Interaction analysis of grapevine MIKC(c)-type MADS transcription factors and heterologous expression of putative véraison regulators in tomato.

MIKC(c)-type MADS-domain transcription factors include important regulators of floral development that interact in protein complexes to control the development of floral organs, as described by the ABC model. Members of the SEPALLATA (SEP) and AGAMOUS (AG) MADS clades include proteins involved in stamen and carpel specification and certain members of these families, such as tomato (Solanum lycopersicon) SlRIN and SlTAGL1, have been shown to regulate fruit development and ripening initiation. A number of expression studies have shown that several floral homeotic MADS genes are expressed during grapevine (Vitis vinifera) berry development, including potential homologues of these characterized ripening regulators. To gain insight into the regulation of berry development and ripening in grapevine, we studied the interactions and functions of grapevine floral homeotic MADS genes. Using the yeast 2- and 3-hybrid systems, we determined that the complexes formed during fruit development and ripening may involve several classes of floral homeotic MADS proteins. We found that a heterologously expressed grapevine SEP gene, VviSEP4, is capable of partially complementing the non-ripening phenotype of the tomato rin mutant, indicating that a role for this gene in ripening regulation may be conserved in fleshy fruit ripening. We also found that ectopic expression of a grapevine AG clade gene, VviAG1, in tomato results in the development of fleshy sepals with the chemical characteristics of tomato fruit pericarp. Additionally, we performed 2-hybrid screens on a library prepared from Pinot noir véraison-stage berry and identified proteins that may interact with the MADS factors that are expressed during berry development and that may represent regulatory functions in grape berry development.

Biosynthesis of wine aroma: transcript profiles of hydroxymethylbutenyl diphosphate reductase, geranyl diphosphate synthase, and linalool/nerolidol synthase parallel monoterpenol glycoside accumulation in Gewürztraminer grapes.

In developing grapevine (Vitis vinifera L.) berries, precursor volatile organic compounds (PVOCs) are largely stored as glycosides which may be hydrolyzed to release VOCs during fruit ripening, wine making, or aging. VOCs can be further transformed by yeast metabolism. Together, these processes contribute to complexity of wine aromas. Floral and citrus odors of many white wine varietals are attributed to monoterpenes and monoterpene alcohols, while phenolic compounds, norisoprenoids, and other volatiles also play important roles in determining aroma. We present an analysis of PVOCs stored as glycosides in developing Gewürztraminer berries during the growing season. We optimized a method for PVOC analysis suitable for small amounts of Muscat grapevine berries and showed that the amount of PVOCs dramatically increased during and after véraison. Transcript profiling of the same berry samples underscored the involvement of terpenoid pathway genes in the accumulation of PVOCs. The onset of monoterpenol PVOC accumulation in developing grapes was correlated with an increase of transcript abundances of early terpenoid pathway enzymes. Transcripts encoding the methylerythritol phosphate pathway gene 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, as well as geraniol diphosphate synthase, were up-regulated preceding and during the increase in monoterpenol PVOCs. Transcripts for linalool/nerolidol synthase increased in later véraison stages.

Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays.

BACKGROUND: Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly 1. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions. RESULTS: We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation. CONCLUSIONS: The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family.

Characterization of a Vitis vinifera cv. Cabernet Sauvignon 3',5'-O-methyltransferase showing strong preference for anthocyanins and glycosylated flavonols.

At ripening initiation in red grapevine (Vitis vinifera) berries, the exocarp turns color from green to red and then to purple due to the accumulation and extent of methylation of anthocyanins. The accumulation of transcripts encoding an O-methyltransferase was recently shown to be closely correlated with the onset of ripening and the degree of blue/purple pigmentation in grapevine berries; however, the biochemical function of this gene has remained uncharacterized. In this study, an O-methyltransferase cDNA that showed a distinct expression pattern when compared to closely related sequences was expressed in Escherichia coli and enzyme assays were carried out with a broad array of anthocyanin and other flavonoid substrates. We demonstrate that this enzyme carries out 3',5'-O-methylation of anthocyanins and flavonol compounds in vitro, which are known to be present in grape berries, with a preference for glycosylated substrates. The highest relative specific activity for the enzyme was found with delphinidin 3-O-glucoside as substrate. The enzyme is not able to methylate flavan type skeletons with chiral centers, such as either catechins or dihydroquercetin. The enzyme showed negligible specific activity for caffeoyl-CoA, compared to flavonol and anthocyanin substrates. Phylogenetic analysis of the O-methyltransferase suggests that it may be a member of a distinct subclass of Type 2 bivalent metal-dependent S-adenosyl-methionine O-methyltransferases.

The bouquet of grapevine (Vitis vinifera L. cv. Cabernet Sauvignon) flowers arises from the biosynthesis of sesquiterpene volatiles in pollen grains.

Terpenoid volatiles are important information molecules that enable pollinators to locate flowers and may protect reproductive tissues against pathogens or herbivores. Inflorescences of grapevine (Vitis vinifera L.) are composed of tiny green flowers that produce an abundance of sesquiterpenoid volatiles. We demonstrate that male flower parts of grapevines are responsible for sesquiterpenoid floral scent formation. We describe temporal and spatial patterns of biosynthesis and release of floral volatiles throughout the blooming of V. vinifera L. cv. Cabernet Sauvignon. The biosynthesis of sesquiterpene volatiles, which are emitted with a light-dependent diurnal pattern early in the morning at prebloom and bloom, is localized to anthers and, more specifically, within the developing pollen grains. Valencene synthase (VvValCS) enzyme activity, which produces the major sesquiterpene volatiles of grapevine flowers, is present in anthers. VvValCS transcripts are most abundant in flowers at prebloom stages. Western blot analysis identified VvValCS protein in anthers, and in situ immunolabeling located VvValCS protein in pollen grains during bloom. Histochemical staining, as well as immunolabeling analysis by fluorescent microscopy and transmission electron microscopy, indicated that VvValCS localizes close to lipid bodies within the maturing microspore.

Generation of a predicted protein database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv. Cabernet Sauvignon) berries at ripening initiation.

BACKGROUND: iTRAQ is a proteomics technique that uses isobaric tags for relative and absolute quantitation of tryptic peptides. In proteomics experiments, the detection and high confidence annotation of proteins and the significance of corresponding expression differences can depend on the quality and the species specificity of the tryptic peptide map database used for analysis of the data. For species for which finished genome sequence data are not available, identification of proteins relies on similarity to proteins from other species using comprehensive peptide map databases such as the MSDB. RESULTS: We were interested in characterizing ripening initiation ('veraison') in grape berries at the protein level in order to better define the molecular control of this important process for grape growers and wine makers. We developed a bioinformatic pipeline for processing EST data in order to produce a predicted tryptic peptide database specifically targeted to the wine grape cultivar, Vitis vinifera cv. Cabernet Sauvignon, and lacking truncated N- and C-terminal fragments. By searching iTRAQ MS/MS data generated from berry exocarp and mesocarp samples at ripening initiation, we determined that implementation of the custom database afforded a large improvement in high confidence peptide annotation in comparison to the MSDB. We used iTRAQ MS/MS in conjunction with custom peptide db searches to quantitatively characterize several important pathway components for berry ripening previously described at the transcriptional level and confirmed expression patterns for these at the protein level. CONCLUSION: We determined that a predicted peptide database for MS/MS applications can be derived from EST data using advanced clustering and trimming approaches and successfully implemented for quantitative proteome profiling. Quantitative shotgun proteome profiling holds great promise for characterizing biological processes such as fruit ripening initiation and may be further improved by employing preparative techniques and/or analytical equipment that increase peptide detection sensitivity via a shotgun approach.

Gene expression analyses in individual grape (Vitis vinifera L.) berries during ripening initiation reveal that pigmentation intensity is a valid indicator of developmental staging within the cluster.

Asynchronous ripening of individual grape berries within clusters can lead to inconsistent organoleptic characteristics for wine making. Ripening initiation in grape berries is non-climacteric and not well understood at the molecular level. Evidence is lacking for a single master switch controlling this process, such as the established role for ethylene in climacteric fruit ripening. We used Affymetrix microarray analyses of 32 individual Vitis vinifera cv. Cabernet Sauvignon berries sampled from two clusters at 50% ripening initiation. By delineating four developmental stages of ripening initiation, we demonstrate that pigmentation is a statistically significant indicator of transcriptional state during ripening initiation. We report on clustered gene expression patterns which were mined for genes annotated with signal transduction functions in order to advance regulatory network modeling of ripening initiation in grape berries. Abscisic acid has previously been demonstrated to be an important signaling component regulating ripening initiation in grapevine. We demonstrate via real-time RT-PCR analyses that up-regulation of a 9-cis-epoxycarotenoid gene family member, VvNCED2, in grape seed and pericarp and a putative ortholog to a reported abscisic acid receptor, VvGCR2, are correlated with ripening initiation. Our results suggest a role for these genes in abscisic acid signaling during ripening initiation.

Generation of ESTs in Vitis vinifera wine grape (Cabernet Sauvignon) and table grape (Muscat Hamburg) and discovery of new candidate genes with potential roles in berry development.

We report the generation and analysis of a total of 77,583 expressed sequence tags (ESTs) from two grapevine (Vitis vinifera L.) cultivars, Cabernet Sauvignon (wine grape) and Muscat Hamburg (table grape) with a focus on EST sequence quality and assembly optimization. The majority of the ESTs were derived from normalized cDNA libraries representing berry pericarp and seed developmental series, pooled non-berry tissues including root, flower, and leaf in Cabernet Sauvignon, and pooled tissues of berry, seed, and flower in Muscat Hamburg. EST and unigene sequence quality were determined by computational filtering coupled with small-scale contig reassembly, manual review, and BLAST analyses. EST assembly was optimized to better discriminate among closely related paralogs using two independent grape sequence sets, a previously published set of Vitis spp. gene families and our EST dataset derived from pooled leaf, flower, and root tissues of Cabernet Sauvignon. Sequence assembly within individual libraries indicated that those prepared from pooled tissues contributed the most to gene discovery. Annotations based upon searches against multiple databases including tomato and strawberry sequences helped to identify putative functions of ESTs and unigenes, particularly with respect to fleshy fruit development. Sequence comparison among the three wine grape libraries identified a number of genes preferentially expressed in the pericarp tissue, including transcription factors, receptor-like protein kinases, and hexose transporters. Gene ontology (GO) classification in the biological process aspect showed that GO categories corresponding to 'transport' and 'cell organization and biogenesis', which are associated with metabolite movement and cell wall structural changes during berry ripening, were higher in pericarp than in other tissues in the wine grape studied. The sequence data were used to characterize potential roles of new genes in berry development and composition.

An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development.

BACKGROUND: Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents. RESULTS: We describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-alpha (elongation factor 1-alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, alpha-tubulin, beta-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series. CONCLUSION: In order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-alpha and SAND as the most relevant reference genes for this purpose.

The molecular basis for wine grape quality--a volatile subject.

Volatile organic compounds are important flavor components of finished wines. In addition to winemaking practices, which shape wine quality, cultivation of the grape berries in the vineyard each season affects the production of volatile organic compounds as well as other chemical components that ultimately contribute to our perception of flavor in finished wines. By studying how berry flavor components are determined by the interplay of vine genotypes, the environment, and cultivation practices at the molecular level, scientists will develop advanced tools and knowledge that will aid viticulturalists in consistently producing balanced, flavorful berries for wine production.

Susceptible to intolerance--a range of hormonal actions in a susceptible Arabidopsis pathogen response.

Ethylene and salicylic acid (SA) are key intermediates in a host's response to pathogens. Previously, we have shown using a tomato compatible interaction that ethylene and SA act sequentially and are essential for disease symptom production. Here, we have examined the relationship between the two signals in the Arabidopsis-Xanthomonas campestris pv. campestris (Xcc) compatible interaction. Preventing SA accumulation by expression of the nahG gene reduced subsequent ethylene production and altered the development of disease symptoms, with plants showing no visible chlorosis. The ethylene insensitive lines, etr1-1 and etr2-1, on the other hand, accumulated SA and exhibited normal but precocious symptom development. Therefore, Arabidopsis, like tomato, was found to exhibit co-operative ethylene and SA action for the production of disease symptoms. However, in Arabidopsis, SA was found to act upstream of ethylene. Jasmonic acid and indole-3-acetic acid levels were also found to increase in response to Xcc. In contrast to ethylene, accumulation of these hormones was not found to be dependent on SA action. These results indicate that the plants response to a virulent pathogen is a composite of multiple signaling pathways.