The evaluation of the impact of microclimatic factors on grapevine berries in a vineyard setting through molecular profiling

Du Plessis, Kari (2017-12)

Thesis (PhDAgric)--Stellenbosch University, 2017.

Thesis

ENGLISH SUMMARY: Grape composition is considered to be the result of the grapevine genotype, the environmental factors the grapes are exposed to and the management practices implemented during their development. However, elucidating how each of these components contributes to the outcome is notoriously difficult under field conditions due to the myriad confounding variables that grapes are influenced by. One of the viticultural management practices frequently implemented in the vineyard is the removal of leaves in the berry bunch zone in order to alter the microclimate of the developing grapes with various potentially advantageous outcomes. However, this common viticultural practice of leaf removal very rarely affects levels of light without elevating bunch temperatures as well. Moreover, definitive links between this treatment and the underlying grape molecular responses are currently lacking, particularly in cause and effect relationships. Utilizing a highly characterized Sauvignon Blanc vineyard, a leaf removal treatment was implemented according to a field-omics experimental approach, in which it was established that light exposure to the developing grapes was the predominant factor modulated by the treatment. A preceding study characterized the physical growth parameters of the developing grapes and targeted specific metabolites in order to determine how elevated light would affect grape development. The results revealed that the growth and development of the grapes were not affected by the treatment, but that specific secondary metabolites with photoprotective abilities were elevated. These results showed that the grapes acclimated to the elevated light exposure, providing the possibility to study the molecular mechanisms associated with this acclimated state in the berries. The aim of this study was therefore to explore the transcriptional responses of the developing grape berries to elevated light exposure to understand how primary metabolism and growth was maintained despite the implementation of stress mitigation strategies. The approach taken to study this transcriptional response involved RNA sequencing (RNASeq) analysis in order to generate a transcriptional snapshot of all the genes expressed in control and light exposed grapes sampled at four developmental stages throughout berry development. This analysis revealed that the green grapes implemented several photoprotective mechanisms simultaneously. Some of these mechanisms involved non-photochemical quenching and the rapid turnover of the proteins of the photosynthetic machinery, much like other foliar photosynthetic tissues, despite the profound differences in photosynthesis dynamics between these tissue types. Additionally, the genes associated with the synthesis of flavonoid compounds were significantly upregulated and these findings were further corroborated by the accumulation of high levels of flavonols that are known to have both light absorbing and antioxidant abilities. In combination, through these photoprotective mechanisms, as well as the synthesis of higher levels of carotenoids in green berries and subsequent apocarotenoids in ripe berries these grapes achieved a state of acclimation. Furthermore, the catabolism of amino acids provided energy precursors and substrates towards the redistribution of energy that contributed to the maintenance of these energetically costly stress mitigation mechanisms. To this end, green, photosynthesizing grapes maintain growth and development at all costs to protect the development and maturation of the grape seed. Therefore, when the berries achieved ripeness, the photoprotective mechanisms associated with photosynthesis had ceased and the upregulation of apocarotenoids and flavonols were no longer effectively mitigating the light stress. A subsequent investigation explored the role that grapevine heat shock factor (Hsf) genes may have played in achieving this acclimated state. The consistent upregulation of three grapevine Hsfs was established and for one of these genes, VviHsfA7a, a unique putative role in photoprotection under elevated light was identified. Furthermore, by utilizing these results, the first putative working model of the expression and regulation of the Hsfs in grapevine berries were proposed.This study further identified two groups of putative developmental stage-specific molecular biomarkers in grape berries. The first group of genes contributed to the current understanding of the underlying molecular mechanisms associated with the coordinated progression of berry development, whereas the other group of genes represented putative light-responsive molecular biomarkers that are developmentally regulated under non-stressed conditions, but that become significantly upregulated by light stress. Further investigation into the effect that the elevated light exposure may have had on the pathways associated with the synthesis of Sauvignon Blanc impact odorants was conducted. These findings provided insights into how leaf removal and elevated light exposure may lower green aroma characteristics in wine by modulating berry metabolism on a molecular level. Taken together, the findings presented in this study provided definitive insights into how light exposure effects grape berry development on a molecular level and the mechanisms that these berries implement in order to ameliorate the potentially harmful affects of light stress. This study further contributed by putting forward the first de novo assembled transcriptome for the Sauvignon Blanc grapevine genotype that can be utilized in future studies in order to draw more conclusive links between genotypic and/or treatment specific expression in grapevine.

AFRIKAANS OPSOMMING: Druif samestelling word bepaal deur die wingerd se genotipe, die omgewigsfaktore waaraan die druiwe blootgestel word en die bestuurspraktyke toegepas tydens die druif se ontwikkeling. Om uit te vind hoe elkeen van hierdie komponte spesifiek bydra tot die finale druifuitkoms is dikwels moeilik onder veldtoestande weens die magdom interafhanklike veranderlikes wat die druiwe beïnvloed. Een van die bestuursgebruike wat algemeen gebruik word, en wat verskeie moontlike voordelige nagevolge kan hê, is die verwydering van blare in die druif trossone ten einde die mikroklimaat van die ontwikkelende druiwe te verander. Hierdie algemene blaar verwyderingstegniek beïnvloed nie net die vlakke van lig blootstelling nie, maar laat ook meestal trostemperature styg. Duidelike skakels tussen hierdie behandeling en die onderliggende molekulêre reaksies van die druiwe ontbreek nog, veral as gesoek word na oorsaak-en-effek verhoudings. In ‘n vorige studie, deur gebruik te maak van ‘n hoogsgekarakteriseerde Sauvignon Blanc wingerd, kon bevestig word dat lig bloostelling aan die druiwe die hoof variërende faktor was in ‘n “field-omics” blaarverwyderingsbehandeling. Die fisiese groei parameters van die ontwikkelende druiwe en geteikende metaboliete is ook reeds gekarakteriseer om te bepaal hoe hoër vlakke van ligblootstelling die druiwe se ontwikkeling sou beïnvloed. Die resultate het onthul dat die algemene groei en ontwikkeling van die druiwe nie beïnvloed was deur die behandeling nie, maar dat die vlakke van spesifieke sekondêre metaboliete wat rolle vervul in fotobeskerming, hoër was. Hierdie resultate het getoon dat die druiwe aangepas het tot die hoër vlakke van ligblootstelling en dit het die bestudering van die molekulêre meganismes onderliggend aan hierdie aangepaste toestand moontlik gemaak. Die doel van hierdie studie was dus om die transkripsionele reaksies van die ontwikkelende druiwe tot hoër ligvlakke te verken om te verstaan hoe primêre metabolisme en groei gehandhaaf kon word ongeag die feit dat die druifkorrels besig was met stres verminderingsstrategië. Die aanslag van die studie was om hierdie transkripsionele reaksiese te bestudeer met RNA sekwensiëringanalise (RNASeq) sodat ‘n transkripsionele oorsig van al die gene en hul uitdrukking in kontrole- en lig-blootgestelde druiwe gegenereer kon word tydens vier spesifieke druif onwikkelingsstadia. Die analise het onthul dat die groen druiwe verskeie fotobeskermingsmeganismes gelyktydig geïmplementeer het. Sommige van hierdie meganismes behels nie-fotochemiese blussing en vinnige omskakeling van die proteïene wat deel vorm van die fotosintetiese masjienerie, soortgelyk aan ander blaaragtige fotosinterende weefsels, ongeag die definitiewe verskille in fotosintetiese dinamika tussen hierdie weefseltipes. Verder was die gene betrokke by die sintese van flavonoïedverbindings beduidend opgereguleer. Hierdie bevindinge was verder ondersteun deur die versameling van hoër vlakke van flavonole wat bekend is vir beide hul ligabsorberende- en antioksidantvermoëns. Deur middel van hierdie fotobeskermingsmeganismes, asook die sintese van hoër vlakke van karotenoïede in groen druiwe; en die gevolglike vorming van apokarotenoïede in ryp druiwe, is ‘n ligstresaangepasing behaal. Verder het die katabolisme van aminosure energie voorgangers en substrate voorsien vir die herverspreiding van energie hulpbronne wat bygedra het tot die handhawing van hierdie stres-verminderingsmeganismes. Dit blyk dus asof die fotosinterende druiwe groei en ontwikkeling gehandhaaf het ten alle koste om sodoende die ontwikkeling en rypwording van die druif-sade te beskerm. Verder, toe die druiwe rypheid behaal het, is die fotobeskermingsmeganismes geassosieërd met fotosintese gestaak en die opregulering van apokarotenoïede en flavonole was nie meer genoegsaam om die ligstres te onderdruk nie. ‘n Opvolgondersoek het die rol van wingerd hitte-skok faktor (Hsf) gene wat moontlik ‘n rol gespeel het in die aangepaste toestand ondersoek. Die volgehoue opregulasie van drie wingerd Hsfs is bevestig en vir een van hierdie gene, VviHsfA7a, is ‘n unieke vermeende rol in fotobeskerming onder hoër ligvlakke geïdentifiseer. Verder, deur die toepassing van hierdie resultate is die eerste vermeende werksmodel van die uitdrukking en regulering van die Hsfs in druiwekorrels saamgestel. Hierdie studie het verder ook twee groepe vermeende ontwikkelingsstadia-spesifieke molekulêre biomerkers in druiwekorrels geïdentifiseer. Die eerste groep gene het bygedra tot die huidige kennis van die onderliggende molekulêre meganismes geassosieër met die gekoördineerde verloop van druifontwikkeling, terwyl die ander groep gene vermeende lig-reaktiewe molekulêre biomerkers aangedui het wat gereguleer word deur ontwikkeling onder nie-stres toestande, maar wat beduidend opgereguleer word as daar ‘n lig stres ervaar word. Ondersoeke na die effek wat hoër ligblootstelling kon hê op die paaie geassosieër met die sintese van Sauvignon Blanc impakgeure is ook uitgevoer. Hierdie bevindinge het insig verskaf in verband met hoe blaarverwydering en hoër vlakke van ligblootstelling groen geur-eienskappe in wyne kan verlaag deur die modulering van druif-metabolisme op ‘n molekulêre vlak. In samevatting, die bevindinge van hierdie studie kon definitiewe insigte gee rakende hoe ligblootstelling druifontwikkeling beïnvloed op ‘n molekulêre vlak en die meganismes wat hierdie druiwe implementeer om die moontlik skadelike nagevolge van lig stres te beperk Hierdie studie het verder bydraes gemaak deur die generering van die eerste de novo saamgestelde transkriptoom vir die Sauvignon Blanc druifgenotipe wat in toekomstige studies toegepas kan word om meer definitiewe skakels te maak tussen genotipe- en/of behandling-spesifieke geenuitdrukkings in druifstudies.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/102890
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