Regulatory ligand binding in plant chalcone isomerase-like (CHIL) proteins.

Chalcone isomerase-like (CHIL) protein is a noncatalytic protein that enhances flavonoid content in green plants by serving as a metabolite binder and a rectifier of chalcone synthase (CHS). Rectification of CHS catalysis occurs through direct protein-protein interactions between CHIL and CHS, which alter CHS kinetics and product profiles, favoring naringenin chalcone (NC) production. These discoveries raise questions about how CHIL proteins interact structurally with metabolites and how CHIL-ligand interactions affect interactions with CHS. Using differential scanning fluorimetry on a CHIL protein from Vitis vinifera (VvCHIL), we report that positive thermostability effects are induced by the binding of NC, and negative thermostability effects are induced by the binding of naringenin. NC further causes positive changes to CHIL-CHS binding, whereas naringenin causes negative changes to VvCHIL-CHS binding. These results suggest that CHILs may act as sensors for ligand-mediated pathway feedback by influencing CHS function. The protein X-ray crystal structure of VvCHIL compared with the protein X-ray crystal structure of a CHIL from Physcomitrella patens reveals key amino acid differences at a ligand-binding site of VvCHIL that can be substituted to nullify the destabilizing effect caused by naringenin. Together, these results support a role for CHIL proteins as metabolite sensors that modulate the committed step of the flavonoid pathway.

Exploring Dihydroflavonol-4-Reductase Reactivity and Selectivity by QM/MM-MD Simulations.

Flavonoids are secondary metabolites ubiquitously found in plants. Their antioxidant properties make them highly interesting natural compounds for use in pharmacology. Therefore, unravelling the mechanisms of flavonoid biosynthesis is an important challenge. Among all the enzymes involved in this biosynthetic pathway, dihydroflavonol-4-reductase (DFR) plays a key role in the production of anthocyanins and proanthocyanidins. Here, we provide new information on the mechanism of action of this enzyme by using QM/MM-MD simulations applied to both dihydroquercetin (DHQ) and dihydrokaempferol (DHK) substrates. The consideration of these very similar compounds shed light on the major role played by the enzyme on the stabilization of the transition state but also on the activation of the substrate before the reaction through near-attack conformer effects.

Nucleoside 5'-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells.

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN's) and adenosine 5'-phosphoramidate (NH2-pA) belonging to nucleoside 5'-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN's induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5'-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5'-phosphoramidates could be considered as new signaling molecules.

Expressional diversity of grapevine 3-Hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) in different grapes genotypes.

BACKGROUND: 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a key enzyme in the mevalonate (MVA) pathway, which regulates the metabolism of terpenoids in the cytoplasm and determines the type and content of downstream terpenoid metabolites. RESULTS: Results showed that grapevine HMGR family has three members, such as VvHMGR1, VvHMGR2, and VvHMGR3. The expression of VvHMGRs in 'Kyoho' has tissue specificity, for example, VvHMGR1 keeps a higher expression, VvHMGR2 is the lowest, and VvHMGR3 gradually decreases as the fruit development. VvHMGR3 is closely related to CsHMGR1 and GmHMGR9 and has collinearity with CsHMGR2 and GmHMGR4. By the prediction of interaction protein, it can interact with HMG-CoA synthase, MVA kinase, FPP/GGPP synthase, diphosphate mevalonate decarboxylase, and participates in the synthesis and metabolism of terpenoids. VvHMGR3 have similar trends in expression with some of the genes of carotenoid biosynthesis and MEP pathways. VvHMGR3 responds to various environmental and phytohormone stimuli, especially salt stress and ultraviolet (UV) treatment. The expression level of VvHMGRs is diverse in grapes of different colors and aroma. VvHMGRs are significantly higher in yellow varieties than that in red varieties, whereas rose-scented varieties showed significantly higher expression than that of strawberry aroma. The expression level is highest in yellow rose-scented varieties, and the lowest in red strawberry scent varieties, especially 'Summer Black' and 'Fujiminori'. CONCLUSION: This study confirms the important role of VvHMGR3 in the process of grape fruit coloring and aroma formation, and provided a new idea to explain the loss of grape aroma and poor coloring during production. There may be an additive effect between color and aroma in the HMGR expression aspect.

Orthogonal Active-Site Labels for Mixed-Linkage endo-ß-Glucanases.

Small molecule irreversible inhibitors are valuable tools for determining catalytically important active-site residues and revealing key details of the specificity, structure, and function of glycoside hydrolases (GHs). ß-glucans that contain backbone ß(1,3) linkages are widespread in nature, e.g., mixed-linkage ß(1,3)/ß(1,4)-glucans in the cell walls of higher plants and ß(1,3)glucans in yeasts and algae. Commensurate with this ubiquity, a large diversity of mixed-linkage endoglucanases (MLGases, EC 3.2.1.73) and endo-ß(1,3)-glucanases (laminarinases, EC 3.2.1.39 and EC 3.2.1.6) have evolved to specifically hydrolyze these polysaccharides, respectively, in environmental niches including the human gut. To facilitate biochemical and structural analysis of these GHs, with a focus on MLGases, we present here the facile chemo-enzymatic synthesis of a library of active-site-directed enzyme inhibitors based on mixed-linkage oligosaccharide scaffolds and N-bromoacetylglycosylamine or 2-fluoro-2-deoxyglycoside warheads. The effectiveness and irreversibility of these inhibitors were tested with exemplar MLGases and an endo-ß(1,3)-glucanase. Notably, determination of inhibitor-bound crystal structures of a human-gut microbial MLGase from Glycoside Hydrolase Family 16 revealed the orthogonal labeling of the nucleophile and catalytic acid/base residues with homologous 2-fluoro-2-deoxyglycoside and N-bromoacetylglycosylamine inhibitors, respectively. We anticipate that the selectivity of these inhibitors will continue to enable the structural and mechanistic analyses of ß-glucanases from diverse sources and protein families.

Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene.

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol's enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

Genome-wide identification of BAM genes in grapevine (Vitis vinifera L.) and ectopic expression of VvBAM1 modulating soluble sugar levels to improve low-temperature tolerance in tomato.

BACKGROUND: Low temperature (LT) is one of the main limiting factors that affect growth and development in grape. Increasing soluble sugar and scavenging reactive oxygen species (ROS) play critical roles in grapevine resistance to cold stress. However, the mechanism of ß-amylase (BAM) involved in the regulation of sugar levels and antioxidant enzyme activities in response to cold stress is unclear. RESULTS: In this study, six BAM genes were identified and clustered into four groups. Multiple sequence alignment and gene structure analysis showed that VvBAM6 lacked the Glu380 residue and contained only an exon. The transcript abundance of VvBAM1 and VvBAM3 significantly increased as temperature decreased. After LT stress, VvBAM1 was highly expressed in the leaves, petioles, stems, and roots of overexpressing tomato lines. The total amylase and BAM activities increased by 6.5- and 6.01-fold in transgenic plants compared with those in wild-type tomato plants (WT) subjected to LT, respectively. The glucose and sucrose contents in transgenic plants were significantly higher than those in WT plants, whereas the starch contents in the former decreased by 1.5-fold compared with those in the latter under LT stress. The analysis of transcriptome sequencing data revealed that 541 genes were upregulated, and 663 genes were downregulated in transgenic plants. One sugar transporter protein gene (SlSTP10), two peroxidase (POD)-related genes (SlPER7 and SlPER5), and one catalase (CAT)-related gene (SlCAT1) were upregulated by 8.6-, 3.6-, 3.0-, and 2.3-fold in transgenic plants after LT stress, respectively. CONCLUSIONS: Our results suggest that VvBAM1 overexpression promotes ROS scavenging and improves cold tolerance ability by modulating starch hydrolysis to affect soluble sugar levels in tomato plants.

Comparative transcriptomic analysis between 'Summer Black' and its bud sport 'Nantaihutezao' during developmental stages.

MAIN CONCLUSION: 4-coumarate-CoA ligase (VIT_02s0109g00250) and copper amine oxidase (VIT_17s0000g09100) played essential roles in contributing to the total soluble solid and total anthocyanin variations induced by bud sport in grape berries. Taste and color, which are important organoleptic qualities of grape berry, undergo rapid and substantial changes during development and ripening. In this study, we used two cultivars 'Summer Black' and its bud sport 'Nantaihutezao' to explore and identify differentially expressed genes associated with total soluble solid and anthocyanin during developmental stages using RNA-Seq. Overall, substantial differences in expression were observed across berry development between the two cultivars. 5388 genes were detected by weighted gene co-expression network analysis (WGCNA) associated with the total soluble solid (TSS) and anthocyanin contents variations. Several of these genes were significantly enriched in the phenylalanine metabolism pathway; two hub genes 4-coumarate-CoA ligase (VIT_02s0109g00250) and copper amine oxidase (VIT_17s0000g09100) played the most essential roles in relating to the total soluble solid and total anthocyanin variations induced by bud sport through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and co-expression network analysis. These findings provide insights into the molecular mechanism responsible for the bud sport phenotype.

Grapevine U-Box E3 Ubiquitin Ligase VlPUB38 Negatively Regulates Fruit Ripening by Facilitating Abscisic-Aldehyde Oxidase Degradation.

The plant U-box E3 ubiquitin ligase-mediated ubiquitin/26S proteasome degradation system plays a key role in plant growth and development. Previously identified as a member of the grape PUB gene family, PUB38 was shown to participate in the berry-ripening progress. Here, we demonstrate that the E3 ligase VlPUB38 mediates abscisic acid (ABA) synthesis via 26S proteasome degradation and its involvement in regulating fruit-ripening processes. Strawberry-overexpressing VlPUB38 lines displayed obvious inhibition of mature phenotype, and this was rescued by exogenous ABA treatment and MG132. Post-ABA treatment, expression levels of ABA response-related genes in VlPUB38-overexpressed Arabidopsis significantly exceeded controls. Strawberry and Arabidopsis ectopic expression assays suggest that VlPUB38 negatively regulates fruit ripening in an ABA-dependent manner. Moreover, VlPUB38 has ubiquitin ligase activity, which depends on the U-box-conserved domain. VlPUB38 interacts with abscisic-aldehyde oxidase (VlAAO), targeting VlAAO proteolysis via the 26S proteasome system. These results indicate that VlPUB38 negatively regulates grape fruit ripening by mediating the degradation of key factor VlAAO in the ABA synthesis pathway.

Brassinosteroid Regulates 3-Hydroxy-3-methylglutaryl CoA Reductase to Promote Grape Fruit Development.

Brassinosteroids (BRs) are known to regulate plant growth and development. However, only little is known about their mechanism in the regulation of berry development in grapes. This study demonstrates that BR treatment enhances the accumulation of fruit sugar components, reduces the content of organic acids (e.g., tartaric acid), promotes coloration, and increases the anthocyanin content in grape berries at the onset of the veraison, half veraison, and full veraison stages at the rate of 0.0998, 0.0560, and 0.0281 mg·g-1, respectively. In addition, BR treatment was also found to accelerate the biosynthesis of terpenoid aroma components, such as α-pinene, d-limonene, and γ-terpinene, which influence the aromatic composition of grapes. BRs can negatively regulate the expression of VvHMGR, a key gene involved in the mevalonate (MVA) pathway, and reduce the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). Inhibiting the expression of HMGR promoted the accumulation of anthocyanins and fruit coloration. Meanwhile, after the inhibition, the contents of auxin indole-3-acetic acid (IAA), abscisic acid (ABA), and brassinosteroid (BR) increased, while gibberellin (GA3) and zeatin riboside (ZR) decreased, and its aromatic composition also changed. Therefore, it may be concluded that BRs inhibited HMGR activity and cooperated with VvHMGR to regulate the formation of color, aroma, and other quality characteristics in fruits.

S-Adenosyl-l-Methionine Promotes Metabolism of Fungicides in Cabernet Sauvignon (Vitis vinifera L.) Berries.

Fungicides are commonly used to prevent and treat grape (Vitis vinifera L.) diseases; however, they are potentially toxic to humans. Herein, we show that the application of S-adenosyl-l-methionine (SAM) accelerated the metabolism of various fungicides in Cabernet Sauvignon berries. The substances and enzymes involved in the metabolism of fungicides were analyzed to elucidate the effects of SAM. Results showed that SAM improved the production rate of superoxide anion, the hydrogen peroxide content, and the activities of superoxide dismutase, catalase, and peroxidase in azoxystrobin-treated berries. Additionally, SAM had a positive effect on the content of reduced glutathione and on the activities of glutathione S-transferase, glutathione reductase, and glutathione peroxidase. Importantly, the stimulatory effect of SAM on fungicide metabolism was also observed for metalaxyl and thiophanate-methyl. These results suggest that SAM can be used to improve food safety.

Chitinase family genes in grape differentially expressed in a manner specific to fruit species in response to Botrytis cinerea.

Chitinases (Chi), an important resistance-related protein, act against fungal pathogens by catalyzing the fungal cell wall, whereas are involved in different biological pathways in grape. In this study, we found 42 Chi family genes in Vitis vinifera L. (VvChis) and evaluated their expression levels after Botrytis infection, stress hormones like ethylene (ETH) and methyl-jasmonate (MeJA), and abiotic stresses like salinity and temperature changes in ripened fruits. VvChis were categorized into five groups including A, B, C, D, and E belonged to glycoside hydrolase family 18 and 19 (GH18 and GH19) according to genes structure, which expression analysis showed distinct temporal and spatial expression patterns changed in different tissues and various development stages. Different responsive elements to biotic and abiotic stresses were determined in the promoter regions of VvChis, specially elicitor-responsive element that was conserved among all VvChis genes. The expression levels of VvChis in groups A, B, and E increased after Botrytis cinerea infection in leaves and berries. Meanwhile, VvChis in glycoside hydrolase family 18 (GH18) were up-regulated under MeJA and ETH treatment, although the induction of VvChis by low temperature was more significant than high temperature. The expression of VvChis was also positively correlated with the concentration of NaCl treatment. Furthermore, differential gene-overexpression of VvChi5, VvChi17, VvChi22, VvChi26, and VvChi31 in strawberry and tomato fruits demonstrated the involvement of various isoforms in resistance to Botrytis infection through antioxidant system and lignin accumulation, which led to a reduction of damage. Among different isoforms of VvChis, we confirmed the interaction of Chi17 with Metallothionein (MTL) as oxidative stress protection, which suggests VvChis can modulate oxidative stress during postharvest storage in ripened fruits.

De novo resveratrol production through modular engineering of an Escherichia coli-Saccharomyces cerevisiae co-culture.

BACKGROUND: Resveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction. RESULTS: Here we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroGfbr) and chorismate mutase/prephenate dehydrogenase (tyrAfbr) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1S659A,S1157A) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved. CONCLUSIONS: This is first study that demonstrates the use of a synthetic E. coli-S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.

Substitution or Dilution? Assessing Pre-fermentative Water Implementation to Produce Lower Alcohol Shiraz Wines.

Changes to regulations by Food Standards Australia New Zealand have permitted the adjustment of must sugar levels with the addition of water in order to ensure a sound fermentation progress as well as mitigating excessive wine-alcohol levels. This study assessed the implications for Shiraz wine quality following a pre-fermentative must dilution (changing liquid-to-solid ratios), in comparison to juice substitution with water (constant liquid-to-solid ratios) that has previously been deemed a promising way to adjust wine-alcohol levels. While working within the legal limit of water addition to grape must, the effects of both approaches on wine quality parameters and sensory characteristics were rather similar, and of negligible nature. However, different implications between substitution and dilution appeared to be driven by grape maturity, and dilution was found to have a greater impact than substitution on some parameters at higher water implementation rates. In line with previous observations, longer hang-time followed by alcohol adjustments via pre-fermentation water addition were of limited merit compared to simply picking grapes earlier. This work provided further knowledge that supports informed decision making regarding the recently permitted approach of using water during winemaking.

Cloning, molecular and functional characterization by overexpression in Arabidopsis of MAPKK genes from grapevine (Vitis vinifera).

BACKGROUND: The mitogen-activated protein kinases (MAPKs), as a part of the MAPKKK-MAPKK-MAPK cascade, play crucial roles in plant development as an intracellular signal transduction pathway to respond various environmental signals. However, few MAPKK have been functionally characterized in grapevine. RESULTS: In the study, five MAPKK (MKK) members were identified in grapevine (cultivar 'Pinot Noir'), cloned and designated as VvMKK1-VvMKK5. A phylogenetic analysis grouped them into four sub-families based on the similarity of their conserved motifs and gene structure to Arabidopsis MAPKK members. qRT-PCR results indicated that the expression of VvMKK1, VvMKK2, VvMKK4, and VvMKK5 were up-regulated in mature leaf and young blades, and roots, but exhibited low expression in leaf petioles. VvMKK2, VvMKK3, and VvMKK5 genes were differentially up-regulated when grapevine leaves were inoculated with spores of Erisyphe necator, or treated with salicylic acid (SA), ethylene (ETH), H2O2, or exposed to drought, indicating that these genes may be involved in a variety of signaling pathways. Over expression of VvMKK2 and VvMKK4 genes in transgenic Arabidopsis plants resulted in the production of seeds with a significantly higher germination and survival rate, and better seedling growth under stress conditions than wild-type plants. Overexpression of VvMKK2 in Arabidopsis improved salt and drought stress tolerance while overexpression of VvMKK4 only improved salt stress tolerance. CONCLUSIONS: Results of the present investigation provide a better understanding of the interaction and function of MAPKKK-MAPKK-MAPK genes at the transcriptional level in grapevine and led to the identification of candidate genes for drought and salt stress in grapes.

Using the Chou's 5-steps rule, transient overexpression technique, subcellular location, and bioinformatic analysis to verify the function of Vitis vinifera O-methyltranferase 3 (VvOMT3) protein.

3-Isobutyl-2-methoxypyrazine (IBMP) is an important odor compound that revives unripe grapes or poor-quality wine. The biosynthesis of IBMP in grape berries is under the catalysis of Vitis vinifera O-methyltranferase 3 (VvOMT3). The homologous verification in this paper was carried out with the transient overexpression technique. The results showed that both the expression levels of the VvOMT3 gene and the IBMP concentration in 'Red globe' grapes increased significantly, which suggested that VvOMT3 could function in the biosynthesis of IBMP. Based on ß-glucuronidase (GUS) staining results, blue color was only observed in grape pulp, not in grape skin, which indicated that VvOMT3 was expressed in grape pulp. The outcomes of the subcellular location examination performed on the protoplasts of Arabidopsis thaliana showed that the VvOMT3 protein was located on the inner surface of the cytoplasmic membrane. In summary, the VvOMT3 enzyme may function at the inner surface of the cytoplasmic membrane of pulp cells during grape development. These results will provide a background for future research on the catalytic mechanisms of VvOMT3.

The grape ubiquitin ligase VpRH2 is a negative regulator in response to ABA treatment.

MAIN CONCLUSION: Ubiquitin ligase VpRH2 is a negative regulator in the grape ABA pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter is inhibited by ABA treatment. In higher plants, ubiquitin ligases play key roles in various cellular processes. As in our previous study (Wang et al. in J Exp Bot 68:1669-1687, 2017), grape RING-H2-type ubiquitin ligase gene VpRH2 and its promoter was induced by powdery mildew and showed resistance to the disease. Diverse small-molecule hormones, like salicylic acid (SA), methyl jasmonate (MeJA) or abscisic acid (ABA), play pivotal roles in plant resistance. Here we found that VpRH2 expression could be induced by SA and MeJA treatment, but inhibited by ABA treatment. The promoter of VpRH2 revealed a similar variation trend under exogenous hormone treatments as the gene expression by GUS activity assay. By a series of deletion fragments, the promoter fragment of VpRH2-P656 to VpRH2-P513 was necessary in response to MeJA treatment, and the inhibition of ABA treatment to the VpRH2 promoter was independent of the ABRE motif. Over-expression of VpRH2 in Arabidopsis thaliana plants displayed ABA-insensitive phenotypes at the germination stage compared to wild type plants. In VpRH2 over-expressing Vitis vinifera cv. Thompson Seedless plants after ABA treatments, the expression of the ABA pathway related genes ABL1 and PYR1 showed a suppresive trend. Moreover, VpGRP2A (an VpRH2-interacting protein) also showed a suppresive trend in response to ABA treatment in VpRH2-overexpressing plants. Our results demonstrate that VpRH2 is a negative regulator in the grape ABA signal pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter was also inhibited by ABA treatment.

Optimization of Catechin and Proanthocyanidin Recovery from Grape Seeds Using Microwave-Assisted Extraction.

Grape seed extract (GSE) is a rich source of condensed flavonoid tannins, also called proanthocyanidins (PACs). The high molecular weight of polymeric PAC limits their biological activity due to poor bioavailability. The present study was undertaken to explore the potential applicability of microwave-assisted extraction (MAE) to convert GSE-PAC into monomeric catechins. A central composite design (CCD) was used to optimize the processing conditions for the MAE. The maximum total yield of monomeric catechins (catechin, epicatechin, and epicatechin gallate) and PAC were 8.2 mg/g dry weight (DW) and 56.4 mg catechin equivalence (CE)/g DW, respectively. The optimized MAE condition was 94% ethanol, 170 °C temperature, and a duration of 55 min. Compared to the results for PACs extracted via conventional extraction (Con) (94% ethanol; shaking at 25 °C for 55 min), MAE yielded 3.9-fold more monomeric catechins and 5.5-fold more PACs. The MAE showed higher antioxidant capacity and α-glucosidase inhibitory activity than that of the conventional extract, suggesting the potential use of the MAE products of grape seeds as a functional food ingredient and nutraceutical.

Molecular Cloning and Characterization of an Acidic Polygalacturonase from Grapes and Its Potential in Industry.

Pectinase enzymes from different plants have many possible biotechnological applications in various industries. Considering industrial significance of pectinolytic enzymes, the polygalacturonase (PG) gene from grapes was cloned into Escherichia coli DH5α using pTZ57R/T vector. Homologous sequences established a close link to Vitis vinifera. Conserve domain analysis predicted PLN02218 domain belongs to the cl31843 superfamily, showing its function as polygalacturonase. After confirmation by PCR and restriction analysis, the PG gene was expressed in E. coli BL21 and induced by IPTG. Expression level was assessed by 12% SDS-PAGE, which showed a 47 kDa protein. High expression level in the soluble fraction indicated that the protein is intracellular or transmembrane. Maximum expression was obtained with 1 mM IPTG and 6 hour induction time, and autoinduction with lactose increased production of the recombinant enzyme. Zymogram analysis revealed that the induced protein was an active enzyme. Expressed PG showed pectinolytic effect on the physiochemical properties of lemon juice. Natural biopolymers are greatly needed because synthetic fibers can negatively affect health. Pectin hydrolysis of banana pseudostem by the PG enzyme produced better quality fiber. Morphological studies by scanning electron microscopy revealed pectin degradation within the fiber cell architecture, showing the effectiveness of PG treatment on banana pseudostem.

Mass spectrometry of peptides and proteins using digestion by a grape cysteine protease at pH 3.

Cysteine protease from grapevine (Vitis vinifera) belongs to those resistant proteins, which survive the process of vinification and can therefore be detected as wine components. Its amino acid sequence shows a homology to other members of the papain family, but the enzyme has only partially been explored so far. In order to get more biochemical information with the help of mass spectrometry (MS), wine proteins were collected by ultrafiltration and separated by gel permeation chromatography. The purified enzyme surprisingly displayed a high molecular mass value of around 200 kDa, indicating a possible oligomeric status and aggregation, as it entered only negligibly the separating 10% gel during polyacrylamide gel electrophoresis. The isoelectric point (pI) value of 3.6 was determined by chromatofocusing. Matrix-assisted laser desorption/ionization (MALDI)-MS was employed to evaluate the cleavage specificity and usefulness of the isolated cysteine protease in protein and peptide research. A potential applicability could be anticipated from the efficient digestion performance in volatile ammonium formate buffers at pH 3. Common peptides were digested and the resulting products analyzed by MS/MS sequencing. Then, mixtures of protein standards and extracted barley nuclear proteins were processed in the same way. Grape cysteine protease is nonspecific but shows a certain preference for Arg, Lys, and also Leu residues. Compared with papain, it seems not to require fully the presence of a large hydrophobic residue adjacent to that at the cleavage site. The enzyme is suitable for protein research as it produces peptides of a reasonable length in acidic pH.