The Evaluation of the Impact of Microclimatic Factors on Grapevine Berries in a Vineyard Setting Through Molecular Profiling
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The evaluation of the impact of microclimatic factors on grapevine berries in a vineyard setting through molecular profiling by Kari du Plessis Dissertation presented for the degree of Doctor of Philosophy (Agricultural Sciences) at Stellenbosch University Institute for Wine Biotechnology, Faculty of AgriSciences Supervisor: Prof Melané A. Vivier Co-supervisor: Dr Philip R. Young December 2017 Stellenbosch University https://scholar.sun.ac.za Declaration By submitting this dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated) that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: December 2017 Copyright © 2017 Stellenbosch University All rights reserved Stellenbosch University https://scholar.sun.ac.za 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. Stellenbosch University https://scholar.sun.ac.za 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. Stellenbosch University https://scholar.sun.ac.za 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 veld- toestande 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 hoogs- gekarakteriseerde 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