Expression and In Situ Localization of Two Major PR Proteins of Grapevine Berries during Development and after UV-C Exposition

In grapevine Vitis vinifera L. cv Pinot noir, the Pathogenesis-Related (PR) proteins CHI4D and TL3 are among the most abundant extractable PR proteins of ripe berries and accumulate during berry ripening from véraison until full maturation. Evidence was supplied in favor of the involvement of these two protein families in plant defense mechanisms and plant development. In order to better understand CHI4D and TL3 function in grapevine, we analyzed their temporal and spatial pattern of expression during maturation and after an abiotic stress (UV-C) by in situ hybridization (ISH) and immunohistolocalization. In ripening berries, CHI4D and TL3 genes were mainly expressed in the exocarp and around vascular bundles of the mesocarp. In UV-C exposed berries, CHI4D and TL3 gene expression was strongly induced before véraison. Corresponding proteins localized in the exocarp and, to a lesser extent, around vascular bundles of the mesocarp. The spatial and temporal accumulation of the two PR proteins during berry maturation and after an abiotic stress is discussed in relation to their putative roles in plant defense.     

Proteomic analysis of the compatible interaction between Vitis vinifera and Plasmopara viticola

We analyzed the proteome of grapevine (Vitis vinifera) leaves 24, 48 and 96 h post infection (hpi) with the downy mildew pathogen Plasmopara viticola. Total proteins were separated on 2-DE gels. By MS analysis, we identified 82 unique grapevine proteins differentially expressed after infection. Upregulated proteins were often included in the functional categories of general metabolism and stress response, while proteins related to photosynthesis and energy production were mostly downregulated. As expected, the activation of a defense reaction was observed more often at the late time point, consistent with the establishment of a compatible interaction. Most proteins involved in resistance were isoforms of different PR-10 pathogenesis-related proteins. Although > 50 differentially expressed protein isoforms were observed at 24 and 96 hpi, only 18 were detected at 48 hpi and no defense-related proteins were among this group. This profile suggests a transient breakdown in defense responses accompanying the onset of disease, further supported by gene expression analyses and by a western blot analysis of a PR-10 protein. Our data reveal the complex modulation of plant metabolism and defense responses during compatible interactions, and provide insight into the underlying molecular processes which may eventually yield novel strategies for pathogen control in the field.     

Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes.     

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.     

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+-pumping pyrophosphatase in pepper plants

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes.     

Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) uses PP(i) as an energy donor and requires free Mg(2+) for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (Asp(253), Lys(261), Glu(263), Asp(279), Asp(283), Asp(287), Asp(723), Asp(727), and Asp(731)) in the putative PP(i)-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PP(i) hydrolysis activity and a complete loss of H(+) transport activity. The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin. Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PP(i), while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys(261) and Glu(263), are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PP(i) hydrolysis but no proton pump activity, we estimated that two residues, Lys(261) and Glu(263), might be related to the energy conversion from PP(i) hydrolysis to H(+) transport. The importance of two residues, Asp(253) and Glu(263), in the Mg(2+)-binding function was also suggested from the trypsin susceptibility in the presence of Mg(2+). Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases.     

iTRAQ-based profiling of grape berry exocarp proteins during ripening using a parallel mass spectrometric method

The 4-plex iTRAQ platform was utilized to analyze the protein profiles in four stages of grapevine berry skin ripening, from pre-veraison to fully ripening. Mass spectrometric data were acquired from three replicated analyses using a parallel acquisition method in an Orbitrap instrument by combining collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) peptide ion fragmentations. As a result, the number of spectra suitable for peptide identification (either from CID or HCD) increased 5-fold in relation to those suitable for quantification (from HCD). Spectra were searched against an NCBInr protein database subset containing all the Vitis sequences, including those derived from whole genome sequencing. In general, 695 unique proteins were identified with more than one single peptide, and 513 of them were quantified. The sequence annotation and GO term enrichment analysis assisted by the automatic annotation tool Blast2GO permitted a pathway analysis which resulted in finding that biological processes and metabolic pathways de-regulated throughout ripening. A detailed analysis of the function-related proteins profiles helped discover a set of proteins of known Vitis gene origin as the potential candidates to play key roles in grapevine berry quality, growth regulation and disease resistance.     

Pollen-specific regulation of vacuolar H+-PPase expression by multiple cis-acting elements

We dissected the regulatory region of the AVP1 gene encoding the vacuolar H⁺-pyrophosphatase (V-PPase) of Arabidopsis thaliana by using a GUS-reporter assay system. The cloned 1.4 kb 5-regulatory region in the GUS-reporter transgenic plants was sufficient for the light-induced repression. Furthermore, the 1.4 kb regulatory region was active in all tissues examined and its activity was especially enhanced in pollen, whereas the shorter 0.4 kb regulatory region was active only in pollen. Further detailed analyses revealed that the GUS activity in pollen was regulated by at least three cis-acting regions in an additive or synergetic manner. These findings establish a distinct mechanism of the tissue-specific regulation of V-PPase expression in developing pollen, and imply the biological significance of the V-PPase in pollen maturation.     

Catalase Is Differentially Expressed in Dividing and Nondividing Protoplasts

Based on our previous results that peroxidase is induced in dividing tobacco protoplasts but it is not expressed in the nondividing grapevine (Vitis vinifera L.) protoplasts during culture (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270), we further tested the hypothesis that oxidative stress may be implicated in the recalcitrance of plant protoplasts. The expression of catalase, a major defense enzyme against cell oxidation, was studied during isolation and culture of mesophyll protoplasts from the recalcitrant grapevine and regenerating tobacco (Nicotiana tabacum L.). Incubation of tobacco leaf strips with cell wall-degrading enzymes resulted in a burst of catalase activity and an increase in its immunoreactive protein; in contrast, no such increases were found in grapevine. The cathodic and anodic catalase isoforms consisted exclusively of subunits [alpha] and [beta], respectively, in tobacco, and of subunits [beta] and [alpha], respectively, in grapevine. The catalase specific activity increased only in grapevine protoplasts during culture. The ratio of the enzymatic activities to the catalase immunoreactive protein declined in dividing tobacco protoplasts and remained fairly constant in nondividing tobacco and grapevine protoplasts during culture. Also, in dividing tobacco protoplasts the de novo accumulation of the catalase [beta] subunit gave rise to the acidic isoenzymes, whereas in nondividing tobacco and grapevine protoplasts, after 8 d in culture, only the basic isoenzymes remained due to de novo accumulation of the [alpha] subunit. The pattern of catalase expression in proliferating tobacco leaf cells during callogenesis was similar to that in dividing protoplasts. The different responses of catalase expression in dividing and nondividing tobacco and grapevine mesophyll protoplasts may indicate a specificity of catalase related to induction of totipotency.     

Profiling of sugar transporter genes in grapevine coping with water deficit

The profiling of grapevine (Vitis vinifera L.) genes under water deficit was specifically targeted to sugar transporters. Leaf water status was characterized by physiological parameters and soluble sugars content. The expression analysis provided evidence that VvHT1 hexose transporter gene was strongly down-regulated by the increased sugar content under mild water-deficit. The genes of monosaccharide transporter VvHT5, sucrose carrier VvSUC11, vacuolar invertase VvGIN2 and grape ASR (ABA, stress, ripening) were up-regulated under severe water stress. Their regulation in a drought-ABA signalling network and possible roles in complex interdependence between sugar subcellular partitioning and cell influx/efflux under Grapevine acclimation to dehydration are discussed.