Structural features in the model of a thermostable and stress-resistant protein, SP1 from aspen

A three dimensional theoretical model of SP1 (stable protein 1), which is resistant to high temperature and biotic-stresses, is presented here. The model was generated by the application of homology modeling technique. The conformational rigidity imparted to the fold by the presence of hydrogen-bonded, C5, C7, C10 and C13 structures in the loop regions, multiple aromatic--aromatic interactions at the protein interior and on the surface, in addition to salt-links and hydrogen-bonds are primarily the major factors, responsible for the increased stability of protein. The putative protein family is characterized by motifs, E-x(0,1)-L-x-[AEGQS] and V-x(2,3)-L-x-[ADEGST] and the active site in the tertiary structure is formed by conserved aromatic and isoleucine clusters.     

Purification of boiling-soluble antifreeze protein from the legume Ammopiptanthus mongolicus

A boiling-soluble antifreeze protein (AFP) was purified from the winter leaves of Ammopiptanthus mongolicus an evergreen legume species surviving in the cold desert of northwest of China. Purification was achieved by using a procedure consisting of a heat treatment step followed by consecutive chromatography, including ion-exchange chromatography (DEAE-Cellulose 52, Source 15Q), molecular exclusion chromatography with Sephacryl S300, and hydrophobic interaction chromatography (Poros 20HP2). This AFP showed thermal hysteresis activity and could modify the normal growth of ice crystals. The thermal hysteresis activity (THA) of this purified antifreeze protein is 0.15 degrees C at a concentration of 10 mg/mL, and its molecular mass is approximately 28 kD by SDS-PAGE analysis.     

Characterization of a stress-responsive ankyrin repeat-containing zinc finger protein of Capsicum annuum (CaKR1)

We isolated many genes induced from pepper cDNA microarray data following their infection with the soybean pustule pathogen Xanthomonas axonopodis pv. glycines 8ra. A full-length cDNA clone of the Capsicum annuum ankyrin-repeat domain C(3)H(1) zinc finger protein (CaKR1) was identified in a chili pepper using the expressed sequence tag (EST) database. The deduced amino acid sequence of CaKR1 showed a significant sequence similarity (46%) to the ankyrin-repeat protein in very diverse family of proteins of Arabidopsis. The gene was induced in response to various biotic and abiotic stresses in the pepper leaves, as well as by an incompatible pathogen, such as salicylic acid (SA) and ethephon. CaKR1 expression was highest in the root and flower, and its expression was induced by treatment with agents such as NaCl and methyl viologen, as well as by cold stresses. These results showed that CaKR1 fusion with soluble, modified green fluorescent protein (smGFP) was localized to the cytosol in Arabidopsis protoplasts, suggesting that CaKR1 might be involved in responses to both biotic and abiotic stresses in pepper plants.     

Characterization of the PL-I-related SP2 protein from Xenopus

The complete cDNA sequence of Xenopus laevis sperm specific proteins SP1 and SP2 has been determined. This information when taken together with N-terminal sequencing and mass spectroscopy data indicates that these two proteins share a product precursor relationship in which SP2 results from cleavage of a short N-terminal peptide of SP1. The secondary and tertiary structures of SP2 have been characterized using circular dichroism and three dimension structure prediction. These structural analyses have conclusively shown that SP1/SP2 proteins are related to proteins of the histone H1 family, particularly to vertebrate histone H1x. Hence, they can be considered bona fide members of the protamine-like- I (PL-I) group of sperm nuclear basic proteins (SNBPs) that have been described in other vertebrate and invertebrate groups. SP2 binds to nucleosomal DNA in a way that is very similar to that of histone H1. However, its interaction with circular DNA does not exhibit an enhanced preference for the supercoiled conformation, and it appears to be mainly driven by ionic interactions.     

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.     

Characterization of phenolic glycosides from quaking aspen

A crude extract quaking aspen Populus tremuloides exhibits differential bioactivity against subspecies of the eastern tiger swallowtail Papilio glaucus. Components were isolated and identified on the basis of NMR and IR spectra, and chemical methods, as the phenolic glycosides salicin, salicortin, tremuloidin, and tremulacin.     

Phosphorylation of RTP, an ER stress-responsive cytoplasmic protein

RTP, also called Drg1/Cap43/rit42/TDD5/Ndr1, was originally identified as a homocysteine-responsive gene product, and is now considered to be involved in stress responses, atherosclerosis, carcinogenesis, differentiation, androgen responses, hypoxia, and N-myc pathways. We raised an antiserum against a recombinant human RTP. Western blot analysis showed that RTP expression was induced in human umbilical vein endothelial cells under conditions causing endoplasmic reticulum stress. RTP was partially phosphorylated at seven or more sites. The phosphorylation was reversible, and was enhanced by an increased level of intracellular cAMP and inhibited by both a protein kinase A inhibitor and a calmodulin kinase inhibitor. Protein kinase A directly phosphorylated recombinant RTP in vitro. The phosphorylated forms were abundant in cells at the early log phase, and then decreased with increasing cell density. These data demonstrated that RTP is a phosphorylated stress-responsive protein, and its phosphorylation may be related to cell growth.     

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.     

Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis

Mitogen-activated protein kinases (MAPKs) are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown because of the lack of information about their substrates. Using a conditional gain-of-function transgenic system, we demonstrated that the activation of SIPK, a tobacco (Nicotiana tabacum) stress-responsive MAPK, induces the biosynthesis of ethylene. Here, we report that MPK6, the Arabidopsis thaliana ortholog of tobacco SIPK, is required for ethylene induction in this transgenic system. Furthermore, we found that selected isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, are substrates of MPK6. Phosphorylation of ACS2 and ACS6 by MPK6 leads to the accumulation of ACS protein and, thus, elevated levels of cellular ACS activity and ethylene production. Expression of ACS6(DDD), a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, confers constitutive ethylene production and ethylene-induced phenotypes. Increasing numbers of stress stimuli have been shown to activate Arabidopsis MPK6 or its orthologs in other plant species. The identification of the first plant MAPK substrate in this report reveals one mechanism by which MPK6/SIPK regulates plant stress responses. Equally important, this study uncovers a signaling pathway that modulates the biosynthesis of ethylene, an important plant hormone, in plants under stress.     

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.     

Characterization of a lignin-specific O-Methyltransferase in aspen wood

O-Methyltransferases were extracted from the differentiating xylem of 10-yr-old Populus euramericana. The enzymes were partially purified by ammonium sulfate precipitation, and column chromatography on DEAE-cellulose, Sephadex G200 and hydroxyapatite. The enzymes were resolved into two peaks by DEAE-cellulose chromatography, and the MWs of the respective enzymes were estimated to be 72 000 and 75 000 by gel filtration chromatography. The enzyme corresponding to the latter peak was unstable and thus only the former peak enzyme was characterized completely. Magnesium ions had no effect, EDTA moderately stimulated and heavy metals and SH group inhibitors strongly inhibited enzyme activity. K_mm values for caffeate and 5-hydroxyferulate were estimated to be 3.8 x 10⁻⁴ and 3.1 x 10⁻⁴ M, respectively. The ratio of V_max/K_m for 5-hydroxyferulate was 5.4 times greater than that for caffeate. The enzyme(s) catalysing the formation of ferulate from caffeate and of sinapate from 5-hydroxyferulate were not separated during the purification or by the disc electrophoresis using polyacrylamide gel. Quercetin, cyanin and catechin were not methylated by the enzyme preparation. The O-methyltransferase of aspen wood, where the phenolic metabolism is almost exclusively directed to lignin biosynthesis, catalyses the methylation of both guaiacyl and syringyl lignin precursors, with preferential utilization of the latter substrate. These findings lead to the conclusion that the enzyme is a typical angiosperm-type O-methyltransferase related to guaiacyl and syringyl lignin biosynthesis in aspen wood.     

Characterization of chlorosis-inducing toxins from a plant pathogenic pseudomonas SP

1.

1. An extracellular, chlorosis-inducing agent and several related, but inactive, products of a Pseudomonas sp. attacking timothy (Phleum pratense L.) were prepared by chromatographic means.     

Characterization of SP41, a surface protein of Brucella associated with adherence and invasion of host epithelial cells

Brucella is an invasive organism that multiplies and survives within eukaryotic cells. The brucellae are able to adhere to the surface of cultured epithelial cells, a mechanism that may facilitate penetration and dissemination to other host tissues. However, no adhesins that allow the bacteria to interact with the surface of epithelial cells before migration within polymorphonuclear leukocytes, monocytes and macrophages have been described. Here, we show that Brucella surface proteins (SPs) with apparent molecular masses of 14, 18 and 41 kDa bound selectively to HeLa cells. However, only antibodies directed against the 41 kDa surface protein (SP41) inhibited in dose-response manner, bacterial adherence and invasion of HeLa cells. HeLa cells treated with neuraminidase did not bind SP41, suggesting the involvement of cellular sialic acid residues in this interaction. Biochemical analysis of SP41 revealed that this protein is the predicted product of the ugpB locus, which showed significant homology to the glycerol-3-phosphate-binding ATP-binding cassette (ABC) transporter protein found in several bacterial species. SP41 appears to be exposed on the bacterial surface as determined by immunofluorescence and immunogold labelling with anti-SP41 antibody. An isogenic DeltaugpB mutant showed a significant inhibitory effect on Brucella adherence and invasion of human cultured epithelial cells and this effect could be reversed by restoration of the ugpB on a plasmid. Lastly, we also show that most of the sera from individuals with acute brucellosis, but not sera obtained from healthy donors or patients with chronic brucellosis, mount antibody reactivity against SP41, suggesting that this protein is produced in vivo and that it elicits an antibody immune response. These data are novel findings that offer new insights into understanding the interplay between this bacterium and host target cells, and identify a new target for vaccine development and prevention of brucellosis.     

Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots

Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.     

Characterization of Grp1p,a novel cis-Golgi matrix protein

A high copy suppressor screen with sec34-2, a temperature-sensitive mutant defective in the late stages of ER to Golgi transport, has resulted in the identification of a novel gene called GRP1 (also called RUD3). GRP1 encodes a hydrophilic yeast protein related to the mammalian Golgi matrix protein golgin-160. A large portion of the protein is predicted to form a coiled-coil structure. Although GRP1 is not essential for growth, the loss of Grp1p results in a growth defect at high temperature. GRP1 genetically interacts with several genes involved in vesicle targeting/fusion stages of ER to Golgi transport. Despite these interactions, pulse chase analysis using Grp1p-depleted cells did not reveal a significant delay in the transit of the vacuolar protease carboxypeptidase Y. Grp1p-depleted cells efficiently secreted invertase which was underglycosylated, suggesting some disturbance of Golgi function. Grp1p-GFP predominantly colocalizes with the cis-Golgi marker Och1p. Despite lacking a signal peptide and a significant stretch of hydrophobic amino acids, Grp1p pellets with membranes. It is extracted with 1M NaCl or 0.1M Na(2)CO(3) (pH 11.0), but is surprisingly insoluble in 1% Triton X-100. Grp1p does not recycle to the ER when forward transport is blocked and a cis-Golgi marker (Och1p-HA), but not a trans-Golgi marker (Chs5p-HA), became dispersed in grp1 Delta cells after 1.5h incubation at 38.5 degrees C. Together, these data suggest that Grp1p is a novel matrix protein that is involved in the structural organization of the cis-Golgi.