Changes in grapevine DNA methylation and polyphenols content induced by solar ultraviolet-B radiation, water deficit and abscisic acid spray treatments.

Environment and crop management shape plant's phenotype. Argentinean high-altitude vineyards are characterized by elevated solar ultraviolet-B radiation (UVB) and water deficit (D) that enhance enological quality for red winemaking. These signals promote phenolics accumulation in leaves and berries, being the responses mediated by abscisic acid (ABA). DNA methylation is an epigenetic mechanism that regulates gene expression and may affect grapevine growth, development and acclimation, since methylation patterns are mitotically heritable. Berry skins low molecular weight polyphenols (LMWP) were characterized in field grown Vitis vinifera L. cv. Malbec plants exposed to contrasting UV-B, D, and ABA treatments during one season. The next season early fruit shoots were epigenetically (methylation-sensitive amplification polymorphism; MSAP) and biochemically (LMWP) characterized. Unstable epigenetic patterns and/or stochastic stress-induced methylation changes were observed. UV-B and D were the treatments that induced greater number of DNA methylation changes respect to Control; and UV-B promoted global hypermethylation of MSAP epiloci. Sequenced MSAP fragments associated with UV-B and ABA showed similarities with transcriptional regulators and ubiquitin ligases proteins activated by light. UV-B was associated with flavonols accumulation in berries and with hydroxycinnamic acids in the next season fruit shoots, suggesting that DNA methylation could regulate the LMWP accumulation and participate in acclimation mechanisms.

The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment.

Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures.

Accumulation of flavanols and expression of leucoanthocyanidin reductase induced by postharvest UV-C irradiation in grape berry.

To examine the effect of postharvest ultraviolet C (UV-C) irradiation on flavanol polyphenol accumulation in the grape berry, we investigated total flavanol polyphenol content, the enzyme activity of leucoanthocyanidin reductase (LAR), and transcription of Vv lar1 and Vv lar2 using spectrophotometry, real-time polymerase chain reaction, and western blot analysis in 5-year-old Vitis vinifera L. cv. Cabernet Sauvignon plants. Our results indicated that the accumulation of flavanol polyphenol reached its highest value when exposed to UV-C irradiation for 30 min. Additionally, UV-C irradiation induced the transcription of Vv lar1 and Vv lar2 and the synthesis of LAR1 and LAR2 proteins, resulting in increased accumulation of flavanol polyphenol in the grape berry. Moreover, these effects were associated with the length of time of UV-C irradiation.

Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures.

BACKGROUND: The aim of the present work was to examine the role of UV-C irradiation on the production of secondary metabolites (total phenolic, total flavanols, total flavonols, catechin, ferulic acid and trans-resveratrol in phenolic compounds and α-, ß-, γ- δ-tocopherols) in callus cultures. Studies on the effects of UV-C treatment on callus culture are seldom and generally focused on UV-B. However UV-C radiation play an important role in accumule secondary metabolites. RESULTS: In this study, callus cultures from Öküzgözü grape cultivar were initiated from leaf petiole explants. Calli formed after 6 weeks on the medium supplemented with 0.5 mg L-1 benzylaminopurine (BA), 0.5 mg L-1 indole acetic acid (IAA) on B5 media. Callus tissues were exposed to UV-C irradiation at 10, 20 and 30 cm distances from the UV source for 5 and 10 minutes and samples were collected at hours 0, 24 and 48. CONCLUSIONS: The greatest total phenolic content (155.14 mg 100 g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. 24 h and 48 h incubation times, 30 cm and 5 min were the most appropriate combination of UV-C application in total flavanol content. Maximum total flavonol content (7.12 mg 100 g-1) was obtained on 0 h, 5 min and 20 cm combination. The highest (+)- catechin accumulation (8.89 mg g-1) was found in calli with 10 min UV-C application from 30 cm distance and sampled after 48 h. Ferulic acid content increased 6 fold in Öküzgözü callus cultures (31.37 µg g-1) compared to the control group. The greatest trans-resveratrol content (8.43 µg g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. The highest α-tocopherol concentration was found in calli exposed to UV-C for 10 min from 30 cm distance and sampled after 24 h. As a conclusion, it was showed that UV-C radiation had remarkable promoting effects on the accumulation of secondary metabolites in the calli of Öküzgözü grape cultivar.

Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures

BACKGROUND: The aim of the present work was to examine the role of UV-C irradiation on the production of secondary metabolites (total phenolic, total flavanols, total flavonols, catechin, ferulic acid and trans-resveratrol in phenolic compounds and α-, ß-, γ- δ-tocopherols) in callus cultures. Studies on the effects of UV-C treatment on callus culture are seldom and generally focused on UV-B. However UV-C radiation play an important role in accumule secondary metabolites. RESULTS: In this study, callus cultures from Öküzgözü grape cultivar were initiated from leaf petiole explants. Calli formed after 6 weeks on the medium supplemented with 0.5 mg L-1 benzylaminopurine (BA), 0.5 mg L-1 indole acetic acid (IAA) on B5 media. Callus tissues were exposed to UV-C irradiation at 10, 20 and 30 cm distances from the UV source for 5 and 10 minutes and samples were collected at hours 0, 24 and 48. CONCLUSIONS: The greatest total phenolic content (155.14 mg 100 g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. 24 h and 48 h incubation times, 30 cm and 5 min were the most appropriate combination of UV-C application in total flavanol content. Maximum total flavonol content (7.12 mg 100 g-1) was obtained on 0 h, 5 min and 20 cm combination. The highest (+)- catechin accumulation (8.89 mg g-1) was found in calli with 10 min UV-C application from 30 cm distance and sampled after 48 h. Ferulic acid content increased 6 fold in Öküzgözü callus cultures (31.37 µg g-1) compared to the control group. The greatest trans-resveratrol content (8.43 µg g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. The highest α-tocopherol concentration was found in calli exposed to UV-C for 10 min from 30 cm distance and sampled after 24 h. As a conclusion, it was showed that UV-C radiation had remarkable promoting effects on the accumulation of secondary metabolites in the calli of Öküzgözü grape cultivar.

Volatile organic compounds characterized from grapevine (Vitis vinifera L. cv. Malbec) berries increase at pre-harvest and in response to UV-B radiation.

Ultraviolet-B solar radiation (UV-B) is an environmental signal with biological effects in plant tissues. Recent investigations have assigned a protective role of volatile organic compounds (VOCs) in plant tissues submitted to biotic and abiotic stresses. This study investigated VOCs in berries at three developmental stages (veraison, pre-harvest and harvest) of Vitis vinifera L. cv. Malbec exposed (or not) to UV-B both, in in vitro and field experiments. By Head Space-Solid Phase Micro Extraction-Gas Chromatography-Electron Impact Mass Spectrometry (HS-SPME-GC-EIMS) analysis, 10 VOCs were identified at all developmental stages: four monoterpenes, three aldehydes, two alcohols and one ketone. Monoterpenes increased at pre-harvest and in response to UV-B in both, in vitro and field conditions. UV-B also augmented levels of some aldehydes, alcohols and ketones. These results along with others from the literature suggest that UV-B induce grape berries to produce VOCs (mainly monoterpenes) that protect the tissues from UV-B itself and other abiotic and biotic stresses, and could affect the wine flavor. Higher emission of monoterpenes was observed in the field experiments as compared in vitro, suggesting the UV-B/PAR ratio is not a signal in itself.

UV-B impairs growth and gas exchange in grapevines grown in high altitude.

We previously demonstrated that solar ultraviolet-B (UV-B) radiation levels in high altitude vineyards improve berry quality in Vitis vinifera cv. Malbec, but also reduce berry size and yield, possibly as a consequence of increased oxidative damage and growth reductions (lower photosynthesis). The defense mechanisms toward UV-B signal and/or evoked damage promote production of antioxidant secondary metabolites instead of primary metabolites. Purportedly, the UV-B effects will depend on tissues developmental stage and interplay with other environmental conditions, especially stressful situations. In this work, grapevines were exposed to high solar UV-B (+UV-B) and reduced (by filtering) UV-B (-UV-B) treatments during three consecutive seasons, and the effects of UV-B, developmental stages and seasons on the physiology were studied, i.e. growth, tissues morphology, photosynthesis, photoprotective pigments, proline content and antioxidant capacity of leaves. The +UV-B reduced photosynthesis and stomatal conductance, mainly through limitation in gas exchange, reducing plant's leaf area, net carbon fixation and growth. The +UV-B augmented leaf thickness, and also the amounts of photoprotective pigments and proline, thereby increasing the antioxidant capacity of leaves. The defense mechanisms triggered by + UV-B reduced lipid peroxidation, but they were insufficient to protect the photosynthetic pigments per leaf dry weight basis. The +UV-B effects depend on tissues developmental stage and interplay with other environmental conditions such as total radiation and air temperatures.

Metabolism of terpenes in the response of grape (Vitis vinifera L.) leaf tissues to UV-B radiation.

This study investigated the terpene profiles as determined by GC-EIMS analysis of in vitro cultured plants of Vitis vinifera exposed to a "field-like" dose of UV-B (4.75 kJ m(-2)d(-1)) administered at two different fluence rates (low, 16 h at 8.25 µW cm(-2), and high 4 h at 33 µW cm(-2)). Low UV-B treatment increased levels of the membrane-related triterpenes sitosterol, stigmasterol and lupeol, more notable in young leaves, suggesting elicitation of a mechanism for grapevine acclimation. By contrast, accumulation of compounds with antioxidant properties, diterpenes α and γ tocopherol and phytol, the sesquiterpene E-nerolidol and the monoterpenes carene, α-pinene and terpinolene had maximum accumulation under high UV-B, which was accentuated in mature leaves. Also the levels of the sesquiterpenic stress-related hormone abscisic acid (ABA) increased under high UV-B, although 24 h post irradiation ABA concentrations decreased. Such increments of antioxidant terpenes along with ABA suggest elicitation of mechanism of defense. The adaptative responses induced by relatively low UV-B irradiations as suggested by synthesis of terpenes related with membrane stability correlated with augments in terpene synthase activity.

Solar UV-B and ABA are involved in phenol metabolism of Vitis vinifera L. increasing biosynthesis of berry skin polyphenols.

It has been previously found that abscisic acid (ABA) participates in the activation of grapevine leaf tissue defense against potentially damaging effects of solar ultraviolet-B radiation (UV-B), apparently by triggering biosynthesis of phenols that filter the harmful radiation and act as antioxidants. The present work studies the effect of solar UV-B and exogenously applied ABA on berry growth, sugar accumulation, and phenol (anthocyanin and nonanthocyanin) profiles across berry development and ripening of Vitis vinifera L. cv. Malbec in a vineyard at 1450 m of altitude. The grapevines were exposed to relatively high UV-B irradiation (normal sunlight; +UV-B) and also to a reduced UV-B treatment (filter exclusion; -UV-B). These two UV-B treatments were combined with weekly spray applications to the leaves and berries of 1 mM ABA (+ABA) or H(2)O (-ABA). Reduction of UV-B delayed berry development and maturation, whereas the +UV-B and +ABA combined treatment hastened berry sugar and phenol accumulation. +UV-B/+ABA treatments also reduced berry growth and decreased sugar per berry without affecting sugar concentration (°Brix) at harvest. Berry skin ABA levels were higher in the +UV-B and +ABA combined treatment, which also hastened the onset of ripening up to 20 days. Berry skin ABA levels then decreased toward harvest, implying a possible role for ABA in the control of ripening in this nonclimacteric fruit. Under both +UV-B and +ABA treatments berry skin phenols were additively increased with a change in anthocyanin and nonanthocyanin profiles and increases in the proportion of phenols with high antioxidant capacity.

Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultraviolet-absorbing compounds, antioxidant enzymes and membrane sterols.

We investigated the interactions of abscisic acid (ABA) in the responses of grape leaf tissues to contrasting ultraviolet (UV)-B treatments. One-year-old field-grown plants of Vitis vinifera L. were exposed to photosynthetically active radiation (PAR) where solar UV-B was eliminated by using polyester filters, or where PAR was supplemented with UV-B irradiation. Treatments combinations included weekly foliar sprays of ABA or a water control. The levels of UV-B absorbing flavonols, quercetin and kaempferol were significantly decreased by filtering out UV-B, while applied ABA increased their content. Concentration of two hydroxycinnamic acids, caffeic and ferulic acids, were also increased by ABA, but not affected by plus UV-B (+UV-B) treatments. Levels of carotenoids and activities of the antioxidant enzymes, catalase, ascorbate peroxidase and peroxidase were elevated by +ABA treatments, but only if +UV-B was given. Cell membrane beta-sitosterol was enhanced by ABA independently of +UV-B. Changes in photoprotective compounds, antioxidant enzymatic activities and sterols were correlated with lessened membrane harm by UV-B, as assessed by ion leakage. Oxidative damage expressed as malondialdehyde content was increased under +UV-B treatments. Our results suggest that the defence system of grape leaf tissues against UV-B is activated by UV-B irradiation with ABA acting downstream in the signalling pathway.

Photoperiod modifies the diurnal expression profile of VvPHYA and VvPHYB transcripts in field-grown grapevine leaves.

Despite the crucial role that phytochromes (Phys) play in light perception and in the entrainment of the circadian clock to local time, the photoperiodic regulation of PHYA and PHYB gene expression has been poorly studied, especially in woody perennials. Here the dynamic of Vitis vinifera PHYA (VvPHYA) and PHYB (VvPHYB) transcript accumulation was studied in field-grown grapevine leaves throughout daily cycles under decreasing natural photoperiods. Given that in grapevine the entrance of buds into endodormancy (ED) is a photoperiod-driven process, increases in BR(50) values, a parameter that measures the depth of dormancy in single bud cuttings assays was used to determine the critical daylength at which grapevine discriminates between long day (LD) and short day (SD) photoperiod. Therefore, we monitored the daily expression profile of VvPHYA and VvPHYB transcripts before, during and after the defined critical daylength. Results showed that under LD photoperiod (21 December, daylength 14 h 40 min) the abundance of both transcripts oscillated with diurnal rhythms, attaining maximum and minimum levels before dawn and after dusk, respectively. However, under SD photoperiod (12 April, daylength 11 h 40 min) the rhythmic expression disappeared, and both transcripts expressed uniformly at high levels. Our results showing that photoperiod regulates VvPHYA and VvPHYB gene expression, contrast with those reported in Arabidopsis and in other herbaceous plants in which PHYA and PHYB gene expression is regulated by the circadian clock.

Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera.

Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.