VvGOLS1 and VvHsfA2 are Involved in the Heat Stress Responses in Grapevine Berries

Among various environmental factors, temperature is a major regulator affecting plant growth, development and fruit composition. Grapevine is the most cultivated fruit plant throughout the world, and grapes are used for wine production and human consumption. The molecular mechanisms involved in grapevine tolerance to high temperature, especially at the fruit level, are poorly understood. To better characterize the sensitivity of berries to the microenvironment, high temperature conditions were locally applied to Vitis vinifera Cabernet Sauvignon clusters. Two genes, VvGOLS1 and VvHsfA2, up-regulated by this treatment, were identified and further characterized. The expression profile of VvGOLS1 correlated positively with galactinol accumulation in heat-stressed berries. However, no galactinol derivatives, such as raffinose and stachyose, accumulated upon heat stress. Heterologous expression of VvGOLS1 in Escherichia coli showed that it encodes a functional galactinol synthase. Transient expression assays showed that the heat stress factor VvHsfA2 transactivates the promoter of VvGOLS1 in a heat stress-dependent manner. Taken together, our results highlight the intrinsic capacity of grape berries to perceive heat stress and to initiate adaptive responses, suggesting that galactinol may play a signaling role in these response

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

<p>Abstract</p> <p>Background</p> <p>Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (<it>Vitis vinifera </it>L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood.</p> <p>Results</p> <p>In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5b^L, the native protein being referred as VvMYB5b^R) was assessed in different <it>in vivo </it>systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5b^Lexhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of <it>VvMYB5b^R</it>and <it>VvMYB5b^L</it>genes in tobacco. Flowers from <it>35S::VvMYB5b^L</it>transgenic plants showed a distinct phenotype in comparison with <it>35S::VvMYB5b^R</it>and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation.</p> <p>Conclusions</p> <p>Taken together, our findings indicate that VvMYB5b^Lis still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5^Lobserved in the tobacco model.</p

Metabolite analysis reveals distinct spatio-temporal accumulation of anthocyanins in two teinturier variants of cv. ‘Gamay’ grapevines (Vitis vinifera L.)

In most red grape cultivars, anthocyanins accumulate exclusively in the berry skin, while ‘teinturier’ cultivars also accumulate anthocyanins in the pulp. Here, we investigated the teinturier cvs. ‘Gamay de Bouze’ and ‘Gamay Fréaux’ (two somatic variants of the white-fleshed cv. ‘Gamay’) through metabolic and transcript analysis to clarify whether these two somatic variants have the same anthocyanin accumulation pattern in the skin and pulp, and whether primary metabolites are also affected. The skin of the three cultivars and the pulp of ‘Gamay de Bouze’ begun to accumulate anthocyanins at the onset of berry ripening. However, the pulp of ‘Gamay Fréaux’ exhibited a distinct anthocyanin accumulation pattern, starting as early as fruit set with very low level of sugars. The highest level of anthocyanins was found in ‘Gamay Fréaux’ skin, followed by ‘Gamay de Bouze’ and ‘Gamay’. Consistently, the transcript abundance of genes involved in anthocyanin biosynthesis were in line with the anthocyanin levels in the three cultivars. Despite no evident differences in pulp sugar content, the concentration of glucose and fructose in the skin of ‘Gamay Fréaux’ was only half of those in the skin of ‘Gamay’ and ‘Gamay de Bouze’ throughout all berry ripening, suggesting an uncoupled accumulation of sugars and anthocyanins in ‘Gamay Fréaux’. The study provides a comprehensive view of metabolic consequences in grape somatic variants and the three almost isogenic genotypes can serve as ideal reagents to further uncover the mechanisms underlying the linkage between sugar and anthocyanin accumulation.This research was supported partly by the National Key R&D Program of China (2018YFD1000200) and was conducted as part of the LIA INNOGRAPE International Associated Laboratory.Peer reviewe

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 9.66 kJ m−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

Sensitivity of grapevine phenology to water availability, temperature and CO2 concentration

In recent decades, mean global temperatures have increased in parallel with a sharp rise in atmospheric carbon dioxide (CO2) levels, with apparent implications for precipitation patterns. The aim of the present work is to assess the sensitivity of different phenological stages of grapevine to temperature and to study the influence of other factors related to climate change (water availability and CO2 concentration) on this relationship. Grapevine phenological records from 9 plantings between 42.75°N and 46.03°N consisting of dates for budburst, flowering and fruit maturity were used. In addition, we used phenological data collected from 2 years of experiments with grapevine fruit-bearing cuttings with two grapevine varieties under two levels of water availability, two temperature regimes and two levels of CO2. Dormancy breaking and flowering were strongly dependent on spring temperature, while neither variation in temperature during the chilling period nor precipitation significantly affected budburst date. The time needed to reach fruit maturity diminished with increasing temperature and decreasing precipitation. Experiments under semi-controlled conditions revealed great sensitivity of berry development to both temperature and CO2. Water availability had significant interactions with both temperature and CO2; however, in general, water deficit delayed maturity when combined with other factors. Sensitivities to temperature and CO2 varied widely, but higher sensitivities appeared in the coolest year, particularly for the late ripening variety, ‘White Tempranillo’. The knowledge gained in whole plant physiology and multi stress approaches is crucial to predict the effects of climate change and to design mitigation and adaptation strategies allowing viticulture to cope with climate change

Development and implementation of an in vitro culture system for intact detached grape berries

Grape composition depends on the metabolites accumulated and synthesized during grape development. It is of paramount importance for grape growers because of its major role in shaping wine quality. Therefore, understanding the regulation mechanisms that control the accumulation of quality-related metabolites in grape is of both scientific and agronomical interests. The composition of grape berry at harvest is under complex regulation and can be affected by many factors (Conde et al., 2007). The study of the effects of these factors on berries still attached to intact plants can be highly challenging because of the large size of the plants, interplant, intercluster and interberry variability; and because it is complicated to precisely control the nutrients and hormones imported by the berries, and the environment. Therefore, in vitro cultured grape berries are a good model system, which better represents berry anatomy structure (skin and flesh) than grape cell suspensions and nevertheless largely reduces the system complexity compared to whole plant (Bravdo et al., 1990; Pérez et al., 2000; Gambetta et al., 2010). To this end, an in vitro culture system of intact detached grape berries has been developed by coupling greenhouse fruiting-cuttings production and in vitro organ culture techniques (Dai et al., 2014). The cultured berries are able to actively absorb and utilize carbon and nitrogen from the culture medium, and exhibit fruit ripening features such as color changing and softening. This in vitro system may serve to investigate the response of berry composition to environmental and nutrient factors

Nested effects of berry half, berry and bunch microclimate on biochemical composition in grape

Aim: Polyphenol composition, an important component of grape quality, is strongly influenced by fruit microclimate. However, information relies exclusively on whole berry data and the underlying response functions to microenvironment variables remain essentially unknown. The aim of this study was therefore to analyze the biochemical composition of grapes at both bunch and berry scales, in relation with microclimate.[br/] Methods and results: Whole berries and berry halves were sampled in mature defoliated bunches from two neighboring Bordeaux vineyards with contrasting row orientations (Vitis vinifera cv. Merlot). Flavonoid and amino-acid contents were analyzed by HPLC methods. The main sources of variation were bunch azimuth, berry exposure and, only in South-exposed bunches, berry side. Models were used to estimate radiation at the berry surface and temperature. Intense effects of bunch side and berry side on total flavonol and anthocyanin concentrations were observed. These results were all consistent at both bunch and berry scales. However, the most intense effects were observed at berry scale and mitigated by scaling up from berry to bunch.[br/] Conclusion: Total flavonol concentrations in the berry skin exhibited a clear positive linear relationship with solar radiation. The large heterogeneity of composition at berry scale is consistent with the better known heterogeneity at bunch scale.[br/] Significance and impact of the study: Models and original response functions to microclimate could help optimize vineyard management and grape ripening

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

34 Pags.- 3 Tabls.- 6 Figs. The definitive version is available at: http://www.sciencedirect.com/science/journal/01689452This 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 9.66 kJ m−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.This work was supported by Fundación Universitaria de Navarra, Navarra-Aquitaine Cooperation Program (Gobierno de Navarra and Conseil Régional d’Aquitaine), Ministerio de Ciencia e Innovación of Spain (MCINN BFU2011-26989), European Union (FP7-KBBE-2012-6-singlestage), Asociación de Amigos de la Universidad de Navarra (grant to J. M-L), UV4growth COST Action FA0906 and Gobierno de Aragón (A03 research group).Peer reviewe

The ACA4 Gene of Arabidopsis Encodes a Vacuolar Membrane Calcium Pump That Improves Salt Tolerance in Yeast

Several lines of evidence suggest that regulation of intracellular Ca(2+) levels is crucial for adaptation of plants to environmental stress. We have cloned and characterized Arabidopsis auto-inhibited Ca(2+)-ATPase, isoform 4 (ACA4), a calmodulin-regulated Ca(2+)-ATPase. Confocal laser scanning data of a green fluorescent protein-tagged version of ACA4 as well as western-blot analysis of microsomal fractions obtained from two-phase partitioning and Suc density gradient centrifugation suggest that ACA4 is localized to small vacuoles. The N terminus of ACA4 contains an auto-inhibitory domain with a binding site for calmodulin as demonstrated through calmodulin-binding studies and complementation experiments using the calcium transport yeast mutant K616. ACA4 and PMC1, the yeast vacuolar Ca(2+)-ATPase, conferred protection against osmotic stress such as high NaCl, KCl, and mannitol when expressed in the K616 strain. An N-terminally modified form of ACA4 specifically conferred increased NaCl tolerance, whereas full-length ATPase had less effect

The grape berry-specific basic helix–loop–helix transcription factor VvCEB1 affects cell size

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