees C) and high temperature (42 degrees C) and high light treatment (1500 micromol m(-2) s(-1)). These results suggest that XVSAP1 may have a significant role to play in the response of X. viscosa to abiotic stresses.     

Proteomic analysis of leaf proteins during dehydration of the resurrection plant Xerophyta viscosa

The desiccation-tolerant phenotype of angiosperm resurrection plants is thought to rely on the induction of protective mechanisms that maintain cellular integrity during water loss. Two-dimensional (2D) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the Xerophyta viscosa Baker proteome was carried out during dehydration to identify proteins that may play a role in such mechanisms. Quantitative analysis revealed a greater number of changes in protein expression levels at 35% than at 65% relative water content (RWC) compared to fully hydrated plants, and 17 dehydration-responsive proteins were identified by tandem mass spectrometry (MS). Proteins showing increased abundance during drying included an RNA-binding protein, chloroplast FtsH protease, glycolytic enzymes and antioxidants. A number of photosynthetic proteins declined sharply in abundance in X. viscosa at RWC below 65%, including four components of photosystem II (PSII), and Western blot analysis confirmed that two of these (psbP and Lhcb2) were not detectable at 30% RWC. These data confirm that poikilochlorophylly in X. viscosa involves the breakdown of photosynthetic proteins during dismantling of the thylakoid membranes. In contrast, levels of these photosynthetic proteins were largely maintained during dehydration in the homoiochlorophyllous species Craterostigma plantagineum Hochst, which does not dismantle thylakoid membranes on drying.     

Protection mechanisms against excess light in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa

Mechanisms of avoidance and protection against light damage were studied in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa.In C. wilmsii, a combination of both physical and chemical changes appeared to afford protection against free radical damage. During dehydration leaves curled inwards, and the abaxial surface became exposed to light. The tissue became purple/brown in colour, this coinciding with a three-fold increase in anthocyanin content and a 30% decline in chlorophyll content. Thus light-chlorophyll interactions are progressively reduced as chlorophyll became masked by anthocyanins in abaxial layers and shaded in the adaxial layers. Ascorbate peroxidase (AP) activity increased during this process but declined when the leaf was desiccated (5% RWC). During rehydration leaves uncurled and the potential for normal light-chlorophyll interaction was possible before full hydration had occurred. Superoxide dismutase (SOD) and glutathione reductase (GR) activities increased markedly during this stage, possibly affording free radical protection until full hydration and metabolic recovery had occurred.In contrast, the leaves of X. viscosa did not curl, but light-chlorophyll interactions were minimised by the loss of chlorophyll and dismantling of thylakoid membranes. During dehydration, free radical protection was afforded by a four-fold increase in anthocyanin content and increased activities of AP, GR and SOD. These declined during rehydration. It is suggested that potential free radical damage may be avoided by the persistence of anthocyanins during the period of thylakoid membrane re-assembly and full chlorophyll restitution which only occurred once the leaves were fully rehydrated.     

The Xerophyta viscosa Aldose Reductase (ALDRXV4) Confers Enhanced Drought and Salinity Tolerance to Transgenic Tobacco Plants by Scavenging Methylglyoxal and Reducing the Membrane Damage

We report the efficacy of an aldose reductase (ALDRXV4) enzyme from Xerophyta viscosa Baker in enhancing the prospects of plant’s survival under abiotic stress. Transgenic tobacco plants overexpressing ALDRXV4 cDNA showed alleviation of NaCl and mannitol-induced abiotic stress. The transgenic plants survived longer periods of water deficiency and salinity stress and exhibited improved recovery after rehydration as compared to the wild type plants. The increased synthesis of aldose reductase in transgenic plants correlated with reduced methylglyoxal and malondialdehyde accumulation and an elevated level of sorbitol under stress conditions. In addition, the transgenic lines showed better photosynthetic efficiency, less electrolyte damage, greater water retention, higher proline accumulation, and favorable ionic balance under stress conditions. Together, these findings suggest the potential of engineering aldose reductase levels for better performance of crop plants growing under drought and salt stress conditions.     

Structural Characterization and Functional Validation of Aldose Reductase from the Resurrection Plant Xerophyta viscosa

Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K _M (1.2 mM) and k _cat (14.5 s^?1) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha–beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl₂), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.     

Isoforms of chalcone synthase in Daucus carota L. and their differential expression in organs from the European wild carrot and in ultraviolet-A-irradiated cell cultures

 Two isoforms of chalcone synthase (CHS) were isolated from cDNA libraries derived from UV-A-irradiated anthocyanin-accumulating (DCb) and non-accumulating (DCs) cell cultures of carrot (Daucus carota L.). The clones designated as DcCHS1, which were present only in the DCb library, had a deduced primary sequence of 389 amino acids and an expected molecular mass of 42.7 kDa, and seem to be alleles of those cloned by Ozeki et al. (1993). The second isoform (DcCHS2) was present in both libraries. It had the highest degree of similarity (97.7%) to parsley CHS over all 397 amino acids. The expected molecular mass of the corresponding protein was 43.6 kDa. Results obtained from Southern blot analysis indicated the existence of at least two CHS genes in carrot. A transient enhancement of the DcCHS1 mRNA level after continuous irradiation with UV-A light could only be observed in anthocyanin-accumulating cultures, whereas an increase in DcCHS2 mRNA was seen in both cell lines. The maximum accumulation of CHS mRNA occurred 48 h after the onset of UV-A irradiation. In the European wild carrot the accumulation of DcCHS1 mRNA was restricted to the red central flowers, whereas the DcCHS2 mRNA was detectable in all red and white petals, as well as leaves, but was absent in stems and roots. The expression of DcCHS1 was restricted to anthocyanin-accumulating cells or organs. The heterologous expression of both cDNAs in Escherichia coli resulted in immunostainable bands of different sizes on the Western blot and high levels of catalytic CHS activity. Received: 2 September 1999 / Accepted: 30 November 1999     

Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa.

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999     

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.     

XVSAP1 from Xerophyta viscosa improves osmotic-, salinity- and high-temperature-stress tolerance in Arabidopsis

XVSAP1, a gene isolated from a dehydrated Xerophyta viscosa cDNA library, was transformed into Arabidopsis thaliana by Ti plasmid-mediated transformation under the control of a cauliflower mosaic virus 35S promoter, a nos terminator and bar gene selection. Expression of XVSAP1 in Arabidopsis led to constitutive accumulation of the corresponding protein in the leaves. Transgenic Arabidopsis grown in tissue culture maintained higher growth rates during osmotic, high-salinity and high temperature stress, respectively. Non-transgenic plants had shorter roots, leaf expansion was inhibited and leaves were more chlorotic than those of the transgenic plants. This study demonstrates that XVSAP1 has a significant impact on dehydration, salinity and high-temperature stress tolerance in Arabidopsis.     

Analysis of the nuclear proteome of the resurrection plant Xerophyta viscosa in response to dehydration stress using iTRAQ with 2DLC and tandem mass spectrometry

Xerophyta viscosa Baker (family Velloziaceae) is a desiccation tolerant plant which survives extremes of dehydration down to 5% relative water content (RWC) and resumes full physiological activity within 80h of rehydration. The nuclear proteome of Xerophyta viscosa and its response to dehydration at 35% RWC as compared to fully hydrated plants was analysed using iTRAQ together with 2DLC and ESI-MS/MS. RWC at 35% is unique for desiccation tolerant species as it represents a distinct phase of the dehydration process where induction of late protection mechanisms are initiated. We reproducibly identified 122 proteins with confidence≥95% (ρ<0.05). In response to dehydration, 65% of the identified proteins had the same protein abundance as the hydrated, 22% were shown to be more abundant while 9.8% were less abundant. Classification of the nuclear proteins according to GO annotation showed that most proteins were part of cellular processes (77.43%) and had binding activity (85.47%) respectively. Ontological classification according to Interpro and Pfam databases categorized most nuclear proteins as part of gene regulation (21%) while the functions of the mapped proteins using MapMan showed involvement in protein synthesis (22%), degradation (9%), DNA structure (8%) and regulation (8%).     

人白细胞介素6cDNA克隆的分离与鉴定

我们从重组的人α干扰素处理的单层HeLa细胞常规提取Poly(A)~+RNA作为逆转录合成cDNA第一链之模板,用引物-适配接头法在噬菌质粒pTz19R中构建cDNA文库。以~(32)p-标记的480bpIL-6cDNA片段作探针进行菌落原位及狭缝印迹杂交,筛选出6个阳性克隆。其中两个克隆并用限制酶切分析及DNA序列测定做进一步鉴定。结果证明,一个克隆的cDNA片段长1.3kb,含有人白介素6全长编码区;另一个的cDNA插入片段为0.9kb,缺乏信号肽及成熟IL-6N端30个残基的编码序列。     

cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)⁺:glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.