Literatura académica sobre el tema "Berry ripening"

The effects of ethephon on arabica coffee (Coffea arabica L.) were tested in experiments conducted over 2 years in North Queensland. Rates tested were 0, 125, 250, 500, 750, 1000, 1500, and 2000 mgkg, applied when 15-20% (early treatment) or 45-50% (late treatment) of fruit were coloured. Measurements were made on the degree of leaf and fruit abscission, berry ripening, and berry weight. Ethephon did not cause significant fruit abscission, but rates >500 mg/kg caused unacceptable leaf abscission. Rates of 250 mg/kg were marginally acceptable in terms of leaf abscission. Excessive defoliation led to dieback, which resulted in a reduced crop the following season. Ethephon concentrated berry ripening, significantly increased the number of red (mature) berries, and decreased the number of green (immature) berries. Rates of >500 mg/kg were effective in accelerating berry ripening, while 125 mg/kg had a limited effect. Maturity stage of the bean at time of application helped to determine the ethephon response; early application of ethephon had a more pronounced effect on berry ripening than late application. Ethephon applied early at rates of 1500 and 2000 mg/kg decreased fresh berry weight compared with all other treatments but parchment weight was unaffected. Selection of a suitable rate of ethephon from these data is difficult because berry ripening is offset by leaf drop. Variability of response was also observed.

Mature field-grown grapevines, Colombard on Ramsey rootstock, grown in a semi-arid climate were irrigated with saline water during any one of 4 growth stages within the season: pre-flowering, during berry development, during berry ripening, and post-harvest. At other times, plots were irrigated with river water (EC 0.5 dS/m) as was the control throughout the season. Saline water (EC 3.5 dS/m) with a high sodium absorption ratio was produced by addition of sodium chloride brine. Soil cation exchange capacity was 14 cmolc/kg, and at the end of the trial, the soil exchangeable sodium percentage in the control was 6%, in the treatment salinised pre-flowering 13%, during berry development 20%, during berry ripening 20%, and post-harvest 19%. Treatments were applied for 6 consecutive seasons. Vines were highly productive, with the average yield in the control equal to 62 t/ha of grapes. Saline irrigation caused significant, but small, declines in yield in 3 seasons, in pruning weights in 2 seasons, and in berry weights in 4 seasons. Effects on growth, once established, often persisted unchanged through one or more subsequent seasons of saline irrigation. The growth stage shown to be most sensitive to saline irrigation was berry development; saline irrigation during berry development reduced the yield by 7% and during berry ripening by 3%, and pre-flowering it reduced the berry weight by 1%, during berry development by 6%, and during berry ripening by 4%. The amounts of irrigation applied in each of the 4 growth stages were not equal, and hence, treatments did not receive equal additions of salt. Normalising data to remove this effect showed that the rate of yield decline per unit dS/m increase in the seasonal average salinity during berry development, 7%/dS.m, was 3-fold greater than the 2%/dS.m during berry ripening. We conclude that this scion/rootstock combination, grown under these conditions, can maintain high productivity despite ‘slugs’ of saline irrigation.

Grape (Vitis vinifera L.) berries are considered to be nonclimacteric fruit as they do not exhibit a large rise in ethylene production or respiration rate at the onset of ripening (veraison). However, ethylene may still play a role in berry development and in ripening in particular. (2-Chloroethyl)phosphonic acid (CEPA), an ethylene-releasing reagent, delayed ripening when applied early in berry development. In agreement with a role for ethylene in controlling the timing of ripening, the application of an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), advanced ripening, as did abscisic acid, when applied during the preveraison period. Applications of CEPA nearer to the time of veraison enhanced berry colouration. Changes in the expression of ethylene biosynthesis and receptor genes were observed throughout berry development. Transcript levels of some of these genes were increased by CEPA and decreased by AVG, suggesting changes in ethylene synthesis and perception during the preveraison period that might contribute to the biphasic response to CEPA (ethylene). The significant delay of ripening in field-grown grapes through the application of CEPA also indicates that this may be useful in controlling the timing of veraison, and therefore harvest date, in warmer climates.

An improved understanding of the hormonal control of grape (Vitis vinifera L.) berry ripening and the ability to manipulate it are of interest scientifically and commercially. Grapes are nonclimacteric fruit with ethylene unlikely to have a principal role in berry ripening but there are several other hormones thought to be involved. In this work, a significant delay in Riesling berry ripening was achieved through preripening treatments with the synthetic auxin 1-naphthaleneacetic acid (NAA). The initiation of sugar accumulation was delayed and the rate of sugar accumulation was lower in NAA-treated fruit, resulting in a 15-day delay in harvest. NAA treatments also reduced the rate of decline in malic acid levels that occurs during ripening, and increased the synchronicity of malic acid and berry sugar accumulation. Sensory panel assessment revealed a significant difference between wine made from control and NAA-treated fruit. Analysis of the volatile composition of the wines’ headspace showed that the concentration of several compounds was altered significantly by the NAA treatment. These data provide further support for the involvement of auxins in inhibiting ripening and suggest that auxin treatments may be useful in controlling both winery intake, and fruit and wine composition.

This study aimed to investigate the possible existence of reproducible aromatic red wine styles, focusing on fresh fruit aromas and mature fruit aromas (i.e., with dark, jammy fruit characteristics) and taking into account both vintage and vineyard.The study was performed on Australian Shiraz and Cabernet‑Sauvignon from three different meso-climate areas and two consecutive vintages. Sequential harvests were carried out based on the plateau of the physiological indicator berry sugar accumulation (mg/berry) in order to obtain fresh fruit and mature fruit wine sensory profiles. There was a predictable aromatic sequence during grape ripening at each of these two distinct maturity stages regardless of grape genotype (variety) and environment (vineyard and vintage). The post-plateau period of berry sugar accumulation was found to be crucial for the evolution of wine aromatic profiles. During this period, wine aromatic and phenolic maturity were uncoupled from technological maturity (i.e., berry sugar concentration). Dimethyl sulfide was found to be the most relevant wine aromatic marker for differentiating the fresh fruit and mature fruit stages irrespective of the variety. Specific cultivar markers with potential sensory contribution were also identified; for example, (Z)‐3‐hexenol, a possible contributor to the aromatic freshness of Shiraz wines from the fresh fruit stage. The evolution of terpenoids appeared to be separate from the dynamics of berry ripening post plateau of fruit sugar accumulation. On the other hand, ester composition was significantly altered during the same ripening period in Shiraz and Cabernet‑Sauvignon wines with a marked grape genotype effect. The results showed that yeast metabolism was also affected by berry ripening evolution from the plateau of berry sugar accumulation onwards.

Abscisic acid (ABA) and auxin are important hormones controlling the ripening progression of grape berry, and both the initiation and duration of ripening can dramatically affect the berry quality. However, the responses of flavor compounds to the hormones are inadequately understood. In this study, ABA and synthetic auxin α-naphthaleneacetic acid (NAA) were sprayed on Cabernet Sauvignon berries before véraison, and comparative transcriptomic and metabolic analysis were conducted to investigate the influence on berry quality-related metabolites. The 1000 mg/L ABA (ABA1000) and 200 mg/L NAA (NAA200) treated grapes exhibited shorter and longer phenological intervals compared to the control, respectively. The transcriptomic comparison between pre-véraison and véraison revealed that the varied ripening initiation and duration significantly affected the expression of genes related to specific metabolism, particularly in the biosynthetic metabolism of anthocyanin and volatile compounds. The up-regulated VviF3’H in both ABA1000-treated and NAA200-treated berries increased the proportion of 3′-substituted anthocyanins, and the 3′5′-substituted anthocyanins were largely reduced in the NAA200-treated berries. Concurrently, VviCCD4a and VviCCD4b were up-regulated, and the norisoprenoids were correspondingly elevated in the NAA200-treated berries. These data suggest that ABA and NAA applications may be useful in controlling the ripening and improving the flavor of the grape berry.

Previous study has demonstrated that the riboflavin treatment promoted the early ripening of the ‘Kyoho’ grape berry. However, the molecular mechanism causing this was unclear. In order to reveal the regulation mechanism of riboflavin treatment on grape berry development and ripening, the different berry developmental stages of the ‘Kyoho’ berry treated with 0.5 mmol/L of riboflavin was sampled for transcriptome profiling. RNA-seq revealed that 1526 and 430 genes were up-regulated and down-regulated, respectively, for the comparisons of the treatment to the control. TCseq analysis showed that the expression patterns of most of the genes were similar between the treatment and the control, except for some genes that were related to the chlorophyll metabolism, photosynthesis–antenna proteins, and photosynthesis, which were revealed by the enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The differentially expressed genes and weighted gene co-expression network analysis (WGCNA) analysis identified some significantly differentially expressed genes and some hub genes, including up-regulation of the photosynthesis-related ELIP1 and growth and development-related GDSL; and down-regulation of the oxidative stress-related ATHSP22 and berry softening-related XTH32 and GH9B15. The results suggested that the riboflavin treatment resulted in the variations of the expression levels of these genes, and then led to the early ripening of the ‘Kyoho’ berry.

Crosses between `Humble' and five other blackberry cultivars were made to determine the efficacy of `Humble' as a source of resistance to rosette (incited by Cercosporella rubi). Seven cultivars and three selections were planted at a disease free location and at a location where rosette is known to occur to determine the effect of double blossom on yield, berry weight, and ripening date. `Cheyenne' X `Humble,' `Humble' X `Brazos,' and `Navaho' X (`Humble' X `Brazos') produced progenies with various levels of resistance. Promising selections were made from the latter two crosses. Genotype X location interaction was significant for yield and not significant for either Julian date of 50% ripe fruit or weight per berry. These results suggest that rosette reduces yield but has little if any effect on weight per berry or ripening date. `Shawnee' and `Choctaw' were the highest yielding cultivars under disease free conditions; however, their yields were low in the test where they were infected with rosette. `Brazos,' `Choctaw,' and `Rosborough' were the earliest ripening and `Navaho' was the latest ripening cultivar.

High temperatures during the growing season characterise many grape growing regions in Australia and elsewhere in the world, and impact on many processes including growth and berry development. To quantify the impact of heat on the Vitis vinifera L. cv. Semillon, potted vines were grown in controlled environments and exposed to a temperature regime of 40/25°C at flowering, fruit set, veraison and mid-ripening stages. Vegetative and reproductive development was measured throughout and leaf photosynthesis and stomatal conductance tracked during heat exposures. Accumulation of soluble solids was determined during ripening. Leaf growth and stem extension were unaffected by heat whereas flowers completely abscised. Berries treated at fruit set developed normally and those treated at veraison and mid-ripening stopped expanding and sugar content stopped increasing. Photosynthesis was also affected on each occasion, with rates declining by 35% and taking 12 days to recover. Up to 10 mg carbon g (berry dry weight)–1 day–1 was required for ripening after veraison. For vines heat treated at veraison and mid-ripening, net carbon acquisition rates fell to below 4 mg carbon g (leaf dry weight)–1 day–1, which is inadequate to supply berry carbon requirements. This suggests that the impacts of heat on the ripening process can be traced back to the supply of carbon.

Thesis (MscAgric (Viticulture and Oenology))--Stellenbosch University, 2008.
The effect of various irrigation strategies on grapevine water relations during the berry ripening period was investigated in a Shiraz/Richter 99 vineyard. Comparisons between different irrigation strategies (full/seasonal, véraison+post véraison, post véraison and no irrigation) were made. During the day, the seasonally irrigated vines experienced less water stress than the deficit treatments. Non-irrigated vines seemed to maintain higher diurnal leaf water potentials. Lower leaf water potentials indicated lower water contents in the vegetative and reproductive tissue. Full irrigation seemed to stimulate primary shoot length. Longer water deficit induced earlier and more complete shoot maturation (reserve accumulation). Re-distribution of leaf area on the shoot may occur when vines are subjected to water deficit. Extended water deficit seemed to induce earlier and restricted water loss from vegetative tissue. The water relations were reflected in the berry size. Irrigation during ripening seemed to induce a continuation of berry water loss. Transpiration losses were apparently much higher in fully irrigated vines whereas stomatal control efficiently maintained water relations in non-irrigated vines. Water deficit seemed to have enhanced the soluble solid accumulation. Irrigation treatments did not seem to affect the titratable acid and pH. The post véraison irrigation in particular seemed to favour a wide window for harvesting. Irrigation at post véraison and especially véraison+post veraison seemed to have a greater effect on the synthesis and extraction of phenolics, anthocyanins and tannins in the berry skins. Different irrigation strategies may affect grapes in such a way that different wine styles are obtained.

La vigne est une plante cultivée dans le monde entier dont l’importance économique est principalement liée à la production de vin. La baie de raisin est également l’un des principaux modèles d’étude pour les fruits non-climatériques notamment pour l’étude des mécanismes contrôlant le mûrissement des baies. Le développement de la baie de raisin est caractérisé par deux phases de croissance séparées par une phase de latence se produisant au moment de la véraison. La baie de raisin est composée de trois tissus principaux: la peau, la pulpe et les graines. La peau et la pulpe présentent une structure et une composition en métabolites distinctes et contribuent de manière différente à la qualité du vin, la pulpe fournissant essentiellement le sucre, les acides aminés et organiques alors que la peau est riche en anthocyanes. A l'heure actuelle, les mécanismes moléculaires impliqués dans le contrôle de la maturation des baies de raisin sont encore mal compris. Si l'ABA, le sucre et différents facteurs de transcription jouent un rôle important dans le contrôle de cette phase de développement, les mécanismes épigénétiques, en particulier la méthylation de l’ADN, apparaissent aussi comme des régulateurs importants du développement et du mûrissement des fruits charnus. Dans ce contexte, Le projet de thèse présenté vise à analyser le rôle de la méthylation de l’ADN (1) dans la maturation des baies de raisin et (2) dans la synthèse des anthocyanes en utilisant comme système modèle des cellules de baie de raisin cultivées in vitro.La culture in vitro de baies de raisin en présence d’inhibiteurs de la méthylation de l'ADN, aboutit à une inhibition de la maturation, suggérant que la méthylation de l’ADN joue un rôle crucial pour cette étape du développement chez la vigne. La pellicule et la chair de baies de raisin récoltées à divers stades de développement ont ensuite été analysées séparément pour déterminer les variations des transcriptomes, de l’abondance de différents métabolites, et de la méthylation de l'ADN. Les principaux résultats indiquent des variations des métabolites et du transcriptome, avec des spécificités liés au tissu analysé. En outre, l'analyse des variations de méthylation de l'ADN à deux stades de développement dans chacun de ces deux tissus révèle l’existence de variations de méthylation spécifiques à chaque tissu, tandis que les variations communes aux deux tissus restent limitées. Ces résultats suggèrent un contrôle de la méthylation de l’ADN spécifique à chaque tissu lors de la maturation de la baie. Cependant les régions différentiellement méthylées identifiées dans chaque tissu, ne sont pas associées à des gènes exprimés différentiellement au cours de la maturation des baies, ce qui pose la question du rôle de la méthylation de l’ADN dans le contrôle de l’expression génique dans les baies.Pour analyser le rôle de la méthylation de l’ADN dans le contrôle de la synthèse des anthocyanes, nous avons utilisé des suspensions de cellules de raisin du génotype Gamay Teinturier (GT), connues pour accumuler des anthocyanes lorsqu’elles sont cultivées à la lumière. L’utilisation de la zébularine, un inhibiteur de la méthylation d’ADN, permet de stimuler l’accumulation d'anthocyanes dans les cellules GT en présence de lumière, et de l’induire à l’obscurité. Les traitements à la zébularine provoquent en outre une limitation de la croissance cellulaire, une modification de l’accumulation des sucres solubles et acides organiques ainsi qu’une reprogrammation importante du transcriptome. Ces résultats suggèrent un effet général de la zébularine sur les cellules GT plutôt qu’un effet spécifique sur l’accumulation d’anthocyanes.Dans l'ensemble, les résultats indiquent que la méthylation de l'ADN est importante pour le contrôle de la maturation des fruits de la vigne, bien que les mécanismes qui sous-tendent les variations de la méthylation et leurs rôles dans les différents tissus de la baie de raisin restent à préciser
Grapevine is a worldwide cultivated fruit crop with high economic importance mainly because of its usage for vine production. Grape berry is also one of the main models for non-climacteric fruits to study the mechanisms controlling the ripening process. Grape berry development is characterized by two phases of rapid size increase separated by a lag phase at the time of ripening induction. Grape berries are composed of three main tissues, the peel, the pulp and the seeds. Peel and pulp present distinct structure and metabolite composition and contribute in a different way to wine quality, the pulp providing sugar, amino and organic acids whereas the peel is important for anthocyanins and other phenolic compound abundance. At the present time, the molecular mechanisms involved in the control of grape berry ripening are still poorly understood. Recent results indicate that both ABA and sugar may be important signals together with various transcription factors. In addition, epigenetic mechanisms are now emerging as important regulators of fleshy fruit development, DNA methylation being critically important for tomato, sweet range and strawberry ripening.The present project aims at analyzing the potential role of DNA methylation in the control grape berry ripening. It also investigates the potential role of DNA methylation in the synthesis of anthocyanins, a compound of primary importance in peel of red grape berries, using in vitro grown fruit cells. To address these questions, grape berries cultivated in vitro were treated with DNA methylation inhibitors. Treatments resulted in delayed and reduced grape berry ripening, therefore sustaining the idea that DNA methylation plays critical roles at this developmental step. Grape berries harvested at various developmental stages were then dissected and each tissue was separately analyzed for transcriptomic, metabolic and DNA methylation variations. Main results indicate significant and distinct metabolic and transcriptomic variations consistent with each tissue following specific modifications during ripening. In addition, analysis of DNA methylation variations at two developmental stages in each tissue indicates both common and tissue specific changes in DNA methylation patterns during fruit ripening. A very small proportion of DMRs is found similarly in the pup and the peel, but most are tissue specific, also consistent with tissue specific control at this developmental phase. Of note, among the different DMRs identified in each tissue, only a few were associated with differentially expressed genes (DEG) during ripening, whereas most were not, questioning the general role of DNA methylation in the control of gene expression at this developmental transition in grape.As Anthocyanins are the most abundant polyphenolic compounds in the skin of red grape berries, we used grape cell suspensions of the Gamay Teinturier genotype, that are known to accumulate anthocyanins when grown in light conditions, to analyze the potential role of DNA methylation in their synthesis. GT cells cultivated in light conditions were treated with the DNA methyltransferase inhibitor zebularine, they accumulate higher quantities of anthocyanins. Of note, GT cells grown in the absence of light do not accumulate anthocyanins. However, zebularine was sufficient to induce anthocyanin accumulation in the absence of light. Zebularine treatments had significant additional effects on grape cells including, cell growth limitation, and modification of soluble sugar, organic acid or stilbene accumulation, together with important transcriptomic reprogramming, consistent with a general effect on cells rather than a specific effect on anthocyanin accumulation.Taken together, results are consistent with DNA methylation being important in the control of grape fruit ripening, although the precise mechanisms underlying methylation variations and roles in grape berries remain to be deciphered

Bibliography: leaves 138-158. This study identified and investigated the properties, functions and patterns of accumulation of prominent berry proteins associated with white wine haze. Detailed analysis was conducted on two PR-like proteins of V. vinifera, VVPR-4a and VVTL1. In vitro fungal growth inhibition assays suggested that berry PR-like proteins may play an important role in plant defence, particularly against fungal attack. Results of this study also have future implications for controlling the ripening process of grapes.

Polyphenols play a crucial role in wine making: they are involved in the oxidation reactions and in the determination of the sensorial quality of wine, particularly the astringency and the structure (tannins) and the color of red wines (anthocyanins). Some polyphenols have nutraceutical properties and are responsible for the benefit of moderate consumption of red wine on human health. Anthocyanins are a very well known group of phenolic compounds responsible for red, blue and purple pigmentation in plants, particularly in some flowers (eg, Petunia) and in many fruits, including red grape berries. Anthocyanins are synthesised in the flavonoid pathway. Previous studies (Downey et al, 2004; Fujita et al 2007; Rustioni et al, 2006) have demonstrated that cluster shading may significantly influence anthocyanin synthesis and, in general, the whole flavonoid pathway. More studies are necessary to elucidate the role of shading in the regulation of the pathway. For this reason we have chosen to study the response to shading in very closely related biotypes of a red berry cultivar of Vitis vinifera. Aglianico is a very famous red cultivar traditionally grown in Southern Italy in many separated areas. Recent study (Costantini et al, 2005) have demonstrated that several byotipes of Aglianico, although showing clearly different phenotypes, they are originated from the same original genotype. For this reason, Aglianico is considered a good model for intra-variety variability. Three main biotypes of Aglianico (Vulture, Taurasi and Taburno) were selected to carry out this work. A vineyard collecting the three biotypes was chosen as experimental site. In the experimental vineyard the biotypes are grown in the same environmental and agronomic conditions. Before veraison, clusters from each biotype underwent two different treatments: 12 clusters were covered with a shading screen designed to exclude light without modifying temperature and relative humidity; other clusters were completely exposed to sunlight through defoliation of the bottom leaves of the canopy. Physiological and technological variables such as sugars, pH and titrable acidity, and the accumulation kinetics of the each polyphenol species were measured. The relative expression of CHS2, F3‘5‘H, F3’H, F3H, DFR1, LDOX1, UFGT, OMT, AM1, AM3, GST4, LAR2, FLS4, MYB5a, MYB5b, MYB12 and MYBA1 was analysed by means of Real Time PCR. This work describes the behaviour of the three biotypes regarding both the accumulation of primary and secondary metabolites as well as the differential expression of the flavonoid biosynthethic genes. In the mean time it describes the accumulation of primary and secondary metabolites and transcriptional expression of the genes of the flavonoid pathway in response to the grape berry shading treatment. This work is the first report about the effect of grape bunch exposure on the expression of the F3’5’H and F3’H genes, and on the AM1, AM3 and GST4 anthocyanin transporters.

Thesis (MSc)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: Grapevine is worldwide an agronomically important crop. Traditionally selective breeding has been used to improve existing cultivars. In the last ten years, however, the advent of biotechnology has shortened these breeding programmes by producing transgenic grapevine. Because this new technology is aimed at the possible genetic manipulation of the ripening process in grape berries, it is important to elucidate all the mechanisms that may be involved in ripening. The aim of the present study was the identification of genes that play an important role during the ripening process in grape berries. This was achieved by investigation of putative differentially expressed genes in ripening Chardonnay berries isolated through subtractive hybridisation. Two subtraction libraries, representing early and late ripening stages were constructed. Four of the ten genes analysed exhibited expression during berry ripening. One of the four genes was expressed in a tissue and stage specific manner. Further characterisation of eight of the DNA and protein sequences revealed that the putative translation products of these clones had homologues that are involved in amongst others cell wall structure in other species. These included UDP-glucose dehydrogenase, which is involved in the synthesis of hemicellulose precursors. The remaining seven clones encoded putative stress response proteins. These included two heat shock proteins, a vacuolar pyrophosphatase and a protein involved in cell division. It is suggested that specific grape mRNAs accumulate in response to stresses such as the storage of high concentrations of sugars and rapid cell expansion. These processes occur rapidly during the ripening of berries. Accumulation of specific mRNAs can be attributed to part of the normal ripening developmental programme.
AFRIKAANSE OPSOMMING: Druiwe is wêreldwyd 'n belangrike landbougewas en kultivars word tradisioneel deur middel van tydsame selektiewe teling verbeter. Die tyd wat hieraan bestee word, kan verkort word deur die implementering van biotegnologie en die produksie van transgeniese duiwe. Omdat hierdie nuwe tegnologie op die moontlike genetiese manipulering van die rypwordingsproses in druiwe gemik is, is dit belangrik dat alle meganismes betrokke by rypwording ondersoek en verstaan word. Die doel van hierdie studie was om gene wat moontlik tydens die rypwordingsproses in druiwe 'n rol kan speel, te identifiseer. Hierdie doel is bereik deurdat differensieel uitgedrukte gene uit die kultivar Chardonnay geïsoleer is met behulp van verrykingsbiblioteke vanuit jong en volwasse druiwekorrels. Vier van die tien gene wat geanaliseer is, word uitgedruk tydens die rypwordingsproses. Verder het een van die vier gene weefsel- en rypwordingstadium- spesifisiteit getoon. Volledige karakterisering van agt van die DNA- en proteïenvolgordes het aangedui dat die proteïenprodukte van hierdie gene homoloog is aan volgordes wat onder andere by selwandstruktuur betrokke is. Dit sluit UDP-glukose dehidrogenase in, wat betrokke is by die sintese van hemi-sellulose boustene. Die ander sewe gene kodeer vir moontlike spanningsproteïene. Twee hitteskokproteïene, 'n vakuolêre pirofosfatase en 'n proteïen wat betrokke is by selverdeling is geïdentifiseer. Daar word voorgestel dat druiwe mRNA versamel in reaksie op spanningsituasies soos die berging van hoë konsentrasies suikers en selvergroting. Hierdie prosesse vind baie vinnig plaas tydens rypwording. Versameling van spesifieke mRNAs kan toegeskryf word as 'n normale deel van die rypwordingsproses.

Mestrado Vinifera Euromaster - Viticulture and Enology - Instituto Superior de Agronomia
Grapevine proteins can play a double role in winemaking. They can have a positive effect in foamability in sparkling wine, but their presence in wine can also be responsible for the wine haze risk (WHR). So, this study is split in two parts as follows: the first part investigated the trend of grape protein content along with the ripening process. Oenological parameters of healthy grapes of cultivars Chardonnay Blanc and Pinot Noir (Vitis vinifera L.), vintages 2012, from the Champagne region at different maturity stages, were collected to investigate the relationships between grape berry maturity degree (MD), and other oenological parameters, such as protein content. The results in both varieties showed an increasing trend of total protein contents (analysed by electrophoresis) during the ripening. Strong correlations have been found among grape berry MD and almost all the parameters in both cultivars. The second part of the work, investigated the use of potential alternative to the use of bentonite for colloidal stability. Indeed, clouding and haze formation are serious aesthetic problems especially in white wines which are caused by the presence of flocculated grape proteins. To overcome this problem and stabilize white wines bentonite is widely used, which allows the easy removal of grape proteins. As known bentonite entails many drawbacks, such as loss of wine volume and stripping of aromas. Thus, this study evaluated the use of an alternative treatment to achieve a colloidal stability without the use of bentonite. Aspergillo-pepsins (AGPs) was added in the aim to stabilize a Sauvignon blanc 2017 grape juices from Amboise (Vallée de la Loire - France), with and without heat treatments (75 °C, 1 min) prior to fermentation. Without heating, AGPs showed a low proteolytic activity. When combined with must heating, more than 90% of the total proteins disappeared in both cases (heating carried out before and after enzyme addition) proving in this case that the heating applied alone was sufficient in fact
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Grapevine represents one of the major economic crop species on a worldwide scale, with a world production approaching 70 million of tons and a harvest area of over 7 million hectares. Amongst the 60 species within the Vitis genus, Vitis vinifera L. is the mostly used for the production of wine and distilled liquors. Before the devastation of European viticulture caused by of the introduction of phylloxera from North America, varieties of V. vinifera used commercially for wine production in Europe were traditionally grown on their own roots. Subsequently, the use of rootstocks from the pest’s origin was introduced to provide resistance to this and other deleterious diseases and to save the fate of European viticulture. Rootstocks have been bred from a number of Vitis species, especially V. berlandieri, V. riparia, and V. rupestris, and are known, in addition to the enhanced resistance to phylloxera and other pathogens, confer tolerance to abiotic stresses (e.g. drought, high salinity and Fe-deficiency), regulate the size of the scion, affected fruit development/ripening, contribute to fruit quality and can alter specific aspects of postharvest fruit quality of a scion. Results presented in this Ph.D thesis are a part of a larger multi-disciplinary project called SERRES (Selection of new grape rootstocks resistant to abiotic stresses through the development and validation of molecular markers) granted by Ager foundation. Selection of resistant rootstocks is crucial for the development of sustainable agricultural models and, at the same time, for inducing a balanced vegetative/productive ratio, a different ripening progression in grape berries and, as well as, differences in their global quality. Improving the knowledge about the molecular, biochemical and physiological bases of stress resistance is an absolute requirement for the selection of genotypes able to cope with stress conditions without any negative consequences on the vegetative growth and production of high quality grape. Drought has an enormous impact on crop production, indeed, it is one of the major factors limiting plant productivity and cause a severe yield reduction. Based on the global climate models, which predict an increase in the aridity in the next future, water deficit may became the major limiting factor. In this context rootstocks may play an important role in limiting crop loss by improving water use efficiency, potential for survival, growth capacity and scion adaptability to stress conditions. Water deficit leads to many morphological and physiological changes across a range of spatial and temporal scales, including reduced expansion of aerial organs, maintenance of root growth, decrease in transpiration and photosynthesis, accumulation of osmotic compounds and ions, activation of detoxifying processes and, in parallel, the transcriptional regulation of a large number of genes. Oxidative stress is related to the accumulation of reactive oxygen species, such as H2O2, O2-, -OH, 1O2, and NO. These ROS are responsible for most of the oxidative damages in biological systems and cellular components. Thus, a strict control of ROS levels, throughout the expression of genes coding for superoxide dismutases (SOD), catalase (CAT), ascorbate peroxidase and glutathione peroxidise ROS scavenging enzymes, is mandatory for plant survival and the cross-talk between ROS accumulation and redox state is integrating part of a fine homeostasis control that plays a pivotal role in the plant response to stresses. Recently, a biochemical and physiological study of the M4 [(V. vinifera x V. berlandieri) x V. berlandieri x cv Resseguier n.1] novel candidate genotype to be used as rootstock in grapevine was performed. This genotype, established from 1985 by the DiSAA research group operating at the Milan University, was selected for its high tolerance to water deficit (WS) and salt exposure (SS). In comparison with the 101.14 commercial genotype, M4 un-grafted plants subjected to water and salt stress showed a greater capacity to tolerate WS and SS maintaining photosynthetic activity also under severe stress conditions and accumulating, especially at the root level, osmotic compounds and ions. In the first part of this thesis were reported results obtained from a large scale whole transcriptome analyses (RNA-seq) performed on root (whole apparatus) and leaf tissues of 101.14 (drought susceptible) and M4 plants sampled in progressive drought (five time points). Physiological analyses were performed on treated (water-stress, WS) and control (well-watered, WW) plants over all the sampling. The multifactorial analysis , which was performed on mRNA -seq data concerning to both the analyzed tissues (leaf and root), allowed us to evaluate the relative weight of the genotype (R: 101.14 and M4), of the type of stress imposed (Treatment, T: WW and WS) and of the time point considered (P: T1-T4), and to identify Differentially expressed Genes (DEGs) that are affected in a specific way or the combined action of these factors (R:T, R:P, T:P and R:T:P). In WS root dataset, all considered components (R, T and P) were found to affect the higher number of genes in comparison to other dataset (WS leaf). A first general observation comparing results of the multifactorial analyses performed on leaves and roots is that in root tissue the “treatment” seems to be the main variable explaining differential gene expression depend on the kind treatment imposed, whereas in leaf tissue the weight of the genotype (rootstock) appear to be the highest. This observation is not surprising, considering that the root system is the first organ perceiving the water deprivation stress and the main one actively responding to it. In this case it’s clear the kind of treatment imposed represent the main variable influencing expression whereas the effect of the genotype is less determinant on differential expression of genes. RNA-seq data were used to performed a Differential Cluster Analysis (DCA), which is based upon comparison of correlation between genes expression of a “reference” and a “target” organism and allowed us to identify conserved and diverged co-expression patterns between related organisms. This analysis allowed us to compared the transcriptomic responses of M4 and 101.14 rootstocks. As concerns plant hormones, it was showed an induction of auxin, JAs and GAs related-genes at the beginning of the stress kinetic in M4 stressed roots, whereas a up-regulation of these transcripts in unstressed root was observed in 101.14. The most interesting metabolic category was the “Secondary metabolism” one because several DEGs belonging to these metabolisms were founded in both root and leaf upon WS, but with a strong specificity of DEGs expression among two considered organs. Indeed, upon WS, roots and leaves of the tolerant genotype M4 exhibit an higher induction of stilbenes (i.e. STS) and flavonoids (e.g. CHS, F3H, LDOX, FLS) biosynthetic genes, respectively. We hypothesized the role of these genes in the control and balance ROS levels, in addition to the others well known ROS scavengers. In presence of water stress, M4 rootstock may acts differential mechanisms in root and leaves which leads to the production of molecules, such as resveratrol and flavonoids and these events may be related to a secondary antioxidant system in this rootstock. The higher resistance of M4 rootstock to water stress, in comparison to what observed in 101.14, should be related to these events. In the second part, in order to evaluate the effects of the rootstocks on grape berry quality and development/ripening, an RNA-seq experiment on Cabernet Sauvignon (CS) grafted onto M4 and 1103 Paulsen rootstocks was carried out. Whole berries were collected from CS/1103P and CS/M4 bunches at 45, 59, 65 days after full bloom (DAFB), in correspondence to the end of lag phase. At this moment most of grape berries reached véraison, the other samples (separating skin and pulp) were collected at 72, 86 and 100 DAFB. On the basis of physical (volume and colour) and chemical (Soluble Solids Concentration, SSC) parameters, the two rootstocks seem to induce a different development and ripening pattern on CS berries. To identify the same developmental phases of berries collected from CS/1103P and CS/M4, the expression profile of genes involved in phenols, sugar and organic acids metabolisms were overlapped. This approach allowed to establish that the green phase occurred at 45 DAFB in both combinations, while véraison happened at 72 and 86 DAFB for CS/M4 and CS/1103P, respectively. An mRNA-seq and a microRNA-seq experiments were carried out on CS berries sampled at pre-véraison (45 DAFB), véraison (72 and 86 DAFB for M4 and 1103P, respectively) and traditional CS vintage date (100 DAFB). For the statistical analyses on RNA-seq data a pairwise comparisons between M4 and 1103P genotypes were accomplished at each time point and a large numbers of DEGs related to auxin metabolisms were identified with enrichment and clustering analysis. It is well known the important role of auxins on grape berry development, so, it was decided to focus our attention on this hormone and to performed a characterization of grape ARF and AUX/IAA gene families. Indeed, in another work presented in this thesis, we showed that an NAA treatment just before véraison caused delayed grape berry ripening at the transcriptional and physiological 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). Considering these results, the differences observed among CS/M4 and CS/1103P in grape berry development and ripening should be related to a different regulation of auxin metabolism. Indeed, all transcripts/miRNAs analyses performed (RNA-seq, microRNA-seq and qPCR) highlighted important differences in the auxin metabolism among the two scion/rootstock combination. Our data suggest an important involvement in the control of grape berry development/ripening of genes that are related, on one hand to auxin action (ARF and AUX/IAA) and, on the other hand, to homeostasis of this hormone through the expression of genes involved in conjugation (GH3) and transport (PIN and ABCB). In this context, also miRNA have an important role, especially by controlling ARF–related genes (e.g. miR160 and miR167). In the case of fruit ripening, auxin acted as a positive regulator of genes that control grape berry size (e.g. expansin-related genes) before the véraison stage; it was indeed observed the up-regulation at the pre-véraison stage, which was different for CS/M4 and CS/1103P, of transcripts that control auxin-responsive genes (e.g. VvARF8A and VvARF1A). The induction of genes that belonged to ARF family was paralleled by the expression of transcripts that control auxin level(e.g. VvGH3-1) and action (VvIAA9, VvIAA15A, VvIAA16), suggesting that an accurate regulation of auxin homeostasis in grape berries at these phases. Moreover, control of auxin levels in grape berry seems pass through other mechanisms which involved control of transport-related genes in the early (ABCBs) and late (PINs) phases of berry development. Taking into accounts that at commercial CS harvest, CS/M4 berries berries were showing differences in some processes ripening-related (e.g. flavonoids metabolism) and a different regulation of auxin metabolisms, when compared to those of CS/1103P, auxin seems to act as negative regulators on some genes related to grape berry ripening but its induction at the pre-véraison stage could be necessary to triggers other metabolism involved in ripening processes.
La vite (genere Vitis) rappresenta una delle principali specie coltivate su scala mondiale , con una produzione che si avvicina ai 70 milioni di tonnellate e una superficie coltivata di oltre 7 milioni di ettari . Tra le 60 specie all'interno del genere Vitis, Vitis vinifera L. è la più utilizzata per la produzione di vino e distillati. Prima della devastazione della viticoltura europea causata dall'introduzione del parassita fillossera dal Nord America, le varietà di V. vinifera usate per la produzione di vino in Europa non erano innestate. Successivamente, l'utilizzo di portinnesti di origine americana ha permesso di fornire una maggiore resistenza al parassita e ad altre malattie che stavano seriamente compromettendo la viticolture Europea. I portinnesti più usati commercialmente derivano da incroci di svariate specie di vite, tra cui V. berlandieri, V. riparia e V. rupestris, e, oltre a migliorare la resistenza alla fillossera e altri patogeni, conferiscono caratteristiche di tolleranza a stress abiotici (come siccità, elevata salinità e Fe-carenza), regolano la crescita dell’acino, contribuiscono alla maturazione e alla qualità dei frutti, possono alterare alcuni aspetti legati alla qualità in post-raccolta dell’acino. I risultati presentati in questa tesi di dottorato sono parte integrante di un progetto multi- disciplinare chiamato SERRES (selezione di nuovi portinnesti di vite resistenti a stress abiotici attraverso lo sviluppo e la validazione di marcatori molecolari) e finanziato dalla fondazione Ager. La selezione e la caratterizzazione di portainnesti che conferiscano un maggiore grado di tolleranza agli stress abiotici è essenziale per lo sviluppo di modelli agricoli sostenibili e, allo stesso tempo, per l’induzione di un rapporto equilibrato tra fase vegetativa e produttiva, una progressione diversa della maturazione dell’uva, così come, differenze a livello qualitativo. Migliorare la conoscenza delle basi molecolari, biochimiche e fisiologiche della resistenza allo stress è un requisito fondamentale per la selezione di genotipi in grado di far fronte alle condizioni di stress senza conseguenze negative su crescita vegetativa e produzione di uva ad alta qualità. Lo stress idrico ha un impatto enorme sulla produzione agricola, infatti, è uno dei principali fattori che limitano la produttività delle piante e causano una grave riduzione della resa. Sulla base dei modelli climatici globali, che prevedono un aumento delle aree aride nel prossimo futuro, la carenza idrica può diventare il principale fattore limitante per la coltivazione. In questo contesto, i portinnesti potrebbero assumere un ruolo importante nel limitare la perdita di raccolto migliorando l'efficienza dell'uso dell'acqua, il potenziale di sopravvivenza della pianta e la capacità di crescita del frutto in presenza di condizioni avverse come siccità ed elevata salinità del suolo (stress osmotici). Lo stress idrico porta a molti cambiamenti morfologici e fisiologici, tra cui ridotta espansione della parte aerea, limitazione della crescita radicale, diminuzione della traspirazione fogliare e dell’efficienza fotosintetica, accumulo di ioni e osmoliti, attivazione di processi di disintossicazione e parallelamente la regolazione a livello trascrizionale di un elevato numero di geni. In seguito allo stress idrico, si innesca uno stress secondario legato all’accumulo di specie reattive dell'ossigeno (ROS), quali H2O2, O2-, -OH, 1O2 e NO. Le ROS sono responsabili della maggior parte dei danni ossidativi nei sistemi biologici e nelle componenti cellulari. Un rigoroso controllo dei livelli delle ROS è obbligatorio per la sopravvivenza delle piante e il cross-talk tra l’accumulo di ROS lo stato redox è parte integrante di un preciso controllo omeostatico che gioca un ruolo fondamentale nella risposta agli stress. Le piante innescano svariati meccanismi di riduzione del livello di ROS (ROS-scavenging) volti all’induzione dell’espressione di geni che codificano per gli enzimi superossido dismutasi (SOD) , catalasi (CAT), ascorbato perossidasi e glutatione perossidasi. Recentemente è stato condotto uno studio di caratterizzazione a livello biochimico e fisiologico di M4 [(V. vinifera x V. Berlandieri) x V. berlandieri cv Resseguier n.1], un nuovo genotipo di vite candidato ad essere utilizzato come portinnesto. Questo genotipo, studiato dal 1985 dal gruppo di ricerca DiSAA dell'Università degli studi di Milano, è stato selezionato per la sua alta tolleranza allo stress idrico (WS) e salino (SS). Se confrontate con il genotipo commerciale 101.14, le piante di M4 sottoposte a deficit idrico hanno mostrato una maggiore capacità di tolleranza e una più elevata attività fotosintetica anche in condizioni di stress gravi. Nella prima parte di questa tesi sono stati osservati i risultati ottenuti da un’analisi trascrittomica condotta su larga scala (RNA -Seq), effettuata su foglie e radici dei portinnesti M4 e 101.14 campionati in condizioni di stress idrico progressivo (5 time-points). Le analisi fisiologiche sono state effettuate sulle piante trattate (deficit idrico, WS) e di controllo (irrigate, WW) lungo tutto il campionamento. L'analisi multifattoriale, che è stata condotta sui dati mRNA-Seq, ci ha permesso di valutare il peso di tre diverse componenti sulla risposta allo stress: genotipo ( R : 101.14 e M4 ), tipo di stress imposto (Trattamento, T : WW e WS) e time-point considerato ( P : T1 - T4 ). Con questa analisi stato inoltre possibile identificare i geni differenzialmente espressi (GDE) legati all’azione specifica o combinata di questi fattori (R:T , R:P , T:P e R:T:P). In WS radice si è sempre osservati un numero maggiore di GDE rispetto alla foglia. Una prima osservazione generale confrontando i risultati delle analisi multifattoriali eseguite su foglie e radici, è che nel tessuto radice il "trattamento" sembra essere la variabile che ha un impatto maggiore sull’espressione genica, mentre nel tessuto fogliare il peso del genotipo (portinnesto) sembra essere il più elevato. Questa osservazione non è sorprendente, considerato che il sistema radicale è il primo organo a percepire lo stress causato dalla carenza idrica e quello principale atto alla risposta. In questo caso è chiaro che il tipo di trattamento imposto rappresenta la variabile principale che influenza l’espressione genica mentre l'effetto del genotipo è meno determinante. Con i dati RNA-seq è stata eseguita una “Differential Cluster Analysis” (DCA), che si basa sul confronto delle correlazioni tra le espressioni dei trascritti di un organismo “reference” e di un “target”. Questa analisi ci ha permesso di identificare i pattern di co-espressione genica (T1-T4) conservati e pattern non-conservati tra M4 e 101.14. Per quanto riguarda gli ormoni vegetali, è stata osservata un’induzione dei geni legati ad auxine, jasmonati ed etilene nelle radici di M4 sottoposte a stress, mentre una sovra-regolazione degli stessi trascritti è stata osservata in 101.14. La categoria metabolica più interessante, emersa dall’analisi DCA, è quella legata ai metaboliti secondari. Infatti sono stati individuati diversi GDE legati a questa categoria sia in radice che in foglia di M4, indotti in condizioni di stress, ed è stata evidenziata una forte specificità di espressione tra i due tessuti. Infatti, in condizioni di carenza idrica, radici e foglie del genotipo tollerante M4 mostrano rispettivamente una maggiore induzione dei geni legati agli stilbeni (i.e. STS) e ai flavonoidi (e.g. CHS, F3H, LDOX, FLS). Il ruolo di questi geni potrebbe essere legato al controllo e al bilanciamento delle specie reattive dell’ossigeno (ROS), in aggiunta ai classici meccanismi di ROS-scaveging (meccanismi antiossidanti primari). In presenza di stress idrico, M4 potrebbe attuare meccanismi differenziali in radice e foglie che portano alla produzione di molecole, come resveratrolo e flavonoidi, correlate ad un sistema antiossidante secondario presente solo nel portinnesto più tollerante. La maggiore tolleranza allo stress idrico di M4, in confronto a quanto osservato in 101.14, potrebbe essere relativo a questi eventi. Nella seconda parte di questa tesi, è stato valutato l’effetto dei portinnesti M4 e 1103P su sviluppo, maturazione e qualità delle bacche di Cabernet Sauvignon (CS). Per questo esperimento sono stati campionati da piante di CS/M4 e CS/1103P acini interi a 45, 59 e 65 giorni dopo la piena fioritura (GDF). Successivamente la maggior parte delle bacche di CS/M4 avevano raggiunto l’invaiatura, si è quindi deciso di separare bucce e polpe per i campionamenti successivi, condotti a 72, 86 e 100 GDF. Sulla base dei parametri fisici (volume e colore) e chimici (solidi solubili totali, SSC), i due portinnesti hanno mostrato una diversa influenza sulla cinetica di sviluppo e maturazione delle bacche di CS. Per identificare le stesse fasi di sviluppo dei frutti raccolti da CS/1103P e CS/M4, è stato condotta un’analisi di espressione preliminare, mediante sistema real-time PCR, sui geni coinvolti nella biosintesi di fenoli, zuccheri e acidi organici. Questo approccio ha permesso di identificare la fase verde a 45 DAFB in entrambe le combinazioni d’innesto, mentre l’invaiatura è stata individuata a 72 e 86 DAFB rispettivamente per CS/M4 e CS/1103P. Le analisi mRNA-seq e micro-RNAseq sono state effettuate sulle bacche in fase di pre-invaiatura (45 GDF), invaiatura (72 GDF per CS/M4 e 86 GDF per CS/1103P) e epoca di raccolta tradizionale di CS (100 GDF). Le analisi statistiche sono state condotte sui dati RNA-seq confrontando il rapporto tra i dati di espressione di CS/M4 e CS/1103P ad ogni punto della cinetica e per entrambi i tessuti. Le analisi di “clusterizzazione” e di arricchimento hanno evidenziato la presenta di un elevato numero di GDE legati a metabolismi auxinici. Le auxine hanno un ruolo fondamentale durante lo sviluppo e sulla maturazione della bacca, si è quindi deciso di concentrare la nostra attenzione su questa classe ormonale e di eseguire una caratterizzazione e un’analsi filogenetica delle famiglie geniche ARF e AUX / IAA sul genoma di PN40024. Il ruolo delle auxine in questi processi è stato studiato anche in un altro un altro lavoro presentato in questa tesi, durante il quale è stato dimostrato che un trattamento sugli acini d’uva in fase di pre-invaiatura con l’auxina sintetica NAA causa un ritardo nella maturazione, che si manifesta a livello fisiologico e di espressione genica, parallelamente alle quali è stata osservata l’induzione di un elevato numero di trascritti atti a controllare l’omeostasi delle auxine. Le analisi condotte con il software HORMONOMETER hanno suggerito che il recupero omeostatico atto a portare i livelli dell’ormone a concentrazioni meno elevate è avvenuto a soli 7 giorni dal trattamento. Questa ipotesi è fortemente supportata dalla sovra-regolazione di geni coinvolti nella coniugazione (GH3 -like) e nell'azione ( IAA4 e IAA31 -like) delle auxine. Considerando questi risultati, le differenze osservate tra CS/M4 e CS/1103P durante lo sviluppo e la maturazione della bacca potrebbero essere collegate ad una diversa regolazione dell’auxina. Infatti, i dati di espressione (mRNA-seq, microRNA-seq e qPCR) evidenziato importanti differenze nel metabolismo auxinico tra le due combinazioni d’innesto. I nostri dati suggeriscono un coinvolgimento importante dell’ormone nel controllo dello sviluppo/maturazione della bacca grazie all’espressione di legati, da un lato all’azione delle auxine (ARF e AUX/IAA) e, dall'altro , all’omeostasi di questo ormone attraverso trascritti coinvolti nella coniugazione (GH3) e nel trasporto (PIN e ABCB). In questo contesto , anche i miRNA hanno un ruolo importante, in particolare esercitando un controllo sulla trascrizione dei geni ARF (e.g. miR160 e miR167). In fase di pre-invaiatura, le auxine hanno un’azione positiva sulla trascrizione dei geni che controllano le dimensioni della bacca (e.g. espansine) e di geni legati alla famiglia delle ARF (ad esempio VvARF8A e VvARF1A ). Parallelamente all'induzione di geni che appartengono alla famiglia ARF, è stata osservata l’induzione di trascritti che controllano i livelli (e.g. VvGH3-1) e l'azione (VvIAA9, VvIAA15A, VvIAA16) dell’ormone, suggerendo un’accurata regolazione dei livelli auxinici in queste fasi importanti dello sviluppo del frutto. Inoltre, il controllo dei livelli di auxina nella bacca d’uva sembra essere legato anche ad altri meccanismi legati all’induzione di geni legati al trasporto ormonale durante le fasi precoci (ABCBs) e tardive (PIN) della maturazione del frutto. Tenendo conto delle differenze osservate tra CS/M4 e CS/1103P nell’espressione di trascritti legati al metabolismo dell’auxina, questo ormone sembra esercitare un’azione negativa su alcuni geni legati alla maturazione della bacca (e.g. flavonoidi), ma la sua induzione nella fase di pre-invaiatura potrebbe essere necessaria per far scattare altri processi metabolici coinvolti nella maturazione dell’acino d’uva.

The Ph.D. research work was aimed at investigating the rootstock influence on grape quality, in conditions of optimal irrigation or water deficit, using an integrated molecular and biochemical approach

Grapevine is one of the most important and cultivated fruit crops in the world. Its economic importance is especially related to winemaking and the production of high-quality grape is one of the major concerns of the viticulturists. In the last years, continuous temperatures increasing have caused an anticipation of the onset of berry ripening, called veraison, modifying the physiological characteristics of grape, its final quality and consequently wine quality. To prevent these negative effects, the interpretation of the molecular mechanisms controlling this process could provide allow the development of more specific and targeted intervention strategies. To this aim, many molecular studies have been performed. One of the most important is represented by the generation of the grapevine gene expression atlas; this study showed a transcriptomic reprogramming during the vegetative-to-mature transition, suggesting the existence of key regulator genes. Further studies showed that this phase transition seem to be regulated by specific genes, defined switch genes; they are mainly transcription factors and the identification of the functions of these genes could provide important details about the molecular mechanism controlling the maturation process in grapevine. Among these transcription factors, five of them, VviNAC33, VviNAC60, VviAGL15, VviWRKY19 and VvibHLH75, have been selected for functional characterization. Their functional analysis in grapevine has been performed using stable genetic transformation and transient gene expression approaches. The application, improvement and development of these approaches has supported the functional characterization of the five selected genes. Regarding the stable transformation, to identify a standard method, 3 different protocols in 3 different cultivars, using GFP as reporter gene, have been tested. The results showed that the regeneration of transgenic somatic embryos and plants occurred only in Shiraz and Garganega cultivars using embryogenic calli as transformation material, indicating that this complex process is cultivar-dependent. Stable genetic transformation was used for the functional analysis of both VviNAC33 and VviNAC60. In a previous work, both NAC genes have been overexpressed in grapevine plants; the overexpression of VviNAC33 has altered the chlorophyll metabolism, while the overexpression of VviNAC60 has caused stunted growth and anthocyanins leaf accumulation, indicating that both genes are involved in the regulation of vegetative-to-mature transition. In this PhD project, both NAC gene have been fused with EAR motif, the strongest transcriptional repression domain in plants, and stably expressed in Garganega and Shiraz plants. The results showed that some putative target genes of both NAC transcription factors are less expressed than WT plants, indicating that EAR motif represents a good approach to study the function of a transcription factor. Regarding transient gene expression, this method was used for the functional analysis of VviAGL15, VviWRKY19 and VvibHLH75. Leaf agroinfiltration was optimized using YFP as reporter gene and tested in different cultivars by a vacuum system. The analysis of YFP transient expression showed that the fluorescence signal is especially localized in the first and second leaf from apex. VviAGL15, VviWRKY19 and VvibHLH75 have been functionally characterized using the improved leaf agroinfiltration protocol. Each transcription factor was co-expressed with YFP gene: the visualization of its expression has allowed to select only agroinfiltrated leaves. Next microarray analysis of overexpressing leaves showed that many upregulated genes are involved in processes associate with ripening, and an exhaustive molecular interpretation of these preliminary results seem to indicate that VviAGL15, VviWRKY19 and VvibHLH75 are master regulators of the onset of berry ripening, controlling many aspects of the maturation programs.

Objectives: The objectives of the proposal were to study how potassium (K) enters the berry and in what tissues it accumulates, to determine what is the sensitive phenological stage that is responsive to K, to study the influence of K on sugar translocation, to determine if K has effects on expression of genes in source and sink organs and to study applied aspects of the responses to K at the vineyard level. During the research it was realized that K acts externally so a major part of the original objectives had to be deserted and new ones, i.e. the role of K in enhancing water loss from the berry, had to be developed. In addition, the US partners developed practical objectives of understanding the interaction of K application and water deficit as well as application of growth regulators. Background: In our preliminary data we showed that application of K at mid-ripening enhanced sugar accumulation of table grapes. This finding is of major implications to both early and late harvested grapes and it was essential to understand the mode of action of this treatment. Our major hypothesis was that K enters the berry and by that increases sugar translocation into the berry. In addition it was important to cover practical issues of the application which may influence its efficacy and its reproducibility. Conclusions: The major conclusion from the research was that our initial hypothesis was wrong. Mineral analysis of pulp tissue indicated that upon application of K there was a significant increase in most of the major minerals. Subsequently, we developed a new hypothesis that K acts by increasing the water loss from the berry. In vitro studies of K-treated berries corroborated this hypothesis showing greater weight-loss of treated berries. This was not necessarily expressed in the vineyard as in some experiments berry weight remained unchanged, suggesting that the vine compensated for the enhanced water loss. Importantly, we also discovered that the efficacy of different K salts was strongly correlated to the pH of the salt solution: basic K salts had better efficacy than neutral or acidic salts and modifying the pH of the same salt changed its efficacy. It was therefore suggested that K changes the properties of the cuticle making it more susceptible to water loss. Of the practical aspects it was found that application of K to the clusters was sufficient to trigger its affect and that dual application of K had a stronger effect than single application. With regard to timing, it was realized that application of K after veraison was affective and the berries responded also when ripe. While the effect of K application was significant at harvest, it was mostly insignificant one week after application, suggesting that prolonged exposure to K was required. Implications: The scientific implications of the study are that the external mineral composition of the berry may have a significant role in sugar accumulation and that water loss may have an important role in sugar accumulation in grapes. It is not entirely clear how K modulates the cuticle but according to the literature its incorporation into the cuticle may increase its polarity and facilitate generation of "water bridges" between the flesh and the environment. The practical implications of this study are very significant because realizing the mode of action of K can facilitate a much more efficient application strategy. For example, it can be understood that sprays must be directed to the clusters rather than the whole vines and it can be predicted that the length of exposure is important. Also, by increasing the pH of simple K salts, the efficacy of the treatment can be enhanced, saving in the costs of the treatment. Finally, the ability of grape growers to apply K in a safe and knowledgeable way can have significant impact on the length of the season of early grape cultivars and improve the flavor of high grape yields which may otherwise have compromised sugar levels.