Comprehensive Genome-Wide Exploration of C2H2 Zinc Finger Family in Grapevine (Vitis vinifera L.): Insights into the Roles in the Pollen Development Regulation.

Some C2H2 zinc-finger proteins (ZFP) transcription factors are involved in the development of pollen in plants. In grapevine (Vitis vinifera L.), it has been suggested that abnormalities in pollen development lead to the phenomenon called parthenocarpy that occurs in some varieties of this cultivar. At present, a network involving several transcription factors types has been revealed and key roles have been assigned to members of the C2H2 zinc-finger proteins (ZFP) family in model plants. However, particularities of the regulatory mechanisms controlling pollen formation in grapevine remain unknown. In order to gain insight into the participation of ZFPs in grapevine gametophyte development, we performed a genome-wide identification and characterization of genes encoding ZFP (VviZFP family). A total of 98 genes were identified and renamed based on the gene distribution into grapevine genome. The analysis performed indicate significant changes throughout VviZFP genes evolution explained by high heterogeneity in sequence, length, number of ZF and presence of another conserved domains. Moreover, segmental duplication participated in the gene family expansion in grapevine. The VviZFPs were classified based on domain and phylogenetic analysis into three sets and different groups. Heat-map demonstrated differential and tissue-specific expression patterns of these genes and k-means clustering allowed to identify a group of putative orthologs to some ZFPs related to pollen development. In transgenic plants carrying the promVviZFP13::GUS and promVviZFP68::GUS constructs, GUS signals were detectable in the anther and mature pollen grains. Expression profiling of selected VviZFP genes showed differential expression pattern during flower development and provides a basis for deepening in the understanding of VviZFPs role on grapevine reproductive development.

Identification of key genes and its chromosome regions linked to drought responses in leaves across different crops through meta-analysis of RNA-Seq data.

BACKGROUND: Our study is the first to provide RNA-Seq data analysis related to transcriptomic responses towards drought across different crops. The aim was to identify and map which genes play a key role in drought response on leaves across different crops. Forty-two RNA-seq samples were analyzed from 9 published studies in 7 plant species (Arabidopsis thaliana, Solanum lycopersicum, Zea mays, Vitis vinifera, Malus X domestica, Solanum tuberosum, Triticum aestivum). RESULTS: Twenty-seven (16 up-regulated and 11 down-regulated) drought-regulated genes were commonly present in at least 7 of 9 studies, while 351 (147 up-regulated and 204 down-regulated) were commonly drought-regulated in 6 of 9 studies. Across all kind of leaves, the drought repressed gene-ontologies were related to the cell wall and membrane re-structuring such as wax biosynthesis, cell wall organization, fatty acid biosynthesis. On the other hand, drought-up-regulated biological processes were related to responses to osmotic stress, abscisic acid, water deprivation, abscisic-activated signalling pathway, salt stress, hydrogen peroxide treatment. A common metabolic feature linked to drought response in leaves is the repression of terpenoid pathways. There was an induction of AL1 (alfin-like), UGKYAH (trihelix), WRKY20, homeobox genes and members of the SET domain family in 6 of 9 studies. Several genes involved in detoxifying and antioxidant reactions, signalling pathways and cell protection were commonly modulated by drought across the 7 species. The chromosome (Chr) mapping of these key abiotic stress genes highlighted that Chr 4 in Arabidopsis thaliana, Chr 1 in Zea mays, Chr 2 and Chr 5 in Triticum aestivum contained a higher presence of drought-related genes compared to the other remaining chromosomes. In seedling studies, it is worth notice the up-regulation of ERF4 and ESE3 (ethylene), HVA22 (abscisic acid), TIR1 (auxin) and some transcription factors (MYB3, MYB94, MYB1, WRKY53 and WRKY20). In mature leaves, ERF1 and Alfin-like 1 were induced by drought while other transcription factors (YABBY5, ARR2, TRFL2) and genes involved phospholipid biosynthesis were repressed. CONCLUSIONS: The identified and mapped genes might be potential targets of molecular breeding activities to develop cultivars with enhanced drought resistance and tolerance across different crops.

Active rearrangements in the cell wall follow polymer concentration during postharvest withering in the berry skin of Vitis vinifera cv. Corvina.

During grape postharvest withering, a worldwide practice used to produce important high-quality wines, the solute concentration increases due to dehydration, and many organoleptic and quality traits, especially related to the berry skin, are affected in a cultivar-specific manner. Nevertheless, a complete comprehension of the underlying processes is still lacking. In this work, we applied ATR-FTIR micro-spectroscopy combined with PCA to monitor cell wall biochemical changes at three stages during postharvest withering on the internal and external sides of the berry skin of the Vitis vinifera cv. Corvina, an important local variety of the Verona province in Italy. The obtained results were integrated by profiling xylogucans and pectins through immunohistochemistry and by genome-wide transcriptomic analysis performed at the same withering stages. Our analysis indicates a gradual passive polymer concentration due to water loss in the first two months of postharvest withering, followed by active structural modifications in the last month of the process. Such rearrangements involve xyloglucans in the internal surface, cuticle components and cellulose in the external surface, and pectins in both surfaces. Moreover, by investigating the expression trend of cell wall metabolism-related genes, we identified several putative molecular markers associated to the polymer dynamics. The present study represents an important step towards an exhaustive comprehension of the postharvest withering process, which is of great interest from both the biological and technological points of view.

Utilization of a freeze-thaw treatment to enhance phenolic ripening and tannin oxidation of grape seeds in red (Vitis vinifera L.) cultivars.

Phenolic ripening represents a major interest for quality wine producers. Nevertheless, climatic or genotypical limitations can often prevent optimal maturation process. During winemaking seeds can be easily separated and technologically processed to improve their quality. Relying on the key role of oxidation for phenolic ripening, a freeze-thaw treatment was proposed to improve the fruit quality for potential use in challenging growing conditions. The experiment was carried on in two distinctive viticultural areas, Michigan and Italy. Five cultivars (Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot noir and Chambourcin) and six cultivars (Cabernet Sauvignon, Sangiovese, Syrah, Croatina, Barbera and Nebbiolo) were used in Michigan and Italy, respectively. Samples were collected at different phenological stages, to describe the natural ripening process and grape seeds were characterized before and after a freeze-thaw treatment. Colorimetric and spectrophotometric data highlighted similarities among natural and artificial seed ripening promising future applications for the wine industries.

Extraction of Pathogenesis-Related Proteins and Phenolics in Sauvignon Blanc as Affected by Grape Harvesting and Processing Conditions.

Thaumatin-like proteins (TLPs) and chitinases are the two main groups of pathogenesis-related (PR) proteins found in wine that cause protein haze formation. Previous studies have found that phenolics are also involved in protein haze formation. In this study, Sauvignon Blanc grapes were harvested and processed in two vintages (2011 and 2012) by three different treatments: (1) hand harvesting with whole bunch press (H-WB); (2) hand harvesting with destem/crush and 3 h skin contact (H-DC-3); and (3) machine harvesting with destem/crush and 3 h skin contact (M-DC-3). The juices were collected at three pressure levels (0.4 MPa, 0.8 MPa and 1.6 MPa), some juices were fermented in 750 mL of wine bottles to determine the bentonite requirement for the resulting wines. Results showed juices of M-DC-3 had significantly lower concentration of proteins, including PR proteins, compared to those of H-DC-3, likely due to the greater juice yield of M-DC-3 and interactions between proteins and phenolics. Juices from the 0.8-1.6 MPa pressure and resultant wines had the highest concentration of phenolics but the lowest concentration of TLPs. This supported the view that TLPs are released at low pressure as they are mainly present in grape pulp but additional extraction of phenolics largely present in skin occurs at higher pressing pressure. Wine protein stability tests showed a positive linear correlation between bentonite requirement and the concentration of chitinases, indicating the possibility of predicting bentonite requirement by quantification of chitinases. This study contributes to an improved understanding of extraction of haze-forming PR proteins and phenolics that can influence bentonite requirement for protein stabilization.

Flowers regulate the growth and vascular development of the inflorescence rachis in Vitis vinifera L.

The rachis, the structural framework of the grapevine (Vitis vinifera L.) inflorescence (and subsequent bunch), consists of a main axis and one or more orders of lateral branches with the flower-bearing pedicels at their fine tips. The rachis is crucial both for support, and transport from the shoot. Earlier suggestions that the flowers per se affect normal rachis development are investigated further in this study. Different percentages (0, 25, 50, 75 or 100) of flowers were removed manually one week before anthesis on field-grown vines. Treatment effects on subsequent rachis development (curvature, vitality, anatomy, starch deposit) were assessed. Sections, both fixed and embedded, and fresh hand-cut were observed by fluorescence and bright-field optics after appropriate staining. Emphasis was on measurement of changes in cross-sectional area of secondary xylem and phloem, and on maturation of fibres and periderm. Specific defects in rachis development were dependent on the percent and location of flower removal one week prior to anthesis. The rachises curved inwards where most of the flowers were removed. When fully de-flowered, they became progressively necrotic from the laterals back to the primary axes and from the distal to the proximal end of those axes, with a concurrent disorganisation of their anatomy. A few remaining groups of flowers prevented desiccation and abscission of the rachis axes proximal to the group, but not distally. Flower removal (50%) reduced rachis elongation, while 75% removal reduced xylem and phloem area and delayed phloem fibre and periderm development. 75% flower removal did not affect starch present in the rachis during berry development. Developing flowers affect the growth and vitality of the rachis and the development of its vascular and support structures. The extent of these effects depends on the cultivar and the number and position of flowers remaining after some are removed one week before anthesis.

Immunolocalization of cell wall polymers in grapevine (Vitis vinifera) internodes under nitrogen, phosphorus or sulfur deficiency.

The impact on cell wall (CW) of the deficiency in nitrogen (-N), phosphorus (-P) or sulphur (-S), known to impair essential metabolic pathways, was investigated in the economically important fruit species Vitis vinifera L. Using cuttings as an experimental model a reduction in total internode number and altered xylem shape was observed. Under -N an increased internode length was also seen. CW composition, visualised after staining with calcofluor white, Toluidine blue and ruthenium red, showed decreased cellulose in all stresses and increased pectin content in recently formed internodes under -N compared to the control. Using CW-epitope specific monoclonal antibodies (mAbs), lower amounts of extensins incorporated in the wall were also observed under -N and -P conditions. Conversely, increased pectins with a low degree of methyl-esterification and richer in long linear 1,5-arabinan rhamnogalacturonan-I (RG-I) side chains were observed under -N and -P in mature internodes which, in the former condition, were able to form dimeric association through calcium ions. -N was the only condition in which 1,5-arabinan branched RG-I content was not altered, as -P and -S older internodes showed, respectively, lower and higher amounts of this polymer. Higher xyloglucan content in older internodes was also observed under -N. The results suggest that impairments of specific CW components led to changes in the deposition of other polymers to promote stiffening of the CW. The unchanged extensin amount observed under -S may contribute to attenuating the effects on the CW integrity caused by this stress. Our work showed that, in organized V. vinifera tissues, modifications in a given CW component can be compensated by synthesis of different polymers and/or alternative linking between polymers. The results also pinpoint different strategies at the CW level to overcome mineral stress depending on how essential they are to cell growth and plant development.

Habitat management of organic vineyard in Northern Italy: the role of cover plants management on arthropod functional biodiversity.

The effect of cover plants on arthropod functional biodiversity was investigated in a vineyard in Northern Italy, through a 3-year field experiment. The following six ground cover plants were tested: Sweet Alyssum; Phacelia; Buckwheat; Faba Bean; Vetch and Oat; control. Arthropods were sampled using different techniques, including collection of leaves, vacuum sampling and sweeping net. Ground cover plant management significantly affected arthropod fauna, including beneficial groups providing ecosystem services like biological control against pests. Many beneficial groups were attracted by ground cover treatments in comparison with control, showing an aggregative numerical response in the plots managed with some of the selected plant species. Alyssum, Buckwheat and 'Vetch and Oat' mixture showed attractiveness on some Hymenoptera parasitoid families, which represented 72.3% of the insects collected by sweeping net and 45.7 by vacuum sampling. Phytoseiidae mites showed a significant increase on leaves of the vineyard plots managed with ground covers, in comparison with control, although they did not show any difference among the treatments. In general, the tested ground cover treatments did not increase dangerous Homoptera populations in comparison with control, with the exception of Alyssum. The potential of ground cover plant management in Italian vineyards is discussed: the overall lack of potential negative effects of the plants tested, combined with an aggregative numerical response for many beneficials, seems to show a potential for their use in Northern Italy vineyards.

Characterization of the adaptive response of grapevine (cv. Tempranillo) to UV-B radiation under water deficit conditions.

This work aims to characterize the physiological response of grapevine (Vitis vinifera L.) cv. Tempranillo to UV-B radiation under water deficit conditions. Grapevine fruit-bearing cuttings were exposed to three levels of supplemental biologically effective UV-B radiation (0, 5.98 and 9.66kJm(-2)day(-1)) and two water regimes (well watered and water deficit), in a factorial design, from fruit-set to maturity under glasshouse-controlled conditions. UV-B induced a transient decrease in net photosynthesis (Anet), actual and maximum potential efficiency of photosystem II, particularly on well watered plants. Methanol extractable UV-B absorbing compounds (MEUVAC) concentration and superoxide dismutase activity increased with UV-B. Water deficit effected decrease in Anet and stomatal conductance, and did not change non-photochemical quenching and the de-epoxidation state of xanthophylls, dark respiration and photorespiration being alternative ways to dissipate the excess of energy. Little interactive effects between UV-B and drought were detected on photosynthesis performance, where the impact of UV-B was overshadowed by the effects of water deficit. Grape berry ripening was strongly delayed when UV-B and water deficit were applied in combination. In summary, deficit irrigation did not modify the adaptive response of grapevine to UV-B, through the accumulation of MEUVAC. However, combined treatments caused additive effects on berry ripening.

Xenia and metaxenia in grapes: differences in berry and seed characteristics of maternal grape cv. 'Narince' (Vitis vinifera L.) as influenced by different pollen sources.

Literature investigations indicate that the grapes have quite complex fertilisation biology. This complexity necessitates extensive investigations to obtain reliable knowledge for both well-organised hybridisation studies and maximising grape yield. Therefore, this study was conducted to investigate the influences of self-, free- and cross-pollination on berry and seed characteristics in grape. Five different pollination treatments were applied to 'Narince', the most widely known and popular white wine grape in Turkey. Pollen tests indicated that all the cultivars had satisfactory in vitro pollen viability percentages. Free-pollination produced a significantly higher percentage berry set. Among the pollinizers, the use of pollen of 'Thompson Seedless' and 'Cardinal' varieties resulted in higher berry set percentage in 'Narince'. The free-pollination was also superior in giving the highest weight, length and width of the berry, as well as number of seeds per berry. These findings revealed that there were strong xenial and metaxenial effects in the studied grape cultivars. Among the pollinizer cultivars, the most effective pollinator was 'Thompson Seedless'. Hence, for better berry set and quality, the use of 'Thompson Seedless' as a pollinizer may be an attractive option in both grape production and breeding studies.

Extraction yields and anti-oxidant activity of proanthocyanidins from different parts of grape pomace: effect of mechanical treatments.

INTRODUCTION: White grape pomace is not subject to maceration, keeping nearly all polyphenols of grapes, so they represent important sources of bioactive compounds such as proanthocyanidins. Preparation of plant polyphenol extracts is usually performed using raw material powder. However, the fine particles make the further extraction procedure steps more difficult. OBJECTIVE: To study the effect of mechanical treatments on extraction yields and anti-oxidant activity from different parts of white grape pomace. METHODS: Skins, stems and seeds were isolated from the pomace of white winemaking process. Sequential solvent extraction of polyphenols, first using 80% methanol in water followed by 75% acetone in water, was carried out on both entire and milled (< 1 mm) grape solids; extraction on seed polyphenols was also performed in its squashed form. The phenolic content of each extract was verified by spectrometric and HPLC methods and its anti-oxidant activity was evaluated by the 1,1-diphenyl-2-picrylhydrazyl test. RESULTS: More total polyphenols can be extracted from each milled tissue than from its entire form. Seeds present the highest total phenol, oligomeric and polymeric proanthocyanidin content, and similar extraction yield was found between milled and squashed tissues. The HPLC analysis showed no difference in extraction yield of low-molecular-weight proanthocyanidins between milled and entire stems. Anti-oxidant activity showed a positive correlation with total phenol content, galloyled oligomers and polymeric proanthocyanidins. CONCLUSION: The use of entire stems and squashed seeds for solvent extraction of polyphenols makes manipulation simpler and more cost-efficient, providing similar extraction yields to using their powdery forms.

Short- and long-term physiological responses of grapevine leaves to UV-B radiation.

The present study aimed at evaluating the short- and long-term effects of UV-B radiation on leaves of grapevine Vitis vinifera (cv. Tempranillo). Grapevine fruit-bearing cuttings were exposed to two doses of supplemental biologically effective UV-B radiation (UV-BBE) under glasshouse-controlled conditions: 5.98 and 9.66kJm(-2)d(-1). The treatments were applied either for 20d (from mid-veraison to ripeness) or 75d (from fruit set to ripeness). A 0kJm(-2)d(-1) UV-B treatment was included as control. The main effects of UV-B were observed after the short-term exposure (20d) to 9.66kJm(-2)d(-1). Significant decreases in net photosynthesis, stomatal conductance, sub-stomatal CO2 concentration, the actual photosystem II (PSII) efficiency, total soluble proteins and de-epoxidation state of the VAZ cycle were observed, whereas the activities of several antioxidant enzymes increased significantly. UV-B did not markedly affect dark respiration, photorespiration, the maximum potential PSII efficiency (Fv/Fm), non-photochemical quenching (NPQ), as well as the intrinsic PSII efficiency. However, after 75d of exposure to 5.98and 9.66kJm(-2)d(-1) UV-B most photosynthetic and biochemical variables were unaffected and there were no sign of oxidative damage in leaves. The results suggest a high long-term acclimation capacity of grapevine to high UV-B levels, associated with a high accumulation of UV-B absorbing compounds in leaves, whereas plants seemed to be tolerant to moderate doses of UV-B.

Trans-acting small interfering RNA4: key to nutraceutical synthesis in grape development?

The facility and versatility of microRNAs (miRNAs) to evolve and change likely underlies how they have become dominant constituents of eukaryotic genomes. In this opinion article I propose that trans-acting small interfering RNA gene 4 (TAS4) evolution may be important for biosynthesis of polyphenolics, arbuscular symbiosis, and bacterial pathogen etiologies. Expression-based and phylogenetic evidence shows that TAS4 targets two novel grape (Vitis vinifera L.) MYB transcription factors (VvMYBA6, VvMYBA7) that spawn phased small interfering RNAs (siRNAs) which probably function in nutraceutical bioflavonoid biosynthesis and fruit development. Characterization of the molecular mechanisms of TAS4 control of plant development and integration into biotic and abiotic stress- and nutrient-signaling regulatory networks has applicability to molecular breeding and the development of strategies for engineering healthier foods.

Mineral stress affects the cell wall composition of grapevine (Vitis vinifera L.) callus.

Grapevine (Vitis vinifera L.) is one of the most economically important fruit crops in the world. Deficit in nitrogen, phosphorus and sulfur nutrition impairs essential metabolic pathways. The influence of mineral stress in the composition of the plant cell wall (CW) has received residual attention. Using grapevine callus as a model system, 6 weeks deficiency of those elements caused a significant decrease in growth. Callus CWs were analyzed by Fourier transform infrared spectroscopy (FT-IR), by quantification of CW components and by immunolocalization of CW epitopes with monoclonal antibodies. PCA analysis of FT-IR data suggested changes in the main components of the CW in response to individual mineral stress. Decreased cellulose, modifications in pectin methyl esterification and increase of structural proteins were among the events disclosed by FT-IR analysis. Chemical analyses supported some of the assumptions and further disclosed an increase in lignin content under nitrogen deficiency, suggesting a compensation of cellulose by lignin. Moreover, polysaccharides of callus under mineral deficiency showed to be more tightly bonded to the CW, probably due to a more extensive cross-linking of the cellulose-hemicellulose network. Our work showed that mineral stress impacts the CW at different extents according to the withdrawn mineral element, and that the modifications in a given CW component are compensated by the synthesis and/or alternative linking between polymers. The overall results here described for the first time pinpoint the CW of Vitis callus different strategies to overcome mineral stress, depending on how essential they are to cell growth and plant development.

Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr.

KEY MESSAGE : VaCDPK3a is actively expressed in leaves, stems, inflorescences, and berries of Vitis amurensis and may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis. Calcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.

Grape berry ripening delay induced by a pre-véraison NAA treatment is paralleled by a shift in the expression pattern of auxin- and ethylene-related genes.

BACKGROUND: Auxins act as repressors of ripening inception in grape (véraison), while ethylene and abscisic acid (ABA) play a positive role as inducers of the syndrome. Despite the increasing amount of information made available on this topic, the complex network of interactions among these hormones remains elusive. In order to shed light on these aspects, a holistic approach was adopted to evaluate, at the transcriptomic level, the crosstalk between hormones in grape berries, whose ripening progression was delayed by applying naphtalenacetic acid (NAA) one week before véraison. RESULTS: The NAA treatment caused significant changes in the transcription rate of about 1,500 genes, indicating that auxin delayed grape berry ripening also at the transcriptional level, along with the recovery of a steady state of its intracellular concentration. Hormone indices analysis carried out with the HORMONOMETER tool suggests that biologically active concentrations of auxins were achieved throughout a homeostatic recovery. This occurred within 7 days after the treatment, during which the physiological response was mainly unspecific and due to a likely pharmacological effect of NAA. This hypothesis is strongly supported by the up-regulation of genes involved in auxin conjugation (GH3-like) and action (IAA4- and IAA31-like). A strong antagonistic effect between auxin and ethylene was also observed, along with a substantial 'synergism' between auxins and ABA, although to a lesser extent. CONCLUSIONS: This study suggests that, in presence of altered levels of auxins, the crosstalk between hormones involves diverse mechanisms, acting at both the hormone response and biosynthesis levels, creating a complex response network.

Adaptation of grapevine flowers to cold involves different mechanisms depending on stress intensity.

Grapevine flower development and fruit set are influenced by cold nights in the vineyard. To investigate the impact of cold stress on carbon metabolism in the inflorescence, we exposed the inflorescences of fruiting cuttings to chilling and freezing temperatures overnight and measured fluctuations in photosynthesis and sugar content. Whatever the temperature, after the stress treatment photosynthesis was modified in the inflorescence, but the nature of the alteration depended on the intensity of the cold stress. At 4°C, photosynthesis in the inflorescence was impaired through non-stomatal limitations, whereas at 0°C it was affected through stomatal limitations. A freezing night (-3°C) severely deregulated photosynthesis in the inflorescence, acting primarily on photosystem II. Cold nights also induced accumulation of sugars. Soluble carbohydrates increased in inflorescences exposed to -3°C, 0°C and 4°C, but starch accumulated only in inflorescences of plants treated at 0 and -3°C. These results suggest that inflorescences are able to cope with cold temperatures by adapting their carbohydrate metabolism using mechanisms that are differentially induced according to stress intensity.

The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program.

We developed a genome-wide transcriptomic atlas of grapevine (Vitis vinifera) based on 54 samples representing green and woody tissues and organs at different developmental stages as well as specialized tissues such as pollen and senescent leaves. Together, these samples expressed ∼91% of the predicted grapevine genes. Pollen and senescent leaves had unique transcriptomes reflecting their specialized functions and physiological status. However, microarray and RNA-seq analysis grouped all the other samples into two major classes based on maturity rather than organ identity, namely, the vegetative/green and mature/woody categories. This division represents a fundamental transcriptomic reprogramming during the maturation process and was highlighted by three statistical approaches identifying the transcriptional relationships among samples (correlation analysis), putative biomarkers (O2PLS-DA approach), and sets of strongly and consistently expressed genes that define groups (topics) of similar samples (biclustering analysis). Gene coexpression analysis indicated that the mature/woody developmental program results from the reiterative coactivation of pathways that are largely inactive in vegetative/green tissues, often involving the coregulation of clusters of neighboring genes and global regulation based on codon preference. This global transcriptomic reprogramming during maturation has not been observed in herbaceous annual species and may be a defining characteristic of perennial woody plants.

Optimization of a method for the extraction and quantification of carotenoids and chlorophylls during ripening in grape berries (Vitis vinifera cv. Merlot).

An extraction method to identify and quantify the carotenoid and chlorophyll profile of lyophilized tissue from unripe (green) to ripe (red) Merlot grape berries was developed. The RP-HPLC method baseline separated all of the carotenoids and chlorophylls and their derivatives. Problems encountered during sample storage and extraction are discussed as well as possible alternative methods. This study confirmed that carotenoids and chlorophylls decreased on a per berry (microg/berry) and concentration (microg/g) basis from veraison to harvest over two growing seasons. The carotenoid 5,8-epoxy-beta-carotene was quantified for the first time in grapes and represents a significant amount of the total carotenoids present at harvest. All the carotenoids and chlorophylls except beta-carotene appeared to be sensitive to seasonal variation in climatic conditions. Lutein and beta-carotene were found to be the most abundant carotenoids present in Merlot grape berries together with chlorophyll a for both seasons studied.

Comparison of three extraction methods used to evaluate phenolic ripening in red grapes.

Because there is not a single method for carrying out phenolic ripening analysis, it is very difficult to compare the results obtained by different researchers. In this study, the three most widely used extraction methods of polyphenols (Glories, AWRI, and ITV) have been analytically compared by evaluating two of the most important parameters for the wine industry: total polyphenols and total anthocyanins. Samples from different grape varieties (Tempranillo, Garnacha, Carinena, Syrah, Merlot, and Cabernet Sauvignon), from three different vintages (2006, 2007, and 2008), and at different ripening states (from the beginning of ripeness until harvest) were analyzed to obtain a wide range of representative phenolic contents. To avoid external interferences on the comparisons, the same grape puree was used to make the maceration assays using the different solvents according to each extraction method. Although every extraction method exhibits a different extraction efficiency, the correlation between the results obtained with each one was very good both for total anthocyanins and for total polyphenols. Thus, after having determined a parameter value of the phenolic ripeness using a specific method, the relationship found can be used to predict the parameter value of the phenolic ripeness provided by the other two methods.