Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine.
Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (≥2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries.
These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing.
The developmental programme of grape berries within a cluster is coordinated to synchronize their ripening. Altered transcriptional events and metabolite accumulation are responsible for the differential progress of ripening.
Transcriptional studies in relation to fruit ripening generally aim to identify the transcriptional states associated with physiological ripening stages and the transcriptional changes between stages within the ripening programme. In non-climacteric fruits such as grape, all ripening-related genes involved in this programme have not been identified, mainly due to the lack of mutants for comparative transcriptomic studies. A feature in grape cluster ripening (Vitis vinifera cv. Pinot noir), where all berries do not initiate the ripening at the same time, was exploited to study their shifted ripening programmes in parallel. Berries that showed marked ripening state differences in a véraison-stage cluster (ripening onset) ultimately reached similar ripeness states toward maturity, indicating the flexibility of the ripening programme. The expression variance between these véraison-stage berry classes, where 11% of the genes were found to be differentially expressed, was reduced significantly toward maturity, resulting in the synchronization of their transcriptional states. Defined quantitative expression changes (transcriptional distances) not only existed between the véraison transitional stages, but also between the véraison to maturity stages, regardless of the berry class. It was observed that lagging berries complete their transcriptional programme in a shorter time through altered gene expressions and ripening-related hormone dynamics, and enhance the rate of physiological ripening progression. Finally, the reduction in expression variance of genes can identify new genes directly associated with ripening and also assess the relevance of gene activity to the phase of the ripening programme.
Enhanced ripening; grape; hormone; plasticity; ripening synchronization; transcriptional programme.
Grapevine (Vitis species) is among the most important fruit crops in terms of cultivated area and economic impact. Despite this relevance, little is known about the transcriptional changes and the regulatory circuits underlying the biochemical and physical changes occurring during berry development.
Fruit ripening in the non-climacteric crop species Vitis vinifera L. has been investigated at the transcriptional level by the use of the Affymetrix Vitis GeneChip® which contains approximately 14,500 unigenes. Gene expression data obtained from berries sampled before and after véraison in three growing years, were analyzed to identify genes specifically involved in fruit ripening and to investigate seasonal influences on the process. From these analyses a core set of 1477 genes was found which was similarly modulated in all seasons. We were able to separate ripening specific isoforms within gene families and to identify ripening related genes which appeared strongly regulated also by the seasonal weather conditions. Transcripts annotation by Gene Ontology vocabulary revealed five overrepresented functional categories of which cell wall organization and biogenesis, carbohydrate and secondary metabolisms and stress response were specifically induced during the ripening phase, while photosynthesis was strongly repressed. About 19% of the core gene set was characterized by genes involved in regulatory processes, such as transcription factors and transcripts related to hormonal metabolism and signal transduction. Auxin, ethylene and light emerged as the main stimuli influencing berry development. In addition, an oxidative burst, previously not detected in grapevine, characterized by rapid accumulation of H2O2 starting from véraison and by the modulation of many ROS scavenging enzymes, was observed.
The time-course gene expression analysis of grapevine berry development has identified the occurrence of two well distinct phases along the process. The pre-véraison phase represents a reprogramming stage of the cellular metabolism, characterized by the expression of numerous genes involved in hormonal signalling and transcriptional regulation. The post-véraison phase is characterized by the onset of a ripening-specialized metabolism responsible for the phenotypic traits of the ripe berry. Between the two phases, at véraison, an oxidative burst and the concurrent modulation of the anti-oxidative enzymatic network was observed. The large number of regulatory genes we have identified represents a powerful new resource for dissecting the mechanisms of fruit ripening control in non-climacteric plants.
Temperature and solar radiation influence Vitis vinifera L. berry ripening. Both environmental conditions fluctuate cyclically on a daily period basis and the strength of this fluctuation affects grape ripening too. Additionally, a molecular circadian clock regulates daily cyclic expression in a large proportion of the plant transcriptome modulating multiple developmental processes in diverse plant organs and developmental phases. Circadian cycling of fruit transcriptomes has not been characterized in detail despite their putative relevance in the final composition of the fruit. Thus, in this study, gene expression throughout 24 h periods in pre-ripe berries of Tempranillo and Verdejo grapevine cultivars was followed to determine whether different ripening transcriptional programs are activated during certain times of day in different grape tissues and genotypes.
Microarray analyses identified oscillatory transcriptional profiles following circadian variations in the photocycle and the thermocycle. A higher number of expression oscillating transcripts were detected in samples carrying exocarp tissue including biotic stress-responsive transcripts activated around dawn. Thermotolerance-like responses and regulation of circadian clock-related genes were observed in all studied samples. Indeed, homologs of core clock genes were identified in the grapevine genome and, among them, VvREVEILLE1 (VvRVE1), showed a consistent circadian expression rhythm in every grape berry tissue analysed. Light signalling components and terpenoid biosynthetic transcripts were specifically induced during the daytime in Verdejo, a cultivar bearing white-skinned and aromatic berries, whereas transcripts involved in phenylpropanoid biosynthesis were more prominently regulated in Tempranillo, a cultivar bearing black-skinned berries.
The transcriptome of ripening fruits varies in response to daily environmental changes, which might partially be under the control of circadian clock components. Certain cultivar and berry tissue features could rely on specific circadian oscillatory expression profiles. These findings may help to a better understanding of the progress of berry ripening in short term time scales.
Circadian; Fruit ripening; Gene expression; Grapevine; Light; Microarray; Phenylpropanoid; Temperature; Terpene; Vitis vinifera
The ripening of fleshy fruits is a complex developmental program characterized by extensive transcriptomic and metabolic remodeling in the pericarp tissues (pulp and skin) making unripe green fruits soft, tasteful and colored. The onset of ripening is regulated by a plethora of endogenous signals tuned to external stimuli. In grapevine and tomato, which are classified as non-climacteric and climacteric species respectively, the accumulation of hydrogen peroxide (H2O2) and extensive modulation of reactive oxygen species (ROS) scavenging enzymes at the onset of ripening has been reported, suggesting that ROS could participate to the regulatory network of fruit development. In order to investigate this hypothesis, a comprehensive biochemical study of the oxidative events occurring at the beginning of ripening in Vitis vinifera cv. Pinot Noir has been undertaken.
ROS-specific staining allowed to visualize not only H2O2 but also singlet oxygen (1O2) in berry skin cells just before color change in distinct subcellular locations, i.e. cytosol and plastids. H2O2 peak in sample skins at véraison was confirmed by in vitro quantification and was supported by the concomitant increase of catalase activity. Membrane peroxidation was also observed by HPLC-MS on galactolipid species at véraison. Mono- and digalactosyl diacylglycerols were found peroxidized on one or both α-linolenic fatty acid chains, with a 13(S) absolute configuration implying the action of a specific enzyme. A lipoxygenase (PnLOXA), expressed at véraison and localizing inside the chloroplasts, was indeed able to catalyze membrane galactolipid peroxidation when overexpressed in tobacco leaves.
The present work demonstrates the controlled, harmless accumulation of specific ROS in distinct cellular compartments, i.e. cytosol and chloroplasts, at a definite developmental stage, the onset of grape berry ripening. These features strongly candidate ROS as cellular signals in fruit ripening and encourage further studies to identify downstream elements of this cascade. This paper also reports the transient galactolipid peroxidation carried out by a véraison-specific chloroplastic lipoxygenase. The function of peroxidized membranes, likely distinct from that of free fatty acids due to their structural role and tight interaction with photosynthesis protein complexes, has to be ascertained.
Chloroplastic lipoxygenase; Fruit ripening; Galactolipids; Hydrogen peroxide; Oxidative stress; Oxylipin; ROS; Singlet oxygen
Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions.
Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented.
These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality.
The complex and dynamic changes during grape berry development have been studied in Vitis vinifera, but little is known about these processes in other Vitis species. The grape variety 'Norton', with a major portion of its genome derived from Vitis aestivalis, maintains high levels of malic acid and phenolic acids in the ripening berries in comparison with V. vinifera varieties such as Cabernet Sauvignon. Furthermore, Norton berries develop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remain susceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit a comprehensive analysis of transcriptional regulation and metabolite pathways.
A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin.
This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.
Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors.
A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal membranes.
A berry specific cDNA clone, Vv-AMP1, was isolated and characterized and shown to encode a plant defensin. Recombinant Vv-AMP1 displayed non-morphogenic antifungal activity against a broad spectrum of fungi, probably altering the membrane permeability of the fungal pathogens. The expression of this peptide is highly regulated in Vitis vinifera, hinting at an important defense role during berry-ripening.
The AP2/ERF protein family contains transcription factors that play a crucial role in plant growth and development and in response to biotic and abiotic stress conditions in plants. Grapevine (Vitis vinifera) is the only woody crop whose genome has been fully sequenced. So far, no detailed expression profile of AP2/ERF-like genes is available for grapevine.
An exhaustive search for AP2/ERF genes was carried out on the Vitis vinifera genome and their expression profile was analyzed by Real-Time quantitative PCR (qRT-PCR) in different vegetative and reproductive tissues and under two different ripening stages.
One hundred and forty nine sequences, containing at least one ERF domain, were identified. Specific clusters within the AP2 and ERF families showed conserved expression patterns reminiscent of other species and grapevine specific trends related to berry ripening. Moreover, putative targets of group IX ERFs were identified by co-expression and protein similarity comparisons.
The grapevine genome contains an amount of AP2/ERF genes comparable to that of other dicot species analyzed so far. We observed an increase in the size of specific groups within the ERF family, probably due to recent duplication events. Expression analyses in different aerial tissues display common features previously described in other plant systems and introduce possible new roles for members of some ERF groups during fruit ripening. The presented analysis of AP2/ERF genes in grapevine provides the bases for studying the molecular regulation of berry development and the ripening process.
Global climate change will noticeably affect plant vegetative and reproductive development. The recent increase in temperatures has already impacted yields and composition of berries in many grapevine-growing regions. Physiological processes underlying temperature response and tolerance of the grapevine fruit have not been extensively investigated. To date, all studies investigating the molecular regulation of fleshly fruit response to abiotic stress were only conducted during the day, overlooking possible critical night-specific variations. The present study explores the night and day transcriptomic response of grapevine fruit to heat stress at several developmental stages. Short heat stresses (2 h) were applied at day and night to vines bearing clusters sequentially ordered according to the developmental stages along their vertical axes. The recently proposed microvine model (DRCF-Dwarf Rapid Cycling and Continuous Flowering) was grown in climatic chambers in order to circumvent common constraints and biases inevitable in field experiments with perennial macrovines. Post-véraison berry heterogeneity within clusters was avoided by constituting homogenous batches following organic acids and sugars measurements of individual berries. A whole genome transcriptomic approach was subsequently conducted using NimbleGen 090818 Vitis 12X (30 K) microarrays.
Present work reveals significant differences in heat stress responsive pathways according to day or night treatment, in particular regarding genes associated with acidity and phenylpropanoid metabolism. Precise distinction of ripening stages led to stage-specific detection of malic acid and anthocyanin-related transcripts modulated by heat stress. Important changes in cell wall modification related processes as well as indications for heat-induced delay of ripening and sugar accumulation were observed at véraison, an effect that was reversed at later stages.
This first day - night study on heat stress adaption of the grapevine berry shows that the transcriptome of fleshy fruits is differentially affected by abiotic stress at night. The present results emphasize the necessity of including different developmental stages and especially several daytime points in transcriptomic studies.
Fruit development is controlled by plant hormones, but the role of hormone interactions during fruit ripening is poorly understood. Interactions between ethylene and the auxin indole-3-acetic acid (IAA) are likely to be crucial during the ripening process, since both hormones have been shown to be implicated in the control of ripening in a range of different fruit species.
Grapevine (Vitis vinifera L.) homologues of the TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR) and YUCCA families, functioning in the only characterized pathway of auxin biosynthesis, were identified and the expression of several TAR genes was shown to be induced by the pre-ripening application of the ethylene-releasing compound Ethrel. The induction of TAR expression was accompanied by increased IAA and IAA-Asp concentrations, indicative of an upregulation of auxin biosynthesis and conjugation. Exposure of ex planta, pre-ripening berries to the ethylene biosynthesis inhibitor aminoethoxyvinylglycine resulted in decreased IAA and IAA-Asp concentrations. The delayed initiation of ripening observed in Ethrel-treated berries might therefore represent an indirect ethylene effect mediated by increased auxin concentrations. During berry development, the expression of three TAR genes and one YUCCA gene was upregulated at the time of ripening initiation and/or during ripening. This increase in auxin biosynthesis gene expression was preceded by high expression levels of the ethylene biosynthesis genes 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase.
In grape berries, members of both gene families involved in the two-step pathway of auxin biosynthesis are expressed, suggesting that IAA is produced through the combined action of TAR and YUCCA proteins in developing berries. The induction of TAR expression by Ethrel applications and the developmental expression patterns of auxin and ethylene biosynthesis genes indicate that elevated concentrations of ethylene prior to the initiation of ripening might lead to an increased production of IAA, suggesting a complex involvement of this auxin and its conjugates in grape berry ripening.
Aminoethoxyvinylglycine; Auxin; Biosynthesis; Ethrel; Ethylene; Interaction; Vitis vinifera; Ripening
Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines.
Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals.
We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between transcript profiles and the kinetics of accumulation of red/cyanidin- and blue/delphinidin-based anthocyanins indicated that VvF3'H and VvF3'5'H expression is consistent with the chromatic evolution of ripening bunches. Local physical maps constructed around the VvF3'H and VvF3'5'H loci should help facilitate the identification of the regulatory elements of each isoform and the future manipulation of grapevine and wine colour through agronomical, environmental and biotechnological tools.
This paper aims at analysing the synthesis of flavonoids, their import and export in plant cell compartments, as well as their involvement in the response to stress, with particular reference to grapevine (Vitis vinifera L.). A multidrug and toxic compound extrusion (MATE) as well as ABC transporters have been demonstrated in the tonoplast of grape berry, where they perform a flavonoid transport. The involvement of a glutathione S-transferase (GST) gene has also been inferred. Recently, a putative flavonoid carrier, similar to mammalian bilitranslocase (BTL), has been identified in both grape berry skin and pulp. In skin the pattern of BTL expression increases from véraison to harvest, while in the pulp its expression reaches the maximum at the early ripening stage. Moreover, the presence of BTL in vascular bundles suggests its participation in long distance transport of flavonoids. In addition, the presence of a vesicular trafficking in plants responsible for flavonoid transport is discussed. Finally, the involvement of flavonoids in the response to stress is described.
flavonoid transport; bilitranslocase; ABC transporters; secondary metabolites; biotic and abiotic stress
The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity.
Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar) grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7), harvest (TH), and 10-days after harvest (TH+10)). Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines.
In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sauvignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase.
This set of up- and down-regulated genes characterize the late stages of berry ripening in the two cultivars studied, and are indirectly linked to wine quality. They might be used directly or indirectly to design immunological, biochemical or molecular tools aimed at the determination of optimal ripening in these cultivars.
Over the course of grape berry development, the tissues of the berry undergo numerous morphological transformations in response to processes such as water and solute accumulation and cell division, growth and senescence. These transformations are expected to produce changes to the diffusion of water through these tissues detectable using diffusion magnetic resonance imaging (MRI). To assess this non-invasive technique diffusion was examined over the course of grape berry development, and in plant tissues with contrasting oil content.
In this study, the fruit of Vitis vinfera L. cv. Semillon at seven different stages of berry development, from four weeks post-anthesis to over-ripe, were imaged using diffusion tensor and transverse relaxation MRI acquisition protocols. Variations in diffusive motion between these stages of development were then linked to known events in the morphological development of the grape berry. Within the inner mesocarp of the berry, preferential directions of diffusion became increasingly apparent as immature berries increased in size and then declined as berries progressed through the ripening and senescence phases. Transverse relaxation images showed radial striation patterns throughout the sub-tissue, initiating at the septum and vascular systems located at the centre of the berry, and terminating at the boundary between the inner and outer mesocarp. This study confirms that these radial patterns are due to bands of cells of alternating width that extend across the inner mesocarp. Preferential directions of diffusion were also noted in young grape seed nucelli prior to their dehydration. These observations point towards a strong association between patterns of diffusion within grape berries and the underlying tissue structures across berry development. A diffusion tensor image of a post-harvest olive demonstrated that the technique is applicable to tissues with high oil content.
This study demonstrates that diffusion MRI is a powerful and information rich technique for probing the internal microstructure of plant tissues. It was shown that macroscopic diffusion anisotropy patterns correlate with the microstructure of the major pericarp tissues of cv. Semillon grape berries, and that changes in grape berry tissue structure during berry development can be observed.
Development; Diffusion anisotropy; Diffusion tensor imaging; Grape berry; Nucellus; Nuclear magnetic resonance imaging; Olive; Seeds; Striation patterns; Vitis vinifera
Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses.
Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening.
Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.
An in vivo experimental system—called the ‘berry‐cup’ technique—was developed to study sugar phloem unloading and the accumulation of sugar in ripening grape berries. The berry‐cup system consists of a single peeled grape berry immersed in a buffer solution in a cup prepared from a polypropylene syringe. A small cross‐incision (2 mm in length) is made on the stylar remnant of a berry during its ripening phase, the skin of the berry then being easily peeled off, exposing the dorsal vascular bundles without damaging either these or the pulp tissue of the berry. The sites of sugar phloem unloading are thus made directly accessible and may be regulated by the buffer solution. In addition, the unloaded photoassimilates are easily transported into the buffer solution in the berry‐cup. With the berry‐cup technique, it takes 60 min to purge the sugar already present in the apoplast, after which the amount of sugar in the buffer solution is a direct measure of the sugar unloading from the grape berry phloem. The optimum times for sampling were 20 or 30 min, depending on the type of experiment. Sugar phloem unloading was significantly inhibited by the inclusion of either 7·5 mm NaF or 2·5 mm PCMB in the buffer solution. This study indicates that sugar phloem unloading in ripening grape berries is via the apoplastic network and that the process requires the input of energy. The system was shown to be an appropriate experimental system with which to study sugar phloem unloading in ripening grape berries, and was applied successfully to the study of berry sugar unloaded from grapevines subjected to water stress. The results showed that water deficiency inhibits sugar unloading in grape berries.
Sugar phloem unloading; ‘berry‐cup’ technique; ripening grape berry; inhibitors; water deficiency
The timing of grape ripening initiation, length of maturation period, berry size and seed content are target traits in viticulture. The availability of early and late ripening varieties is desirable for staggering harvest along growing season, expanding production towards periods when the fruit gets a higher value in the market and ensuring an optimal plant adaptation to climatic and geographic conditions. Berry size determines grape productivity; seedlessness is especially demanded in the table grape market and is negatively correlated to fruit size. These traits result from complex developmental processes modified by genetic, physiological and environmental factors. In order to elucidate their genetic determinism we carried out a quantitative analysis in a 163 individuals-F1 segregating progeny obtained by crossing two table grape cultivars.
Molecular linkage maps covering most of the genome (2n = 38 for Vitis vinifera) were generated for each parent. Eighteen pairs of homologous groups were integrated into a consensus map spanning over 1426 cM with 341 markers (mainly microsatellite, AFLP and EST-derived markers) and an average map distance between loci of 4.2 cM. Segregating traits were evaluated in three growing seasons by recording flowering, veraison and ripening dates and by measuring berry size, seed number and weight. QTL (Quantitative Trait Loci) analysis was carried out based on single marker and interval mapping methods. QTLs were identified for all but one of the studied traits, a number of them steadily over more than one year. Clusters of QTLs for different characters were detected, suggesting linkage or pleiotropic effects of loci, as well as regions affecting specific traits. The most interesting QTLs were investigated at the gene level through a bioinformatic analysis of the underlying Pinot noir genomic sequence.
Our results revealed novel insights into the genetic control of relevant grapevine features. They provide a basis for performing marker-assisted selection and testing the role of specific genes in trait variation.
The selective removal of grapevine leaves around berry clusters can improve the quality of ripening fruits by influencing parameters such as the berry sugar and anthocyanin content at harvest. The outcome depends strongly on the timing of defoliation, which influences the source–sink balance and the modified microclimate surrounding the berries. We removed the basal leaves from Vitis vinifera L. cv Sangiovese shoots at the pre-bloom and veraison stages, and investigated responses such as shoot growth, fruit morphology and composition compared to untreated controls. Moreover, we performed a genome-wide expression analysis to explore the impact of these defoliation treatments on berry transcriptome.
We found that pre-bloom defoliation improved berry quality traits such as sugar and anthocyanin content, whereas defoliation at veraison had a detrimental effect, e.g. less anthocyanin and higher incidence of sunburn damage. Genome-wide expression analysis during berry ripening revealed that defoliation at either stage resulted in major transcriptome reprogramming, which slightly delayed the onset of ripening. However, a closer investigation of individual gene expression profiles identified genes that were specifically modulated by defoliation at each stage, reflecting the uncoupling of metabolic processes such as flavonoid biosynthesis, cell wall and stress metabolism, from the general ripening program.
The specific transcriptional modifications we observed following defoliation at different time points allow the identification of the developmental or metabolic processes affected in berries thus deepening the knowledge of the mechanisms by which these agronomical practices impact the final berry ripening traits.
Vitis vinifera; Defoliation; Berry ripening; Transcriptome; Flavonoid; Source-sink balance
This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first.
In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions.
The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening.
The ripening of grape berry is generally regulated by abscisic acid (ABA), and has no relationship with ethylene function. However, functional interaction and synergism between ABA and ethylene during the beginning of grape berry ripening (véraison) has been found recently.
The expressions of VvNCED1 encoding 9-cis-epoxycarotenoid dioxygenase (NCED) and VvGT encoding ABA glucosyltransferase were all increased rapidly at the stage of véraison and reached the highest level at 9th week after full bloom. However, VvCYP1 encoding ABA 8'-hydroxylase and VvβG1 encoding berry β-glucosidase are different, whose expression peak appeared at the 10th week after full bloom and in especial VvβG1 remained at a high level till harvest. The VvACO1 encoding 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, the VvETR2 (ethylene response 2) and VvCTR1 (constitutive triple response 1) had a transient expression peak at pre-véraison, while the VvEIN4 (ethylene insensitive 4) expression gradually increased from the véraison to one week before harvest stage. The above mentioned changes happened again in the berry after harvest. At one week before véraison, double block treatment with NiCl2 plus 1-methylcyclopropene (1-MCP) not only inhibited the release of ethylene and the expression of related genes but also suppressed the transcription of VvNCED1 and the synthesis of ABA which all might result in inhibiting the fruit ripening onset. Treatment with ABA could relieve the double block and restore fruit ripening course. However, after harvest, double block treatment with NiCl2 plus 1-MCP could not suppress the transcription of VvNCED1 and the accumulation of ABA, and also could not inhibit the start of fruit senescence.
The trace endogenous ethylene induces the transcription of VvNCED1 and then the generation of ABA followed. Both ethylene and ABA are likely to be important and their interplaying may be required to start the process of berry ripening. When the level of ABA reached the peak value, part of it will be stored in the form of ABA-GE. While after harvest, abiotic stresses principally (such as dehydration, harvest shock) could induce the transcription of VvNCED1 and the accumulation of ABA, thus starting the process of fruit senescence.
Grape ripening represents the third phase of the double sigmoidal curve of berry development and is characterized by deep changes in the organoleptic characteristics. In this process, the skin plays a central role in the synthesis of many compounds of interest (e.g. anthocyanins and aroma volatiles) and represents a fundamental protective barrier against damage by physical injuries and pathogen attacks. In order to improve the knowledge on the role of this tissue during ripening, changes in the protein expression in the skin of the red cultivar Barbera at five different stages from véraison to full maturation were studied by performing a comparative 2-DE analysis.
The proteomic analysis revealed that 80 spots were differentially expressed throughout berry ripening. Applying a two-way hierarchical clustering analysis to these variations, a clear difference between the first two samplings (up to 14 days after véraison) and the following three (from 28 to 49 days after véraison) emerged, thus suggesting that the most relevant changes in protein expression occurred in the first weeks of ripening. By means of LC-ESI-MS/MS analysis, 69 proteins were characterized. Many of these variations were related to proteins involved in responses to stress (38%), glycolysis and gluconeogenesis (13%), C-compounds and carbohydrate metabolism (13%) and amino acid metabolism (10%).
These results give new insights to the skin proteome evolution during ripening, thus underlining some interesting traits of this tissue. In this view, we observed the ripening-related induction of many enzymes involved in primary metabolism, including those of the last five steps of the glycolytic pathway, which had been described as down-regulated in previous studies performed on whole fruit. Moreover, these data emphasize the relevance of this tissue as a physical barrier exerting an important part in berry protection. In fact, the level of many proteins involved in (a)biotic stress responses remarkably changed through the five stages taken into consideration, thus suggesting that their expression may be developmentally regulated.
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.
Auxin; GH3 proteins; ripening; Vitis vinifera
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.
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.
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.
A homologue of the mammalian bilirubin transporter bilitranslocase (BTL) (TCDB 2.A.65.1.1), able to perform an apparent secondary active transport of flavonoids, has previously been found in carnation petals and red grape berries. In the present work, a BTL homologue was also shown in white berries from Vitis vinifera L. cv. Tocai/Friulano, using anti-sequence antibodies specific for rat liver BTL. This transporter, similarly to what found in red grape, was localized in the first layers of the epidermal tissue and in the vascular bundle cells of the mesocarp. In addition, a strong immunochemical reaction was detected in the placental tissue and particularly in peripheral integuments of the seed. The protein was expressed during the last maturation stages in both skin and pulp tissues and exhibited an apparent molecular mass of c. 31 kDa. Furthermore, the transport activity of such a carrier, measured as bromosulphophthalein (BSP) uptake, was detected in berry pulp microsomes, where it was inhibited by specific anti-BTL antibodies. The BTL homologue activity exhibited higher values, for both Km and Vmax, than those found in the red cultivar. Moreover, two non-pigmented flavonoids, such as quercetin (a flavonol) and eriodictyol (a flavanone), inhibited the uptake of BSP in an uncompetitive manner. Such results strengthen the hypothesis that this BTL homologue acts as a carrier involved also in the membrane transport of colourless flavonoids and demonstrate the presence of such a carrier in different organs and tissues.
Bilitranslocase; flavonoid transport; immunohistochemistry; seed; Vitis vinifera L.; white berry cultivar