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Tese de doutoramento em Biology, A cultura da videira tem uma elevada relevância económica e cultural. Atualmente, esta espécie enfrenta desafios difíceis pois, num contexto de alterações climáticas, a severidade dos stresses abióticos está a aumentar, causando impactos negativos na fisiologia da videira, em particular na atividade fotossintética e na regulação do estado hídrico. Desta forma, uma gestão adequada da luz/radiação na copa é essencial para garantir uma boa produção de uvas e vinho. Previamente, mostrámos que o exocarpo e os tegumentos das sementes de bagos de uva de uma casta branca (cv. Alvarinho) foram os tecidos mais fotossinteticamente ativos, que essa atividade variou ao longo das fases de desenvolvimento (verde, véraison e madura) e em resposta ao microclima de luz (LL - luz baixa; e HL - luz alta). No entanto, a função da fotossíntese da uva ainda é amplamente desconhecida. Neste trabalho pretendemos estudar os efeitos desses dois microclimas de luz e de duas estratégias de mitigação do stresse climático - aplicação foliar de caulino e irrigação - na atividade fotossintética, perfil de metabolitos e nos transcritos de genes-alvo desses tecidos do bago de uva, colhidos nessas três fases de desenvolvimento. Estudos por fluorometria de pulso de amplitude modulada (PAM) mostraram que HL aumentou a eficiência quântica máxima (Fv/Fm) e a atividade fotossintética (rETR200) de ambos os tecidos da fase verde. Curiosamente, a aplicação foliar de caulino aumentou a atividade fotossintética dos exocarpos LL da fase verde em comparação com o controlo, enquanto que a irrigação diminuiu a atividade fotossintética das sementes HL nas fases véraison e madura, especialmente nas videiras de parcelas pulverizadas com caulino. Espectrometria de massa por cromatografia líquida (LCMS) revelou que apenas a “irrigação” e o “microclima de luz” levaram a diferenças significativas no perfil de metabolitos dos tecidos do bago. Análises transcricionais por reação em cadeia da polimerase (qPCR) mostraram que os níveis de transcrição de genes codificadores de elementos associados à fotossíntese, clorofila sintetase (VvChlSyn) e ribulose-1,5-bisfosfato carboxilase/oxigenase (VvRuBisCO), foram regulados positivamente pelo microclima HL. Paralelamente, o estudo de lipidómica mostrou que as sementes LL tiveram níveis mais altos de ácidos gordos livres, enquanto que HL levou à regulação positiva de ceramidas na fase verde e triglicerídeos e glicerofosfolípidos na fase madura. Globalmente, este trabalho fornece evidências sobre a contribuição da fotossíntese para a fisiologia do exocarpo e da semente do bago de uva, bem como novos conhecimentos para uma gestão adequada das práticas vitícolas., Grapevine is an agriculture crop with high economic and cultural relevance. Currently, this plant species faces a difficult challenge, as in the context of climate changes, the severity of abiotic stresses is increasing, causing negative impacts on grapevine physiology, namely on photosynthetic activity and water status regulation. Therefore, an appropriate management of the light/radiation intercepted by the canopy is essential to ensure a proper grape and wine production. Previously, we showed that grape berry exocarp or skins and seed integuments from a white variety (cv. Alvarinho) were photosynthetically active and that this activity varied along grape berry developmental stages (green, véraison and mature) and was responsive to the light microclimate that clusters experienced in the canopy (LL - low light; and HL - high light). However, the function of grape berry photosynthesis is still largely unknown. In this work we intended to study the effects of these two contrasting light microclimates under two short-term climate stress mitigation strategies - foliar kaolin application and irrigation - on the photosynthetic activity, metabolite profile and transcripts of target genes of the same two grape berry tissues, sampled at the same three developmental stages. Pulse amplitude modulation (PAM) fluorometry showed that HL increased the maximum quantum efficiency (Fv/Fm) and photosynthetic activity (rETR200) of both tissues at the green stage. Interestingly, kaolin applied to leaves increased the photosynthetic activity of LL exocarps at green stage as compared with control, while the irrigation decreased the photosynthetic activity of HL seeds at véraison and mature stages, especially in those grapevine parcels sprayed with kaolin. Untargeted liquid chromatography mass spectrometry revealed that only “irrigation” and “light microclimate” led to significant differences in the metabolite composition of the berry tissues. Transcriptional analysis by real-time quantitative polymerase chain reaction showed that the transcript levels of genes encoding photosynthesis-related elements, chlorophyll synthase (VvChlSyn) and ribulose 1,5-bisphosphate carboxylase/oxygenase (VvRuBisCO), were up-regulated by HL microclimate. In parallel, lipidomics analysis showed that LL seeds had higher relative levels of free fatty acids, while HL led to up-regulation of ceramides at green stage and triacylglycerols and glycerophospholipids at mature stage. Overall, this work provides insights for the contribution of tissue-specific photosynthesis to grape berry’s skin and seed physiology and metabolome, as well as new knowledge for a good management of viticultural practices., Andreia Raquel Martins Garrido acknowledges the financial support provided by national funds through FCT - Portuguese Foundation for Science and Technology (PD/BD/128275/2017), under the Doctoral Programme “Agricultural Production Chains – from fork to farm” (PD/00122/2012) and from the European Social Funds and the Regional Operational Programme Norte 2020. This study was also supported by Centre for the Research and Technology of Agro-Environmental and Biological Sciences (UIDB/04033/2020) and by Centre of Molecular and Environmental Biology (UIDB/04050/2020). The work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project “MitiVineDrought—Combining “omics” with molecular, biochemical, and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use” with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI 01-0145-FEDER-030341, respectively; and through the research project “BerryPlastid” with ref. POCI 01-0145-FEDER-028165 and ref. PTDC/BIA-FBT/28165/2017, respectively. The work was also support by project I&D&I “AgriFood XXI”, ref. NORTE-01-0145-FEDER-000041, co-financed by the European Regional Development Fund (FEDER), through NORTE 2020 (Northern Regional Operational Program 2014/2020). This work also benefited from the networking activities within the European Union-funded COST Action CA17111“INTEGRAPE—Data Integration to maximize the power of omics for grapevine improvement”.

Viticulture practices that change the light distribution in the grapevine canopy can interfere with several physiological mechanisms, such as grape berry photosynthesis and other metabolic pathways, and consequently impact the berry biochemical composition, which is key to the final wine quality. We previously showed that the photosynthetic activity of exocarp and seed tissues from a white cultivar (Alvarinho) was in fact responsive to the light microclimate in the canopy (low and high light, LL and HL, respectively), and that these different light microclimates also led to distinct metabolite profiles, suggesting a berry tissue-specific interlink between photosynthesis and metabolism. In the present work, we analyzed the transcript levels of key genes in exocarps and seed integuments of berries from the same cultivar collected from HL and LL microclimates at three developmental stages, using real-time qPCR. In exocarp, the expression levels of genes involved in carbohydrate metabolism (VvSuSy1), phenylpropanoid (VvPAL1), stilbenoid (VvSTS1), and flavan-3-ol synthesis (VvDFR, VvLAR2, and VvANR) were highest at the green stage. In seeds, the expression of several genes associated with both phenylpropanoid (VvCHS1 and VvCHS3) and flavan-3-ol synthesis (VvDFR and VvLAR2) showed a peak at the véraison stage, whereas that of RuBisCO was maintained up to the mature stage. Overall, the HL microclimate, compared to that of LL, resulted in a higher expression of genes encoding elements associated with both photosynthesis (VvChlSyn and VvRuBisCO), carbohydrate metabolism (VvSPS1), and photoprotection (carotenoid pathways genes) in both tissues. HL also induced the expression of the VvFLS1 gene, which was translated into a higher activity of the FLS enzyme producing flavonol-type flavonoids, whereas the expression of several other flavonoid pathway genes (e.g., VvCHS3, VvSTS1, VvDFR, and VvLDOX) was reduced, suggesting a specific role of flavonols in photoprotection of berries growing in the HL microclimate. This work suggests a possible link at the transcriptional level between berry photosynthesis and pathways of primary and secondary metabolism, and provides relevant information for improving the management of the light microenvironment at canopy level of the grapes., This research and the APC was funded by FCT—Portuguese Foundation for Science and Technology, grant provided to Andreia Garrido (PD/BD/128275/2017), under the Doctoral Programme “Agricultural Production Chains—from fork to farm” (PD/00122/2012), and by the European Social Funds and the Regional Operational Programme Norte 2020., info:eu-repo/semantics/publishedVersion