The impact of UV-light on grapevine berry and wine metabolites.

Honeth, Chandré (2018-12)

Thesis (PhD)--Stellenbosch University, 2018.


ENGLISH ABSTRACT: Within their natural environments, plants are constantly challenged by a multitude of stress factors and have therefore evolved different adaptive strategies to mitigate potential damage as well as to optimise resource utilisation. Sunlight, being one of these abiotic factors, is fundamental to plant functioning, however also represents a potential source of stress and damage. Understanding light stress and consequent plant responses have therefore received considerable attention. The impacts of light on plant development have been studied comprehensively in model systems as well as crop plants. As one of the most commercially important fruit crops, grapevine has received considerable attention and significant headway has been made in recent years to profile the potential responses of grapevine tissues to light quantity and quality, specifically UVB. Despite this progress, scope for further exploration into the impacts of varying light quality exposure on berry growth and composition, as well as the extended effects into the wine matrices still exists. The purpose of this study was therefore to examine the impacts of a modulated exposure level (quantity of light) in combination with modulated quality of light (UVB presence or attenuation) on Sauvignon Blanc grape berry growth and metabolite composition during the development and ripening processes, as well throughout the wine-processing steps, ending with a sensorial description of the wines. The distinctive varietal style of Sauvignon Blanc has been well characterised in relation to light exposure, making this cultivar an ideal study system for evaluating the impacts of UVB radiation. The trial was designed using a field-omics approach where an experimental system in a cool-climate Sauvignon Blanc vineyard was previously validated to study berry metabolism under high and low light exposure in the bunch zones. This provided an advantageous base from which to evaluate the grape berry responses to UVB radiation under these two light regimes by strategically installing UVB-attenuating acrylic sheets over the bunch zone, thereby creating the following four distinctive bunch microclimates, namely high light and low light microclimates, which served as the controls for the high and low light -UVB microclimates, respectively. Meso- and microclimatic monitoring confirmed that the intended light conditions were indeed achieved in the various microclimates. When evaluating the high light and low light environments separately, the data confirmed the successful attenuation of UVB in each condition while light exposure remained unaffected by the UVB attenuating sheets. The metabolic responses of the berries under the different microclimates were evaluated by profiling and quantifying primary and secondary metabolites in the whole berries during the developmental and ripening period by sampling at the green, véraison and ripe berry stages over three consecutive seasons. Major sugars and organic acids, photosynthetic pigments, volatile organic compounds, amino acids and polyphenolics were profiled and quantified in the samples and subjected to statistical and multivariate data analyses to reveal developmentally responsive metabolites, and/or metabolites that responded to the variable light quantity and quality exposure level. It was clear that in addition to developmental patterns, variations in exposure and UVB levels lead to particular changes in berry metabolite compositions. The results extended the current understanding of UVB responses in grapevine berries by showing that during the green developmental stage, certain carotenoids implicated in photoprotection responded to the variation in light exposure and that the UVB signal specifically was implicated in the photoinhibition response linked to the violaxanthin cycle. Interestingly, under lower light conditions, a similar UVB dependency was seen for the accumulation of lutein epoxide, a xanthophyll linked to acclimation in shade conditions. The primary metabolites as well as the chlorophylls and major carotenoids were mostly unaffected by UVB radiation, indicating that the berries successfully acclimated to their different microclimates. The metabolic profiles of the photoprotective compounds however suggested that the berries in the UVB attenuation microclimates were possibly more prone to stress, particularly in the low light UVB attenuated environment. The ripe berries also responded to UVB attenuation, but in a different way to the green berries. These responses were furthermore influenced by the level of light exposure. In the ripe berries, the formation of compounds known to have antioxidant and/or “sunscreening” properties were negatively impacted when UVB was attenuated. This was most notable in the high light environment where ambient UVB levels lead to an increase in polyphenolics as well as in certain berry volatile compounds including monoterpenes and norisoprenoids. Similarly, the amino acid composition of the ripe berries was differentially modulated by UVB, specifically regarding the branched chain amino acids and GABA, which may be implicated in the mitigation of stress through their roles as metabolites or signalling compounds. Overall, the results indicated a switch in berry employed acclimation strategies to UVB between the early and late stages of development. The primary objective of the green berries appeared to be the maintenance of photosynthetic activity, whereas the ripe berries shifted their metabolism to accumulate compounds involved in blocking UVB and maintaining the antioxidant status of the tissues as protective measures. The skins and pulp tissues of the ripe berries were also subjected to cell wall profiling techniques, but no indication of altered cell wall monomer or polymer profiles were detected for the different microclimates. The ripe grapes from the four microclimates were used to conduct a grape to juice to wine metabolite profiling analyses, using a typical Sauvignon Blanc vinification work-flow and including a sensory description of the resultant wines. Juice samples were generated at three pre-fermentation processing steps and evaluated for amino acids, polyphenolics, volatile compounds and glutathione. The results firstly confirmed that the four microclimates yielded four unique juice matrices. Secondly, by tracking the metabolites through the three juice possessing steps, evidence of the inherent dynamic nature of the juice matrices was revealed, implying the presence of chemical or biological processes which influence susceptible compounds during processing. Additionally, the variations in both light quantity and quality altered the juice environment by possibly changing the juice oxidation status and the juice lipidome, which also impacted the outcome of certain compounds in the wine. The chemical analysis of the wine focused on the fermentation derived compounds and the results confirmed the significant influence of the microclimate on the chemical compositions of the wines. The most notable impacts were noted in the young wines where a higher content of esters was seen with ambient UVB exposure in both the high light and low light microclimates. These results could potentially be related to amino acid composition of the juices, however significant changes occurred in the finished wines during aging. Sensorial analysis of the final bottled wines following aging revealed perceptible differences associated with the four different microclimates. The results reiterated the characteristic aromatic changes which occur in Sauvignon Blanc wines in relation to the variability of light quantity, but also highlighted the significant impact of specifically UVB on wine sensorial characteristics. In the high light microclimates, ambient UVB exposure was strongly associated with tropical aromatic wines, while the attenuation of UVB generated wine with certain similarities to those of the low light microclimate. This indicated that the UVB component of light was necessary for the formation of compounds responsible for the tropical aromas. Furthermore, the low light microclimate wines were generally described as more green in character, however the attenuation of UVB significantly intensified these aromas. Overall, the results show the significant influence of berry microclimate on grape berry composition, leading to altered juice and wine matrices and ultimately perceivable differences in the wines. The findings of this study therefore provided new insights into the underlying metabolic mechanisms employed by grape berries to acclimate to UVB radiation, revealing the employment of phenotypic plasticity by Sauvignon Blanc. The results furthermore highlighted the influence of UVB on juice and wine compositional properties and also provided novel insights into the grape-juice-wine transitions of certain metabolites in Sauvignon Blanc.

AFRIKAANSE OPSOMMING: Plante word voortdurend binne hulle natuurlike omgewing uitgedaag deur 'n verskeidenheid stresfaktore en het dus verskillende strategieë ter aanpassing ontwikkel om potensiële skade te verminder en ook om die gebruik van hulpbronne te optimaliseer. Sonlig, ‘n abiotiese faktor, is fundamenteel belangrik vir plantfunksionering, maar kan ook 'n potensiële bron van stres en skade wees as dit oormatig voorkom. Dit lei daartoe dat die onderwerp van ligstres en die gevolglike reaksie van die plant, aansienlike aandag geniet. Die impak van lig op plantontwikkeling is reeds omvattend in modelsisteme sowel as gewasplante bestudeer. Omdat druiwe een van die kommersieel belangrikste vrugte gewasse is, het dit aansienlike aandag ontvang en is daar in onlangse jare groot vooruitgang gemaak om die potensiële reaksie van wingerdweefsels op ligkwaliteit en kwantiteit aan te dui, ook spesifiek op UVB bestraling. Ten spyte van hierdie vordering bestaan daar nog steeds ‘n behoefte om meer te weet van die impak van blootstelling aan verskillende ligfrekwensies op korrelgroei en samestelling, asook die gevolglike impakte op die wynmatrikse. Die doel van hierdie studie was dus om die impak van 'n gemoduleerde vlak van beligting (lig kwantiteit), in kombinasie met ‘n gemoduleerde lig kwaliteit (gemodifiseerde frekwensiespektrum ten opsigte van UVB-teenwoordigheid) op die Sauvignon Blanc druiwekorrel se groei en metabolietsamestelling te ondersoek. Die analises het gefokus op die ontwikkelings- en rypwordingsprosesse, asook gedurende die wyn voorbereidingsstappe en die uiteindelike sensoriese beskrywing van die wyne. Die kenmerkende kultivar-gekoppelde styl van Sauvignon Blanc, wat beïnvloed word deur blootstelling aan lig, is goed gekarakteriseer en maak dus hierdie kultivar 'n ideale kandidaat om te bestudeer vir die evaluering van die impak van UVB-bestraling. 'n Veld-omika (“Field-omics”) -benadering is gebruik om die studie te ontwerp en deur te voer op ‘n eksperimentele perseel van ‘n koel-klimaat Sauvignon Blanc-wingerd wat reeds voorheen gevalideer is om korrelmetabolisme, onder hoë en lae ligblootstelling, in die trossone te bestudeer. Hierdie gevalideerde perseel is gepas bevind vir die evaluering van die druifkorrels se reaksies op UVB-bestraling onder die hoë en lae ligkondisies. Deur die strategiese installering van akriel UVB-uitsluitingspanele oor die trossones, is vier kenmerkende tros mikroklimate geskep, naamlik ‘n hoë lig en ‘n lae lig mikroklimaat, wat dan ook onderskeidelik die kontroles was vir die hoë en lae lig-sonder-UVB mikroklimate. Meso- en mikroklimaat monitering het bevestig dat die beplande ligtoestande inderdaad in die verskillende mikroklimate bereik is. Die evaluering van die hoë- en lae lig-sonder UVB omgewings het bevestig dat UVB bestraling feitlik afwesig in die trossones agter die akrielpanele was, terwyl die res van die ligfrekwensies nie deur die UVB uitsluitings panele beïnvloed is nie. Die metaboliese reaksies van die korrels op die verskillende mikroklimate is geëvalueer deur die primêre- en sekondêre metabolietprofiele van die heel korrels te kwantifiseer gedurende die ontwikkelings- en rypwordingstydperk. Monsters is geneem tydens die groen, véraison- en ryp stadia, oor drie opeenvolgende seisoene. Die primêre suikers en organiese sure, fotosintetiese pigmente, vlugtige organiese verbindings, aminosure en polifenoliese komponente is geïdentifiseer en gekwantifiseer in die monsters en statistiese en multiveranderlike data-analise is gedoen om ontwikkelings-responsiewe metaboliete en/of metaboliete te identifiseer wat op die veranderlike ligkwantiteit en -kwaliteit gereageer het. Dit was duidelik dat verskille in ligkwantiteit en UVB vlakke tot veranderings in ontwikkelingspatrone gelei het, asook tot spesifieke veranderinge in die metabolietsamestelling van die druifkorrels. Die resultate lei tot ‘n beter begrip van die reaksie van druiwekorrels op UVB bestraling deur te wys dat sekere karotenoïede wat by fotobeskerming betrokke is op die variasie in ligblootstelling reageer tydens die groen ontwikkelingsfase en dat die UVB-sein spesifiek met die foto-inhibisie-reaksie, gekoppel aan die vioolaksien-siklus, geassosieer is. Interessant genoeg, onder laer ligstoestande, is 'n soortgelyke UVB-afhanklikheid gesien vir die opeenhoping van luteïenepoksied, 'n xantofil betrokke by aanpassings by skaduwee toestande. Die primêre metaboliete, asook die chlorofille en primêre karotenoïede was meestal nie deur UVB-bestraling beïnvloed nie, wat aandui dat die korrels suksesvol ge-akklimatiseer het tot hulle verskillende mikroklimate. Die metaboliese profiele van die fotobeskermingsverbindings het egter aangedui dat die korrels in die mikroklimate waar UVB uitgesluit was moontlik meer geneig was tot stres, veral in die lae lig sonder-UVB omgewing. Die ryp korrels het op ‘n ander manier as die groen korrels op UVB-uitsluiting gereageer en die reaksie was ook beïnvloed deur die vlak van ligblootstelling. In die ryp korrels is die vorming van verbindings wat optree as antioksidante en/of "sonskerm" eienskappe het, negatief beïnvloed wanneer UVB uitgesluit was. Dit was veral opvallend in die hoë-lig omgewing waar die ongemoduleerde UVB-vlakke gelei het tot hoër vlakke van polifenoliese verbindings en volataliele organiese komponente soos monoterpene en norisoprenoïede in die korrel. Net so was die aminosuursamestelling van die ryp korrels differensieel gemoduleer deur UVB, spesifiek ten opsigte van die vertakte-ketting aminosure en GABA, wat by stresverligting betrokke kan wees deur middel van hul rolle as metaboliete of seinverbindings. Algeheel dui die resultate daarop dat die druifkorrel verskillende UVB akklimasie strategieë aanwend tussen die vroeë en laat stadiums van ontwikkeling. Die primêre doelwit van die groen korrels blyk die instandhouding van fotosintetiese aktiwiteit te wees, terwyl die ryp korrels hul metabolisme verskuif deur beskermende verbindings te produseer wat UVB kan uitblok en ook die antioksidant status van die weefsel kan onderhou as beskermingsmaatreëls. Die selwandprofiele van die dop- en pulpweefsels van die ryp korrels is ook bepaal, maar daar was geen aanduiding van ‘n verandering in selwandmonomeer of -polimeerprofiele in reaksie tot die verskillende mikroklimate nie.

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