Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Grape quality is important for the producer, exporter and the consumer. Consumers judge table grapes according to their size, colour, taste and shelf life. The consumer’s prerequisites will influence the producer. Therefore, it is essential to know how the table grape berry develops so that it can be manipulated, favouring the postharvest quality and shelf life. This study was performed on Prime and Crimson Seedless, both grafted onto Ramsey, in the Paarl district of South Africa. The aim of this study was to describe and quantify table grape berry development and compositional changes taking place throughout growth and ripening. The effect of sugar:acid ratio on postharvest shelf life was also evaluated. To evaluate whether berry size influenced the measured development parameters, three berry sizes were induced for both cultivars by using plant bioregulators such as gibberellic acid (GA3) and forchlorfenuron – synthetic cytokinin (CPPU) or girdling. The following sizes were obtained for Prime: (i) small berries (<20 mm) with no treatment, which acted as the control; (ii) medium berries (20-24 mm) obtained by 15 ppm GA3 application at 8 mm berry size; (iii) large berries (>24 mm) obtained by combination of 15 ppm GA3 and 1 ppm CPPU application at 8 mm berry size. Crimson Seedless berry sizes were as follows: (i) small berries (<18 mm) with no treatment, which acted as the control; (ii) medium berries (18-22 mm) treated with 10 ppm GA3 at 7 mm berry size; (iii) large berries (>22 mm) treated with 10 ppm GA3 and vines were girdled at 7 mm berry size. To evaluate the effect of sugar:acid ratio on postharvest shelf life, grapes were stored for five weeks at -0.5 ˚C and another week at 7.5˚C. The bunches were evaluated for loose berries, browning, soft tissue breakdown, decay and berry split. The following components were analysed for both cultivars to determine changes in berry composition throughout the season: berry fresh weight, total soluble solids (TSS), glucose, fructose, titratable acidity (TA), tartaric acid, malic acid, abscisic acid (ABA) and total phenols. Total and individual anthocyanins were analysed for Crimson Seedless. Differences were obtained for the three berry sizes for both cultivars. Véraison, representing the start of ripening, started at the same time in successive seasons: 21 days after pea size berry (5 mm berry diameter) for Prime and 28 days after pea size berry (5 mm berry diameter) for Crimson Seedless. A lag stage was not observed, at seven day sampling intervals, for either of the cultivars. Components such as TSS, glucose, fructose and TA content per berry were influenced by berry size in either one or in both seasons for both cultivars. Significant changes in component concentration were detected at the start of, or around véraison. Sugar concentrations (TSS) already started to increase for both cultivars before the start of véraison. At véraison, concentrations of glucose, fructose and ABA increased while concentrations of TA, tartaric acid, malic acid and total phenols decreased. Total anthocyanins in Crimson Seedless started to increase one week after véraison commenced. The main anthocyanin found in Crimson Seedless was peonidin-3-glucoside. During ripening a 1:1 glucose:fructose ratio was detected in both cultivars. Prime tartaric:malic acid ratio was lower than Crimson Seedless tartaric:malic acid ratio in both seasons. Tartaric acid was the main organic acid found in Prime, while malic acid was the main organic acid found in Crimson Seedless. No significant differences were found in the postharvest defects between the different berry sizes. However, tendencies for differences were observed which led to the assumption that medium size berries were more prone to loose berries in both cultivars. Large berries showed a higher percentage berry split for both cultivars. Crimson Seedless second harvest date took place 24 hours after rainfall which could have very likely led to the higher percentages berry defects compared to the first season. Greater berry decay was found with later harvest dates for both cultivars. No significant differences were found for the TSS:TA ratio between the three berry sizes for both cultivars. Postharvest defects were therefore found not only to be influenced by TSS:TA ratio but rather by harvest date and packing procedures. Environmental conditions prior to harvest also had an impact on postharvest shelf life. / AFRIKAANSE OPSOMMING: Druif kwaliteit is belangrik vir die produsent, uitvoerder en verbruiker. Tafeldruiwe word gekeur deur die verbruiker volgens grootte, kleur, smaak en raklewe. Die verbruiker se voorkeure sal dus die produsent beïnvloed. Daarom is dit belangrik om te weet hoe tafeldruiwe ontwikkel ten einde korrelsamestelling te manipuleer om na-oes kwaliteit en raklewe te kan bevoordeel. Hierdie studie is uitgevoer op Prime en Crimson Seedless, beide geënt op Ramsey, in die Paarl distrik van Suid Afrika. Die doel van die studie is om vas te stel hoe korrelsamestelling gedurende groei en rypwording verander. Die effek van suiker:suurverhouding op na-oes raklewe is ook geëvalueer. Om te kan meet of korrel grootte die gemete parameter beïnvloed is drie korrelgroottes verkry vir albei kultivars deur die gebruik van plant bioreguleerders, te wete gibbereliensuur (GA3) en sintetiese sitokiniene (CPPU), of ringelering. Die volgende korrelgroottes is verkry vir Prime: (i) klein korrels (<20 mm) d.m.v. geen behandeling, geklassifiseerd as kontrole; (ii) medium korrels (20-24 mm) d.m.v. ‘n 15 dpm GA3 behandeling by 8 mm korrelgrootte; (iii) groot korrels (>24 mm) d.m.v. ‘n kombinasie van 15 dpm GA3 en 1 dpm CPPU by 8 mm korrelgrootte. Crimson Seedless korrelgroottes was soos volg: (i) klein korrels (<18 mm) d.m.v. geen behandeling, wat as kontrole gedien het; (ii) medium korrels (18-22 mm) d.m.v. ‘n 10 dpm GA3 behandeling by 7 mm korrelgrootte; (iii) groot korrels (>22 mm) d.m.v. ‘n 10 dpm GA3 behandeling en gelyktydige ringelering by 7 mm korrelgrootte. Om die effek van suiker:suur verhouding op na-oes houvermoë te kon evalueer was druiwe gestoor vir vyf weke by -0.5˚C en ‘n verdere week by 7˚C. Die trosse is geëvalueer vir loskorrels, verbruining, sagte weefsel afbreek, verval en korrelbars. Die volgende komponente is geanaliseer vir albei kultivars om veranderinge in korrelsamestelling gedurende die seisoen te bepaal: vars korrelgewig, totale oplosbare vaste stowwe (suikerinhoud), glukose, fruktose, titreerbare sure, wynsteensuur, appelsuur, absisiensuur en totale fenole. Die totale en individuele antosianiene is ook vir Crimson Seedless gemeet. Beduidende verskille tussen die drie korrelgroottes vir albei kultivars is verkry. Deurslaan, naamlik die begin van rypwording, het op dieselfde dag in opeenvolgende seisoene plaasgevind: 21 dae na ertjiekorrel grootte (5 mm korrel deursnee) vir Prime en 28 dae na ertjiekorrel grootte (5 mm korrel deursnee) vir Crimson Seedless. In teenstelling met die tipiese korrel ontwikkelingspatroon is ‘n rusfase nie waargeneem by beide kultivars nie. Komponente soos suikerinhoud, glukose, fruktose en titreerbare suur inhoud per korrel is deur korrelgrootte beïnvloed in een of albei seisoene vir beide kultivars. Suiker konsentrasie van albei kultivars het reeds voor deurslaan begin toeneem. By deurslaan het die konsentrasies van glukose, fruktose en absisiensuur inhoud toegeneem, terwyl die konsentraies van titreerbare sure, wynsteensuur, appelsuur en totale fenole gedaal het. Totale antosianiene in Crimson Seedless het ‘n week na deurslaan begin toeneem. Die hoof antosianien in Crimson Seedless is peonidien-3-glukosied. Gedurende rypwording was daar ‘n 1:1 glukose:fruktose verhouding gevind vir beide kultivars. In terme van sure is Prime se wynsteensuur:appelsuur verhouding laer as in Crimson Seedless vir albei seisoene. Wynsteensuur is die hoof organiese suur in Prime terwyl appelsuur die hoof organiese suur in Crimson Seedless is. Geen betekenisvolle verskille vir na-oes houvermoë tussen korrelgroottes is waargeneem vir beide kultivars nie. Daar was egter tendense wat aanleiding gegee het in die aanname dat medium grootte korrels geneig is tot loskorrels in albei kultivars. Groot korrels het ‘n hoër korrelbars persentasie getoon vir beide kultivars. Crimson Seedless se tweede oes het plaasgevind 24 uur na reënval, wat aanleiding gegee het tot hoër persentasies korrelbederf. Hoër persentasie korrelbederf was ook gevind met later oesdatums. Geen beduidende verskille is gevind vir suiker:suur verhouding tussen die drie korrelgroottes vir beide kultivars nie. Dus word na-oes houvermoë nie net deur suiker:suur verhouding beïnvloed nie, maar ook deur oestyd en verpakkingsprodsedures. Omgewingsomstandighede voor oes kan ook na-oes houvermoë beïnvloed.

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.

Hormones control plant development by coordinating changes in the expression of numerous genes at crucial times in a tissue and organ-specific manner. They have been implicated in controlling various aspects of grape berry development, in particular, the important process of ripening and are used in some crops to control growth and ripening. Abscisic acid (ABA), is associated in grapevine with the response to water stress but may also have a role in berry ripening. We have shown over three seasons that ABA levels in Cabernet Sauvignon berries increase dramatically at veraison, consistent with it being involved either as a trigger for ripening or as a response to the increase in sugars that occurs at this time. Net ABA accumulation doesn't occur until veraison, the decrease in ABA concentration in the first phase of berry development being due to berry expansion. The decrease in ABA that occurs later in development is likely to be due to a combination of catabolism and sequestration into the bound form. The genes crucial to ABA synthesis, 9-cis-epoxycarotenoid dioxygenase (NCED) and zeaxanthin epoxidase (ZEP), were expressed throughout berry development and no clear correlation was found between their levels and that of ABA. Laboratory studies have shown that isolated berries respond to the presence of sucrose through an increase in ABA biosynthesis pathway gene expression (NCED and ZEP). This resulted in de novo synthesis of ABA as inhibition of the carotenoid synthesis pathway by a phytoene desaturase inhibitor prevented ABA accumulation. Replicated field trials clearly showed that ABA treatments can be effective in significantly enhancing ripening when applied in at or near the end of the first period of berry expansion. Colour accumulation in the skins commenced earlier in ABA-treated fruit as did the increase in sugar levels. ABA treatment also advanced the timing of the second phase of berry expansion as it appeared to eliminate the lag phase of berry growth. Taken together these data demonstrate that ABA is likely to play some part in the control of berry ripening and can be used to advance the timing of ripening. Further investigation into the characteristics of ABA-treated fruit will be needed to investigate the compositional character of treated fruit and to gauge its suitability for winemaking. An ability to control the timing of ripening may provide considerable benefits to the wine industry in terms of wine style/quality and for winery scheduling. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1278807 / Thesis (Ph.D.) - The University of Adelaide, School of Agriculture, Food & Wine, 2007.

Hormones control plant development by coordinating changes in the expression of numerous genes at crucial times in a tissue and organ-specific manner. They have been implicated in controlling various aspects of grape berry development, in particular, the important process of ripening and are used in some crops to control growth and ripening. Abscisic acid (ABA), is associated in grapevine with the response to water stress but may also have a role in berry ripening. We have shown over three seasons that ABA levels in Cabernet Sauvignon berries increase dramatically at veraison, consistent with it being involved either as a trigger for ripening or as a response to the increase in sugars that occurs at this time. Net ABA accumulation doesn't occur until veraison, the decrease in ABA concentration in the first phase of berry development being due to berry expansion. The decrease in ABA that occurs later in development is likely to be due to a combination of catabolism and sequestration into the bound form. The genes crucial to ABA synthesis, 9-cis-epoxycarotenoid dioxygenase (NCED) and zeaxanthin epoxidase (ZEP), were expressed throughout berry development and no clear correlation was found between their levels and that of ABA. Laboratory studies have shown that isolated berries respond to the presence of sucrose through an increase in ABA biosynthesis pathway gene expression (NCED and ZEP). This resulted in de novo synthesis of ABA as inhibition of the carotenoid synthesis pathway by a phytoene desaturase inhibitor prevented ABA accumulation. Replicated field trials clearly showed that ABA treatments can be effective in significantly enhancing ripening when applied in at or near the end of the first period of berry expansion. Colour accumulation in the skins commenced earlier in ABA-treated fruit as did the increase in sugar levels. ABA treatment also advanced the timing of the second phase of berry expansion as it appeared to eliminate the lag phase of berry growth. Taken together these data demonstrate that ABA is likely to play some part in the control of berry ripening and can be used to advance the timing of ripening. Further investigation into the characteristics of ABA-treated fruit will be needed to investigate the compositional character of treated fruit and to gauge its suitability for winemaking. An ability to control the timing of ripening may provide considerable benefits to the wine industry in terms of wine style/quality and for winery scheduling. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1278807 / Thesis (Ph.D.) - The University of Adelaide, School of Agriculture, Food & Wine, 2007.

Hormones control plant development by coordinating changes in the expression of numerous genes at crucial times in a tissue and organ-specific manner. They have been implicated in controlling various aspects of grape berry development, in particular, the important process of ripening and are used in some crops to control growth and ripening. Abscisic acid (ABA), is associated in grapevine with the response to water stress but may also have a role in berry ripening. We have shown over three seasons that ABA levels in Cabernet Sauvignon berries increase dramatically at veraison, consistent with it being involved either as a trigger for ripening or as a response to the increase in sugars that occurs at this time. Net ABA accumulation doesn't occur until veraison, the decrease in ABA concentration in the first phase of berry development being due to berry expansion. The decrease in ABA that occurs later in development is likely to be due to a combination of catabolism and sequestration into the bound form. The genes crucial to ABA synthesis, 9-cis-epoxycarotenoid dioxygenase (NCED) and zeaxanthin epoxidase (ZEP), were expressed throughout berry development and no clear correlation was found between their levels and that of ABA. Laboratory studies have shown that isolated berries respond to the presence of sucrose through an increase in ABA biosynthesis pathway gene expression (NCED and ZEP). This resulted in de novo synthesis of ABA as inhibition of the carotenoid synthesis pathway by a phytoene desaturase inhibitor prevented ABA accumulation. Replicated field trials clearly showed that ABA treatments can be effective in significantly enhancing ripening when applied in at or near the end of the first period of berry expansion. Colour accumulation in the skins commenced earlier in ABA-treated fruit as did the increase in sugar levels. ABA treatment also advanced the timing of the second phase of berry expansion as it appeared to eliminate the lag phase of berry growth. Taken together these data demonstrate that ABA is likely to play some part in the control of berry ripening and can be used to advance the timing of ripening. Further investigation into the characteristics of ABA-treated fruit will be needed to investigate the compositional character of treated fruit and to gauge its suitability for winemaking. An ability to control the timing of ripening may provide considerable benefits to the wine industry in terms of wine style/quality and for winery scheduling. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1278807 / Thesis (Ph.D.) - The University of Adelaide, School of Agriculture, Food & Wine, 2007.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Maceration enzymes for the wine industry are preparations containing mainly pectinases, cellulases and hemicellulases, used during wine making to degrade the berry cell walls and release polyphenolic and aroma molecules to increase wine quality. These types of enzymes are also used for the harvesting of revenue-generating molecules from pomace (skins, pulp and seeds from grape processing waste), or as processing aids when used in the production of bioethanol. Grape berry cell walls are recalcitrant towards degradation, therefore knowledge about their structures and compositions, as well as how the application of enzymes modify these structures is essential in order to optimise these processes. The aim of this study was to extend current knowledge by using a mixture of existing and novel methodologies to study grape berry cell walls by focusing on the profiles of polymers present in the walls. Cell wall profiling techniques used in this study include the Comprehensive Microarray Polymer Profiling (CoMPP) method that employs monoclonal antibodies and Carbohydrate Binding Modules (CBM) which specifically recognise the polymers in the plant cell wall. With this method we measured the abundance of specific polymers and traced the fluctuation in their levels of abundance as influenced by external factors such as enzyme hydrolysis. The CoMPP method was coupled with monosaccharide profile analysis by GC-MS to determine the building blocks of the cell wall polymers, as well as with Infrared Spectroscopy to monitor the changes in the bulk chemistry profile. Data sets generated by the cell wall profiling methods were analysed with uni- and multivariate statistical methods to detect the major patterns in the data. This study highlighted the cell wall differences on the polymer level, in the berry skin cells of Pinotage grapes at different ripeness levels and how it changes during a standard wine fermentation, leading to the release of homogalacturonans and the exposing of arabinogalactan proteins. When maceration enzymes were added, further depectination was evident and the enzymes unravelled the cell wall of the ripe grapes. In overripe grapes no additional degradation could be observed due to maceration enzyme actions, presumably indicating that the endogenous grape enzymes already caused extensive degradation. When purified enzymes were incubated under buffered conditions with isolated skin cell walls from Pinotage grapes or with Chardonnay grape pomace, different levels of enzymatic hydrolysis were observed and defined. The sequence in which cell wall polymers were extracted, and the influence of specific enzymes in facilitating the extraction process, provided important information on the accessibility of specific cell wall polymers. Synergistic action between, for example an endo-polygalacturonase (EPG) and an endo-glucanase (EG) was demonstrated with CoMPP. This EPG and EG synergism was also demonstrated with a yeast strain (a Saccharomyces paradoxus x S. cerevisiae hybrid) fermented in a buffered pomace suspension. This yeast strain has a native EPG and was engineered to also express a recombinant EG from a genome integrated cassette. The cell walls isolated from the pomace after fermentation were unravelled and depectination took place, as evident from CoMPP data. The cell wall profiling techniques used in this study were proven to be fast and sensitive. It provided insights into the structure of grape cell walls and was used to evaluate the changes due to ripening, fermentation, enzymatic hydrolysis and a heat pre-processing treatment. In addition to the knowledge gained, we also demonstrated that these techniques can be used to evaluate different enzymes and enzyme combinations as well as the potential of microorganisms to degrade grape tissue. / AFRIKAANSE OPSOMMING: Maserasie ensieme vir die wynindustrie is ensiem mengsels wat hoofsaaklik pektinases, sellulases en hemisellulases bevat en word tydens wynbereiding gebruik om die druifkorrel se selwand af te breek, die polifenole en aroma molekules vry te stel en sodoende die wyn kwaliteit te verbeter. Hierdie soort ensieme word ook gebruik om inkomste-genererende molekules vanuit druiweprosesserings afval (doppe, pulp en pitte) te isoleer, en ook as prosesserings hulpmiddels in die produksie van bioetanol. Druifkorrel selwande is weerstandig teen ensiem afbraak en daarom is kennis oor die struktuur en samestelling van die selwand, asook hoe die selwand strukture deur die toediening van ensieme verander word noodsaaklik om sodoende hierdie prosesse te optimaliseer. Die doel van hierdie studie was om die huidige kennis uit te brei deur bestaande asook nuwe metodes te gebruik om die druifkorrel selwand te bestudeer met die fokus op die polimeerprofiel van die selwande. Selwand karakteriserings tegnieke wat in hierdie studie gebruik is sluit in die Comprehensive Microarray Polymer Profiling (CoMPP) metode wat monoklonale teenliggaampies en koolhidraat bindende modules (Carbohydrate binding modules, CBMs) wat spesifiek die selwandpolimere van die plant selwand herken, gebruik. Met hierdie metode het ons die vlakke van spesifieke polimere gemeet asook die skommeling in hulle vlakke soos dit beïnvloed is deur eksterne faktore soos ensiem hidroliese. Die CoMPP metode is tesame met monosakkaried profiel analise, met behulp van GC-MS, wat die boublokke van die selwand polimere bepaal, asook infrarooi spektroskopie om die veranderinge in die oorhoofse chemiese profiel te bepaal, gebruik. Datastelle wat met die selwand karakteriserings tegnieke gegenereer is, is ontleed met een- en multiveranderlike statistiese metodes om die hoof tendense in die data op te spoor. Hierdie studie het die selwand verskille, op die polimeervlak, van Pinotage druiwe uitgelig. Verskillende rypheidsgrade asook hoe dit verander tydens ‘n standaard wynfermentasie is gevolg. Laasgenoemde het die vrystelling van homogalakturonaan en die ontbloting van arabinogalaktoproteïene tot gevolg gehad. Met die byvoeging van maserasie ensieme was dit duidelik dat addisionele pektienverwydering plaasgevind het en dat die ensieme die selwand van die ryp druiwe ontrafel het. In oorryp druiwe was daar geen addisionele selwand afbreking sigbaar as gevolg van die aksie van maserasie ensieme nie, wat moontlik aandui dat die inherente druif ensieme reeds uitgebreide selwand afbraak versoorsaak het. Wanneer gesuiwerde ensieme met geïsoleerde selwande van Pinotage druiwedoppe en met Chardonnay druiweprosesserings afval geïnkubeer is onder gebufferde kondisies, is verskillende vlakke van ensiematiese hidroliese waargeneem en geklassifiseer. Die volgorde waarin die selwand polimere geëkstraheer is, asook die invloed van spesifieke ensieme in die bevordering van die ekstraksie proses, het belangrike inligting verskaf oor die toeganglikheid van spesifieke selwand polimere. Sinergistiese aksie tussen, byvoorbeeld ‘n endo-poligalakturonase (EPG) en ‘n endo-glukanase (EG) is geidentifiseer met behulp van die CoMPP data. Hierdie EPG en EG sinergisme is ook geïllustreer met ‘n gisras (‘n Saccharomyces paradoxus x S. cerevisiae hibried) wat in ‘n gebufferde druifprosesserings afval suspensie gefermenteer het. Hierdie gisras het ‘n endogene EPG en is ontwerp om ook ‘n rekombinante EG uit te druk vanaf ‘n genoom geïntegreerde kasset. Die selwande van die druiweprosesserings afval wat na die fermentasie geïsoleer is, was ontrafel en pektienverwydering het plaasgevind, soos bevestig met CoMPP data. In hierdie studie is bewys dat die selwand karakteriserings tegnieke vinnig en sensitief is. Dit het insigte verskaf oor die struktuur van die druifselwand en is gebruik om die veranderinge as gevolg van rypheidsverskille, wynfermentasie, ensiem hidroliese en hitte prosessering te evalueer. Buiten die bydraes tot kennis oor hierdie onderwerpe, is die bruikbaarheid van hierdie tegnieke ook aangetoon, veral in die evaluasie van verskillende ensieme en ensiemkombinasies, asook mikroörganismes vir die afbraak van druifweefsel.

Thesis (M.S.)--Michigan State University. Dept. of Entomology, 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references (p. 106-117). Also issued in print.

Les flavonoïdes (anthocyanes, flavonols et proanthocyanidines) sont des éléments clés de la qualité organoleptique des baies de raisin. Chez les végétaux, l’expression des gènes de la voie de biosynthèse de ces composés est contrôlée par des complexes protéiques organisés autour des facteurs de transcription de type MYB. Dans le cadre de cette thèse, une première approche s’est intéressée aux mécanismes de régulation de l’expression du gène VvMyb5a et de l’activité biologique de la protéine codée par ce gène. L’analyse du promoteur VvMyb5a a montré que son activité au cours du développement de la baie serait plutôt placée sous contrôle hormonal. Des expériences de double hybride ont révélé que la protéine VvMyb5a pouvait interagir avec une protéine kinase de type GAMYB et une protéine WD40. Une deuxième approche, basée sur l’analyse globale du transcriptome de mutants naturels de vigne affectés dans la biosynthèse des anthocyanes, a permis d’identifier deux nouveaux gènes MYB nommés VvMybPA1 et VvMyb24. L’expression différentielle de ces gènes dans des baies de cépages rouges et blancs a été confirmée et leurs caractérisations fonctionnelles ont été engagées chez Arabidopsis thaliana. / Flavonoids, like anthocyanins, flavonols and condensed tannins, are key elements of he organoleptic quality of grape berries. In plants, expression of genes encoding enzymes of he flavonoid biosynthetic pathway is controlled by small protein complexes organised around MYB transcription factors. In the present work, we first focused on the regulatory mechanisms of VvMyb5a expression and on the biological activity of the corresponding protein. Promoter analysis indicated that VvMyb5a expression is probably mainly controlled by hormones. A yeast two-hybrid screen revealed that VvMyb5a can interact with a GAMYB ype protein kinase and a WD40 protein. In a second time, global transcriptome analysis of grapevine natural mutants deficient in anthocyanin biosynthesis led to the identification of wo new MYB genes, named VvMybPA1 and VvMyb24. Differential expression of these two genes in red and white berry skins was confirmed by RT-PCR and their functional characterizations have been initiated in Arabidopsis thaliana.

La masse de la baie est le résultat de l’effet intégré de plusieurs facteurs. La recherche a été dessinée afin d’étudier l’effet simultané des facteurs majeurs influençant la masse et la composition de la baie, de les hiérarchiser selon leur degré d’impact à des échelles différentes, de séparer leur effet direct et indirect sur la composition du raisin et de comparer le profil de vins élaborés à partir de petites et grosses baies. L’étude a été conduite sur deux sites expérimentaux, localisés dans les régions de Saint-Emilion (France) et Alcamo (Italie), pendant les années 2014 et 2015. Sur le premier site, les vignes sont plantées sur deux types de sols, tandis que sur le deuxième, deux traitements hydriques étaient appliqués. A l’échelle intra-parcellaire, l’état hydrique de la vigne représente le facteur le plus important, tandis que l’effet du nombre de pépins par baie n’est pas significatif. Des résultats opposés sont obtenus lorsque les relations sont étudiées à l’échelle de la grappe et de la plante. A large échelle, les facteurs impactent directement et indirectement la composition du raisin et les petites baies produisent des moûts et des vins plus concentrés. A l’inverse, à l’échelle de la grappe et de la plante, la masse de la baie n’influence pas la composition du raisin. Seule la concentration en anthocyanes est significativement liée à la masse à toutes les échelles. Cette relation est particulièrement évidente sous conditions hydriques limitantes. Un déficit hydrique augmente le ratio pellicule/pulpe, indépendamment de la masse de la baie. Petites et grosses baies d’une parcelle ayant une condition hydrique homogène, tendent à avoir un profil similaire. / Final berry mass is the result of the integrated effect of several factors. They also influence berry composition. The present work was designed to study the simultaneous effect of major factors influencing berry mass and composition, to hierarchize their impact at different scales, to distinguish their direct and indirect effect on berry composition and to compare the profile of wines made from large and small berries. The study was carried out simultaneously on two vineyards located in the Saint Emil ion (France) and Alcamo (Sicily) areas, during 2014 and 2015. On the first site, vines were planted on two soil types, while on the second site two different irrigation treatments were applied. Depending on the scale, some factors homogeneously impacted the berry mass and composition. At the intra-parcel scale, vine water status represented the most impacting factor, while berry seed number did not have significant effect. Opposite results were obtained when the investigation was carried out at the intra-bunch and intra-plant scales. At large scale, factors impacted directly and indirectly berry compounds and grape juices and wines produced from smaller berries were more concentrated. Neither at intra-bunch, nor at intra-plant scales, berry size effect on juice composition was significant. Only anthocyanin concentration was related to berry size at all scales. This fact was particularly obvious in berries produced under limited water conditions. Water deficit increased the skin to flesh ratio, independently of berry size. This means that small and large berries, produced from a single parcel with homogenous water uptake conditions, tend to have similar enological profiles.

Les changements climatiques mondiaux ont déjà affecté et continueront d’affecter la physiologie de la vigne, en particulier le rendement et la composition des baies à la récolte, et donc, en définitive, la qualité et la typicité des vins produits. Parmi les différentes stratégies possibles d’adaptation de la viticulture au changement climatique (modifications de l’encépagement, création de nouvelles variétés mieux adaptées aux conditions futures,...), le contrôle de la composition des baies à la récolte par le biais d’une modification des pratiques culturales et notamment par des manipulations de la canopée (rapport feuilles/fruits (Fe/Fr), manipulation de l’exposition des grappes, échardage) est certainement celle qui serait la plus rapide à mettre en œuvre.Ma thèse a pour but de rechercher le meilleur point de compromis entre diminution du taux de sucres et maintien de l’acidité, de l’accumulation des composés phénoliques et d’une complexité aromatique proche de l’actuelle, en réponse aux manipulations de canopée (rapport Fe/Fr, échardage) couplées ou non à l’application d’acide abscissique (ABA).Différents ratio Fe/Fr ont ainsi été étudiés sur le cépage d’étude Cabernet-Sauvignon en conditions semi-contrôlées en serre sur boutures fructifères puis au vignoble au sein d’une parcelle de production. L’analyse de la composition des baies en métabolites primaires et secondaires, réalisée lors de ces deux expériences, a permis de déterminer que le ratio 6 feuilles restantes par grappe était la valeur seuil en dessous de laquelle un effet sur les métabolites primaires est visible. Une diminution du taux de sucres a notamment été observée, également associée à la diminution de la concentration en anthocyanes totales qui sont particulièrement importantes dans la définition de la typicité des vins de Bordeaux. L’application d’ABA exogène sur les baies avant la véraison a permis de diminuer les effets négatifs de la modification du ratio Fe/Fr sur l’accumulation des anthocyanes et n’a pas montré d’impact sur la typicité des vins lors des dégustations des microvinifications réalisées au cours de cette étude. Ces résultats analytiques obtenus ont été couplés à des analyses transcriptomiques de type RNAseq et PCR quantitative en temps réel (Fluidigm) afin d’identifier les gènes clefs associés au voies métaboliques primaires et secondaires réagissant aux modulations du ratio Fe/Fr couplées ou non à l’ajout d’ABA. Ainsi, le couplage modulation du ratio Fe/Fr et application d’ABA exogène sur les baies serait une méthode potentiellement utilisable afin de réduire le taux de sucres et de maintenir une typicité bordelaise proche de l’actuelle.La caractérisation fonctionnelle d'un gène potentiellement associé à la voie de biosynthèse des méthoxypyrazines, impliquées dans le caractère végétal du raisin et du vin, a également été mise en œuvre. Différentes approches ont été testées et ont fourni des indices utiles, cependant les résultats obtenus sont encore en cours de confirmation. / Global climate changes have already affected and will continue to affect the physiology of grapevine, in particular the yield and berry composition at harvest, and thus ultimately the quality and typicality of the wines produced. Among the different possible strategies of adapting viticulture to climate change (changes varieties, creation of new varieties better adapted to future conditions,...), modulating berry composition at harvest through changing cultural practices, such as manipulations of the canopy (leaf / fruit ratio, grape exposure), may be a fast and efficient option as a short-term adaptation strategy.My thesis aims to find the best point of compromise between reducing the sugar level and, maintaining the acidity, phenolic compounds, and the aromatic complexity by the canopy manipulations (leaves / fruits ratio, secondary shoot removal), coupled or not with the application of abscisic acid.Different leaf / fruit ratios were studied on the Cabernet-Sauvignon cultivar in greenhouse and in vineyard within a plot of production. The berry composition analysis of primary and secondary metabolites, performed in these two experiments, made it possibleto determine that the ratio of 6 leaves remaining per cluster was the threshold value below which an effect on the primary metabolites is visible. A decrease in the sugar content was especially observed, also associated with a decrease in the concentration of total anthocyanins which are particularly important in the definition of the typicality of Bordeaux wines. The application of exogenous abscisic acid to the berries before veraison permitted to reduce the negative effects of the modification of the leaf-to-fruit ratio on the accumulation of anthocyanins and did not affect the typicality of the wines during the tastings of the microvinifications carried out during this study. These analytical results were coupled with transcriptomic analyses of RNAseq and real-time quantitative PCR (Fluidigm) to identify key genes associated with the primary and secondary metabolic pathways reacting to modulations of leaf-to-fruit ratio coupled or not with the addition of abscisic acid.Thus, the coupling modulation of the leaf-to-fruit ratio to the application of exogenous abscisic acid on the berries would be a usable method to reduce the sugar content and maintain a typicality of Bordeaux wines close to the current one.The functional characterization of a gene potentially associated with the biosynthetic pathway of methoxypyrazines, which are involved in vegetable character of grapes and wine, was also conducted. Different approaches have been tested and provided useful clues but the conclusive results are still under further confirmation.