The diversity of pathogenesis-related proteins decreases during grape maturation

It was recently shown that wines contain typically a huge diversity of structurally similar polypeptides that exhibit a high degree of homology to pathogenesis-related (PR) proteins. This observation suggested the existence of one or a few precursors in mature grapes, common to most or all the wine PR proteins. Limited proteolysis and chemical modification of the precursor(s) during fruit ripening and winemaking could then generate the large number of distinct wine polypeptides. However, the patterns of PR proteins extracted from grape berries regularly harvested from the onset of development until maturity did not confirm the previous hypothesis. Two different methodologies, involving 2-D immunoblotting and a combination of FPLC cation/anion exchange chromatographies with 1-D immunoblotting, indicate that the total concentration of PR proteins is increased but its diversity is reduced from the early stages of berry development until maturity. These results indicate that PR proteins are synthesized in a wide variety of forms from the early stages of grape development, eliminating the hypothesis previously formulated on the existence of one or few precursors common to the wine proteins.     

The metacaspase gene family of Vitis vinifera L.: Characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1–VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape.     

Morphological variation and relationships of Georgian populations of Vitis vinifera L. subsp. sylvestris (C.C. Gmel.) Hegi

Georgia is considered as one of the regions where for the first time wild growing grapevine plants were used and taken into cultivation by men. An ampelographic and ampelometric study of leaf characters and flower landmark morphometrics was carried out in seven populations of wild grapevine located in four river basins of three geographic regions – Western (1 popul.), Eastern (5 popul.) and Southern (1 popul.) Georgia, and then compared using principal component analysis and multivariate discriminant analysis allowing the populations to be distinguished. Lengths of main leaf veins and nectaries in male flowers were the variables, which allowed the best discrimination among populations. A mutation leading to white-fruited grapevine is found in one population. Pubescence varies within populations and even within an individual genome and could be used as a discriminant trait only in combination with shapes and sizes of leaves and other morphological characters. The results revealed significant differences among populations located in different geographic regions of the Southern Caucasus area, but the five investigated populations from Eastern Georgia differ only insignificantly. The high morphological variability of wild grapevine in Georgia might be considered as characterizing the large gene pool that contributed to the domestication process of grapevine.     

Neuroprotection of the leaf and stem of Vitis amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons

Vitis amurensis (Vitaceae) has been reported to have anti-oxidant and anti-inflammatory activities. The present study investigated a methanol extract from the leaf and stem of V. amurensis for neuroprotective effects on cerebral ischemic damage in rats and on excitotoxicity induced by glutamate in cultured rat cortical neurons. Transient focal cerebral ischemia was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion (MCAO/reperfusion) in rats. Orally administered V. amurensis (25-100 mg/kg) reduced MCAO/reperfusion-induced infarct and edema formation, neurological deficits, and neuronal death. Depletion of glutathione (GSH) level and lipid peroxidation induced by MCAO/reperfusion was inhibited by administration of V. amurensis. The increase of phosphorylated mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 (COX-2), and pro-apoptotic proteins and the decrease of anti-apoptotic protein in MCAO/reperfusion rats were significantly inhibited by treatment with V. amurensis. Exposure of cultured cortical neurons to 500 μM glutamate for 12 h induced neuronal cell death. V. amurensis (1-50 μg/ml) and (+)-ampelopsin A, γ-2-viniferin, and trans-?-viniferin isolated from the leaf and stem of V. amurensis inhibited glutamate-induced neuronal death, the elevation of intracellular calcium ([Ca²⁺]_i), the generation of reactive oxygen species (ROS), and changes of apoptosis-related proteins in cultured cortical neurons, suggesting that the neuroprotective effect of V. amurensis may be partially attributed to these compounds. These results suggest that the neuroprotective effect of V. amurensis against focal cerebral ischemic injury might be due to its anti-apoptotic effect, resulting from anti-excitotoxic, anti-oxidative, and anti-inflammatory effects and that the leaf and stem of V. amurensis have possible therapeutic roles for preventing neurodegeneration in stroke.     

Crystal Structure and Catalytic Mechanism of Leucoanthocyanidin Reductase from Vitis vinifera

Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 3₁₀ helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.     

施氮时期对酿酒葡萄叶片氮代谢酶及相关基因表达的影响

以10年生‘蛇龙珠’葡萄为材料,在萌芽期(S1)、新梢旺长期(S2)、开花期(S3)、果实第一次膨大期(S4)、副梢生长旺期(S5)和果实第二次膨大期(S6)分别一次性施入尿素300kg·hm-2,以不施氮肥为对照(CK),分析了花前5d(DBF5)、花后25d(DAF25)、花后55d(DAF55)和花后85d(DAF85)叶片的各项指标,以明确氮素施用时期对葡萄叶片氮代谢的调控与影响。结果表明:(1)S1和S2处理葡萄叶片中总氮及可溶性蛋白含量在DAF25时显著增加。(2)S3和S4处理的NR及GS活性在DAF85时显著高于其他处理;在DAF25时,S1和S2处理的GOGAT活性,以及S3和S4处理的GDH活性均显著高于同期对照和其他施肥处理。(3)各施肥处理叶片VvNR表达水平在不同时期均高于同期对照,S3处理VvNR表达水平在DAF25和DAF85时分别为对照的3.4倍和2.7倍;S3和S4处理的VvGS表达水平分别在DAF55和DAF85时达到最高值,S3处理的VvGOGAT和S4处理的VvGDH表达水平在DAF55和DAF85均显著高于其他处理,S3处理的VvGDH表达水平在DAF55和DAF85仅次于S4处理。研究表明,氮素通过诱导叶片氮素代谢基因的响应,从而调控叶片中氮素代谢酶活性增加,促进了氮素的积累,S3和S4处理在不同时期氮代谢酶活性和对应的基因表达水平均较高,更有利于叶片中氮素的转化和代谢。     

Proteomic identification of extracellular proteins regulated by the Gna1 Gα subunit in Stagonospora nodorum

The fungus Stagonospora nodorum is the causal agent of stagonospora nodorum blotch (syn. leaf and glume blotch) disease of wheat. The Gna1-encoded Gα protein is an important signal transduction component in the fungus, which is required for full pathogenicity, sporulation and extracellular depolymerase production. In this study, we sought to gain a better understanding of defects associated with the gna1 mutant by using two-dimensional gel electrophoresis to analyse the extracellular proteome for differences to the wildtype. Mass spectrometry analysis of altered abundant protein spots and peptide matching to the Stagonospora nodorum genome database have led to the identification of genes implicated in cell wall degradation, proteolysis, RNA hydrolysis and aromatic compound metabolism. In addition, quantitative RT-PCR has demonstrated that some of the encoding genes showed differential expression throughout host infection. Implications of these proteins and their corresponding genes in fungal virulence are discussed.     

Galloylated catechins and stilbene diglucosides in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes. When cells were grown in a medium inducing polyphenol synthesis, (−)-epicatechin-3-O-gallate, dimeric procyanidin B-2 3′-O-gallate and two resveratrol diglucosides were isolated, together with a new natural compound that was identified as cis-resveratrol-3,4′-O-β-diglucoside by spectroscopical methods.     

Quantifying the trans influence of triphenylarsine. Crystal and molecular structures of cis-[PtCl2(SMe2)(AsPh3)] and cis-[PtCl2(AsPh3)2]·CHCl3

Reaction between a mixture of cis-trans-[PtCl₂(SMe₂)₂] and 1 equiv. AsPh₃ in chloroform gives cis-[PtCl₂(SMe₂)(AsPh₃)] crystallizing in P2₁/n with a=10.397(2), b=14.876(3), c=13.956(3) Å, β=90.86(3)° and Z=4. Selected geometrical parameters are PtAs 2.3531(10), PtS 2.262(2), PtCl (trans to S) 2.301(2), PtCl (trans to As) 2.328(2) Å and SPtAs 88.85(6), SPtCl(2) 90.77(8), AsPtCl(1) 91.07(6) and ClPtCl 89.42(7)°. cis-[PtCl₂(AsPh₃)₂]·CHCl₃ crystallizes in P2₁/c with a=20.557(4), b=9.5951(19), c=20.147(4) Å, β=96.77(3)° and Z=4. Selected geometrical parameters are PtAs(1) 2.3599(9), PtAs(2) 2.3770(9), PtCl(1) (trans to As(1)) 2.3515(18), PtCl(2) (trans to As(2)) 2.3251(18) Å and AsPtAs 97.87(3), As(1)PtCl(2) 88.67(5), As(2)PtCl(1) 84.30(5) and ClPtCl 89.32(7)°. By comparison with related structures from the literature the following trans influence series was established PMe₂Ph>PPh₃>AsPh₃≈SbPh₃>Me₂SO≈SMe₂≈SPh₂>NH₃≈olefin>Cl⁻>MeCN.     

Biosynthesis of salicylic acid in fungus elicited Catharanthus roseus cells

Feeding experiments using [1-¹³C]-d-glucose to Catharanthus roseus (L.) G.Don cell suspension cultures followed by elicitation with Pythium aphanidermatum extract were performed in order to study the salicylic acid (SA) biosynthetic pathway and that of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a comparison. A strongly labeled C-7 and a symmetrical partitioning of the label between C-2 and C-6 would occur if SA was synthesized from phenylalanine. In case of the isochorismate pathway, a relatively lower incorporation at C-7 and a non-symmetrical incorporation at C-2 and C-6 would be obtained. Relatively, high- and non-symmetrical enrichment ratios at C-2 and C-6, and a lower enrichment ratio at C-7 were observed in both SA and 2,3-DHBA detected by ¹³C NMR inverse gated spectrometry leading to the conclusion that the isochorismate pathway is responsible for the biosynthesis of both compounds. However, different enrichment ratios of the labeled carbons in SA and 2,3-DHBA indicate the use of different isochorismate pools, which means that their biosynthesis is separated in time and/or space.     

Quantification of cis and trans influences in [PtX(PPh3)3] complexes. A P NMR study

In [PtX(PPh₃)₃]⁺ complexes (X = F, Cl, Br, I, AcO, NO₃, NO₂, H, Me) the mutual cis and trans influences of the PPh₃ groups can be considered constants in the first place, therefore the one bond Pt-P coupling constants of P_(cis) and P_(trans) reflect the cis and trans influences of X. The compounds [PtBr(PPh₃)₃](BF₄) (2), [PtI(PPh₃)₃](BF₄) (3), [Pt(AcO)(PPh₃)₃](BF₄) (4), [Pt(NO₃)(PPh₃)₃](BF₄) (5), and the two isomers [Pt(NO₂-O)(PPh₃)₃](BF₄) (6a) and [Pt(NO₂-N)(PPh₃)₃](BF₄) (6b) have been newly synthesised and the crystal structures of 2 and 4·CH₂Cl₂·0.25C₃H₆O have been determined. From the ¹J_PtP values of all compounds we have deduced the series: I > Br > Cl > NO₃ > ONO > F > AcO > NO₂ > H > Me (cis influence) and Me > H > NO₂ > AcO > I > ONO > Br > Cl > F > NO₃ (trans influence). These sequences are like those obtained for the (neutral) cis- and trans-[PtClX(PPh₃)₂] derivatives, showing that there is no dependence on the charge of the complexes. On the contrary, the weights of both influences, relative to those of X = Cl, were found to depend on the charge and nature of the complex.     

Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes

The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes.     

Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium

Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p<0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.Contribution Number 217.     

Endometrial inflammation and abnormal expression of extracellular matrix proteins induced by Mycoplasma bovis in dairy cows

Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.     

Gibberella fujikuroi侵染果蔗叶片病程相关蛋白编码基因的表达分析

利用实时荧光定量RT-PCR,用梢腐病病原菌(Gibberella fujikuroi)侵染不同果蔗品种叶片,对病程相关蛋白编码基因SoSOD、SoCHIT、SoPOD与SoTPS6P的转录水平进行了分析。结果表明,果蔗的SoSOD、SoCHIT、SoPOD与SoTPS6P受到梢腐病病原菌的诱导表达,丰城紫皮、白鳝、福安与拔地拉果蔗叶片中它们的表达量较高,而在温岭、宁德与歪干担叶片中的表达量较低。这说明这些病程相关蛋白编码基因表达水平与不同果蔗品种的梢腐病抗病性存在一定的关联。