Ilaria Filippetti

Whole Plant Temperature Manipulation Affects Flavonoid Metabolism and the Transcriptome of Grapevine Berries

Among environmental factors, temperature is the one that poses serious threats to viticulture in the present and future scenarios of global climate change. In this work, we evaluated the effects on berry ripening of two thermal regimes, imposed from veraison to harvest. Potted vines were grown in two air-conditioned greenhouses with High Temperature (HT) and Low Temperature (LT) regimes characterized by 26 and 21°C as average and 42 and 35°C as maximum air daily temperature, respectively. We conducted analyses of the main berry compositional parameters, berry skin flavonoids and berry skin transcriptome on HT and LT berries sampled during ripening. The two thermal conditions strongly differentiated the berries. HT regime increased sugar accumulation at the beginning of ripening, but not at harvest, when HT treatment contributed to a slight total acidity reduction and pH increase. Conversely, growing temperatures greatly impacted on anthocyanin and flavonol concentrations, which resulted as strongly reduced, while no effects were found on skin tannins accumulation. Berry transcriptome was analyzed with several approaches in order to identify genes with different expression profile in berries ripened under HT or LT conditions. The analysis of whole transcriptome showed that the main differences emerging from this approach appeared to be more due to a shift in the ripening process, rather than to a strong rearrangement at transcriptional level, revealing that the LT temperature regime could delay berry ripening, at least in the early stages. Moreover, the results of the in-depth screening of genes differentially expressed in HT and LT did not highlight differences in the expression of transcripts involved in the biosynthesis of flavonoids (with the exception of PAL and STS) despite the enzymatic activities of PALs and UFGT being significantly higher in LT than HT. This suggests only a partial correlation between molecular and biochemical data in our conditions and the putative existence of post-transcriptional and post-translational mechanisms playing significant roles in the regulation of flavonoid metabolic pathways and in particular of anthocyanins.

Transcriptional Responses to Pre-flowering Leaf Defoliation in Grapevine Berry from Different Growing Sites, Years, and Genotypes

Leaf removal is a grapevine canopy management technique widely used to modify the source–sink balance and/or microclimate around berry clusters to optimize fruit composition. In general, the removal of basal leaves before flowering reduces fruit set, hence achieving looser clusters, and improves grape composition since yield is generally curtailed more than proportionally to leaf area itself. Albeit responses to this practice seem quite consistent, overall vine performance is affected by genotype, environmental conditions, and severity of treatment. The physiological responses of grape varieties to defoliation practices have been widely investigated, and just recently a whole genome transcriptomic approach was exploited showing an extensive transcriptome rearrangement in berries defoliated before flowering. Nevertheless, the extent to which these transcriptomic reactions could be manifested by different genotypes and growing environments is entirely unexplored. To highlight general responses to defoliation vs. different locations, we analyzed the transcriptome of cv. Sangiovese berries sampled at four development stages from pre-flowering defoliated vines in two different geographical areas of Italy. We obtained and validated five markers of the early defoliation treatment in Sangiovese, an ATP-binding cassette transporter, an auxin response factor, a cinnamyl alcohol dehydrogenase, a flavonoid 3-O-glucosyltransferase and an indole-3-acetate beta-glucosyltransferase. Candidate molecular markers were also obtained in another three grapevine genotypes (Nero d’Avola, Ortrugo, and Ciliegiolo), subjected to the same level of selective pre-flowering defoliation (PFD) over two consecutive years in their different areas of cultivation. The flavonol synthase was identified as a marker in the pre-veraison phase, the jasmonate methyltransferase during the transition phase and the abscisic acid receptor PYL4 in the ripening phase. The characterization of transcriptome changes in Sangiovese berry after PFD highlights, on one hand, the stronger effect of environment than treatment on the whole berry transcriptome rearrangement during development and, on the other, expands existing knowledge of the main molecular and biochemical modifications occurring in defoliated vines. Moreover, the identification of candidate genes associated with PFD in different genotypes and environments provides new insights into the applicability and repeatability of this crop practice, as well as its possible agricultural and qualitative outcomes across genetic and environmental variability.

Influence of berry ripeness on accumulation, composition and extractability of skin and seed flavonoids in cv. Sangiovese (Vitis vinifera L.).

BACKGROUND The anthocyanin and tannin concentration and composition of Vitis vinifera L. cv. Sangiovese berries were investigated from post-veraison to harvest. Exhaustive extraction with methanol and acetone was performed to determine the total flavonoid concentration, while a model hydroalcoholic solution was used to prepare extracts representing the winemaking process. The aim of this study was to improve the knowledge of the phenolic maturity of Sangiovese grape. RESULTS The total anthocyanin concentration increased during ripening, but the quantity of extractable anthocyanins increased more rapidly than the total. The total skin tannin concentration declined from post-veraison to harvest, whereas the extractable portion increased, with little difference in the composition of the fractions. Both the total and extractable seed tannin concentration diminished rapidly just after veraison, and only small fluctuations were detected until harvest. CONCLUSION These results indicate that the extractability of anthocyanins and skin tannins increases during ripening, whereas there is no clear trend for seed tannins during the same period. This is the first survey to study the behavior of phenolic compounds during different steps of ripening of Sangiovese grape.

Post-veraison trimming slow down sugar accumulation without modifying phenolic ripening in Sangiovese vines: Post-veraison trimming of Sangiovese vines

BACKGROUND Vineyard strategies have recently been developed to combat the effects of global warming on grapevines, which is causing grapes to ripen quickly, excessive sugar accumulation in berries, and high alcohol levels in wines. We evaluate the effectiveness of post-veraison trimming as a means of slowing down sugar accumulation, without modifying phenolic ripening, in Sangiovese vines grown in highly fertile and well watered soil. RESULTS The removal of about two-thirds of the leaf area by shoot trimming after veraison led to a reduction in sugar content without affecting yield. Total and extractable anthocyanins, skin, and seed tannins showed no significant variation at harvest after the treatment during the three-year trial, while the replenishment of carbohydrates in canes at the end of the trial was negatively affected. CONCLUSIONS These results indicate that, in highly fertile and well-watered soil, post-veraison trimming may represent a powerful tool for decreasing sugar concentration during harvest without affecting yield or total and extractable phenolic compounds. However, the reduction in starch reserves compared to the control serves as a warning about repeated trimming over the years.

Impact of Flavonoid and Cell Wall Material Changes on Phenolic Maturity in cv. Merlot (Vitis vinifera L.)

In black berry varieties, the changes in flavonoid concentration and composition that occur in the last weeks before harvest only partially explain the improved sensory attributes linked with the progression of ripening. To better understand the factors involved in phenolic maturity of cv. Merlot, total and extractable anthocyanins and tannins were analyzed during late ripening, and the properties of skin cell wall material were determined. Over two consecutive years, both total and extractable anthocyanins increased until harvest, while minor variations in tannin concentration were found. A greater affinity of cell wall material for a commercial seed tannin was observed in the last ripening phases, preferentially involving high molecular mass tannins and galloylated forms. The increased anthocyanins could allow a more highly colored wine to be obtained with the progression of ripening, and the increasing affinity of skin cell wall material for seed tannins may play a role in the reduction of astringency. This research shows how different factors could influence phenolic maturity, confirming the original insights in Merlot.

The Effectiveness of Basal Shoot Mechanical Leaf Removal at the Onset of Bloom to Control Crop on cv. Sangiovese (V. vinifera L.): Report on a Three-year Trial

Vine basal shoot leaves are known to be the primary nutritional source for fruit set at the onset of bloom. The effectiveness of mechanical removal of this foliage at that date was tested from 2012 to 2014 to control the cropping of high-yielding cv. Sangiovese in a “Toscana rosso” TGI (Typical Geographic Indication) district in Tuscany, where the yield threshold is 16 t/ha. A tractor-mounted leaf remover featuring sideby- side rotary suction and feed rollers was employed along a 50 cm basal area of cropping shoots at the beginning of bloom; control was the usual manual thinning of clusters carried out at veraison in the same vineyard. Mechanical leaf removal eliminated about 30% of leaf area and some shoots and inflorescences, thus reducing cropping potential and even resulting in a physiological effect, as the resulting clusters were composed of lower numbers of berries and were less compact and less susceptible to mould than the control. By harvest, the defoliated vines showed higher leaf area, most likely because leaf removal at the onset of bloom may have triggered compensatory new growth, and their grapes had a higher content of soluble solids than the thinned control. While both treatments kept the yield below the 16 t/ha threshold – leaf removal at an estimated 15.1 t/ha and manual cluster thinning at 15.6 t/ha – mechanical defoliation notably reduced yearly labour input: an estimated 4 h/ha against the 38 h/ha from thinning.