Physiology of photosynthetic grape berry tissues: the effects of canopy light microclimate and climate stress mitigation strategies

Universidade do Minho Escola de Ciências Andreia Raquel Martins Garrido July 2021 Physiology of photosynthetic grape berry tissues: the effects of canopy light microclimate and climate stress mitigation strategies Andreia Raquel Martins Garrido Physiology of photosynthetic grape berry tissues: the effects of canopy light microclimate and climate stress mitigation strategies UMinho|2021 Universidade do Minho Escola de Ciências Andreia Raquel Martins Garrido July 2021 Physiology of photosynthetic grape berry tissues: the effects of canopy light microclimate and climate stress mitigation strategies Work supervised by Professor Doctor Ana Cristina Gomes da Cunha Doctor Artur Jorge da Silva Conde Doctor Ric Cornelis Hendricus De Vos Doctoral Thesis PhD Thesis in Biology __________________________________________________________________________________ ii DECLARATION COPYRIGHT AND TERMS OF USE OF THE WORK BY THIRD PARTIES This is an academic work that can be used by third parties as long as the internationally accepted rules and best practices are respected, with regard to copyright and related rights. Thus, this work can be used under the terms of the license below. If the user needs permission to be able to use the work under conditions not foreseen in the above mentioned licensing, he/she should contact the author, through the RepositóriUM of the University of Minho. License granted to users of this work Attribution CC BY https://creativecommons.org/licenses/by/4.0/ __________________________________________________________________________________ iii Acknowledgments First and foremost, I want to deeply thank Professor Ana Cunha for giving me the opportunity to work together along the last few years. I thank the sharing of scientific and practical knowledge, as well as her support, dedication and effort on this project. It was a great privilege to work with her and I am very truly grateful for her guidance. I also deeply thank Doctor Artur Conde for his supervision, dedication, concern, availability to help and kindness. I am very thankful for his useful scientific contribution and suggestions during the preparation of this work. I would like to deeply thank the supervision of Doctor Ric De Vos from Business Unit Bioscience of Wageningen Plant Research Center at Wageningen University and Research (WUR). I thank for giving me the possibility to spend six months at his laboratory, which allow me to acquire new scientific and technical knowledge and many other interpersonal skills. It was a very rewarding experience, both professionally and personally. I deeply thank for his sharp and rigorous scientific analysis of the manuscripts. I am truly honored for his guidance. The scientific collaboration of Professor João Serôdio, from the Center for Environmental and Marine Studies (CESAM) at the University of Aveiro, was also important for this work. I am thankful for his availability, patient and kindness. I want to deeply thank the owners of Quinta Cova da Raposa, Manuel Taxa and his wife, for their availability and sympathy. I thank for kindly allowing me to implement and conduct the experimental design in their vineyards and for providing me the grape berry samples used in this work. I thank the support given by the direction of this Doctoral Programme “Agricultural Production Chains – From Fork to Farm (AgriChains)”, as well as given by the secretariat. I also deeply thank all the Professors involved on this PhD, especially Professor Teresa Lino Neto, for her tutoring and curricular guidance, and Professor Hernâni Gerós, for his sympathy and kindness. I would like to thank the support given by all members of Business Unit Bioscience of Wageningen Plant Research Center (at WUR), in particular all from Plants Metabolomics group: Professor Doctor Robert Hall for his kindness and Roland Mumm, Bert Schipper, Henriette van Eekelen and Jeroen van Arkel for their help in the chemical analysis and data processing. A special thanks to Jasper Engel, for his help in the statistical analysis. I also want to thank the PhD colleagues, Giusi, Cristina and specially Sena for their companionship, help and for making me feel at home during my stay at Wageningen. __________________________________________________________________________________ iv I also acknowledge the technical support given by the staff members of Biology Department of the School of Sciences from the University of Minho, namely Cristina Ribeiro, Luís Correia, Manuela Rodrigues, Amaro Rodrigues and Inês Pinheiro. I thank the support and companionship of all members of Plant Biology laboratory: Henrique Noronha, Viviana Martins, António Teixeira, Richard Breia, Mariana Vale, Angélica Silva, Hélder Badim, António Freitas and Jorge Rodrigues. I also want to thank the Agrichains colleagues, namely Eunice Santos, Luis Giraldo for their sympathy and specially Ana Sofia Freitas for her support and friendship. Finally, I would like to express my unmeasured gratitude to my parents and my brother for their unmatched support, patient and help. Financial support 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. POCI01-0145-FEDER-030341, respectively; and through the research project “BerryPlastid” with ref. POCI01-0145-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”. __________________________________________________________________________________ v STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. __________________________________________________________________________________ vi Fisiologia dos tecidos fotossintéticos do bago de uva: os efeitos do microclima de luz na copa e das estratégias de mitigação do stress climático Resumo 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. Palavras chave: expressão de genes, fotossíntese, medidas de mitigação de curto-prazo - irrigação e caulino, microclima de luz, metabolismo. __________________________________________________________________________________ vii Physiology of photosynthetic grape berry tissues: the effects of canopy light microclimate and climate stress mitigation strategies Abstract 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 ribulose1,5-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. Keywords: gene expression, irrigation and kaolin short-term measures, light microclimate, metabolism, photosynthesis. __________________________________________________________________________________ xiv PAR Photosynthetically active PCA Principal component analysis PEP Phosphoenolpyruvate PEPC Phosphoenolpyruvate carboxylase PFD Photon flux density PFR Photosynthetic photon fluence rates PK Polyketides PSI Photosystem I PSII Photosystem II PUFA Polyunsaturated fatty acids PVP Polyvinylpyrrolidinone qPCR quantitative Polymerase Chain Reaction RC Reaction centers rETR Relative electron transport rate through PSII RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase RuBP Ribulose 1,5-bisphosphate SL Saccharolipids SP Saturation pulse SP Sphingolipids SPS Sucrose-phosphate synthase STS Stilbene synthase SUC Sucrose/H+ symporters SuSy Sucrose synthase TAG Triacylglycerides or triacylglycerols TBA 2-thiobarbituric acid TBARS Thiobarbituric acid-reactive-substances TCA Tricarboxylic acid UDP-G Uridine diphosphate glucose WAA Weeks after anthesis ΦII Effective quantum yield of PSII __________________________________________________________________________________ xv List of publications and communications Papers in the international peer-review journals Published: Garrido, A., Engel, J., Mumm, R., Conde, A., Cunha, A.* & De Vos, R. C. H.* (2021). Metabolomics of Photosynthetically Active Tissues in White Grapes: Effects of Light Microclimate and Stress Mitigation Strategies. Metabolites , 11 (4), 205. https://doi.org/10.3390/metabo11040205 *Equal senior authorship. Garrido, A., Serôdio, J., De Vos, R., Conde, A., & Cunha, A. (2019). Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries. Agronomy , 9 (11), 685. https://doi.org/10.3390/agronomy9110685 Submitted: Garrido, A., De Vos, R. C. H., Conde, A.* & Cunha, A.* (submitted). Light microclimate-driven changes at transcriptional level in photosynthetic grape berry tissues. Plants , 10 , x, https://doi.org/10.3390/xxxxx. (26th June 2021). *Equal senior authorship. Under final revision by the co-authors: Garrido, A., Conde A., De Vos, R. C. H & Cunha, A. Fruit Photosynthesis: where, how and why? (Provisional tittle) . Garrido, A., Conde A., De Vos, R. C. H & Cunha, A. The influence of light microclimate on the lipid profile and associated transcripts of photosynthetically active grape berry seeds. (Provisional tittle) . Garrido, A., De Vos, R. C. H., Conde, A. and Cunha, A. Establishment of callus cultures from white grape berry tissues. (Provisional tittle) . __________________________________________________________________________________ xvi Communications in international and national congresses Oral presentations: Garrido, A., Conde, A., De Vos, R. C. H. & Cunha, A. (2021) Grapevine canopy light microclimate effects on the metabolism and gene expression of two photosynthetically active grape berry tissues. In II Plant Abiotic Stress Forum - An integrative lens over plant adaptation, 29th of May, Organized by iB2 Laboratory and PlantStress Laboratory PhD Students, University of Porto (online version), Portugal. Garrido, A., Conde, A., Cunha, A. & De Vos, R. (2019). Phytochemicals and Photosynthesis of Grape Berry Exocarp and Seed ( Vitis vinifera , cv. Alvarinho): Effects of Foliar Kaolin and Irrigation. In 21st International Conference on Grapevine Physiology and Biotechnology, 16th – 17th of December, Barcelona, Spain. (Note: This presentation allowed to receive the “Best Presentation Award”). Garrido, A., Serôdio, J., De Vos R., Conde, A. & Cunha, A. (2019). Photosynthesis of grape berry tissues under kaolin film application at canopy. In XXIII Meeting of the Spanish Society of Plant Physiology/ XVI Hispano-Portuguese Congress of Plant Physiology, 26th – 28th of June, Pamplona, Spain. Garrido, A., Serôdio, J. & Cunha, A. (2018). Photosynthetic phenotype adaptation of grape berry ( Vitis vinifera ) to canopy light microclimate and acclimation to short-term light challenges: how to cope with Mediterranean summer stress? In National Congress on Climate Change, 19th – 21st of February, UTAD, Vila Real, Portugal. pp. 43. ISBN 978-989-704-259-1. Garrido, A., Serôdio, J. & Cunha, A. (2017). Impact of the light microenvironment on photosynthetic activity of grape berry ( Vitis vinifera ): insights for light absorption mitigations measures. In 2nd World Symposium on Climate Change Adaptation, 6th – 8th of September, Coimbra, Portugal. Garrido, A., Pimentel, D., Serôdio, J. & Cunha, A. (2017). Photosynthesis and phenylalanine ammonia lyase activity in grape berry ( Vitis vinifera ). In XV Spanish-Portuguese Congress of Plant Physiology, 26th – 29th of June, Barcelona, Spain. Poster presentation: Garrido, A., Conde, A., De Vos, R. C. H. & Cunha, A. (2021). Metabolome and transcriptional changes in ripening grapes in relation to berry tissue-specific photosynthesis. In Plant Biology Europe 2021 Congress, 28th of June – 1st of July (online version). Torino. Italy. Chapter 1 General Introduction Chapter 1 – General Introduction __________________________________________________________________________________ 18 1.1. Vitis vinifera : a plant species with high social, cultural and economic relevance Grapevine ( Vitis vinifera L.) is a perennial woody species belonging to the Vitaceae family. Currently, more than 70 species grow in different geographical areas, being Vitis vinifera the most renowned one, with domestication of Vitis vinifera sylvestris beginning in Transcaucasia (today’s Georgia, Armenia and Azerbaijan) over 8000 years ago, and subsequently spread to other countries (Estreicher, 2017). According to the latest report of the International Organization of Vine and Wine (OIV) (OIV, 2019), world vineyards covered an area of approximately 7.449 million hectares (ha) in 2018, and 5 countries represented 50 % of this area, including, Spain (13 %) China (12 %), France (11 %), Italy (9 %), and Turkey (6 %). The same report indicated a record-breaking value of the grape production of 77.8 million tons, considering its all uses, namely, its production in the form of wine grapes (57 % of all grapes), table grapes (36 %), or dried grapes (7 %). Regarding to the production of wine grapes (in relation to the total), countries such as Germany (99.6 %), France (99.6%), Spain (96 %), Argentina (93.7 %), Romania (93.1 %), Australia (90.9 %), Italy (86.5 %) are the highest producers. Concerning the winemaking sector, the global wine production remained stable over the last two decades, with 292 million hectoliters (hl) in 2018, being the most important wine producers, Italy (54.8 million hl), France (48.6), Spain (44.4) and the USA (23.9). According to the same OIV 2018 report, Portugal was ranked as the 11th world and 5th European wine producer. The vineyard area in Portugal, in 2018, was 192,000 ha, which contributed for 6.1 million hl of wine production. The world trade, that is, the sum of exports from all countries, and considering the monetary value, was approximately EUR 30 billion in 2018. Currently, grapevine is considered one of the most important agricultural crops cultivated in the world with high socioeconomic relevance due to the great diversity of modes of its fruit consumption and use: table grapes, raisins, juices, jellies, wine vinegar, food additives and most importantly, wine (Conde et al., 2007). Furthermore, other grape by-products (e.g., pomace, stems, leaves) have been used for pharmaceutical, nutraceutical and cosmetic purposes, due to their cardioprotective, antioxidant and antiinflammatory properties, which are mainly associated with the high phenolic content (Teixeira et al., 2014). For instance, grape seed extracts have become popular in recent years, being used as nutritional supplement (Waterhouse et al., 2000). Nowadays, wine is considered an integral component of the culture of many countries and, when moderately consumed, has health benefits. In addition to water, wine is composed by several compounds, as sugars, alcohol, phenolics, organic acids and mineral salts. The grapevine variety, the edaphoclimatic conditions and the enological practices are examples of factors that affect the wine chemical composition and its complexity. The main constituent of the wine is water, accounting for 75 to 90 % (v/v), followed by ethyl alcohol, which, Chapter 1 - General Introduction __________________________________________________________________________________ 19 according to the type of wine, varies from 8 % to 13 % (v/v) and can achieves 15 % (v/v) due to climate changes (van Leeuwen et al., 2019). The content in sugar is important for the growth of fermentative yeasts, being directly responsible for the final alcoholic content of the wine. In addition, the higher content of phenolic compounds present in the wine, confers antioxidant features, which are beneficial for the human health, for instance, in preventing cardiovascular diseases (Cordova et al., 2005) and diabetes (Caimi et al., 2003). 1.2. Grape berry: histology, development and composition Grape berry, a non-climacteric fruit, is comprised of different tissues and layers of cells (e.g., skin or exocarp, flesh or mesocarp and seeds) (Figure 1.1), with different anatomical characteristics and biochemical profiles, which can play distinct roles during the development and ripening of the fruit (Hardie et al., 1996). The growth and development of the grape berry presents a double sigmoid pattern (Coombe, 1992), that is, two successive growth phases separated by a phase where no increase in volume occurs (Figure 1.2). Figure 1.1. Anatomy and histology of a mature grape berry and close-up of a seed viewed in a transverse section. Adapted from Famiani et al., (2000), Kennedy (2002a). Illustration by Jordan Koutroumanidis. The first stage (stage I), from flowering to approximately 60 days after, is characterized by rapid cell division and cell expansion, and in which the berry is formed and the seed embryos are produced (Kennedy, 2002a). Several organic compounds are accumulated in the berry during this first growth period, such as tartaric and malic acids. Tartaric acid accumulates at the beginning of this phase and its concentration is highest at the skin of the developing berry. By contrast, malic acid is accumulated in the Chapter 1 – General Introduction __________________________________________________________________________________ 20 flesh at the end. Hydroxycinnamic acids (HcA) and tannins are also accumulated during this first growth stage. Hydroxycinnamic acids are accumulated in flesh and skin, while tannins accumulate in skin and seed tissues (Kennedy et al., 2000a; 2000b; 2001). In addition, at this early stage of development, some grape berry tissues, namely the exocarp and seed outer integument, present photosynthetic activity, as we demonstrated before (Breia et al., 2013; Garrido et al., 2018), and as it will be exposed in more detail further ahead (in Chapter 2). Figure 1.2. Structural, metabolic and physiological changes during the development and ripening of grape berry. Grape berry development occurs in three stages: stage I (green stage), stage II (lag phase) and stage III (ripening), which starts at véraison . Adapted from Kennedy, 2002a. Illustration by Jordan Koutroumanidis. The second stage (stage II) is known as lag phase and it is characterized by an arrest in growth and grape berries begin to lose chlorophyll. After this lag phase, the beginning of second growth phase (stage III), which is known as véraison , coincides with the onset of ripening, and in red berries is when a change in the skin color is observed (in Portuguese this transition phase is called “pintor” (meaning painter) due to this changing in color). Besides the increase in volume, at this stage III berries become less acidic and sweeter due to the beginning of sugar accumulation. Sugars are transported to the grapes since the first stage, through the capillary vascular bundles, peripheral and central (Figure 1.1). A dorsal bundle network extends at the periphery of the fruit, and central vascular bundles connected to the seeds Chapter 1 - General Introduction __________________________________________________________________________________ 21 irrigate the central flesh (Zhang et al., 2006). Some compounds produced and accumulated during the stage I subsist only in lower concentrations in the stage III. These compounds include malic acid, which is metabolized and used as a source of carbon and energy (Conde et al., 2007). The total tannin content also decreases during this stage in seed coat and in the exocarp (Kennedy et al., 2002a; 2000b). In the seed coat this decline accompanies the color changes that occur in seeds during ripening, suggesting that this seed browning represents oxidation of tannins during ripening (Kennedy et al., 2000a; 2000b). Despite of this decrease, in the stage III other metabolites determinants for the quality of the wine increase including, anthocyanins (in red grape varieties) and flavor compounds, which are important to the pleasant aroma of many varieties (Kennedy, 2002a). 1.3. Photosynthesis, photoassimilate distribution and plant growth Photosynthesis is a physiological process performed by plants, algae and photosynthetic bacteria, crucial for all life on Earth. These photoautotrophic organisms are able to capture the energy of sunlight and use it to transform inorganic compounds (water and carbon dioxide) into highly energetic organic compounds (sugars). In eukaryotes, this process is divided into two distinct phases, spatially and functionally distinguished within the chloroplasts: the light-dependent photochemical reactions located in the thylakoid membranes and the carbon-reducing phase or Calvin-Benson cycle, which occurs in the stroma. In the photochemical phase, bound pigment-protein complexes, the photosystems II and I work in series to intercept and convert sunlight energy into chemical energy stored in adenosine triphosphate (ATP) and reducing power in nicotinamide adenine dinucleotide phosphate (NADPH) (Keller, 2015) (Figure 1.3). The process begins with the energy of the photons being absorbed by the photosynthetic pigments (chlorophyll a and b and carotenoids) present in light-harvesting complexes (LHC) of photosystems II (PSII) and photosystem I (PSI), and channeled, by inductive resonance, to the respective reaction centers (RC). Here, the excitation energy (or exciton) is absorbed by the chlorophyll a molecules ( chl.a *), and the excited electron will eventually leave the molecule being transferred to an electron acceptor. In PSII, the strong oxidant produced when the excited chlorophyll a from the RC loses the electron ( chl.a +), promotes the separation of the water molecule into molecular oxygen, protons and electrons in the oxygen evolving complex (OEC). The high-energy electrons leaving both PS are transferred to other molecules creating a chain of redox reactions known as electron transport chain (ETC), leading to the formation of NADPH (Nelson and Ben-Shem, 2004). The transthylakoidal proton gradient (∆pH) Chapter 1 – General Introduction __________________________________________________________________________________ 22 generated by the operation of the ETC in favor of a redox potential and the oxidation of water in the lumen, allows the formation of ATP in the stroma by the ATP synthase (Figure 1.3). Figure 1.3. The main pathways of plant photosynthesis. Electron transport, driven by the excitation of photosystem I (PSI) and photosystem II (PSII), results in the reduction of NADP+ to NADPH and the accumulation of protons in the thylakoid lumen, which are used to make ATP. The Calvin-Benson cycle proceeds in three stages: carboxylation, reduction and regeneration. Abbreviations: 3-PGA, 3-phosphoglycerate; Cyt bf, cytochrome b6f complex; Fd, ferredoxin; G-3-P, glyceraldehyde-3-phosphate; PC, plastocyanin; PQ, plastoquinone; PQH2, plastoquinol; RuBP, ribulose 1,5-Bisphosphate. Adapted from Baker (2008). In the reactions of the Calvin-Benson cycle, the ATP and NADPH formed in the photochemical phase are used to assimilate CO2, diffusing from the atmosphere through the stomata to the chloroplasts, into sugars. The Calvin-Benson cycle proceeds in three main phases: carboxylation, reduction and regeneration (Figure 1.3). In the first phase, the enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO) combines the CO2 with ribulose 1,5-Bisphosphate (RuBP) to produce two molecules of 3phosphoglycerate (3-PGA). In the reduction phase, 3-PGA is transformed into glyceraldehyde-3-phosphate (G-3-P) using ATP and NADPH. Most of the G-3-P molecules (5/6) are used to regenerate RuBP allowing Chapter 1 - General Introduction __________________________________________________________________________________ 23 to start the cycle again. The remaining G-3-P molecules can be used in the chloroplast or exported from the chloroplast, for instance to produce translocable sugars, like sucrose, and distributed among a significant number of pathways providing carbon compounds essential for plant growth and development (Smith and Stitt, 2007). In plants, in addition to the leaves, other green-structures (e.g., fruits, stems, flower organs and roots) may present photosynthetic activity at some point of development, as reviewed by Aschan and Pfanz (2003) and Brazel and Ó’Maoileídigh (2019). Thus, they also might contribute partially for the carbon and energy budget, necessary for its own metabolism, growth and development (Cipollini and Levey, 1991; Cocaliadis et al., 2014; Ollat and Gaudillere, 2000). 1.4. Major compounds from primary metabolism in grape berries Primary metabolism plays an essential role in grape berry development. During development, fruits act as strong sinks importing massive amounts of photoassimilates from the main photosynthesizing organs (Nath et al., 2014). These photoassimilates are translocated via phloem and used for growth and also as precursors of secondary metabolites (Nath et al., 2014). The products from primary metabolism are not only crucial for grape development and survival, but also endow grape berry specific characteristics decisive for its market value. Sugars, organic acids and the grape seed oils are among the most relevant primary metabolites in grape berries, as discussed below. 1.4.1. Sugars Fruit sweetness is an essential characteristic of fruit quality and is determined by the total sugar content. In fact, for winemaking, the accumulation of sugars in mature grapes (65 to 91 % of mature grape berry dry weight is glucose and fructose) is important for the production of ethanol (Conde et al., 2007). Sucrose is the main sugar transported at the plant level, being produced in leaf mesophyll cells, with a vital role of the sucrose-phosphate synthase (SPS). Sucrose is then transported to the companion cells-sieve elements (cc-se) complex of the phloem either by a symplastic (passive transport via plasmodesmata) or apoplastic pathway (reviewed by Boss and Davies, 2001) (Figure 1.4). The unloading of sucrose from cc-se to the fruit, can occur also by these two different pathways (Figure 1.4). It was demonstrated that during stages I and II of grape berry development, the phloem unloading was predominantly symplastic, but at the onset of ripening (with the beginning of stage III) the apoplastic pathway dominated (Zhang et al., 2006). This apoplastic mechanism is explained by the high sugar Chapter 1 – General Introduction __________________________________________________________________________________ 30 Kennedy et al., 2006). In Figure 1.6 an hypothetical tetramer condensed tannin is composed by an extension of the subunits catechin, epicatechin, epigallocatechin and the terminal epicatechin gallate (Adams, 2006). These subunits are linked by C4-C6 and C4-C8 interflavan bonds. In addition, the structures of tannin polymers, which are composed by monomeric flavan-3-ols, suggests a precursor product relationship (Adams, 2006). In the grape berries, tannins are present in hypodermal layers of the skin and the soft parenchyma of the seed coat, inside the vacuole or bound to cell wall polysaccharides, being the degree of polymerization much larger in the skin than in seed (Adams, 2006; Hanlin et al., 2010; Kennedy et al., 2006). However, it is important to understand how tannins are transported across vacuole membranes and accumulated in these tissues. A study with mutants of Arabidopsis with a defective plasma membrane H+-ATPase demonstrated that they were unable to accumulate tannins in vacuoles of the seed coat endothelial cells (Baxter et al., 2005), suggesting that tannin accumulation requires proper endomembrane trafficking and that at least one of the requisite H+-ATPases plays a specific role in tannin biogenesis and vacuole accumulation. Figure 1.6. Hypothetical condensed tannin made up of four subunits: catechin, epicatechin, epigallocatechin, epicatechin gallate. The first subunit (catechin) is bound to the second (epicatechin) by an interflavan bond between carbon 4 of catechin and carbon 8 of epicatechin. Adapted from Adams (2006). 1.5.3. Biosynthesis pathways of phenolic compounds Erythrose 4-phosphate and phosphoenolpyruvate, derivatives of the primary metabolism, are the initial precursors of the shikimate pathway. This biosynthetic pathway is responsible for the production of phenylalanine, as well as other aromatic amino acids, such as tyrosine and tryptophan. The biosynthetic pathway of soluble phenolic compounds, more specifically the phenylpropanoid pathway, begins with the aromatic amino acid phenylalanine (Figure 1.7) (Vogt, 2010). The first enzyme responsible for the synthesis of phenolic compounds is phenylalanine ammonia lyase (PAL), which converts phenylalanine to cinnamic acid. Cinnamic acid is then converted into p -coumaric acid by a hydroxylation at the 4-position by cinnamate-4-hydroxylase (C4H). The third step of the phenylpropanoid pathway is the esterification of p -coumaric acid with coenzyme A (CoA) by 4-coumaroyl:CoA-ligase (4CL) that produces 4-coumaroyl-CoA. Chapter 1 - General Introduction __________________________________________________________________________________ 31 Figure 1.7. Biosynthetic pathways of phenolic compounds in grape berry. Abbreviations: PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl:CoA-ligase; CHS, chalcone synthase; STS, stilbene synthase; CHI, chalcone isomerase; F3H, flavonone 3-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; LDOX, leucoanthocyanidin dioxygenase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, flavonoid glucosyltransferase. Full lines represent direct enzymatic conversion and dashed lines represent omitted intermediates. Adapted from Ageorges et al. (2014) and Teixeira et al. (2013). The end-product of phenylpropanoid pathway, 4-coumaroyl-CoA, is used as substrate by stilbene synthase (STS) and chalcone synthase (CHS), beginning the stilbene and flavonoid pathways, respectively. In the stilbene pathway, STS enzyme catalyzes three reactions of condensation of 4-coumaroyl-CoA with 3 molecules of malonyl-CoA producing resveratrol. In the STS reaction, the terminal carboxyl group is removed prior to closure of the A ring, so resveratrol has a different ring-folding compared to the CHS product naringenin chalcone. Chapter 1 – General Introduction __________________________________________________________________________________ 32 1.5.3.1. Flavonoid pathway The flavonoid pathway leads to the synthesis of different classes of metabolites such as flavonols, flavan-3-ols, proanthocyanidins, and anthocyanins (Figure 1.7). In tissue-specific mRNA expression analysis it was verified that many of the genes encoding flavonoid biosynthetic enzymes were specifically expressed in the skin or seed (Grimplet et al., 2007). In grape berry, at least three genes encoding CHS exist, Chs1 (AB015872), Chs2 (AB066275), and Chs3 (AB066274), which are transcribed under different controls (Goto-Yamamoto et al., 2002). After that, the naringenin results from the formation of the C ring by chalcone isomerase (CHI). The characterization of grape transcriptome allows to verified that a gene encoding a putative CHI is expressed strongly at the onset of véraison (da Silva et al., 2005). The next step of flavonoid pathway corresponds to hydroxylation of naringenin into different dihydroflavonols (He et al., 2010). When the hydroxylation occurs in the position 3, catalyzed by flavanone3-hydroxylase (F3H), the dihydrokaempferol is formed. In addition, naringenin can also suffer hydroxylation at the 3’ and 3’,5’ positions by the activity of flavonoid-3’-hydroxylase (F3’H) and flavonoid3’,5’-hydroxylase (F3’5’H), which catalyze the conversion into eriodictyol and pentahydroxyflavanone, respectively. These compounds are then converted by F3H into another dihydroflavonols, which are dihydromyricetin or dihydroquercetin. The dihydroflavonols mentioned above can be oxidized into flavonols, like kaempferol, quercetin, and myricetin by flavonol synthase (FLS). In grapevine genome, five FLS genes were identified (Fujita et al., 2006). Three different isogenes encoding FLS were identified with each being expressed in different tissues, such as, skin (TC46143), pulp (TC46972) and seed (TC40373) (Grimplet et al., 2007). The putative flavonol regulator VvMYBF1 , which activate FLS expression, was identified and functionally validated (Matus et al., 2008). Its expression in the berry was strongly reduced as a result of shading (Matus et al., 2009) and was induced by light (Czemmel et al., 2009). On the other hand, the dihydroflavonols can also be reduced by dihydroflavonol reductase (DFR) to their corresponding leucoanthocyanidins (leucocyanidin, leucopelargnidin and leucodelphindin) (He et al., 2010). Recently, studies have been done in order to understand the structural and biochemical properties of the DFR, namely in grape berries, where the region of substrate binding and recognition was confirmed (Petit et al., 2007). It was also shown that DFR gene expression is induced by white light, calcium and sucrose (Gollop et al., 2002). After this step, the leucoanthocyanidins are converted into the corresponding anthocyanidins (cyanidin, pelargonidin and delphinidin) by the action of leucoanthocyanidin dioxygenase (LDOX), also Chapter 1 - General Introduction __________________________________________________________________________________ 33 called as anthocyanidin synthase (ANS). The promoter of the grape Ans gene (CAA53580) have several putative DNA binding motifs and can be induced by the same factors as DFR gene (Gollop et al., 2001). The leucoanthocyanidins and anthocyanidins are considered as potential substrates for flavan-3ols and consequently for proanthocyanidins biosynthesis. Leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) convert, respectively, leucoanthocyanidin and anthocyanidins into the flavan-3-ols catechin and epicatechin (Bogs et al., 2005). These flavan-3-ols monomers are required for the tannin polymerization process, which is not yet fully understood (Watrelot and Norton, 2020). In grape berry, two genes encoding LAR ( VvLAR1 and VvLAR2 ) and one encoding ANR ( VvANR ) were identified (Bogs et al., 2005). The genes encoding LAR were expressed in developing fruit, but had different patterns of expression in skin and seeds (Grimplet et al., 2007). VvANR was expressed only in seeds and detectable after véraison (Grimplet et al., 2007). In addition, those expressions were consistent with the accumulation of proanthocyanidins in the berry, suggesting that they are responsible for determining tannin composition of the fruit during ripening (Bogs et al., 2005). Furthermore, MYB transcription factors, VvMYBPA1 and VvMYBPA2 , were recognized in berry seeds and skin, respectively, and are expressed in parallel with proanthocyanidins accumulation during the early stages of grape berry development (Bogs et al., 2007; Terrier et al., 2008). The last phase of the flavonoid pathway consists of the formation of anthocyanins. Anthocyanidins are inherently unstable under physiological conditions, and thus the glycosylation is an important modification for increasing their hydrophilicity and stability (He et al., 2010). The glycosylation catalyzed by flavonoid glucosyltransferase (UFGT) stabilizing cyanidin, pelargonidin and delphinidin, leads to formation of the anthocyanins cyanidin-3O -glucoside, pelargnidin-3O -glucoside and delphinidin-3O - glucoside, respectively (He et al., 2010). Normally, UFGT expression is only detected in red grape varieties skin after the onset of véraison (Boss et al., 1996). 1.6. Impact of environmental conditions on grape berry physiology, development and quality: the microclimate concept Grapevine physiology and metabolism are affected by several environmental conditions (Blancquaert et al., 2019; Poni et al., 2018). Each wine-growing area presents a complex and interacting system, commonly called as terroir , which includes specific soil, topography, climate, landscape characteristics and biodiversity features, and its interaction with applied viticultural practices (Leeuwen and Seguin, 2006). In this way, the terroir determines grapevine development and physiology, and therefore grape berry composition and wine quality. On a finer scale at the grapevine level, grape berry Chapter 1 – General Introduction __________________________________________________________________________________ 34 clusters and leaves have a microclimate, which is characterized by specific biotic and abiotic factors, including light and temperature. The concept microclimate was adopted by Smart et al. (1985) to define the environmental conditions in the vicinity of leaves and fruits. In this way, the structural organization of grapevines can influence the microclimate conditions that fruits and leaves experience, and consequently grape berry productivity and quality (Kraus et al., 2018). Water availability is one of the most important factors for the growth and quality of the grapes. In general, water deficit, in particular associated with extreme temperatures, strongly affects key metabolic pathways of primary and secondary metabolism, like sugars and phenolics (Teixeira et al., 2013). In fact, in response to water stress, grape berries produce and accumulate osmotically active solutes, including sugars, amino acids, fatty acids, potassium ions, varying this response between grapevine varieties and stress intensity (Koundouras et al., 2006). For instance, the sugar content increased in Cabernet Sauvignon berries under water deficit, but no significant differences were observed in Chardonnay, Merlot and Shiraz (Castellarin et al., 2007a; Castellarin et al., 2007b; Ojeda et al., 2002). In addition, malate concentrations decreased under water stress conditions, while the anthocyanins and total phenols increased (Downey et al., 2006; Kennedy et al., 2002b; Matthews and Anderson, 1989). The increase in anthocyanin accumulation is due to up-regulation of genes, like VvLDOX , VvDFR , VvUFGT1 , and transcription factors (e.g., VvMYBA1 ), which are responsible for encoding enzymes involved in this pathway (Castellarin et al., 2007a; Deluc et al., 2009). Water deficit also increased the flavonol content in a white grapevine variety Chardonnay, but this was not observed in a red variety (Cabernet Sauvignon), in which the contents were similar between irrigation treatments (Deluc et al., 2009). However, in another red variety (Aragonez), total flavonols content was higher in skins of berries from irrigated vines than from non-irrigated ones (Zarrouk et al., 2012). Light and temperature are abiotic factors in close association, and both influence the overall grapevine physiology and berry composition (as reviewed by Palliotti and Poni (2016)). In vineyards, the increase of the number of days with high temperatures is particularly relevant. The production and quality of grape berries are sensitive to heat waves, especially at certain phenological stages, such as flowering and maturation. In fact, when the environment is warmer than the ideal for a given grapevine variety, it may result in a faster than desired phenological development (Gerós et al., 2016). The effects of light microclimate conditions during growth on grape berry metabolism has been studied (Friedel et al., 2015; Koyama et al, 2012; Plessis et al., 2017; Reshef et al., 2017; Young et al., 2016). In general, grapes exposed to light have higher concentrations in sugars, anthocyanins and phenolic compounds and lower values of titratable acidity and malate, when compared to mature grapes Chapter 1 - General Introduction __________________________________________________________________________________ 35 grown in the shade (Dokoozlian and Kliewer, 1995). This can be explained by a delay of grape berry maturation in the shade condition, as proposed by Zha et al. (2019) in a study testing different treatments of bagging. However, excessive higher temperatures can lead to a decline in sugar and anthocyanin content due to an increase in its degradation (Spayd et al., 2002). It was verified that light modulates the expression of flavonol synthase ( VvFLS ) and of VvMYBF1 , a transcriptional regulator, being the flavonol content reduced in shaded berries (Azuma et al., 2012; Koyama et al, 2012). In addition, the shade can also induce lower anthocyanidin content in berries, due to down-regulation of genes/ transcription factors like VvUFGT , VvMYBA1 , and VvMYBA2 (Azuma et al., 2012; Koyama and Goto-Yamamoto, 2008; Matus et al., 2009). 1.7. Climate changes effects on viticulture and mitigation strategies Climate changes effects are becoming a real concern for the agriculture sector in general, including viticulture, since its deeply dependent on weather and climate conditions (Santos et al., 2020). Indeed, the climate changes projections for the European viticulture, point to an intensification of summer-related environmental constraints, such as an increase in temperature and solar radiation, and a reduction in water availability (Fraga et al., 2013; Fraga et al., 2020). Projections for Portugal indicated that Douro, Alentejo and Minho regions will also be affected by these climate adversities (Fraga et al., 2014a; 2014b), thus imposing new challenges for these winemaking regions. In fact, several studies have already reported impacts of climate changes on grapevine phenology (Fraga et al., 2016) and physiology, as well as on grape berry composition and wine quality (Leeuwen and Darriet, 2016; Mira de Orduña, 2010). Moreover, it also enhances the severity of grapevine diseases caused by the biotic factors (Caffarra et al., 2012). Stress mitigation strategies emerge as a solution to reduce the impacts caused by climate changes on grapevine physiology and can be classified as shortor long-term (Santos et al., 2020). Short-term mitigation strategies include, for instance: smart irrigation (Koech and Langat, 2018), training systems (i.e., grapevine canopy management) (Reynolds, 2010), application of leaf sunscreens (e.g., the mineral kaolin) (Brito et al., 2019), vine shadings (Caravia et al., 2016), and cover crops and soil tillage to maintain the water capacity in the soil (Parpinello et al., 2019). Long-term mitigation strategies, include relocations of vineyards to cooler sites, varietal selection and genetic breeding (Duchêne et al., 2012). In particular, the white mineral kaolin (Al2Si2O5(OH)4), has become a cost-efficient mitigation strategy in vineyards, mainly due to its reflective properties, in alleviating the stress associated to excessive heat/radiation absorbed by leaves and grape berry clusters (Brito et al., 2019). In fact, recent investigations showed the positive effects of foliar kaolin application at the whole grapevine physiology, Chapter 1 – General Introduction __________________________________________________________________________________ 36 as well as at the grape berry level (Conde et al., 2016; Conde et al., 2018; Dinis et al., 2016; Dinis et al., 2018; Frioni et al., 2019). Additionally, other studies also focused on the interaction between foliar kaolin application with irrigation treatments (Cooley et al., 2008; Glenn et al., 2010; Shellie and Glenn, 2008; Shellie and King, 2013). However, it is important to state that these strategies may have implications on the light received by leaves and fruits. On one hand, foliar kaolin will directly alter light reflection both outwards and into the canopy (Wünsche et al., 2004), while irrigation may indirectly lead to more shading due to an enhanced vegetative growth (Keller et al., 2016). Previously, we studied the response of grape berry photosynthetic tissues to long-term light microclimate conditions and to short-term light acclimation, giving insights for possible effects of mitigation measures on the light microclimate of grape berries (Garrido et al., 2018). In summary, these mitigation measures aiming reducing radiation absorption by the vine canopy may also interfere with light spectrum and intensity for the grape berry photosynthesis, and thus, more investigations on that respect are required. 1.8. Research objectives The main objective of this PhD dissertation was to study aspects of the grape berry physiology under different growing conditions, with a focus on the light microclimate experienced by the berry clusters during their development. In particular, we intended to investigate the effects of: i) two contrasting light microclimates within the canopy of grapevines, namely low light (LL) and high light (HL); and ii) two climate stress mitigation strategies, namely foliar kaolin application and grapevine irrigation, on various physiological and biochemical aspects of the two photosynthetically active tissues in developing grape berries (exocarp and seed) from the white grapevine variety Alvarinho. A set of complementary methodologies and approaches were used, including the assessment of the photosynthetic activity of the two grape berry tissues, sampled at three developmental stages in all treatments and microclimates, their respective metabolomic profiling, as well as the transcriptional analysis of target genes of key metabolic pathways. With this broad approach we also aim to unveil the potential functions of photosynthesis of these grape berry tissues on metabolic pathways/fingerprint metabolites and on the expression pattern of genes coding for respectively involved enzymes. Ultimately, we expect to contribute with knowledge that may help farmers on their decisions concerning viticultural practices, like sustainable adaptation strategies. Firstly, in this Chapter 1 we intended to expose a general introduction. In the next chapter (Chapter 2) we will thoroughly review what is so far known about fruit photosynthesis. In that regard, histological Chapter 1 - General Introduction __________________________________________________________________________________ 37 and physiological characteristics of fleshy fruits, with a particular emphasis on the grape berry will be addressed. Also, a special focus on photosynthesis in seeds will be given. Finally, the potential functions of photosynthesis in fruits and other organs, will be discussed. The subsequent five chapters (Chapter 3 to 7) concern the research work carried out to meet the objectives of the thesis, as briefly described here: In the Chapter 3 it is presented the influence of two mitigation strategies (foliar kaolin application and irrigation) on the photosynthetic activity of grape berry tissues (exocarp and seeds), grown in two light microclimates of the grapevine canopy. The photosynthetic activity (photochemical and nonphotochemical parameters) of grape berry tissues was analysed using the Pulse amplitude modulated (PAM) chlorophyll fluorescence imaging technique (Baker, 2008). This analysis was performed in collaboration with Professor João Serôdio from the Center for Environmental and Marine Studies (CESAM) laboratory at the University of Aveiro. The characterization of canopy microclimates in terms of light intensity received by the clusters and associated grape berry temperatures, as well as results obtained from spectral analysis regarding transmittance and reflectance properties of leaves with and without kaolin are also presented here. In the Chapter 4, metabolomic techniques were used to analyze the effects of tested factors - mitigation strategies and light microclimates - on the metabolic profile of photosynthetically berry tissues, aiming to evaluate the potential role of tissue-specific photosynthesis in their metabolomes. This part of the work was developed during a six-month internship at the Bioscience Department, Wageningen Plant Research, from Wageningen University & Research, The Netherlands, under the supervision of Doctor Ric C. H. De Vos. The untargeted metabolomics by Liquid Chromatography Mass Spectrometry (LCMS) and Gas Chromatography Mass Spectrometry (GCMS) allowed to find the global effects of all the treatments of this study, on the exocarp and seed metabolome. The observations were also complemented with targeted analysis, for instance, by High Performance Liquid Chromatography (HPLC) with PhotoDiode Array (PDA) and Fluorescence (Fl) detectors, for the detection of chlorophylls, carotenoids and tocopherols. Chapter 5 is devoted to study the expression patterns of selected genes coding for enzymes involved in primary and secondary metabolism that are directly or indirectly influenced by light exposure. This part of the work was carried out under supervision of Doctor Artur Conde from Biology Department at University of Minho. In this case, the photosynthetic grape berry tissues from the two light microclimates were considered for the gene expression analysis by real-time qPCR. Chapter 1 – General Introduction __________________________________________________________________________________ 38 Chapter 6 is dedicated to study the effects of light microclimate on seed lipid profile, determined by LCMS analysis, since it is an important factor for the final quality of seeds, and to the best of our knowledge, there are no known studies on that respect. In Chapter 7, it will be presented a sequence of different optimization protocols, which were tested in order to establish in vitro cultures from grape berry tissues. In particular, we tested: grape berry surface disinfection treatments, types of culture medium, hormonal combinations, special additives, among others. At the end, we obtained callus from exocarp. The main objective with this task was to establish cell lines with different photochemical competences allowing to relate photosynthetic activity, under control conditions, with metabolites and gene expression levels selected from previous results. Unfortunately, due to lack of time we were not able to perform it completely. Finally, in the Chapter 8 we enclose a general integrative discussion of the present work, highlighting our main achievements and prospects of future work. 1.9. References Adams, D. O. (2006). Phenolics and ripening in grape berries. In American Journal of Enology and Viticulture (Vol. 57, pp. 249–256). Ageorges, A., Cheynier, V., & Terrier, N. (2014). Polyphenols. In Fruit Ripening: Physiology, Signalling and Genomics (pp. 151–177). France. Ali, K., Maltese, F., Choi, Y. H., & Verpoorte, R. (2010). Metabolic constituents of grapevine and grape-derived products. Phytochemistry Reviews , 9 (3), 357–378. Aschan, G., & Pfanz, H. (2003). Non-foliar photosynthesis – a strategy of additional carbon acquisition. Flora , 198 (2), 81–97. Azuma, A., Yakushiji, H., Koshita, Y., & Kobayashi, S. (2012). Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions. Planta , 236 (4), 1067–1080. Baker, N. R. (2008). Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annual Review of Plant Biology , 59 , 89–113. Baur, J. A., & Sinclair, D. A. (2006). Therapeutic potential of resveratrol: the in vivo evidence. Nature Reviews Drug Discovery , 5 (6), 493–506. Bavaresco, L., Fregoni, C., Van Zeller De Macedo Basto Gonçalves, M. I., & Vezzulli, S. (2009). Physiology & molecular biology of grapevine stilbenes: An update. In Grapevine Molecular Physiology and Biotechnology: Second Edition (pp. 341–364). Baxter, I. R., Young, J. C., Armstrong, G., Foster, N., Bogenschutz, N., Cordova, T., Harper, J. F. (2005). A plasma membrane H+ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana, 102 (15). Baydar, N. G., & Akkurt, M. (2001). Oil Content and Oil Quality Properties of Some Grape Seeds. Turkish Journal of Agriculture and Forestry , 25 (3), 163–168. Retrieved from http://journals.tubitak.gov.tr/agriculture/issues/tar-01-25-3/tar-25-3-3-9909-25.pdf Baydar, N. G., Özkan, G., & Sema Çetin, E. (2007). Characterization of grape seed and pomace oil extracts. Grasas y Aceites , 58 (1), 29–33. Blancquaert, E. H., Oberholster, A., Ricardo-da-Silva, J. M., & Deloire, A. J. (2019). Effects of abiotic factors on phenolic compounds in the grape berry - A review. South African Journal of Enology and Viticulture , 40 (1), 1–14. Bogs, J, Jaffe, F. W., Takos, A. M., Walker, A. R., & Robinson, S. P. (2007). The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol , 143 (3), 1347– Chapter 1 - General Introduction __________________________________________________________________________________ 39 1361. Bogs, Jochen, Downey, M. O., Harvey, J. S., Ashton, A. R., Tanner, G. J., & Robinson, S. P. (2005). Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiology , 139 (2), 652– 63. Boss, P., & Davies, C. (2001). Molecular biology of anthocyanin accumulation in grape berries. In R.-A. KA (Ed.), Molecular Biology and Biotechnology of the Grapevine (pp. 1–33). Dordrecht, The Netherlands: Kluwer Academic. Boss, P. K., K, P., Robinson, S. P., Davies, C., Robinson, S. P., Osmond, G., & Scientific, C. (1996). Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape berries and the implications for pathway regulation. Plant Physiology , 111 (1 996), 1059–1066. Brazel, A. J., & Ó’Maoileídigh, D. S. (2019). Photosynthetic activity of reproductive organs. Journal of Experimental Botany , 70 (6), 1737–1753. Breia, R., Vieira, S., Da Silva, J. M., Gerós, H., & Cunha, A. (2013). Mapping grape berry photosynthesis by chlorophyll fluorescence imaging: The effect of saturating pulse intensity in different tissues. Photochemistry and Photobiology , 89 (3), 579–585. Brito, C., Dinis, L. T., Moutinho-Pereira, J., & Correia, C. (2019). Kaolin, an emerging tool to alleviate the effects of abiotic stresses on crop performance. Scientia Horticulturae , 250 (November 2018), 310–316. Caffarra, A., Rinaldi, M., Eccel, E., Rossi, V., & Pertot, I. (2012). Modelling the impact of climate change on the interaction between grapevine and its pests and pathogens: European grapevine moth and powdery mildew. Agriculture, Ecosystems and Environment , 148 , 89–101. Cahoon, E. B., Hall, S. E., Ripp, K. G., Ganzke, T. S., Hitz, W. D., & Coughlan, S. J. (2003). Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nature Biotechnology , 21 (9), 1082–1087. Caimi, G., Carollo, C., & Presti, R. Lo. (2003). Diabetes mellitus: oxidative stress and wine. Current Medical Research and Opinion , 19 (7), 581–586. Caravia, L., Collins, C., Petrie, P. R., & Tyerman, S. D. (2016). Application of shade treatments during Shiraz berry ripening to reduce the impact of high temperature. Australian Journal of Grape and Wine Research , 22 (3), 422–437. Castellarin, S. D., Bavaresco, L., Falginella, L., Gonçalves, M. I. V. Z., & Di Gaspero, G. (2012). Phenolics in Grape Berry and Key Antioxidants | BenthamScience. In The Biochemistry of the Grape Berry (pp. 89–110). Castellarin, S. D., Matthews, M. A., Di Gaspero, G., & Gambetta, G. A. (2007a). Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries. Planta , 227 (1), 101–112. Castellarin, S. D., Pfeiffer, A., Sivilotti, P., Degan, M., Peterlunger, E., & Di Gaspero, G. (2007b). Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit. Plant, Cell and Environment , 30 (11), 1381–1399. Chong, J., Poutaraud, A., & Hugueney, P. (2009). Metabolism and roles of stilbenes in plants. Plant Science , 177 (3), 143–155. Cipollini, M. L., & Levey, D. J. (1991). Why some fruits are green when they are ripe: carbon balance in fleshy fruits. Oecologia , 88 (3), 371–377. Cocaliadis, M. F., Fernández-Muñoz, R., Pons, C., Orzaez, D., & Granell, A. (2014). Increasing tomato fruit quality by enhancing fruit chloroplast function. A double-edged sword? Journal of Experimental Botany , 65 (16), 4589–4598. Conde, A., Pimentel, D., Neves, A., Dinis, L.-T., Bernardo, S., Correia, C. M., Moutinho-Pereira, J. (2016). Kaolin foliar application has a stimulatory effect on phenylpropanoid and flavonoid pathways in grape berries. Frontiers in Plant Science , 7 , 1–14. Conde, Artur, Neves, A., Breia, R., Pimentel, D., Dinis, L. T., Bernardo, S., Moutinho-Pereira, J. (2018). Kaolin particle film application stimulates photoassimilate synthesis and modifies the primary metabolome of grape leaves. Journal of Plant Physiology , 223 , 47–56. Conde, C., Agasse, A., Glissant, D., Tavares, R., Hernâni, G., & Delrot, S. (2006). Pathways of Glucose Regulation of Monosaccharide Transport in Grape Cells. Plant Physiology , 141 (4), 1563–1577. Conde, C., Silva, P., Fontes, N., Dias, A. C. P., Tavares, R. M., Sousa, M. J., Gerós, H. (2007). Biochemical changes Chapter 2 Fruit Photosynthesis: where, how and why? The information presented in this Chapter 2 is being prepared for publication: Garrido, A., Conde A., De Vos, R. C. H. and Cunha, A. Fruit Photosynthesis: where, how and why? Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 47 Abstract Photosynthesis is a key physiological process for life on Earth. In addition to leaves, other plant organs, such as fruits and particularly their seeds can exhibit photosynthetic activity. There are several fruit-related aspects that influence fruit photosynthesis like anatomical and physiological characteristics, fruit developmental stage, but also the microenvironment where the fruits grow in the plant canopy - their microclimate. The main objective of this review was not only to compile the most recent information about these aspects, but also to address the challenging biological questions: why a sink organ has photosynthetic activity and what may be the main function(s) of fruit photosynthesis? Keywords: fruit characteristics, photosynthetic activity, fruit tissues, roles and functions. 2.1. Introduction Fruit is an important product derived from agriculture and its consumption is indispensable for the human diet. In addition to vitamins, inorganic minerals and fibers, the antioxidant capacity of several phytonutrients present in the fruits have beneficial effects in health, such as, to reduce the risk of a wide range of cancers, cardiovascular diseases and other diet-related diseases (Rodriguez-Casado, 2016). Due to the high economic value of fruits – a perishable staple – several studies have been carried out at the biochemical, physiological and molecular levels, namely to improve its nutritional quality and shelf life (Nath et al., 2014). Evolutionary pressures have resulted in a diversity of fruits, ranging from small dry seed capsules that burst to allow seed dispersal, to relatively large complex fleshy fruits, that have evolved bright colors and complex aromas to attract seed-dispersing birds and animals (Lorts et al., 2008). That diversity can be organized under the following dichotomies: and fleshy or dry (without/with a soft succulent pericarp), and the dry fruits in dehiscent or indehiscent (open/not open to discharge seeds). For instance, capsules, siliques and legumes are dehiscent and dry; achenes, nuts and caryopsis of cereal grains are indehiscent and dry; drupes, pomes and berries are indehiscent and fleshy. Regarding to the type of fruit ripening, the classification in climacteric and non-climacteric is based on ethylene production and respiration rate (Kou and Wu, 2018). The onset of ripening in climacteric fruits is characterized by an increase in respiration with a simultaneous and a well-characterized peak of ethylene production (e.g., apple - Busatto et al., 2017; and tomato - Alexander and Grierson, 2002), while in non-climacteric fruits the ripening process occurs without sudden changes (e.g., strawberry - Symons et al., 2012; and grape - Chervin et al., 2004). Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 48 Photosynthesis depends essentially on the irradiance, on diffusive resistance to CO2 - from the atmosphere to the sites of assimilation in the chloroplasts - and from the surface containing chlorophyll. In plants, it occurs predominantly in green leaves, which are the primary sources of photoassimilates to the whole plant. It was demonstrated, however, that throughout the life cycle of the higher plants, other vegetative and reproductive structures can be photosynthetically active, such as fruits, green stems, green flower organs and even roots (as reviewed by Aschan and Pfanz, 2003; Brazel and Ó’Maoileídigh, 2019) (Table 2.1). The photosynthetic activity of these organs and structures may be seasonal, expressed at specific developmental stages. Table 2.1. Net photosynthetic (PN) rates in different species and plant structures. Species Structure P N (µmol CO2 m-2 s-1) References Fruit Cucumis sativus L. Cucumber 2.1 – 2.4 Sui et al. (2017) Helleborus viridis L. agg. - 0.1 Aschan et al. (2005) Olea europaea L. (cv. Leccino) Olive approx. 9a Proietti et al. (1999) Ficus carica L. Figs 18.2 (cv. Kalamon) 12.9 (cv. Fracasana and Mission Vemmos et al., (2013) Fragaria L. Strawberry 1-4 Blanke (2002) Floral Parts Helleborus viridis L. agg. Sepals 2.3 Aschan et al. (2005) Lilium hybrid L. (cv. Enchantment) Anther 2.3 Clément et al. (1997a, b) Tepals 1.8 Spiranthes cernua L. Flower 2.5 Bud 3.7 Antlfinger and Wendel (1997) Inflorescence 0.2 Caesalpinia virgata Torr. Stem 7.8 Senna armafa L. 5.8 Nilsen and Sharifi (1994) Alessio et al. (2005) Prunus persica L. 0.4 - 1.0 Spartium junceum L. approx. 8 Nilsen et al. (1993) Vitis vinifera L. approx. 0.8 (Fv/Fm values) Tikhonov et al. (2017) Roots Sonneratia alba Sm. Pneumatophores 0.6 Kitaya et al. (2002) Avicennia marina (Forssk.) 0.2 Tecticornia pergranulata (J.M.Black) K.A.Sheph. & Paul G.Wilson Aquatic adventitious 0.5 Rich et al. (2008) a Measured as gross photosynthetic rate. Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 49 Concerning to fruit photosynthesis, some early studies were reviewed by Blanke and Lenz (1989) in climacteric apple fruit, non-climacteric grape berry (both fleshy fruits), indehiscent fruits (e.g., cereal grain) and dehiscent fruits (e.g., pea pod). They reported that the photosynthetic profile of each type of fruit was dependent on some morphological and anatomical characteristics, as for instance, pea pods have much less resistance to CO2 diffusion than fleshy fruits (Blanke and Lenz, 1989). There are still few works about this research topic, many using different experimental approaches. In addition, relevant lateral information is dispersed in the literature and should be recruited for a better understanding of the drivers and constraints for fruit photosynthesis to occur. Therefore, this review aims to compile and combine information focusing on characteristics of fruits, including, anatomical, physiological and biochemical, as well as raising discussion on the possible functions of photosynthesis on fruit metabolism and development. 2.2. Anatomical and physiological characteristics of fruits 2.2.1. Cuticular structure Fleshy fruits are covered by an outer epidermis coated with a cuticle of variable thickness, which is composed by cutin and impregnated with waxy or greasy layers (Lara et al., 2015). During the development of fleshy fruits, the biosynthesis of cuticular wax is regulated by environmental factors like drought/humidity, light, temperature and pathogens (as reviewed by Trivedi et al., 2019). As in leaves, fruit cuticle provides a waterproof barrier between the epidermal cells and the relatively dry environment (Zarrouk et al., 2018). In addition, during fruit growth and development, the cuticle maintains its integrity with increasing volume and turgor pressure and also plays a central role in protecting the fruit against biotic stresses (e.g., insects and fungi) and abiotic stresses (e.g., UV radiation) (Lara et al., 2015). Several studies have been carried out with the aim of understanding cuticle formation in fruits, including apple (Albert et al., 2013), grape (Becker and Knoche, 2012) and tomato (Segado et al., 2016). In general, with the growth of fruits, there is a rapid accumulation of wax in the cuticle, which makes it thicker and hinders the diffusion of gases (Blanke and Lenz, 1989). For instance, the cuticle of Riesling grape berries is present at early stages, with an increasing rate of cutin and wax deposition at prevéraison , but after that, with the very rapid expansion of fruit surface area, the cuticular material flattens out (Casado and Heredia, 2001). In oranges from later developmental stages, the genes involved in the biosynthesis of wax, cutin and lignin were significantly induced, while genes involved in photosynthesis were repressed (Wang et al., 2016). In pepper fruit, it was verified that cuticle removal increased the gas permeability of Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 50 the surface (Banks and Nicholson, 2000), suggesting that this structure is crucial in determining compositional differences between the external and internal atmosphere. The pea pod ( Pisum sativum L.) have two distinct photosynthetic layers, the outer (exocarp) and the inner (endocarp) epidermis, containing a thick and thin cuticle, respectively, the first allowing a controlled CO2 diffusion from the outside atmosphere by stomata and the second allowing the photoassimilation of CO2 diffusing from the inner fruit cavity, and that is mainly released from seeds respiration process (Atkins et al., 1977). 2.2.2. Stomata frequency and functionality Although stomata are present in the outer epidermal layers of fruits, its density is 10 to 100 times lower than in the abaxial epidermis of the respective leaves (Aschan and Pfanz, 2003). Despite this, in young fruits stomata are as sensitive as in leaves and regulate the rate of CO2 exchange to a certain extent. However, with fruit growth the surface expands and the frequency of stomata decreases, after which lenticels (small, round or elliptical, pore-like structures that can be derived from nonfunctional stomata) dominate the diffusive resistance to CO2 (Blanke and Lenz, 1989). In grape berries, there are functional stomata till véraison , but after that the frequency decreases to less than one stoma per mm2 (Blanke and Leyhe, 1987) and they become nonfunctional (lenticel covered by wax), causing a decrease in the transpiration rate and loss of water, but also higher CO2 and lower O2 concentrations in the berry core (Palliotti and Cartechini, 2001). Similarly, in Citrus unshiu about 300 stomata mm-2 are present on fruit surface during early stages (∼10-30 mm diameter fruit) collapsing steadily thereafter (Hiratsuka et al., 2015). In cucumber fruit, the stomatal frequency is only 1.58 % and 0.91 % of the upper and lower surfaces of leaves, respectively (Sui et al., 2017). Currant ( Ribes species) varieties exhibited between 4 and 18 stomata per single fruit, corresponding to 0.2–0.3 stomata mm-2 (Blanke, 1993). Stomata on a ripe apple are 30 times scarcer than on the abaxial surface of the respective leaf (Blanke and Lenz, 1989). In avocado fruit, the stomata are present in large number, that is, 20,000 to 30,000 per fruit, which corresponds between 50 to 75 stomata mm-2, value that decrease with fruit expansion during ontogeny (Blanke, 1992). Equally, the number of stomata per strawberry decreased with growth and surface expansion from 6 stomata mm-2 to 1-3 mm-2 (Blanke, 2002). The high stomata density on the surface of young peach fruits leads to a high conductance, but at maturity stomata lose their function and differentiate into lenticels (de Oliveira Lino et al., 2016). Also, stomata are absent from the inner and outer epidermis of mature chili pepper fruits, and the gas exchange occurs only through Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 51 the cuticle (Blanke and Holthe, 1997). In mature olive fruit, scanning electron micrographs demonstrated that the stomata are covered by wax of a complex architecture (Proietti et al., 1999). Stomata were also observed in the external surface of the pods, but again were fewer in number when comparing to leaves. For instance, the chickpea external pod surface had a stomatal density of 31±3 mm-2 compared to 126±6 mm-2 in leaves (Ma et al., 2001). The same study demonstrated that the higher rates of transpiration and the poorer water use efficiencies in old compared to young pods was due to the increased leakiness of stomata with age. The inner epidermis of endocarp of pea pods have thin cuticles and no stomata, while the outer epidermis, with thick cuticles, present stomata with a density approximately 25 % of the leaflet, suggesting its importance for the atmospheric CO2 uptake (Atkins et al., 1977), and regulation of water losses. In soybean pods, the stomata were present at early stages of growth, being open and able for gas exchange (Andrews and Svec, 1975). Similarly, the stomata of Brassica pods were more functional at initial developmental stages with stomatal conductance reaching a maximum 30 days after anthesis and decreasing during the later phases (Singal et al., 1995). The grain of cereals has a green pericarp where stomata are occasionally present in the external surface. In fact, there are contradictory data concerning this issue. Cochrane and Duffus (1979) reported very few stomata in wheat pericarp, and probably insufficient for gas exchange, while in another study, Tambussi et al. (2007) did not find stomata in the wheat pericarp. These latter authors suggested that the photosynthetic activity of the green pericarp is dependent of CO2 internally generated by the respiration of endosperm cells. 2.2.3. Light diffusion inside the fruits The morphology and anatomy of fleshy fruits (e.g., large volumetry) impose physical constraints to light penetration into the inner tissues, eventually reducing the photic zone to the outermost layers (Breia et al., 2013). Besides intensity, the quality of light reaching the inner regions is influenced by the cells of the outer pericarp. The presence of chlorophylls in green fruit can strongly influence the spectral composition of the light filtered through the fruit pericarp, as reviewed by Llorente et al. (2016). The photon flux density (PFD) transmission through the skin ranges from 1 to 47 % of the incident PFD and, generally, only 2 % reach the internal regions (Aschan and Pfanz, 2003). For instance, the exocarp of grape berry transmitted 47.1 % of incident PFD and of apple Golden Delicious about 31.4 % (Aschan and Pfanz, 2003). In Citrus unshiu , photosynthesis was greater in fruits than in leaves under considerably low PFD (13.5 to 68 µmol m-2 s-1) (Hiratsuka et al., 2015). Green peel avocado fruit transmitted only 1.5 % of the incident light at 660 nm, but increase to 8.4 % at 730 nm (Blanke, 1992), Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 52 consistent with a selection of the spectrum by chlorophylls present in the peel. In inner tomato tissues, such as the locules, with low access to light, a high expression of genes associated with photosynthesis was observed (Lemaire-Chamley et al., 2005). Under a constant photosynthetically active radiation (PAR) of 1750 µmol m-2 s-1, the peel of apple fruit transmitted 1-3 % of incident PAR at 400 nm, increasing to 10-12 % at 850 nm (Blanke and Notton, 1992), and with fruit development this light transmission into the fruit core decreased by up to 80 %. Similarly, in olive fruits, PAR diffusion into internal tissue layers is very problematic, due to the increase in volumetry (Proietti et al., 1999). Chen and Cheng (2007) observed that sun-exposed peel of apple fruit had higher photosynthetic O2 evolution capacities, as well as higher activities of enzymes from Calvin-Benson cycle. Additionally, in exocarp of cucumber fruit it was noted the expression of other key genes involved in the photochemical phase, for example, associated with light-harvesting complexes (Lhca) of photosystems I (PSI) and light-harvesting proteins (Lhcb) of photosystems II (PSII) (Sui et al., 2017). In the same fruit the decreased of PAR from 200 to 50 µmol m2 s-1 led to a reduction of photosynthetic rate by 60-65 % (Marcelis and Hofman-Eijer, 1995). In pea pods, up to 27 days after anthesis, under an incident PFD of 2200 µmol m-2 s-1, the exocarp and mesocarp (pericarp) absorbed around 67 %, the endocarp received 10 % and the remaining 23 % was transmitted to seeds in the pod cavity (Atkins et al., 1977). In later stages of pea pod development, a decline in chlorophyll content in outer layers allows an increase in PAR reaching the endocarp and seeds. In this way, there is a temporal separation in the loss of photosynthetic capacity between pods and seeds. Developing soybean embryos receive moderate levels of light (5–30 µmol m-2 s-1), but the amount of light transmitted by seed coat to the embryo is high (approx. 15 %), which influences seed photosynthesis (Allen et al., 2009). Similarly, in pod of chickpea, the seed coat light transmission increased with development leading to a greater light utilization by the embryo (Furbank et al., 2004). The complex structure of the ear in C3 cereals, implies difficulties in transmission of light into the grains. In fact, the grain is surrounded by the lemma and palea, and it is shaded by the glume, which imposes low PFD levels reaching the green pericarp and endosperm (Tambussi et al., 2007). 2.2.4. Chloroplasts and photosynthetic pigments Microscopic observations revealed the presence of chloroplasts in different fruits, as reviewed by Blanke and Lenz (1989). In fruit tissues, the density of chloroplasts is much lower as compared to the leaves, and thus the photosynthetic rate per unit of area (or per fresh or dry weight) is reduced (Aschan and Pfanz, 2003). For instance, strawberry contains 0.2-0.6 mg chlorophyll g-1 fresh weight, i.e., 7-fold less chlorophyll than in the respective leaves (Blanke, 2002). But, differently from the leaves, where most Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 53 of the organ consist of chlorophyllin cells, generally in fruits only specific tissues or cells are chlorophyllin (e.g., Breia et al., 2013). In apple, for instance, chloroplasts can be only found at the hypodermal and inner perivascular tissue (Phan, 1973). Also, chloroplasts from different tissues may differ in structure, composition and function. Resorting to the apple example, in the hypodermal layers the chloroplasts are smaller than those from the inner tissue, exhibit grana throughout fruit development and contain starch granules, being more closely related with those found in leaves and perform photosynthesis-like C3 pathway (Phan, 1973). On the other hand, chloroplasts from perivascular tissue are larger and in a relatively small number compared with the respective leaf and with apparent absence of starch grains, being characterized by a C4-type photosynthesis (Phan, 1973). In the case of cucumber, the chloroplasts (with grana stacks 1.7-fold larger than in leaves) appear in inner walls of fleshy parenchyma cells, but its quantity per unit area is lower than in leaves (Sui et al., 2017). Avocado fruit contains sun-type chloroplasts that retain its structural integrity until the harvest. They are comprised by grana (with few thylakoids), with starch and lower chlorophyll content than the respective leaves on a per surface area basis (Blanke, 1992). The pericarp of grape berries, at day zero after anthesis, have small plastids with their loosely arranged inter-granal lamellae and granal thylakoids, and with abundance of starch grains, and for that they can be regarded as amyloplasts (Hardie et al., 1996). During the post-anthesis period (i.e., between day 0 and approx. until day 42 after anthesis), it was observed an increase in chlorophyll content, as well as in plastid density within the pericarp tissues, as a result of the preceding period of cell division and enlargement of the fruit (Hardie et al., 1996). Thereafter, plastids acquire a larger pleomorphic form, being largely devoid of starch granules until the last stages of ripening, but contain large lipid-like globules (Hardie et al., 1996). In the same study, it was suggested that the plastids of the grape pericarp play a central role in the isoprenoid synthesis, such as monoterpenes, and thus in grape and wine flavor and aroma. Chlorophyll fluorescence measurements showed that fruits have its photochemical machinery activated by the energy received from light – e.g., mango (Hetherington, 1997), lemon (Nedbal et al., 2000), tomato, (Carrara et al., 2001), papaya (Bron et al., 2004), eggplant (Calvo et al., 2017) and grape berry (Breia et al., 2013; Garrido et al., 2018; Garrido et al., 2019). In fact, the light growing conditions of fruits is a determinant factor affecting pigment concentration, the photosynthetic rate and the level of carbohydrates, as demonstrated for Nules Clementine mandarin fruit, that when growing inside the canopy had lower chlorophyll and carotenoid contents and lower carbohydrate levels (Cronje et al., 2013). Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 54 Similarly, in eggplant fruit, it was verified that different environmental light conditions influenced the fluorescence ratio of each photosystem (Calvo et al., 2017). Throughout fruit development and maturation, the granal structure of the internal chloroplasts disintegrates and the chlorophyll content decreases as result of its substitution by other pigments, like carotenoids, or due to the activity of chlorophyllases (Hörtensteiner and Kräutler, 2011; reviewed by Seifert et al., 2014). For instance, in tomatoes there is fragmentation of the thylakoid membrane of the green chloroplasts and formation of colored chromoplasts with new carotenoid-bearing structures (Egea et al., 2011), as detected by confocal laser scanning microscopy (D’Andrea et al., 2014). The decrease in chlorophyll content during ripening, was observed in several fruits, such as papaya (Sanxter et al., 1992), olive (Proietti et al., 1999), tomato (Kozukue and Friedman, 2003), mandarin (Cronje et al., 2013), apple (Nagy et al., 2016) and grape berry (Garrido et al., 2018; Garrido et al., 2019). In grape berry tissues, while in seed integuments chlorophylls content decreased from 80 to 40 μg g-1 fresh weight, in exocarps it decreased from 120 to 20 μg g-1 fresh weight (Garrido et al., 2018). Our results of transcriptional analysis in exocarp and seed showed, however, that the Chlorophyll Synthase gene ( VvChlSyn ) keeps its relative expression of transcripts until later stages of development (Garrido et al., submitted). The regulation of carotenoids in fruits can be rather complex due to the dramatic changes in content and composition during ripening, which are also dependent on the fruit tissue and the developmental stage. Recently, Lado et al. (2016) provided a comprehensive overview concerning the main carotenoid profiles in fleshy fruits and pattern of changes during ripening and of the different regulatory levels responsible for the diversity of carotenoid accumulation in fruit tissues. In grape berries, β-carotene and lutein are the predominant carotenoids, and there is a steady decline after véraison , which appears to be related to chloroplast disappearance and to the formation of carotenoid-derived norisoprenoid volatiles (β-ionone and β-damascenone) (Crupi et al., 2010; Young et al., 2012; Joubert et al., 2016), all important for wine aroma, because of their low olfactory perception threshold (MendesPinto, 2009). In agreement with this, in our previous work the total carotenoids content (i.e., the sum values of α-, βand lutein) decreased in grape berry exocarp and seed during development (Garrido et al., 2019). Pea pod walls also have chloroplasts in different cells layers. The exocarp outer epidermis contains few chloroplasts, but they are abundant in the inner epidermis of the endocarp (with smaller starch grains), as well as in the parenchyma layers of mesocarp (Atkins et al., 1977). In the case of chickpea pod walls, low levels of chlorophyll were localized at the embryo and evenly distributed throughout the Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 55 cotyledons, but the seed coat contained a layer of chloroplast-rich cells directly below the epidermis (Furbank et al., 2004). The variation of chlorophyll concentration in pods is similar to that of fleshy fruits, with a decrease through development, as demonstrated for soybean pods (Andrews and Svec, 1975). These pods had 9.2-12.4 times less chlorophyll per gram fresh weight than leaves. However, the gross photosynthetic rates in pods were greater than in leaves on a per mg chlorophyll basis, which may be important for the rapid seed filling period of pods (Andrews and Svec, 1975). The total chlorophyll content on a dry weight basis is lower on ear parts when compared with flag leaf (Lu and Lu, 2004). This differences in pigment composition can be explained by the distinct light environment between these two structures, being the ear localized at top of the canopy. In fact, the authors Lu and Lu (2004) verified that lumes, lemmas and awns of the wheat ears had a lower chlorophyll/carotenoid ratio, that reflect the required photoprotection. During the development of wheat grain, the maximum of chlorophyll content in the pericarp green layer was observed at 20 days after anthesis, when there was also a peak of photosynthetic activity (Caley et al., 1990). 2.2.5. Assimilation and refixation of internal CO2 Fruit gas exchange with the external atmosphere takes place mainly through stomata and depends on a diversity of morphological and physiological aspects, namely: fruit type and size, fruit ontogeny stage, fruit temperature, shading and incident PFD, and chlorophyll content (Wahid et al., 2005). For instance, Aschan and Pfanz (2003) mentioned that fleshy fruits perfom basically internal CO2 refixation, but dry fruits, during the younger stages, are able to assimilate atmospheric CO2. Besides that, the incident light also influences the rate of CO2 fixation, as demonstrated for mandarin fruit, in which the light-saturated net CO2 assimilation rate (Amax) of fruit in the outer canopy was significantly higher than in the inner canopy (Cronje et al., 2013). Overall, fruits assimilate less atmospheric CO2 via ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) when compared to the respective leaves (Blanke and Lenz, 1989), as observed in oranges (with 50 % to 75 % less) (Cronje et al., 2013). As exposed above, at early stages, the cuticular and stomata characteristics enable gas exchange in fruits. Then, during fruit ontogeny, the great accumulation of wax in its surface leads to a reduction of CO2 atmospheric exchange rate by 10-fold (Blanke and Lenz, 1989), resulting in an increase in internal CO2 concentration and a simultaneous reduction of water loss through transpiration. Moreover, the increase in mitochondrial respiratory processes, fueled predominantly by imported photoassimilates from leaves to these sink organs at this later stages, results in a rise of internal CO2 concentration (Blanke and Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 62 al., 2011; Wang et al., 2017), metabolomic (Dai et al., 2013; Degu et al., 2014; Wang et al., 2017), and of these data integration in network analysis for identification of putative stage-specific biomarkers (Serrano et al., 2017; Zamboni et al., 2010), as well as data from co-expression gene networks (Zou et al., 2019), confirmed the presence of components directly related with photosynthetic activity, namely before véraison (e.g., light harvesting complexes, photosystem II oxygen evolving complex, Calvin-Benson cycle enzymes). In particular, the down-regulation of photosynthetic genes after véraison was first analysed by expressed sequence tag (EST) profiling (Terrier et al., 2001; Silva et al., 2005) and confirmed by microarray analysis in grape berries (Terrier et al., 2005; Deluc et al., 2007; Pilati et al., 2007; Zamboni et al., 2010), and specifically in berry skins (Waters et al., 2005), wherein photosynthesis-related transcripts are more abundant (Grimplet et al., 2007). All those investigations support our results obtained by chlorophyll fluorescence analysis by imaging-PAM fluorometry (Breia et al., 2013; Garrido et al., 2018; Garrido et al., 2019). The Calvin-Benson cycle can supply precursors for pathways of primary and secondary metabolism (as reviewed for tomato by Cocaliadis et al., 2014). Secondary metabolites, like phenolic compounds, play important roles in plant defense against biotic and abiotic factors (Cheynier, 2012). Also, the diversity of compounds contributes for the nutritional quality of fruits and for its organoleptic properties, which are important to make them attractive and palatable. For instance, in grape berries, phenolics contribute to the color, taste, texture and astringency of the wine, as well as to its antioxidant properties and beneficial effects on health (Shrikhande, 2000; Weston, 2005). These compounds are mainly present in the exocarp and seeds of the grape berries (Garrido et al., 2021; Montealegre et al., 2006), both photosynthetically active (Breia et al., 2013; Garrido et al., 2018; Garrido et al., 2019). 2.4.2. The particular case of photosynthesis in seeds In addition to fruit tissues, some seeds can perform photosynthesis at least during part of their development period. Investigations have been developed to understand the specific functions of photosynthesis in these organs (Ruuska et al., 2004; Rolletschek et al., 2005b; Tschiersch et al., 2011; Galili et al., 2014). In general, these investigations showed that seed photosynthesis may contribute in three distinct ways: 1) supply of oxygen to prevent and/or reduce hypoxia; 2) production of nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP) both fundamental for energetically demanding biosynthetic pathways in the chloroplast, such as fatty acid synthesis; 3) provision of C-intermediates for primary and secondary metabolism by the Calvin-Benson cycle; and 4) re-fixation of respiratory CO2 by RuBisCO, what can improve the energy efficiency of seeds. Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 63 2.4.2.1. Possible functions of O2, ATP and NADPH from the photochemical phase Most seeds have some peculiarities that hinders the absorption of oxygen, namely a thick coat and the accumulation of proteins and oils that become a glassy matrix during desiccation (Buitink and Leprince, 2008). Thus, during the maturation and desiccation process there is a decrease in oxygen diffusion into the dense inner seed tissues. This situation of hypoxia causes restrictions to the production of ATP by mitochondria (oxidative phosphorylation), which is pivotal for various metabolic pathways during seed development and embryo maturation (van Dongen et al., 2004; Vigeolas et al., 2011, 2003). Hypoxia can directly or indirectly affect several other processes in seeds, such as, nutrient uptake (e.g., wheat – van Dongen et al., 2004), storage activity and metabolite distribution (e.g., soybean – Rolletschek et al., 2005b), assimilate partitioning between endosperm and embryo (e.g., maize – Rolletschek et al., 2005a), and enzymatic activities associated with lipid metabolism (e.g., rapeseed – Vigeolas et al., 2003). It is impelling to relate seed photosynthesis – generally, a hidden structure deep in the fruit volume –, the production of O2 in the photochemical phase and the benefits of avoiding hypoxia conditions inside of seeds. Monocotyledonous barley caryopsis ( Hordeum vulgare L.) has a green pericarp with chlorophyll (called chlorenchyma) (Figure 2.3A), where photosynthesis occurs in the mid-storage stage (Wobus et al., 2005). Chlorophyll fluorescence images of the effective quantum yield of PSII allowed to verify that the photosynthetic activity was restricted to the chlorenchymatic regions of the pericarp (Figure 2.3B) (Tschiersch et al., 2011). Besides that, the photosynthetic activity in these regions is responsible for the production of 3.5 µmol NADPH h-1 and 2.3 µmol ATP h-1, both important for storage (Tschiersch et al., 2011). The oxygen distribution (‘oxygen maps’) in barley allow to understand better the photosynthetic activity in its tissues (Figure 2.3C) (Rolletschek et al., 2004). In general, the pericarp exhibits high levels of oxygen, while at the central regions there is an oxygen deficiency. In the dark experiments, the oxygen levels decreased dramatically in the inner endosperm region and in the transfer cells (transport pathway of assimilates to endosperm) (Patrick and Offler, 2001). On the other hand, in the light assays, this reduction was not so great, suggesting that the oxygen released by photosynthesis may play an important role in nutrient transport to the endosperm. In addition, the expression of photosynthesis-related genes peaks before the assimilate storage phase (Sreenivasulu et al., 2004). Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 64 Figure 2.3. Representative oxygen maps and photosynthetic parameters of green seeds (A to C - barley; D and E - soybean; F - grape berry seeds). (A) Chlorophyll red auto-fluorescence in a barley seed cross-section performed by Confocal Laser Scanning Microscopy. (B) Image of effective quantum yield of PSII of a cross-section of barley caryopsis 12 days after pollination. Oxygen maps for barley seeds (C) and for soybean (D), measured in either light (red circles) or dark (black circles), throughout pericarp (pe) and endosperm (en) and seed coat (sc). (E) Image of the effective quantum yield of PSII measured in soybean. (F) Image of maximum fluorescence of a grape berry cross-section. Adapted from Borisjuk and Rolletschek (2009), Breia et al. (2013) and Tschiersch et al. (2011). Legume seeds, such as soybean [ Glycine max (L.) Merril], present an embryo that turns green at the early stages of development. Approximately only 10 % of incident light is available to embryo surface (Rolletschek et al., 2005b). However, soybean embryos exhibit specialized chloroplasts with high grana stacking. The oxygen maps of soybean (Figure 2.3D) shows that the seed coat presents high concentrations of oxygen (at early storage stage), but these values decrease to minimum levels within the endospermal liquid. Furthermore, and when measured in the dark, the oxygen concentration within embryo is much lower (2 µM) comparatively to that under light conditions (220 µM) (Figure 2.3D) (Rolletschek et al., 2005b). At late-storage stage, soybean embryos have less oxygen concentration and a lower variation (in the same conditions), suggesting a decline in the capacity to balance oxygen consumption with its supply (Rolletschek et al., 2005b). Pulse amplitude modulated (PAM) fluorescence analysis allowed to confirm this situation. In fact, the effective quantum yield of PSII showed a homogeneous pattern for small embryos at the early storage stage, but at the mid-/late storage stage Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 65 (Figure 2.3E), there was a gradient declining towards the interior of the embryo, suggesting a gradual loss of photosynthetic ability (Borisjuk et al., 2005). More recently, studies in grape berry using chlorophyll fluorescence imaging PAM demonstrate that, besides the exocarp, also the outer integument of the green seed had a very high fluorescence signal (Figure 2.3F) corresponding to high effective quantum efficiencies of PSII (Breia et al., 2013). Later on, it was shown that the seed integument had higher values for photochemical efficiency and capacity at green stages of development and then suffer a decrease in these parameters at later stages (Garrido et al., 2018; Garrido et al., 2019). Thus, and despite the increase in volume of the grape berry along developmental stages, seeds can receive diffuse transmitted light, as already reported by Aschan and Pfanz (2003), allowing photosynthetic activity at later stages even if at lower levels. This photosynthetic activity can provide the O2 necessary to avoid the hypoxia that exist in grape berries (Xiao et al., 2018). Additional research in developing seeds (soybean, rapeseed and oilseed rape) suggested that the photosynthetic activity can supply the energy (ATP) and reduction power (NADPH) necessary for lipid biosynthesis, storage metabolism and redox modulation of biosynthetic enzymes (Ruuska et al., 2004; Borisjuk et al., 2005; Goffman et al., 2005; Rolletschek et al., 2005b). 2.4.2.2. Intermediates from photosynthesis used on seeds’ metabolism and RuBisCO as a CO2 rescue mechanism Seeds have also high concentrations of internal CO2 (Goffman et al., 2004). During embryogenesis, seeds receive photoassimilates from the phloem that are used for the synthesis of reserves, being this metabolic pathway characterized by the conversion of sucrose to pyruvate, through glycolysis, which is then transformed by pyruvate dehydrogenase (PDH) in acetyl-CoA. This is the main precursor of fatty acid biosynthesis, which are then used towards triacylglycerides or triacylglycerols synthesis (TAG, or storage lipids or oils) (Schwender et al., 2004). This conversion of sugars results in the loss of carbon, in the form of CO2, for each acetyl-CoA unit produced. According to Ruuska et al. (2004), RuBisCO provides another route for fixation of CO2 released by the PDH. Corroborating this view, from a study with embryos of Brassica napus L. (oilseed rape), Schwender et al. (2004) described a new metabolic pathway, in which RuBisCO acts without the Calvin-Benson cycle, in a mechanism previously unknown (Figure 2.4). Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 66 Figure 2.4. Metabolic pathway of transformation of sugars into fatty acids and highlighting the possible contributes of photosynthesis. Abbreviations: Glc-6-P, glucose-6-phosphate; GAP, glyceraldehydes-3-phosphate; PEP, phosphoenolpyruvate; Fru-6P, fructose-6-phosphate; PGA, 3-phosphoglyceric acid; PDH, pyruvate dehydrogenase; AcCoA, acetyl-CoA; FAS, fatty acid synthesis; OPPP, oxidative pentose phosphate pathway; PRK, phosphoribulokinase; E4P, erythrose-4-phosphate; RuBP, ribulose-1,5-bisphosphate. TAGs, triacylglycerides. Adapted from Schwender et al. (2004), Ruuska et al. (2004) and Allen et al. (2009). This metabolic pathway involves three main steps. The first, is the conversion of hexoseand triosephosphates to ribulose-1,5-bisphosphate by the non-oxidative reactions of the oxidative pentose phosphate pathway (OPPP) together with phosphoribulokinase (PRK); secondly, the conversion of RuBP and CO2 (most of which is produced by pyruvate dehydrogenase - PDH) to 3-phosphoglyceric acid (PGA) by RuBisCO; and third, the metabolism of PGA to pyruvate and acetyl-CoA, and then to fatty acids. Therefore, this mechanism avoids loss of carbon by recycling internal CO2, as well as provides several intermediates of the Calvin-Benson cycle for distinct pathways of metabolism. Schwender et al. (2004) showed that this new pathway provides 20 % more acetyl-CoA for fatty acid synthesis and resulted in 40 % less loss of carbon as CO2, comparatively to glycolysis. Similarly, Allen et al. (2009) verified that in soybean embryos, RuBisCO re-fixed 11 % of the CO2 released by lipid synthesis and TCA cycle, and consequently the Calvin-Benson cycle contributed for the carbon economy. Moreover, in rapeseeds, at the early stage of oil accumulation, the fatty acids content was higher in seeds exposed to light when compared to those from dark conditions (Ruuska et al., 2004). Likewise, our recent results Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 67 showed that seeds from grape berries grown at two contrasting light microclimates at canopy, had distinct photosynthetic activity specially at green stage (Garrido et al., 2019), and also had different lipid profile (Garrido et al., manuscript in preparation). In addition, the relative expression of VvRuBisCO in those seeds was maintained in high levels during berry ripening, in values similar to than seen for exocarps (Garrido et al., submitted), suggesting that RuBisCO may have a function in re-assimilating the locally released CO2 in these photosynthetic grape berry tissues, and thus contributing with intermediates for several metabolomic pathways, like storage lipids. Additionally, intermediates of OPPP pathway, as erythrose-4-phosphate, together with PEP can be used for amino acid synthesis, which in turn can contribute as precursors for the shikimate pathway. This biosynthetic pathway is responsible for the production of phenylalanine, as well as other aromatic amino acids, such as tyrosine and tryptophan. Phenylalanine in the first substrate of a key secondary metabolic pathway, the phenylpropanoid pathway (Vogt, 2010). 2.4.3. Photosynthesis and the vascular system of fruits Until later stages of development, sugars are transported from source organs to the fruits, through the phloem. The unloading of these sugars can occur by symplastic or apoplastic pathways, being the latter a mechanism dependent of energy (Lemoine et al., 2013). The photochemical phase of photosynthesis can provide the energy (ATP) necessary for the apoplastic unloading (Keller, 2015), a energetically demanding process. Hibberd and Quick (2002) verified that the cells surrounding the peripheral vascular system of stems and petioles of tomato, have chlorophyll and photosynthetic activity (C4-type). Similar results were observed in young shoots and chlorenchyma of lignified shoots of grapevine (Tikhonov et al., 2017). Although it was refereed that, in grape berries, the unloading is predominantly symplastic in early stages of development, becoming the apoplastic pathway dominant with the onset of ripening (Zhang et al., 2006), our previous work in white grape berries tissues using the chlorophyll fluorescence imaging technique (Figure 2.3F), showed that there was a high concentration of chlorophyll/photochemical activity in perivascular cells (peripheral dorsal system) (Breia et al., 2013), consistent with a role of photosynthesis in the process. Another interesting putative role for photosynthesis in the vascular bundles was observed in cucumber fruit, where the PEPC is present (Sui et al., 2017). The refixation of respiratory CO2 by PEPC, followed by the synthesis of organic acids that can accumulate in the vacuole, can provide the turgor pressure necessary for cell expansion and fruit growth (Sui et al., 2017). The same was suggested for Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 68 tomato (Carrara et al., 2001; Guillet et al., 2002). In grape berry, immunohistochemical studies verified that PEPC is present in the vasculature, in the parenchyma cells of the pericarp and within the developing seeds, leading to the hypothesis that PEPC may play a role in the metabolism of the assimilates after their delivery to the fruit (Famiani et al., 2000), and also into the seed at the appropriate time during its development (Walker et al., 1999). 2.4.4. Ecological advantages of green fruits and seeds The advantages or “services” of fruit photosynthesis can also be examined from an ecological point of view, and we can start this topic by saying that a green-colored fruit, located among green leaves, does not facilitate zoochoric seed dispersal. In fact, fruit color influences their ability to be dispersed by animals, namely birds (Cazetta et al., 2009). In this manner, the ripening process composes the mutualistic relationship between fleshy-fruit plants and seed-disperser animals (Duan et al., 2014). Besides the visibility conditions and the visual aptitude of the receiver, the visual signal detectability is determined by its contrast against the background, that is, the conspicuousness of the signal (Cazetta et al., 2009). Young fruits are usually green, but upon ripening they range from red, blue, yellow and orange to green and brown. Indeed, chlorophyll degradation is accompanied by a conversion of chloroplasts into chromoplasts that progressively accumulate high levels of carotenoids. However, some fruits are green when they are ripe (“green-ripe” or chlorophyllous), for example, cucumber, kiwi, pea, pepper and green apple varieties (Cipollini and Levey, 1991). The dispersion of seeds of fleshy fruits is commonly done by animals (zoochory), that follow their visual and olfactory senses (Schaefer, 2011). For diurnal seed dispersers, such as birds, the visual stimuli are particularly important, whereas many of the nocturnal seed dispersers, such as bats and other mammals, rely to a large extent on olfactory stimuli. In case of the “green-ripe” fruits they are less conspicuous than fruits of other colors, being only dispersed by mammals, especially bats (Cipollini and Levey, 1991). In this way, the fruits that maintained green until maturation, tend to be dispersed by a limited variety of frugivores. Cipollini and Levey (1991) suggested that the ecological advantage of the “green-ripe” fruits consist in their ability to photosynthesize, reducing costs of production and enriching the pulp in nutrient rewards for frugivores. The same authors verified that at high light levels the greenripe fruits have a positive carbon balance, but at low light levels the high rates of respiration often result in net CO2 losses. These high respiration rates could be due to the maintenance of photosynthetic pigments and proteins, but also because these fruits are generally larger (Cipollini and Levey, 1991). The “green-ripe” fruits also present higher seed and pulp mass, fact that evidence an offer quantitatively larger Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 69 for dispersers. In addition, the presence of strong odors in these fruits, it is also indicative that some secondary metabolic pathways may be active, eventually fueled by photosynthesis. The photosynthetic tissues of the grape berries, that is, the exocarp and seed outer integument (Breia et al., 2013), contain high levels of tannins, especially accumulated at green stage, decreasing afterwards (Garrido et al., 2021). Moreover, the two distinct light microclimate at canopy (LL and HL, low and high light, respectively) also led to a differences in the total flavan-3-ols in the exocarp at green and mature stage (Garrido et al., 2021). From a sensory standpoint, these compounds are correlated with astringency and bitterness of the wine (Ma et al., 2014), and in terms of ecophysiological functions, they confer protection against fungal and bacterial pathogens, insect pests and larger herbivores (as reviewed by Barbehenn and Constabel, 2011). 2.5. Concluding remarks Fruits are vital organs in plant sexual reproduction and indispensable foods in our diet. Its quality depends on the physiological and biochemical mechanisms, that at the end contribute for the accumulation of several compounds. The main purpose of the present review was to compile and integrate information on anatomical, physiological and biochemical features and constraints of different types of fruits and of their seeds, in order to unveil potential functions of the photosynthesis performed by some of their green tissues. With this bibliographic research work, a diversity of photosynthetic mechanisms, or in some cases, the utilization of part of “old” photosynthetic routes in new solutions to meet tissue-specific demands or alleviate biochemical pressures were discussed. The relevance of photosynthesis in fruits is clearly supported by many findings. Overall, the evidences point to some roles and functions, such as: firstly, the importance of energy (ATP) and reducing power (NADPH), both produced during the photochemical phase, and which can be important for energy-dependent biochemical processes, like the unloading of sugars from the vascular system or even the synthesis of fatty acids; secondly, the production of oxygen that can prevent and/or reduce the hypoxia inside of seeds; thirdly, the re-fixation of respiratory CO2 by RuBisCO in the CalvinBenson cycle or by PEPC (C4-type photosynthesis); and finally, the carbon skeletons, derived from CalvinBenson cycle, that can fuel pathways of primary and secondary metabolism, and thus, contribute for the organoleptic properties of fruits. This is a fascinating topic studied for over 40 years and from very different perspectives, from the molecular to the ecological one. However, due to fruit photosynthesis’ complexity, in space (different tissues, compartments, fruit geometries), in time (variation associated with development) and in its Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 70 dependency on environmental factors, information is still lacking or conflicting. If using controlled simple systems is needed to better identify cause and effect mechanistic relationships, integrative global approaches like systems biology and omic networks are increasingly crucial. As an example of the first, in vitro cultures established from different tissues with distinct photosynthetic competences, would allow to study a more direct link between photosynthetic activity and specific changes at molecular and biochemical levels. Moreover, it is also important to understand the complexities of coordination between the environmental stresses, photosynthesis and stress responses, since it will represent key information for crops improvement in the context of the ongoing climatic changes. 2.6. References Albert, Z., Ivanics, B., Molnár, A., Miskó, A., Tóth, M., & Papp, I. (2013). Candidate genes of cuticle formation show characteristic expression in the fruit skin of apple. Plant Growth Regulation , 70 (1), 71–78. Alessio, G. A., Pietrini, F., Brilli, F., & Loreto, F. (2005). Characteristics of CO2 exchange between peach stems and the atmosphere. Functional Plant Biology , 32 (9), 787–795. Alexander, L., & Grierson, D. (2002). Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. Journal of Experimental Botany , 53 (377), 2039–2055. Allen, D. K., Ohlrogge, J. B., & Shachar-Hill, Y. (2009). The role of light in soybean seed filling metabolism. Plant Journal , 58 (2), 220–234. Andrews, A. K., & Svec, L. V. (1975). Photosynthetic activity of soybean pods at different growth stages compared to leaves. Canadian Journal of Plant Science , 55 (2), 501–505. Antlfinger, A. E., & Wendel, L. F. (1997). Reproductive effort and floral photosynthesis in Spiranthes cernua (Orchidaceae). American Journal of Botany , 84 (6), 769–780. Araus, J. L., Bort, J., Brown, R. H., Bassett, C. L., & Cortadellas, N. (1993). Immunocytochemical localization of phosphoenolpyruvate carboxylase and photosynthetic gas-exchange characteristics in ears of Triticum durum Desf. Planta , 191 (4), 507–514. Aschan, G., Pfanz, H., Vodnik, D., & Batič, F. (2005). Photosynthetic performance of vegetative and reproductive structures of green hellebore ( Helleborus viridis L. agg.). Photosynthetica , 43 (1), 55–64. Aschan, Guido, & Pfanz, H. (2003). Non-foliar photosynthesis – a strategy of additional carbon acquisition. Flora , 198 (2), 81–97. Atkins, C. A., Kuo, J., & Pate, J. S. (1977). Photosynthetic Pod Wall of Pea ( Pisum sativum L.): Distribution of Carbon Dioxide-fixing Enzymes in Relation to Pod Structure. Plant Physiology , 60 (5), 779–786. Banks, N. H., & Nicholson, S. E. (2000). Internal atmosphere composition and skin permeance to gases of pepper fruit. Postharvest Biology and Technology , 18 (1), 33–41. Barbehenn, R. V., & Peter Constabel, C. (2011). Tannins in plant-herbivore interactions. Phytochemistry , 72 (13), 1551–1565. Becker, T., & Knoche, M. (2012). Deposition, strain, and microcracking of the cuticle in developing “Riesling” grape berries. Vitis - Journal of Grapevine Research , 51 (1), 1–6. Beriashvili, T.V. and Beriashvili, L. (1996). Metabolism of malic and tartaric acids in grape berries. Biochemistry , 61 , 1316–1321. Birkhold, K. T., Koch, K. E., & Darnell, R. L. (1992). Carbon and Nitrogen Economy of Developing Rabbiteye Blueberry Fruit. Journal of the American Society for Horticultural Science , 117 (1), 139–145. Blanke, M. (2002). Photosynthesis of strawberry fruit. IV International Strawberry Symposium , 567 (567), 373– 376. Blanke, M M. (1993). Stomata of Currant Fruits. Angewandte Botanik , 67 (1–2), 1–2. Blanke, M M, & Lenz, F. (1989). Fruit photosynthesis. Plant, Cell and Environment , 12 , 31–46. Blanke, M. (1998). Fruit photosynthesis and pome fruit quality. In Acta Horticulturae (Vol. 466, pp. 19–22). Chapter 2 - Fruit Photosynthesis: where, how and why? __________________________________________________________________________________ 71 Blanke, Michael M., & Holthe, P. A. (1997). Bioenergetics, maintenance respiration and transpiration of pepper fruits. Journal of Plant Physiology , 150 (3), 247–250. Blanke, Michael M., & Leyhe, A. (1987). Stomatal Activity of the Grape Berry cv. Riesling, Müller-Thurgau and Ehrenfelser. Journal of Plant Physiology , 127 (5), 451–460. Blanke, Michael M., & Notton, B. A. (1992). Light transmission into apple fruit and leaves. Scientia Horticulturae , 51 (1–2), 43–53. Blanke, Michael M. (1992). Photosynthesis of Avocado Fruit. In World Avocado Congress (pp. 179–189). Borisjuk, L., Nguyen, T. H., Neuberger, T., Rutten, T., Tschiersch, H., Claus, B., Rolletschek, H. (2005). Gradients of lipid storage, photosynthesis and plastid differentiation in developing soybean seeds. New Phytologist , 167 (3), 761–776. Borisjuk, L., & Rolletschek, H. (2009). The oxygen status of the developing seed. New Phytologist , 182 (1), 17–30. Bort, J., Brown, R. H., & Araus, J. L. (1996). Refixation of respiratory CO2 in the ears of C3 cereals. Journal of Experimental Botany , 47 (303), 1567–1575. Brazel, A. J., & Ó’Maoileídigh, D. S. (2019). Photosynthetic activity of reproductive organs. Journal of Experimental Botany , 70 (6), 1737–1753. Breia, R., Vieira, S., Da Silva, J. M., Gerós, H., & Cunha, A. (2013). Mapping grape berry photosynthesis by chlorophyll fluorescence imaging: The effect of saturating pulse intensity in different tissues. Photochemistry and Photobiology , 89 (3), 579–585. Bron, I. U., Ribeiro, R. V., Azzolini, M., Jacomino, A. P., & Machado, E. C. (2004). Chlorophyll fluorescence as a tool to evaluate the ripening of “Golden” papaya fruit. Postharvest Biology and Technology , 33 (2), 163–173. Buitink, J., & Leprince, O. (2008). Intracellular glasses and seed survival in the dry state. Comptes Rendus - Biologies , 331 (10), 788–795. Busatto, N., Tadiello, A., Trainotti, L., & Costa, F. (2017). Climacteric ripening of apple fruit is regulated by transcriptional circuits stimulated by cross-talks between ethylene and auxin. Plant Signaling and Behavior , 12 (1), e1268312. Caley, C. Y., Duffus, C. M., & Jeffcoat, B. (1990). Photosynthesis in the Pericarp of Developing Wheat Grains. Journal of Experimental Botany , 41 (3), 303–307. Calvo, B. O., Parapugna, T. L., & Lagorio, M. G. (2017). Variability in chlorophyll fluorescence spectra of eggplant fruit grown under different light environments: a case study. Photochem. Photobiol. Sci. , 16 (5), 711–720. Carrara, S., Pardossi, A., Soldatini, G. F., Tognoni, F., & Guidi, L. (2001). Photosynthetic activity of ripening tomato fruit. Photosynthetica , 39 (1), 75–78. Carrari, F., Baxter, C., Usadel, B., Urbanczyk-Wochniak, E., Zanor, M.-I., Nunes-Nesi, A.,Fernie, A. R. (2006). Integrated Analysis of Metabolite and Transcript Levels Reveals the Metabolic Shifts That Underlie Tomato Fruit Development and Highlight Regulatory Aspects of Metabolic Network Behavior. Plant physiology . Casado, C. G., & Heredia, A. (2001). Ultrastructure of the cuticle during growth of the grape berry ( Vitis vinifera ). Physiologia Plantarum , 111 (2), 220–224. Cazetta, E., Schaefer, H. M., & Galetti, M. (2009). Why are fruits colorful? The relative importance of achromatic and chromatic contrasts for detection by birds. Evolutionary Ecology , 23 (2), 233–244. Chen, L. S., & Cheng, L. (2007). The sun-exposed peel of apple fruit has a higher photosynthetic capacity than the shaded peel. Functional Plant Biology , 34 (11), 1038–1048. Chervin, C., El-Kereamy, A., Roustan, J. P., Latché, A., Lamon, J., & Bouzayen, M. (2004). Ethylene seems required for the berry development and ripening in grape, a non-climacteric fruit. Plant Science , 167 (6), 1301–1305. Cheynier, V. (2012). Phenolic compounds: From plants to foods. Phytochemistry Reviews , 11 (2–3), 153–177. Cipollini, M. L., & Levey, D. J. (1991). Why some fruits are green when they are ripe: carbon balance in fleshy fruits. Oecologia , 88 (3), 371–377. Clément, C., Mischler, P., Burrus, M., & Audran, J.-C. (1997a). Characteristics of the Photosynthetic Apparatus and CO2 -Fixation in the Flower Bud of Lilium. I. Corolla. International Journal of Plant Sciences , 158 (6), 794– 800. Clément, C., Mischler, P., Burrus, M., & Audran, J.-C. (1997b). Characteristics of the Photosynthetic Apparatus and CO2 -Fixation in the Flower Bud of Lilium. II. Anther. International Journal of Plant Sciences , 158 (6), 801–810. Cocaliadis, M. F., Fernández-Muñoz, R., Pons, C., Orzaez, D., & Granell, A. (2014). Increasing tomato fruit quality by enhancing fruit chloroplast function. A double-edged sword? Journal of Experimental Botany , 65 (16), Chapter 3 Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries The work presented in this chapter was published: Garrido, A., Serôdio, J., De Vos, R., Conde, A. and Cunha, A. (2019). Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries. Agronomy , 9 (11), 685. https://doi.org/10.3390/agronomy9110685 Supplementary Materials are available online: https://www.mdpi.com/2073-4395/9/11/685/s1 Author Contributions: Conceptualization, A.G., A.C. (Artur Conde), R.V., and A.C. (Ana Cunha) Methodology, A.G., J.S., and A.C. (Ana Cunha) Formal analysis, A.G. and A.C. (Ana Cunha). Investigation, A.G. and A.C. (Ana Cunha). Resources, J.S. and R.V. Writing—original draft preparation, A.G. Writing— review and editing, A.C. (Ana Cunha), J.S., R.V., and A.C. (Artur Conde). Supervision, A.C. (Artur Conde), R.V., and A.C. (Ana Cunha) Project administration, A.C. (Ana Cunha). Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 79 Abstract Climate changes may cause severe impacts both on grapevine and berry development. Foliar application of kaolin has been suggested as a mitigation strategy to cope with stress caused by excessive heat/radiation absorbed by leaves and grape berry clusters. However, its effect on the light microenvironment inside the canopy and clusters, as well as on the acclimation status and physiological responses of the grape berries, is unclear. The main objective of this work was to evaluate the effect of foliar kaolin application on the photosynthetic activity of the exocarp and seeds, which are the main photosynthetically active berry tissues. For this purpose, berries from high light (HL) and low light (LL) microclimates in the canopy, from kaolin-treated and non-treated, irrigated and non-irrigated plants, were collected at three developmental stages. Photochemical and non-photochemical efficiencies of both tissues were obtained by a pulse amplitude modulated chlorophyll fluorescence imaging analysis. The maximum quantum efficiency (Fv/Fm) data for green HL-grown berries suggest that kaolin application can protect the berry exocarp from light stress. At the mature stage, exocarps of LL grapes from irrigated plants treated with kaolin presented higher Fv/Fm and relative electron transport rates (rETR200) than those without kaolin. However, for the seeds, a negative interaction between kaolin and irrigation were observed especially in HL grapes. These results highlight the impact of foliar kaolin application on the photosynthetic performance of grape berries growing under different light microclimates and irrigation regimes, throughout the season. This provides insights for a more case-oriented application of this mitigation strategy on grapevines. Keywords: light microclimates, mitigation strategies, kaolin, irrigation, Vitis vinifera L., grape berry tissues, pulse amplitude modulated (PAM) fluorometry, photosynthesis, photosynthetic pigments. 3.1. Introduction Viticulture is a historically important agronomic and socio-economic sector in Portugal. According to the last report from the International Organization of Vine and Wine (OIV), Portugal is the 11th world and 5th European wine producer (OIV, 2018). With 14 winemaking regions distributed throughout the country, the Vinhos Verdes or Minho region, as well as the Douro and Alentejo, are the major contributors for national exports and growth of this sector (Lavrador da Silva et al., 2018). Grapevine is influenced by a complex and interacting system commonly called terroir , which, according to the OIV (OIV, 2010), includes specific soil, topography, climate, landscape characteristics and biodiversity features, and interaction with applied vitivini-cultural practices. This complex system Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 80 influences the canopy microclimate and grapevine physiology and development and, consequently, grape berry quality and the organoleptic properties of its wine, which is typical of each region. Currently, climate change projections point to a particularly pronounced temperature variation, with an overall increase of up to 3.7 °C by the end of this century, compared to the 1985–2005 reference period (IPCC, 2019). These temperature changes will have great impacts in the Mediterranean wine regions (Ferrise et al., 2013). According to recent investigations using very high resolution bioclimatic zoning, both temperature and dryness are predicted to increase in several economically important Portuguese viticulture regions, including the Vinhos Verdes region (Fraga et al., 2014). Therefore, Portuguese vineyards will be subject to increased stress due to the interaction of the existing high radiation levels with the foreseen elevated air temperature and drought, which, all together, can have high impact on grapevine phenology, physiology, and productivity. Several of these climate impacts have already been reported, such as: earlier phenological timings and shortenings of the grapevine growing season (Fraga et al., 2016), sunburns in leaves and grape berries (Leeuwen and Darriet, 2016), reduction of stomatal conductance and decrease of photosynthetic rates, either by stomatal and non-stomatal limitations (Moutinho-Pereira et al., 2007), appearance and/or intensification of grapevine-related pests and diseases (Bois et al., 2017; Caffarra et al., 2012), increased grape sugar concentrations that lead to higher wine alcohol levels, lower acidities, and modification of varietal aroma compounds (Mira de Orduña, 2010), and higher inter-annual yield and wine production variability (Cunha and Richter, 2016). In order to mitigate these adverse climate effects, new short-term measures have recently been implemented in Portuguese vineyards, such as smart irrigation (Costa et al., 2016; Fraga et al., 2018) and foliar application of kaolin (Brito et al., 2019). Vineyards are not traditionally irrigated and there are even restrictions on this practice in some regions, such as the Douro region (Costa et al., 2016). However, according to a recent projection model, a 10 % reduction in grapevine yield is expected in the Minho region if irrigation is not applied (Fraga et al., 2018). Kaolin is a white, chemically inert, and non-toxic clay material (Al₂Si₂O₅(OH)₄) that can reflect radiation, including photosynthetically active (PAR), ultraviolet (UV), and infrared radiation (IR) (Brito et al., 2019). Foliar application of this mineral has become a cost-efficient mitigation strategy to cope with water stress and excessive heat/radiation absorbed by leaves and grape berry clusters, which also proves effective in alleviating negative impacts on grapevines (Conde et al., 2016, 2018; Dinis et al., 2016a, 2016b, 2018). However, the amount and spectral quality of light intercepted by leaves and transmitted/ reflected into the canopy, crucial factors for leaf, and fruit physiology and development (Poni et al., 2018) are also important aspects to consider when mitigation practices are used. Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 81 Previous work done by our group, using pulse amplitude modulated (PAM) chlorophyll fluorescence imaging, has mapped grape berry photosynthesis at a histological level, and revealed both the exocarp and the seed outer integument as the main photosynthetically competent tissues (Breia et al., 2013). More recently, we have studied the photosynthetic performance of grape berry tissues from clusters growing at three distinct light microclimates in the canopy and observed microclimate-related differences in their photosynthetic capacity and acclimation status (Garrido et al., 2018). This led to the hypothesis that, if a specific viticulture practice changes the light reaching the clusters, and alters its light microclimate, it may impact the photosynthetic activity of berry tissues and associated tissue-specific biochemical processes. In fact, foliar kaolin application may have direct implications on light distribution at the whole canopy level, and irrigation is an indirect one, through increased vegetative growth. For instance, it has already been shown that kaolin application generally reduces the photosynthetic rates of individual leaves in other agricultural crops (e.g., apple, almond, and walnut canopies) (Le Grange et al., 2004; Wünsche et al., 2004), due to a 20–40 % increase in the reflection of PAR (Rosati et al., 2006). However, the photosynthesis of the whole canopy remained unaffected or even increased (9 %), because of the better light distribution within the canopy (Glenn and Puterka, 2007; Glenn, 2009; Rosati et al., 2007). In another study, decreased photosynthesis was observed in the inner leaves of irrigated grapevines due to higher vegetative growth (Escalona et al., 2003). While the function of photosynthesis in fruits is still poorly understood, it can be linked with primary and secondary metabolomic pathways (Cocaliadis et al., 2014; Obiadalla-Ali et al., 2004). Therefore, any effect on photosynthesis may impact grape berry development and composition. Therefore, the main objective of the present work was to evaluate the effects of foliar kaolin application on the photosynthetic activity of grape berry tissues from clusters growing at two distinct microclimates, which include high light (HL) and low light (LL) microclimates, of irrigated and non-irrigated grapevines, during the season. 3.2. Materials and Methods 3.2.1. Site Description, Applied Treatments, and Sampling Grape berry samples were collected in 2018 from field-grown ‘Alvarinho’ cultivar grapevines ( Vitis vinifera L.) in the commercial vineyard Quinta Cova da Raposa in the Demarcated Region of Vinho Verde, Braga, Portugal (41°34’16.4” N, 8°23’42.0” W). The vineyard is managed by following standard cultural practices applied in organic farming, and is arranged in terraces along a granitic hillside with high drainage. The vine training system applied for this cultivar follows the settings of Sylvoz (Simple Ascending Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 82 and Recumbent Cord). The sector selected for the trial was located on a hill with NW-SE orientation and the vineyard rows with a NE-SW orientation. The treatments applied were: kaolin (K) and non-kaolin (NK) application on leaves, and irrigation (I) and non-irrigation (NI), in a complete factorial design (four treatment combinations) with two blocks, each with three to four vines per combination treatment (Figure 3.1B). A suspension of 5 % (w/v in water) kaolin (EPAGRO®, Sunprotect, Alverca do Ribatejo, Portugal) was applied twice on leaves on both sides of the rows. On July 6 and 27, corresponding to four weeks after anthesis (WAA) or BBCH-73 (BBCH-scale used for grapes by Lorenz et al. (1995) and seven WAA or BBCH-77, respectively. Irrigation of half of the plants, started on July 26 (seven WAA, BBCH-77), (Figure 3.1A,B). Water was applied by drip irrigation with one dripper per vine and a drip line placed approximately 80 cm above the soil. Irrigation occurred every three days, once a day either early in the morning or late in the afternoon, for 2 h, with an average dripper capacity of 5.5 ± 1.6 L h−1 ( n = 12 randomly selected drippers, ± SD). Clusters with contrasting light exposure were also selected to harvest grape berries during their development. These were called low light (LL) and high light (HL) clusters. LL clusters grew in the shaded inner zones of the canopy, which were exposed only to diffuse, reflected, and transmitted light, while HL clusters were exposed to direct or reflected sunlight most of the day. Six independent subclusters (three per block), each containing 15–20 grape berries, were collected randomly from clusters growing at each of the experimental conditions (four treatments × 2 microclimates) from the southeast side of rows. Berries were harvested in the morning (9–10 a.m.) at three distinct developmental stages: Green (16 July, 6 WAA, BBCH-75), Véraison (29 August, 12 WAA, BBCH-83), and Mature (17 September, 15 WAA, BBCH-89). The material was transported in refrigerated boxes to the Center for Environmental and Marine Studies (CESAM) laboratory and used within 2–6 h for imaging fluorometry experiments. For other assays, berries were immediately frozen in liquid nitrogen and stored at −80 °C. Figure 3.1. (A) Timeline of the grape growing season depicting the sampling times, foliar kaolin application dates, and the onset of irrigation. (B) Scheme of treatment combinations applied in the field: irrigation (I)/ nonirrigation (NI) x kaolin (K)/ non-kaolin (NK). (WAA - weeks after anthesis). Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 83 3.2.2. Light Intensity and Temperature Measurements for Microclimate Characterization In order to characterize the microclimates in the vicinity of the growing clusters of all experimental conditions, light intensities and temperatures were registered on cloudless days (mean of 1500 ± 300 µmol photons m−2 s−1), between 15 h and 17 h, at green and mature stages, as described by Garrido et al. (2018). The light intensity (µmol photons m−2 s−1) was measured with a radiometer (LI-COR, LI-250 Light Meter, Lincoln, NE, USA) and the temperature (°C) was measured with an infrared digital thermometer (Infrared, DT8380, Beijing, China). Both parameters were determined in the frontal region of the clusters (LL and HL), at the southeast side of the row, and in full sun-exposed leaves. The devices were placed perpendicularly to the plant organ (cluster or leaf). The light sensor was placed on the organ surface facing outward, which registered the light intensity reaching at this point, and the thermometer was pointing to the organ at a distance of about 15 cm, which registered an average organ temperature. Sixteen replicate measurements per treatment were considered on randomly selected vines. 3.2.3. Kaolin Film Transmittance and Reflectance Transmittance and reflectance spectra were obtained using a spectrometer (USB2000-VIS-NIR, grating #3, Ocean Optics, Duiven, The Netherlands), connected to a 400 mm-diameter fiber optic (QP4002-VIS/NIR-BX; Ocean Optics), and recorded using the spectral acquisition software Spectra Suite (Ocean Optics, https://oceanoptics.com/). The transmittance spectrum was obtained by spreading a 5 % (w/v) kaolin suspension prepared in 70 % ethanol (allowing fast evaporation to prevent solvent interference), over a glass plate, which simulated particle distributions similar to those observed in the field. A halogen lamp was placed underneath to illuminate the spectrometer sensor positioned 3 cm above the glass plate. Different spectra were obtained from different areas randomly ( n = 3). The reflectance spectrum was obtained, according to Dinis et al. (2018), by pointing the fiber optics perpendicularly to the surface of collected leaves illuminated by the same halogen lamp. Three independent spectra were obtained from different leaf regions of both kaolin-treated and non-treated leaves. Transmittance and reflectance spectra were recorded for the 370–900 nm spectral range, with a spectral resolution of 0.33 nm. The transmittance spectra were expressed as a percentage of the controls (glass). Reflectance spectra were normalized to the spectrum reflected from a reference white panel (WS-1-SL Spectralon Reference Standard, Ocean Optics). Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 84 3.2.4. Chlorophyll Fluorescence Analysis The photosynthetic activity of grape berry tissues was assessed as described by Garrido et al. (2018). For this, an imaging chlorophyll fluorometer was used ( Open FluorCAM 800 MF; Photon Systems Instruments, Drásov, Czech Republic), which was comprised of four 13 × 13 cm LED panels emitting red light (emission peak at 621 nm, 40-nm bandwidth) and a 2/3 inch CCD camera (CCD381, Beijing, China) with a F1.2 (2.8–6 mm) objective. Two of the LED panels provided modulated measuring light (<0.1 μmol m−2 s−1) and the other two provided saturating pulses (>7500 μmol m−2 s−1, 0.8 s). Chlorophyll fluorescence images were captured and processed using FluorCam7 software (Photon Systems Instruments, Drásov, Czech Republic). In a dark cabinet, exocarps and seeds were separated from dark-adapted berries and disposed in 8 × 8-well plates filled with water. Two independent plates were prepared for each microclimate (LL and HL), with each comprising all treatments and tissues. Exocarps and seeds were placed alternately in three rows each, using two columns per treatment, in a total of 12 biological replicates per condition and tissue ( n = 3 × 2 × 2 = 12). Each plate was subjected to the experiments described below. The maximum quantum efficiency of photosystem II [Fv/Fm = (Fm − F0)/Fm], which is a chlorophyll fluorescence parameter that reflects the probability of electrons being transferred from the PSII reaction center for the transport chain of electrons by quanta absorbed (Baker, 2008; Schreiber, 2004), was computed following a saturation pulse (SP). The isolated tissues were then acclimated to an actinic light (AL) of 200 µmol photons m−2 s−1 for 15 min, and, after a new SP, the effective quantum yield of PSII [ΦII = (F′m − Fs)/F′m] was computed. This parameter correlated with the quantum yield of CO2 fixation in a wide range of physiological conditions (Genty et al., 1989). From ΦII and PFR (photosynthetic photon fluence rates) (200 µmol photons m−2 s−1), the relative electron transport rate through PSII (rETR200 = ΦII × PFR) was calculated. Then, tissues were exposed to 1500 µmol photons m−2 s−1 for 15 min, with an SP being applied every 3 min. The last F’m values (at 15 min) were used to calculate the non-photochemical quenching [NPQ = (Fm − F’m)/F’m]. 3.2.5. Analysis of Chlorophylls and Carotenoids by High Performance Liquid Chromatography Coupled to A Photodiode Array Detector (HPLC-PDA) The extraction procedure was adapted from Fraser et al. (2000). Freeze-dried material (20 mg) of grape berry tissues, which includes exocarp and seed, was extracted in 1.8 mL of chloroform/methanol (1:1) (chloroform - Emsure®, Darmstadt, Germany, methanol - Biosolve®, Dieuze, France) with both 0.1 % (w/v) butylated hydroxytoluene (BHT, Sigma®, Zwijndrecht, The Netherlands) as an antioxidant and Sudan Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 85 1 (0.5 μg mL−1) as the internal standard (IS). The samples were vortexed (10 s), kept on ice for 30 min (vortexed in between), and then sonicated for 15 min (Branson®, 3510 Ultrasonic Cleaner, Danbury, CT, USA). These steps were performed twice. After that, the samples were centrifuged at 16,100× g (Eppendorf®, Centrifuge 5415 R, Hamburg, Germany) and the supernatant (approx. 1200 μL) was transferred to a new Eppendorf tube with a perforated lid. The samples were dried for 1 h in a Speedvac (Savant®, SC100, Schiphol, The Netherlands) and then stored at −80 °C until the next steps. Prior to high performance liquid chromatography (HPLC) analysis, samples were dissolved in 200 μL ethylacetate solution containing 0.1 % (w/v) BHT, sonicated (10 min), and then centrifuged as above. Samples were protected from light and kept on ice during all of these procedures. The supernatant (180 μL) was transferred to amber-colored 2 mL HPLC vials with a glass insert and sealed. The HPLC-PDA procedure was adapted from Mokochinski et al. (2018). The samples (20 μL) were analyzed using an HPLC (Waters Alliance e2695 Separations Module, Milford, MA, USA) coupled to a photodiode array detector (PDA) (Waters 2996) over the 240 to 700 nm UV/Vis range. Separation was performed on a reverse-phase C30 column (250 × 4.6 mm i.d., S-5 μm - YMC Carotenoid, Komatsu, Japan) kept at 35 °C with a flow rate of 1.0 mL min−1. The compounds were identified based on comparisons of retention times and absorption spectra (240 to 700 nm) with authentic standards. 3.2.6. Statistical Analysis Results were statistically analyzed using Analysis of Variance tests (two-way ANOVA), followed by post hoc multiple comparisons using the Bonferroni test whenever the factors had significant effects (GraphPad Prism version 5.00 for Windows, GraphPad Software, La Jolla, CA, USA). Significant differences ( p ≤ 0.05) between sample groups are indicated with different letters. Notation with an asterisk means that only one factor (kaolin or irrigation) was significant. Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 86 3.3. Results and Discussion 3.3.1. Climatic Conditions and Microclimate Characteristics In order to characterize the climate during the growing season at the study site (Braga), we used the official information available from the Instituto Português do Mar e da Atmosfera (IPMA) (IPMA, 2019), to determine the temperatures and total precipitation during 2018 (Figure S3.1). This growing season was atypical from a climatic point of view, with a relatively cold and extremely dry winter, which caused a delay of sprouting/flowering for two to three weeks (IVV, 2018), and a relatively cold spring with rainy periods during the vegetative growth of the grapevines. To characterize the microclimates for the LL and HL berry clusters (two a priori selected distinct light microclimates within the canopy), measurements of light intensities and temperatures were performed at the cluster level, at two time points during the growing season, i.e., when the berries were still green (green stage) and, two months later, when the berries were at their mature stage of ripening (Figure 3.2 and Figure 3.3). Figure 3.2 depicts the average light intensities at LL and HL clusters growing under the different experimental conditions: i.e., irrigation/non irrigation (Figure 3.2a,c) and with kaolin/without kaolin (Figure 3.2b,d). The two microclimates were clearly distinct at both ripening stages, with HL clusters receiving about three-fold more light than LL clusters. At the green stage, i.e., before the onset of irrigation (Figure 3.1A), no significant differences were detected between the two sets of plots assigned to the subsequent irrigation experiment (four plots for irrigated (I) plants, i.e., 2 × NK-I and 2 × K-I) and four plots for non-irrigated (NI) control plants (2 × NK-NI and 2 × K-NI), see Figure 3.1B) (Figure 3.2a), which reveals that there were no plot-related effects on microclimate light intensity. At this early ripening stage, and with the adopted measurement procedure, no differences were detected with respect to light intensities reaching the berry clusters due to foliar kaolin application (Figure 3.2b). However, at a mature stage, both irrigation and kaolin had a small but significant effect on the light intensity reaching the HL clusters (Figure 3.2c,d). Irrigation slightly reduced the light intensity, likely due to the better vegetative growth of the plants, while foliar kaolin application increased it, likely due to an increased light reflection to both the interior and lower levels of the canopy. In the LL clusters, these effects of irrigation and kaolin on light intensity were not observed, at this time of day. HL grapes consistently experienced higher temperatures than LL ones (Figure 3.3), and both I and K treatments exerted significant and contrasting effects on this microclimate parameter, mainly at the mature stage. Again, and consistent with what was observed and discussed above for light intensity, no effect was detected for I treatment on the grape berry temperature at the green stage (before the onset of irrigation) (Figure 3.3a). Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 87 Figure 3.2. Light intensities received by LL and HL clusters at the green stage (a, b) and the mature stage (c, d), for plants with irrigation (blue columns, note: the textured blue columns at green stage i.e., before the onset of irrigation, represent the measurements in the plots that were later irrigated) and foliar kaolin application (white columns). Black columns correspond to the respective controls. Values represent means with a standard deviation ( n = 16 plants). Statistical notation: per ripening stage, different capital letters refer to significant differences (twoway ANOVA, p ≤ 0.05) between the two light microclimates within the same plant treatment, and different lowercase letters for differences between treatments within each light microclimate. If the respective factor did not have a significant effect, the lowercase letters were omitted. Figure 3.3. Temperatures of LL and HL clusters at the green stage (a, b) and the mature stage (c, d), for plants with irrigation (blue columns, note: the textured blue columns at the green stage i.e., before the onset of irrigation, represent the measurements in the plots that were later irrigated) and foliar kaolin application (white columns). Black columns correspond to the respective controls. Values represent means with standard deviation Kaolin treatment LL HL 0 50 100 150 200 B A B A (b) Light intensity (mol photons m-2s-1) Irrigation treatment LL HL 0 50 100 150 200 B A B A (a) Light intensity (mol m-2s-1) LL HL 0 50 100 150 200 B A,a B A,b (c) Light intensity (mol m-2s-1) LL HL 0 50 100 150 200 B A,b B A,a (d) Light intensity (mol photons m-2s-1) Green Mature Irrigation treatment LL HL 20 25 30 35 40 B A B A (a) Temperature (ºC) Kaolin treatment LL HL 20 25 30 35 40 B A,a B A,b (b) Temperature (ºC) LL HL 20 25 30 35 40 B,b A,b B,a A,a (c) Temperature (ºC) LL HL 20 25 30 35 40 B,a A,a A,b A,b (d) Temperature (ºC) Green Mature Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 94 Figure 3.7. Non-photochemical quenching (NPQ) mean values ( n = 12–24 berries, +SD). All the microclimate conditions, treatment combinations, and statistical information are the same as in Figure 3.5. 3.3.3.4. Photosynthetic Pigments To better evaluate the impact of foliar kaolin application on the light microclimate of grape berry clusters and its relationship with berry photosynthesis, and non-photochemical mechanisms, photosynthetic pigments were quantified in exocarps and seeds of both LL and HL-exposed grapes. Results obtained for the green stage are depicted in Figure 3.8 (for later stages, see Supplementary Materials). At control conditions, the HL berries had higher levels of both chlorophylls and carotenoids than LL berries, in both tissues. Additionally, and in line with the rETR200 results (Figure 3.6a), kaolin application resulted in a marked increase by 26 % in chlorophylls and 82 % in carotenoids content in exocarps from LL berries (Figure 3.8a,c), which support the idea that more light reached the inner parts of the kaolin-sprayed canopy. This is fundamental to build the photosynthetic machinery (Tikkanen et al., 2012). During ripening, the photosynthetic pigments decrease in both tissues and especially in the seed integuments (Figure S3.3 and Figure S3.4) and no consistent and conspicuous effects by combined mitigation treatments were observed (Figure S3.3). Exocarp LL HL 0 2 4 6 ab (a) NPQ Seed LL HL 0 2 4 6(b) LL HL 0 1 2 3 5 6 A bB bA b A aAAAA (c) * NPQ LL HL 0.0 0.2 0.4 0.6 0.8 1.0 5 6 AABA AAAB (d) * LL HL 0 1 2 3 5 6 AAAA B A AA (e) * NPQ LL HL 0.0 0.2 0.4 0.6 0.8 1.0 5 6 AAAA BBBB (f) Control Kaolin Non-Kaolin, Non-Irrigation (NK,NI) Non-Kaolin, Irrigation (NK,I) Kaolin, Non-Irrigation (K, NI) Kaolin, Irrigation (K, I) Green Véraison Mature Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 95 Figure 3.8. Chlorophylls (a, b) and carotenoids (c, d) concentration mean values ( n = 3, +SD) of exocarps and seeds obtained from LL and HL grape berries grown under non-kaolin (black columns) and kaolin (white columns) application, and collected at the green stage. Statistical notation: different capital letters refer to significant differences (two-way ANOVA, p ≤ 0.05) between the two light microclimates within the same plant treatment, and different lowercase letters to differences between treatments within each light microclimate. If the respective factor did not have a significant effect, the letters were omitted. In addition, and supporting the view discussed above, the higher grape berry temperature was registered in irrigated treatments at later developmental stages (Figure 3.3c), by imposing physiological impairments. The temperature recruits more energy-dissipation by NPQ (Figure 3.7), which is the fact that carotenoids contents (Figure S3.4), but not chlorophylls (Figure S3.3), were also increased by irrigation treatment, for both tissues at the véraison stage. Overall, the results obtained by pulse amplitude modulated fluorometry showed that, for the external tissue, exocarp, and foliar kaolin application led to an increase of Fv/Fm (Figure 3.5a, HL), rETR200 (Figure 3.6a,e LL), and a reduction in non-photochemical quenching (Figure 3.7a, HL). To our best knowledge, this is the first work assessing the impact of foliar kaolin application on photochemical and non-photochemical functions in grape berries. Recently, it was verified that grapevine leaves with kaolin display the same response, i.e., an increase in Fv/Fm, Φll, and ETR, and a decrease in NPQ (Dinis et al., 2018; Frioni et al., 2019). Similar results were also reported for olive leaves (Brito et al., 2019). In this way, and in terms of photochemical processes, those kaolin-treated leaves have lower photo-inhibitory damage (Dinis et al., 2016b; Maxwell and Johnson, 2000), and the open PSII reaction centers captured the light absorbed by PSII antenna more efficiently (Baker, 2008; Dinis et al., 2016b). This response was LL HL 0 50 100 150 200 250 B,b A,a (c) AA Total Carotenoids (g/g DW) Exocarp LL HL 0 250 500 750 1000 1250 B,b A,a (a) AA Total Chlorophylls (g/g DW) Seed LL HL 0 250 500 750 1000 1250 A,a A,b (b) A B Control Kaolin LL HL 0 50 100 150 200 250 (d) BA AA Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 96 likely due to a reduced loss of excitation energy by thermal dissipation, which could compete with its transference to PSII reaction centers, as shown by the lower NPQ values (Baker, 2008; Dinis et al., 2016b). For exocarps of grape berries growing in inner parts of the canopy (LL microclimate), the photosynthetic results revealed that foliar kaolin application, may cause an extra “sunscreen” effect, and did not have a negative effect on those parameters, which we conjectured in our previous work (Garrido et al., 2018). The increased reflection provided by this mineral to inner parts of the canopy allowed good photochemical performance of LL exocarps, which is contrary to what we hypothesized in our previous work (Garrido et al., 2018). This contributes to higher carbon gains at the whole canopy level and also at the fruit level. Regarding the results for the seed integument (internal organ), the positive effects of kaolin were observed mainly in non-irrigated plants such as an increase in Fv/Fm (Figure 3.5f, HL) and a decrease in NPQ (Figure 3.7d, HL). In more temperate or Mediterranean regions, this seems like a positive effect, but these results also show the importance of the irrigation system. The interaction between kaolin application and irrigation treatments on grapevine leaves have been studied before (Cooley et al., 2008; Glenn et al., 2010; Shellie and Glenn, 2008; Shellie and King, 2013a; 2013b). However, based on our knowledge, no study has approached the impacts on photosynthetic activity at the grape berry level, using chlorophyll fluorescence analysis. 3.4. Conclusions The purpose of the current study was to assess the effects of foliar application of kaolin and irrigation, as abiotic stress mitigation strategies, on the photosynthetic activity of exocarps (skins) and seeds of grape berries growing under different light microclimates in the canopy. One of the most relevant findings was that the kaolin applied to leaves increased the photosynthetic activity of both exocarps and seed integuments of berries growing under low light conditions in the canopy. This is likely due to higher reflection of PAR to the inner zones. We believe, though, that the beneficial effects will depend on the canopy structure and on the incident radiation, with denser canopies and higher radiations conferring higher overall photosynthetic gains. Somewhat puzzling was the observation that seeds of irrigated plants showed lower photosynthetic activities, in the véraison and mature stages, especially under kaolin treatment. Several causes may explain this unexpected phenomenon, so more detailed and ad-hoc design studies should be conducted to address this relevant finding. Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 97 This comprehensive study provides the first evidence of foliar kaolin application as a procedure allowing the modulation of photosynthesis in the grape berry, but also calls attention to the importance of the irrigation system. In this way, this knowledge can be used by farmers to support their decisions concerning sustainable adaptation strategies applied on vineyards. Research to unveil the function of berry tissues’ photosynthesis on the metabolome of the grapes is already underway, which ultimately contributes to the final quality of the fruit and wine. 3.5. Supplementary Materials The following are available online at https://www.mdpi.com/2073-4395/9/11/685/s1. Jan Feb Mar Apr May Jun Jul Aug Sep Out Nov Dec 0 10 20 30 40 Minimal Average Maximal (a) Temperature (C) Jan Fev Mar Abr Maio Jun Jul Ago Set Out Nov Dez 0 100 200 300 400 (b) Total Precipitation (mm) Figure S3.1. Meteorological elements from IPMA institute from Braga city. (a) Temperature (°C) maximal, average and minimal. (b) Total precipitation (mm). Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 98 Non-Irrigation Irrigation 20 25 30 35 40 (a) Temperature (ºC) Non-Kaolin Kaolin 20 25 30 35 40 (b) Temperature (ºC) Non-Irrigation Irrigation 20 25 30 35 40 (c) Temperature (ºC) Non-Kaolin Kaolin 20 25 30 35 40 (d) ab Temperature (ºC) Green Mature Figure S3.2. Temperatures of full exposed leaves at the green stage (a, b) and the mature stage (c, d), for plants with irrigation (blue columns; note: the textured blue columns at green stage i.e. before the onset of irrigation, represent the measurements in the plots that were later irrigated) and foliar kaolin application (white columns). Black columns correspond to the respective controls. Values represent means with standard deviation ( n = 16 plants). Statistical notation: per ripening stage, different lowercase letters refer to significant differences ( p ≤ 0.05) between treatments. Whenever letters are omitted it means that the respective factor did not have a significant effect. Exocarp LL HL 0 250 500 750 1000 1250 (a) Total Chlorophylls (g/g DW) Seed LL HL 0 25 750 1000 1250 A a A bA b,c A c A aA bA b B a,b (b) LL HL 0 50 100 150 200 250 750 1000 1250 A aA bA b A a,b (c) BBAA Total Chlorophylls (g/g DW) LL HL 0 10 20 750 1000 1250 (d) AAAB AAAA Véraison Mature Non-Kaolin, Non-Irrigation (NK, NI) Non-Kaolin, Irrigation (NK, I) Kaolin, Non-Irrigation (K, NI) Kaolin, Irrigation (K, I) Figure S3.3. Chlorophylls concentration mean values ( n = 3, +SD) of exocarp and seed obtained from LL and HL grape berries grown under the four combinations of the two treatments applied: irrigation (I)/ non-irrigation Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 99 (NI) x kaolin (K)/ non-kaolin (NK). Samples were collected at three development stages (green, véraison and mature). Statistical notation: for each developmental stage, capital letters refer to differences between light microclimates within the same treatment combination, and lowercase letter refers to differences between treatment combinations within each light microclimate (mean values with a common letter were not significantly different). When capital and lowercase letters are omitted, the respective factor did not have a significant effect (two-way ANOVA, p ≤ 0.05). Exocarp LL HL 0 50 100 150 200 250 ba ba,b (a) Total Carotenoids (g/g DW) Seed LL HL 0 50 100 200 250 B b A a A aA bA bA b A a,b A a,b (b) LL HL 0 50 100 150 200 250 A aA bA bA b (c) BBBA Total Carotenoids (g/g DW) LL HL 0 50 100 200 250 (d) Non-Kaolin, Non-Irrigation (NK, NI) Non-Kaolin, Irrigation (NK, I) Kaolin, Non-Irrigation (K, NI) Kaolin, Irrigation (K, I) Véraison Mature Figure S3.4. Carotenoids concentration mean values ( n = 3, +SD) of exocarp and seeds. All the microclimate conditions, treatment combinations, and statistical information are the same as in Figure S3.3. 3.6. Funding The FCT-Portuguese Foundation for Science and Technology by the grant provided to Andreia Garrido (PD/BD/128275/2017), under the Doctoral Program “Agricultural Production Chains – from fork to farm” (PD/00122/2012), funded this research and APC. 3.7. Acknowledgments The National Funds by FCT - Portuguese Foundation for Science and Technology, under the strategic programmes UID/AGR/04033/2019 and UID/BIA/04050/2019, and the project “INTERACT - VitalityWine - NORTE-01-0145-FEDER-000017 – funded by Norte2020 supported the work. The FCT and FEDER/COMPETE/POCI - Operational Competitiveness and Internationalization Program, under Project the projects MitiVineDrought – PTDC/BIA-FBT/30341/2017 (POCI-01-0145-FEDER-030341), and POCI01-0145-FEDER-006958 also supported this work. Artur Conde was supported with a post-doctoral fellow Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 100 of the mentioned INTERACT/VitalityWine project with the Reference BPD/UTAD/INTERACT/VW/218/2016, and also supported by a post-doctoral researcher contract/position within the project “MitiVineDrought” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145FEDER-030341). This work also benefited from the networking activities within the European Unionfunded COST Action CA17111 – “INTEGRAPE - Data Integration to maximize the power of omics for grapevine improvement”. Authors acknowledge the owner from Quinta Cova da Raposa , Manuel Taxa, who provided the samples, Susana Chaves (from CBMA) for her English grammar revision, and also all support given by the Biology Department of the School of Sciences from the University of Minho. 3.8. References Aschan, G., & Pfanz, H. (2003). Non-foliar photosynthesis – a strategy of additional carbon acquisition. Flora , 198 (2), 81–97. Baker, N. R. (2008). Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annual Review of Plant Biology , 59 , 89–113. Bois, B., Zito, S., Calonnec, A., & Ollat, N. (2017). Climate vs grapevine pests and diseases worldwide: The first results of a global survey. Journal International Des Sciences de La Vigne et Du Vin , 51 (2), 133–139. Breia, R., Vieira, S., Da Silva, J. M., Gerós, H., & Cunha, A. (2013). Mapping grape berry photosynthesis by chlorophyll fluorescence imaging: The effect of saturating pulse intensity in different tissues. Photochemistry and Photobiology , 89 (3), 579–585. Brito, C., Dinis, L. T., Luzio, A., Silva, E., Gonçalves, A., Meijón, M., Correia, C. M. (2019). Kaolin and salicylic acid alleviate summer stress in rainfed olive orchards by modulation of distinct physiological and biochemical responses. Scientia Horticulturae , 246 , 201–211. Brito, C., Dinis, L. T., Moutinho-Pereira, J., & Correia, C. (2019). Kaolin, an emerging tool to alleviate the effects of abiotic stresses on crop performance. Scientia Horticulturae , 250 , 310–316. Caffarra, A., Rinaldi, M., Eccel, E., Rossi, V., & Pertot, I. (2012). Modelling the impact of climate change on the interaction between grapevine and its pests and pathogens: European grapevine moth and powdery mildew. Agriculture, Ecosystems and Environment , 148 , 89–101. Cocaliadis, M. F., Fernández-Muñoz, R., Pons, C., Orzaez, D., & Granell, A. (2014). Increasing tomato fruit quality by enhancing fruit chloroplast function. A double-edged sword? Journal of Experimental Botany , 65 (16), 4589–4598. Conde, A., Pimentel, D., Neves, A., Dinis, L.-T., Bernardo, S., Correia, C. M., Moutinho-Pereira, J. (2016). Kaolin foliar application has a stimulatory effect on phenylpropanoid and flavonoid pathways in grape berries. Frontiers in Plant Science , 7 , 1–14. Conde, Artur, Neves, A., Breia, R., Pimentel, D., Dinis, L. T., Bernardo, S., Moutinho-Pereira, J. (2018). Kaolin particle film application stimulates photoassimilate synthesis and modifies the primary metabolome of grape leaves. Journal of Plant Physiology , 223 , 47–56. Cooley, N. M., Glenn, D. M., Clingeleffer, P. R., & Walker, R. R. (2008). The Effects of Water Deficit and Particle Film Technology Interactions on Cabernet Sauvignon Grape Composition. Acta Horticulturae , (792), 193–200. Costa, J. M., Vaz, M., Escalona, J., Egipto, R., Lopes, C., Medrano, H., & Chaves, M. M. (2016). Modern viticulture in southern Europe: Vulnerabilities and strategies for adaptation to water scarcity. Agricultural Water Management , 164 , 5–18. Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 101 Cunha, M., & Richter, C. (2016). The impact of climate change on the winegrape vineyards of the Portuguese Douro region. Climatic Change , 138 (1–2), 239–251. Dinis, L. T., Bernardo, S., Conde, A., Pimentel, D., Ferreira, H., Félix, L., Moutinho-Pereira, J. (2016a). Kaolin exogenous application boosts antioxidant capacity and phenolic content in berries and leaves of grapevine under summer stress. Journal of Plant Physiology , 191 , 45–53. Dinis, L. T., Ferreira, H., Pinto, G., Bernardo, S., Correia, C. M., & Moutinho-Pereira, J. (2016b). Kaolinbased, foliar reflective film protects photosystem II structure and function in grapevine leaves exposed to heat and high solar radiation. Photosynthetica , 54 (1), 47–55. Dinis, L. T., Malheiro, A. C., Luzio, A., Fraga, H., Ferreira, H., Gonçalves, I., & Moutinho-Pereira, J. (2018). Improvement of grapevine physiology and yield under summer stress by kaolin-foliar application: water relations, photosynthesis and oxidative damage. Photosynthetica , 56 (2), 641-651. dos Santos, T. P., Lopes, C. M., Lucília Rodrigues, M., de Souza, C. R., Ricardo-da-Silva, J. M., Maroco, J. P., Manuela Chaves, M. (2007). Effects of deficit irrigation strategies on cluster microclimate for improving fruit composition of Moscatel field-grown grapevines. Scientia Horticulturae , 112 (3), 321– 330. Escalona, J. M., Flexas, J., Bota, J., & Medrano, H. (2003). Distribution of leaf photosynthesis and transpiration within grapevine canopies under different drought conditions. Vitis , 42 (2), 57–64. Ferrise, R., Moriondo, M., Trombi, G., Miglietta, F., & Bindi, M. (2013). Climate Change Impacts on Typical Mediterranean Crops and Evaluation of Adaptation Strategies to Cope With. In Advances in Global Change Research . Fraga, H., García de Cortázar Atauri, I., & Santos, J. A. (2018). Viticultural irrigation demands under climate change scenarios in Portugal. Agricultural Water Management , 196 , 66–74. Fraga, H., Malheiro, A. C., Moutinho-Pereira, J., Jones, G. V., Alves, F., Pinto, J. G., & Santos, J. A. (2014). Very high resolution bioclimatic zoning of Portuguese wine regions: Present and future scenarios. Regional Environmental Change , 14 , 295–306. Fraga, H., Santos, J. A., Moutinho-Pereira, J., Carlos, C., Silvestre, J., Eiras-Dias, J., Malheiro, A. C. (2016). Statistical modelling of grapevine phenology in Portuguese wine regions: Observed trends and climate change projections. Journal of Agricultural Science , 154 (5), 795–811. Fraser, P. D., Elisabete S Pinto, M., Holloway, D. E., & Bramley, P. M. (2000). Application of highperformance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant Journal , 24 (4), 551–558. Frioni, T., Tombesi, S., Luciani, E., Sabbatini, P., Berrios, J. G., & Palliotti, A. (2019). Kaolin treatments on Pinot noir grapevines for the control of heat stress damages. BIO Web of Conferences , 13 , 04004. Garrido, A., Breia, R., Serôdio, J., & Cunha, A. (2018). Impact of the Light Microclimate on Photosynthetic Activity of Grape Berry ( Vitis vinifera ): Insights for Radiation Absorption Mitigations’ Measures. In Theory and Practice of Climate Adaptation (pp. 419–441). Springer, Cham. Genty, B., Briantais, J.-M., & Baker, N. R. (1989). The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta (BBA) - General Subjects , 990 (1), 87–92. Glenn, D. Michael, Prado, E., Erez, A., McFerson, J., & Puterka, G. J. (2002). A Reflective, ProcessedKaolin Particle Film Affects Fruit Temperature, Radiation Reflection, and Solar Injury in Apple. Journal of the American Society for Horticultural Science , 127 (2), 188–193. Glenn, D. Michael, & Puterka, G. J. (2007). The use of plastic films and sprayable reflective particle films to increase light penetration in apple canopies and improve apple color and weight. HortScience , 42 (1), 91–96. Glenn, David M. (2009). Particle Film Mechanisms of Action That Reduce the Effect of Environmental Stress in ‘Empire’ Apple. Journal of the American Society for Horticultural Science , 134 (3), 314– Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 102 321. Glenn, M. D. (2012). The mechanisms of plant stress mitigation by kaolin-based particle films and applications in horticultural and agricultural crops. HortScience , 47 (6), 710–711. Glenn, M., NicolaCooley, Walker, R., Clingeleffer, P., & Shellie, K. (2010). Impact of kaolin particle film and water deficit on wine grape water use efficiency and plant water relations. HortScience , 45 (8), 1178–1187. Greer, D. H., & Weston, C. (2010). Heat stress affects flowering, berry growth, sugar accumulation and photosynthesis of Vitis vinifera cv. Semillon grapevines grown in a controlled environment. Functional Plant Biology , 37 (3), 206–214. IPCC (International Panel on Climate Change). (2019). Special Report on Climate Change, Desertification, Land Degradation, Sustainable Land Management, Food Security, and Greenhouse gas fluxes in Terrestrial Ecosystems. Summary for Policymakers . IPMA. (2019). Instituto Português do Mar e da Atmosfera. Retrieved June 2, 2019, from http://www.ipma.pt/pt/otempo/obs.superficie/index-map-dia-chart.jsp#Braga, Merelim IVV, Instituto da Vinha e do Vinho, I. P. I. de mercado. (2018). Nota Informativa, No3/2018, 30/07/2018, Previsão de Colheita - Campanha 2018/2019. Retrieved July 1, 2019, from https://www.ivv.gov.pt/np4/8955.html Joubert, C., Young, P. R., Eyéghé-Bickong, H. A., & Vivier, M. A. (2016). Field-grown grapevine berries use carotenoids and the associated xanthophyll cycles to acclimate to UV exposure differentially in high and low light (Shade) conditions. Frontiers in Plant Science , 7 , 786. Kadir, S., Von Weihe, M., & Al-Khatib, K. (2007). Photochemical Efficiency and Recovery of Photosystem II in Grapes After Exposure to Sudden and Gradual Heat Stress. Journal of the American Society for Horticultural Science , 132 (6), 764–769. Kraus, C., Pennington, T., Herzog, K., Hecht, A., Fischer, M., Voegele, R. T., Kicherer, A. (2018). Effects of canopy architecture and microclimate on grapevine health in two training systems. Vitis - Journal of Grapevine Research , 57 (2), 53–60. Krause, G. H., & Jahns, P. (2007). Non-photochemical Energy Dissipation Determined by Chlorophyll Fluorescence Quenching: Characterization and Function. In Chlorophyll a Fluorescence . Lavrador da Silva, A., João Fernão-Pires, M., & Bianchi-de-Aguiar, F. (2018). Portuguese vines and wines: heritage, quality symbol, tourism asset. Ciência e Técnica Vitivinícola , 33 (1), 31–46. Le Grange, M., Wand, S. J. E., & Theron, K. I. (2004). Effect of kaolin applications on apple fruit quality and gas exchange of apple leaves. XXVI International Horticultural Congress: Key Processes in the Growth and Cropping of Deciduous Fruit and Nut Trees , 636 , 545–550. Leeuwen, C. Van, & Darriet, P. (2016). The Impact of Climate Change on Viticulture and Wine Quality. Journal of Wine Economics , 11 (01), 150–167. Lorenz, D. H., Eichhorn, K. W., Bleiholder, H., Klose, R., Meier, U., & Weber, E. (1995). Growth Stages of the Grapevine: Phenological growth stages of the grapevine ( Vitis vinifera L. ssp. vinifera )—Codes and descriptions according to the extended BBCH scale. Australian Journal of Grape and Wine Research , 1 (2), 100–103. Luo, H. B., Ma, L., Xi, H. F., Duan, W., Li, S. H., Loescher, W., Wang, L. J. (2011). Photosynthetic responses to heat treatments at different temperatures and following recovery in grapevine ( Vitis amurensis L.) leaves. PLoS ONE , 6 (8). Maxwell, K., & Johnson, G. N. (2000). Chlorophyll fluorescence--a practical guide. Journal of Experimental Botany , 51 (345), 659–668. Medrano, H., Bota, J., Abadía, A., Sampol, B., Escalona, J. M., & Flexas, J. (2002). Effects of drought on light-energy dissipation mechanisms in high-light-acclimated, field-grown grapevines. In Functional Plant Biology . Mira de Orduña, R. (2010). Climate change associated effects on grape and wine quality and production. Chapter 3 - Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries __________________________________________________________________________________ 103 Food Research International , 43 (7), 1844–1855. Mokochinski, J. B., Mazzafera, P., Sawaya, A. C. H. F., Mumm, R., de Vos, R. C. H., & Hall, R. D. (2018). Metabolic responses of Eucalyptus species to different temperature regimes. Journal of Integrative Plant Biology . Moutinho-Pereira, J. ., Magalhães, N. ., Gonçalves, B. ., Bacelar, E. ., Brito, M. ., & Correia, C. . (2007). Gas exchange and water relations of three Vitis vinifera L. cultivars growing under Mediterranean climate. Photosynthetica , 45 (2), 202–207. Obiadalla-Ali, H., Fernie, A. R., Lytovchenko, A., Kossmann, J., & Lloyd, J. R. (2004). Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size, but only small changes in carbohydrate metabolism. Planta , 219 (3), 533–540. OIV. (2010). Definition of vitivinicultural “terroir.” OIV. (2018). International Organisation of Vine and Wine Statistical Report on World Vitiviniculture . France. Oliveira, M. (2018). Viticulture in Warmer Climates: Mitigating Environmental Stress in Douro Region, Portugal. In Grapes and Wines - Advances in Production, Processing, Analysis and Valorization . Poni, S., Gatti, M., Palliotti, A., Dai, Z., Duchêne, E., Truong, T. T., Tombesi, S. (2018). Grapevine quality: A multiple choice issue. Scientia Horticulturae , 234 , 445–462. Reynolds, A. G., & Heuvel, J. E. V. (2009). Influence of grapevine training systems on vine growth and fruit composition: A review. American Journal of Enology and Viticulture , 60 (3), 251–268. Rosati, A., Metcalf, S. G., Buchner, R. P., Fulton, A. E., & Lampinen, B. D. (2006). Physiological effects of kaolin applications in well-irrigated and water-stressed walnut and almond trees. Annals of Botany , 98 (1), 267–275. Rosati, Adolfo, Metcalf, S. G., Buchner, R. P., Fulton, A. E., & Lampinen, B. D. (2007). Effects of Kaolin application on light absorption and distribution, radiation use efficiency and photosynthesis of almond and walnut canopies. Annals of Botany , 99 (2), 255–263. Schreiber, U. (2004). Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview. In Chlorophyll a Fluorescence: A Signature of Photosynthesis (George C., pp. 279–319). Dordrecht, The Netherlands: Kluwer Academic. Sharma, R. R., Vijay Rakesh Reddy, S., & Datta, S. C. (2015). Particle films and their applications in horticultural crops. Applied Clay Science , 116 – 117 , 54–68. Shellie, K. C., & King, B. A. (2013a). Kaolin particle film and water deficit influence malbec leaf and berry temperature, pigments, and photosynthesis. American Journal of Enology and Viticulture , 64 (2), 223–230. Shellie, K. C., & King, B. A. (2013b). Kaolin particle film and water deficit influence red winegrape color under high solar radiation in an arid climate. American Journal of Enology and Viticulture , 64 (2), 214–222. Shellie, K., & Glenn, D. M. (2008). Wine grape response to foliar kaolin particle film under differing levels of preveraison water stress. HortScience , 43 (5), 1392–1397. Szabó, I., Bergantino, E., & Giacometti, G. M. (2005). Light and oxygenic photosynthesis: Energy dissipation as a protection mechanism against photo-oxidation. EMBO Reports . Tikkanen, M., Grieco, M., Nurmi, M., Rantala, M., Suorsa, M., & Aro, E. M. (2012). Regulation of the photosynthetic apparatus under fluctuating growth light. Philosophical Transactions of the Royal Society B: Biological Sciences , 367 (1608), 3486–3493. Wünsche, J.-N., Lombardini, L., Greer, D. H., & Palmer, J. W. (2004). “Surround” particle film applications - the effect on whole canopy physiology of apple. XXVI International Horticultural Congress: Key Processes in the Growth and Cropping of Deciduous Fruit and Nut Trees , 636 , 565–571. Xiao, F., Yang, Z. Q., & Lee, K. W. (2017). Photosynthetic and physiological responses to high temperature in grapevine ( Vitis vinifera L.) leaves during the seedling stage. The Journal of Horticultural Science and Biotechnology , 92 (1), 2–10. Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 110 Figure 4.1. Principal component analysis (PCA) score plots of the liquid chromatography mass spectrometry (LCMS) metabolite data for the exocarp (a) and seed (b) in berries at three developmental stages (green, véraison , mature), including both microclimates (high light (HL) and low light (LL)) and both mitigation treatments. Colored ellipses represent 95 % confidence interval ( n = 4 for green stage and n = 3 for véraison and mature stages). Analysis of Variance (ANOVA) in combination with false discovery rate (FDR) correction indicated that 362 metabolites in the exocarp (i.e., 91.6 % of the metabolites detected by LCMS in this tissue) and 388 metabolites in seeds (i.e., 95.4 % of the detected seed metabolites) differed significantly between the three developmental stages. A heatmap plot was constructed based on the 25 top-ranking metabolites according to the ANOVA test (Figure S4.3). For both tissues, the heatmap shows two main blocks of metabolites, i.e., a group of metabolites with higher abundance in the green stage and another group of metabolites higher in the later stages. The FDR corrected p -values and the fold change (FC) values between the averages of the green and mature groups for both the exocarp (Supplemental File S1, Table S2) and seed (Supplemental File S2, Table S2) of untreated control samples grown in a HL microclimate (as an example) were calculated to select those compounds that were most influenced by ripening (PC1). In fact, 17.2 % of the total of LCMS-compounds detected in the exocarp tissue appeared to be uniquely present (i.e., above detection threshold) in either green or mature grapes (Supplemental File S1, Table S2), while this was 19.8 % in seeds (Supplemental File S2, Table S2). In addition, among the metabolites present at both developmental stages, 39.4 % and 43.2 % were significantly different ( p < 0.05) between these developmental stages in the exocarp and seed, respectively. In the exocarp, a range of procyanidins (also called flavan-3-ols) were higher in green than in mature grapes (Supplemental File S1, Table S2) including Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 111 procyanidin trimers (e.g., ID 308, 71-fold), dimers (e.g., ID 189, 4.5-fold and ID 140, epicatechin– gallocatechin, 17-fold) and the monomer catechin (ID 206, 4.2-fold), as well as stilbenes like resveratrol (ID 653, 58-fold) and piceid (ID 601, 2.8-fold). Their decrease upon ripening is in accordance with previous results with the exocarp of red grape berries (Jeandet et al., 1991; Jordão et al., 2001). Resveratrol in green berries has been suggested to play a role in preventing fungal infection and damage by UV irradiation (Hasan and Bae, 2017). On the other hand, a series of flavonol glycosides significantly increased upon ripening, such as quercetin 3O -glucoside (ID 492, 5.4-fold), kaempferol-3-glucoside (ID 545, 46-fold) and an isorhamnetin-hexoside (ID 583, 49-fold), in accordance with previous results obtained with the skin of both white and red grapes (Downey et al., 2003). In the seed (Supplemental File S2, Table S2), there was a similar decrease in various procyanidins, such as gallocatechin (ID 229, 18-fold) and other polymeric compounds from the same class (e.g., ID 150, 28-fold; ID 377, 19-fold; ID 579, 12-fold), which is in accordance with previous results with seeds of red grape berries (Jordão et al., 2001). On the other hand, seed ripening coincided with an increase in resveratrol (ID 979, 7.2-fold) and several of its putatively-identified oligomers including a dimer (ID 926, 36-fold), a trimer-hexoside (ID 990, 33-fold) and a tetramer (ID 1074, 169-fold). Subsequently, PCA was performed for each developmental stage and for each grape berry tissue separately (Figure 4.2 and Figure S4.4). For the PCA of the green stage, only the effects of microclimates and foliar kaolin application could be assessed, since at this developmental stage, no irrigation was yet applied to the vineyards. At the green stage, the PCA result plots indicated a separation of samples mainly according to the canopy microclimate (LL vs. HL), most specifically in exocarp samples (PC1 explaining 41.3 % and 29.3 % of total variance in exocarp and seed, respectively), while no clear sample grouping was observed for kaolin-treated versus untreated plants in either tissue, neither based on the first two PCs (Figure 4.2a,b) nor upon considering subsequent PCs (PC3 and PC4; data not shown). At the véraison stage (Figure S4.4), the berry exocarp metabolome (Figure S4.4a) was mainly influenced by microclimate as well (PC1, 24.9 %) and secondly also by irrigation (PC2, 18.9 %). In contrast, at this developmental stage, the seed metabolome (Figure S4.4b) was mainly affected by the irrigation treatment (PC1, 38.0 %). At the mature stage, the exocarp metabolome (Figure 4.2c) was primarily influenced by the canopy microclimate (PC1, 35.5 %) and secondly by the irrigation treatment (PC2, 11.5 %), while for mature seeds (Figure 4.2d) no clear grouping of the differently treated berries was detected. Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 112 Figure 4.2. Principal component analysis (PCA) score plots based on the LCMS metabolite data for both exocarp (a, c) and seed (b, d) at the two most contrasting ripening stages (a, b—green and c, d—mature), including all microclimates and treatments ( n = 4 for green stage and n = 3 for mature stage). The abbreviations in the legend represent: NI—Non-Irrigation; I—Irrigation; NK—Non-kaolin; K—Kaolin; LL—Low Light microclimate; HL—High Light microclimate. To assess the composition of primary metabolites in exocarps, gas chromatography mass spectrometry (GCMS) analysis was performed. This was done for mature berries only, since this stage is most directly related to the quality of grapes and wine. The unbiased processing of the 24 exocarp samples resulted in the relative abundances of 99 metabolites, mainly sugars, amino acids and organic acids (Supplemental File S1, Table S3, Figure S4.5). In contrast to the PCA based on LCMS metabolites (Figure 4.2c), the PCA based on these GCMS compounds did not reveal clear effects of either microclimate, kaolin or irrigation on the metabolic composition of these mature exocarp samples (Figure Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 113 S4.6). The lack of irrigation effects suggests that the accumulation of primary compounds is unrelated to the effect of irrigation on the photosynthetic activity in these mature exocarps (Garrido et al., 2019). ANOVA Simultaneous Component Analysis (ASCA) was subsequently used to determine which of the growth conditions, as well as their possible interactions, exerted a significant effect on the metabolome of the exocarp and seeds at each of the three berry developmental stages, based on either the LCMS and GCMS analysis (Table 4.1). In addition, we applied a N-way ANOVA to study the effect of the growth conditions on each metabolite in more detail (Table 4.1: numbers between brackets indicate numbers of significant metabolites). The significant compounds (all, or top 20) following from the ANOVA analysis were subsequently manually annotated (for exocarp—Supplemental File S1, Tables S4-S9; for seed— Supplemental File S2, Tables S3-S5; Files available on this link). Table 4.1. Levels of significance ( p values) obtained by ANOVA simultaneous component analysis (ASCA), for the effects of the various growth conditions and their interactions on the exocarp and seed metabolite composition, based on either the LCMS or GCMS analysis, during berry ripening (G—Green, V— Véraison , M— Mature). Significant effects ( p ≤ 0.05) are highlighted by the grey color; interactions that appear insignificant were omitted. The numbers between brackets indicate the total number of significant (FDR-adjusted p ≤ 0.05) metabolites from N-way ANOVA. LCMS Data GCMS Data Exocarp Seed Exocarp Growth Conditions G V M G V M M Soil irrigation - 0.001 (78) 0.002 (48) - 0.016 (30) 0.264 0.147 Kaolin 0.472 0.112 0.165 0.262 0.197 0.145 0.036 (0) Berry microclimate (HL/LL) 0.001 (95) 0.001 (88) 0.001 (154) 0.012 (26) 0.115 0.006 (31) 0.003 (3) Irrigation × Kaolin - 0.044 (0) 0.043 (0) - 0.084 0.194 0.001 (10) It is worth noting that the p -values of the univariate tests were adjusted for multiplicity by the Benjamini–Hochberg false discovery rate (FDR) procedure to control (in expectation) the proportion of false positive differential metabolites. Nevertheless, there is still a chance for false positive results, due to a relatively large number of variables of both the treatments tested and metabolites detected compared to the low number of biological replicates per group sample. Therefore, in the subsequent part we only focus on those significantly differing metabolites with the lowest p -values and for which the size of the effect (i.e., the fold change) was much larger than the overall technical variation for that specific compound (as determined from the quality control samples) (Supplemental Files S1 and S2, Table S1). Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 114 This ASCA approach identified the berry microclimate as the main growth condition influencing the LCMS-metabolites in both berry tissues at all three developmental stages, except for seeds at the véraison stage (Table 4.1), which is in accordance with the PCA results based on these LCMSmetabolites (Figure 4.2). In addition, the ASCA results for the GCMS-metabolites in the mature exocarp indicated that the irrigation treatment itself has no significant impact (cf. Figure S4.6); in contrast, here the berry microclimate has a significant impact, which was undetectable by the PCA model (Figure S4.6). Soil irrigation had a significant impact on the LCMS profiles: for exocarp at both véraison and mature stages and for seeds at the véraison stage only. In contrast, kaolin did not significantly influence the LCMS-profiles at either developmental stage or tissue, while it did significantly impact the GCMS profiles in the mature exocarp, but no significant differences could be shown for the individual metabolites. Previous studies with red grape varieties showed that kaolin application had a positive influence on both phenylpropanoids and flavonoids (Conde et al., 2016), while it only had a minor effect on both free and bound volatile organic compounds in the berries (Song et al., 2012). Our ASCA models also showed a significant interaction effect between irrigation and kaolin on both LCMS and GCMS compounds in the berry exocarp, with the ANOVA model indicating a few significantly differing compounds in the GCMS-profiles only. The size of this interactive effect on individual GCMS compounds was rather small, i.e., less than 40 % change in abundance (Supplemental File S1, Table S4), while the direction of this effect differed between compounds: kaolin application reduced the irrigation-induced increase and decrease in L-alanine and quininic acid, respectively, while the (small but significant) increases in the abundance of several sugars induced by either kaolin or irrigation alone were counteracted when both treatments were applied together (Figure S4.7). Previous studies using whole red grape berries did not observe any significant interactive effect of kaolin and irrigation on free and bound volatile compounds (Song et al., 2012), or total soluble solids, total organic acids, anthocyanins and phenolics (Cooley et al., 2008). Altogether it seems likely that the interaction of these two mitigation treatments does not exert a large, if any, effect on the global metabolome of mature grapes in practice. 4.2.1.1. Specific Effects of Microclimate By comparing the fold change (FC) values of the average metabolite abundances in the HL and LL groups, i.e., the intensity ratio between the average of the HL and LL samples irrespective of mitigation treatment, we identified those metabolites that were most strongly affected by microclimate (for exocarps— Supplemental File S1, Tables S5-S7; for seeds— Supplemental File S2, Table S3 and S4; Files available on this link). Overall, the HL exocarps were characterized by a consistently higher level of several Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 115 flavonol conjugates (Figure 4.3), except for isorhamnetin hexoside at the green stage (Figure 4.3f). During berry development, the flavonol conjugates showed differential accumulation patterns (Figure 4.3). On the one hand, the relative intensities of quercetin-3O -rutinoside (rutin) and quercetin 3O -glucuronide decreased in HL exocarps during development, while in LL, exocarps kept their values constant (Figure 4.3a,c). The remaining flavonols showed an increase in intensity during development for both microclimates (Figure 4.3b,d–f). These results suggest that there was a development-specific flavonol composition and that this was significantly influenced by the light microclimate. If the relative abundance values of these six compounds are added up, notwithstanding their potentially differential ionization efficiencies in the MS source, the mean value of the total of these flavonols are significantly higher in HLexocarps than in LL ones at all developmental stages (Figure S4.8a). These results suggest that HL berries had their maximum level of total flavonols already at the green stage and this high level was maintained upon subsequent ripening; in contrast, during ripening of LL berries, their flavonol content was continuously increasing to a level that at the mature stage was still lower than that of HL berries. By using a calibration curve of authentic standard, absolute quantities of the main flavonol quercetin 3O -glucoside were obtained (Figure S4.8b), and these absolute values showed the same pattern as the relative peak values (Figure 4.3b). Green Véraison Mature 0 1.0106 2.0106 3.0106 A,a A,b A,a A,B b A,a B,b Rutin (ID 462) (a) Relative Intensity Green Véraison Mature 0 5.0106 1.0107 1.5107 2.0107 2.5107Quercetin 3-O-glucoside (ID 492) (b) A,a A,b B,a B,b C,a C,b Green Véraison Mature 0 5.0106 1.0107 1.5107 2.0107 2.5107Quercetin 3-O-glucuronide (ID 505) (c) A,a A,b A,a B,b A,a B,b Green Véraison Mature 0 1.0106 2.0106 3.0106 4.0106Kaempferol-3-glucoside (ID 545) (d) A,a A,b B,a B,b C,a C,b Relative Intensity Green Véraison Mature 0 2.0105 4.0105 6.0105 Kaempferol glucuronide (ID 588) (e) A,a A,b B,a B,b C,a C,b Green Véraison Mature 0 2.0104 4.0104 6.0104 Isorhamnetin hexoside (ID 583) (f) A,a A,a A,a B,b B,a B,b Exocarp LL HL Figure 4.3. Relative intensities of the six main flavonol compounds as detected by LCMS (means and SD of n = 8–12) for exocarp tissue at two canopy microclimates (LL and HL; independent of mitigation treatment) and at three developmental stages (green, véraison and mature). The flavonols considered are: (a) rutin (quercetin-3O -rutinoside) (ID 462), (b) quercetin 3O -glucoside (ID 492), (c) quercetin 3O -glucuronide (ID 505), (d) kaempferol3-glucoside (ID 545), (e) kaempferol glucuronide (ID 588) and (f) isorhamnetin hexoside (ID 583) (Supplemental File S1, Tables S5-S7). Statistical analysis (two-way ANOVA, p ≤ 0.05) was applied after data Log2 transformation. Statistical notation above the bars: the capital letters refer to differences between developmental stages for the Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 116 same microclimate, while the lowercase letters refer to differences between the two light microclimates for each stage. The increase in flavonols by HL compared to LL is in accordance with previous reports on the microclimate effects on both white grape berries (du Plessis et al., 2017; Friedel et al., 2015; Joubert et al., 2016) and red varieties (Koyama et al., 2012; Reshef et al., 2018). This increase, especially relevant in the green stage when the total amount of flavonols peaks in exposed clusters, had a parallel with the increased photosynthetic activity of exocarps under HL conditions in the green stage (Garrido et al., 2019). Flavonols are generally considered to have antioxidant and/or “sunscreen” abilities, thereby protecting the photosynthetic apparatus as well as other macromolecules from excess solar radiation in situ (Agati and Tattini, 2010). Thus, the higher levels of flavonols in HL exocarps may represent an acclimation response to the higher intensity light microclimate, possibly to protect their photosynthetic system from radiation-mediated oxidative damage (Joubert et al., 2016), and therefore keeping its photosynthetic activity until the later stages of development (Garrido et al., 2019). In HL exocarps, we also observed higher levels of some putatively annotated glycosylated aroma compounds, such as a vanillyl alcohol hexoside (ID 136) and geraniol-hexose-pentose (ID 685), as compared to LL berries (Supplemental File S1, Tables S6 and S7). Another class of grape flavonoids, the flavan-3-ols or procyanidins, comprising both monomers and a range of oligomers/polymers of (epi)catechin and (epi)gallocatechins, are key to wine quality as they confer astringency and bitterness (Ma et al., 2014). In addition, they protect the plant and its fruits against pathogens, pest insects and herbivores (Barbehenn and Peter Constabel, 2011). Absolute quantities of six selected flavan-3-ols were obtained by using calibration curves of authentic standards; since their abundance patterns were more or less similar across samples (data not shown), their levels were added up to calculate total monomers and total procyanidins (Figure 4.4). At the green stage, HL berry exocarps had significantly higher contents of total flavan-3-ols, i.e., both (epi)catechin monomers (Figure 4.4a) and procyanidin dimers (Figure 4.4b), compared with LL ones (Figure 4.4a). During ripening, these contents decreased in both microclimates, but more so in HL than in LL. In addition, the HL microclimate led to an up-regulation of several other compounds including flavan-3-ols in the berry exocarp, when compared to LL; at the green stage, the HL exocarps contained more hydroxy-procyanidin trimers (e.g., ID 147 and ID 151) and a procyanidin conjugate (e.g., ID 310) (Supplemental File S1, Table S5). However, at the mature stage, the flavan-3-ols monomers, e.g., (+)-catechin (ID 206), dimers e.g., procyanidin B1 (ID 174) and trimers, (e.g., ID 215) were lower in HL exocarps (Supplemental File S1, Table S7). The analysis Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 117 by GCMS confirmed that mature exocarps accumulated less catechin monomers in HL than in LL conditions (ID 13276, FC HL/LL = 0.8). Figure 4.4. Total flavanols in exocarp tissue at two canopy microclimates (LL and HL; independent of mitigation treatment) and at three developmental stages (green, véraison and mature). (a) Total flavan-3-ols monomers levels (mg/g of dry weight (DW)): catechin, epicatechin, epicatechin-3O -gallate. (b) Total procyanidin dimer levels (µg/g DW): procyanidin B1, B2 and B3. Statistical analysis with two-way ANOVA ( n = 8–12, +SD, p ≤ 0.05). Statistical notation is the same as in Figure 4.3. There are yet unexplained and conflicting results reported on the influence of light on the accumulation of flavan-3-ols in white grape berries. On the one hand, one study showed that the total flavan-3-ol content was affected neither by shading treatments nor by more incoming light due to leaf removal (Friedel et al., 2015). On the other hand, at the green stage, shaded white grapes contained more total flavan-3-ols monomers than exposed ones (du Plessis et al., 2017), while in another study with two different white varieties, the amount of various flavan-3-ols was greater in sun-exposed berries than in shaded ones (Rocchi, 2015), in line with our results at the green stage (Figure 4.4). However, in the skin of red mature grapes, shading resulted in a decrease in flavan-3-ols monomers and, subsequently, in a decreased level of condensed tannins (procyanidins) (Fujita et al., 2007); these results are in contrast to those previously obtained with white grapes and presented here for LL mature berry exocarps (du Plessis et al., 2017) (Figure 4.4). The microclimate also affected the metabolic profile of seeds, at both green and mature stages (Supplemental File S2, Tables S3 and S4). At the green stage, seeds from HL showed an up-regulation of hydroxycinnamic acid compounds, i.e., upstream from the flavonoid pathway (Supplemental File S2, Table S3), including a coumaroyl conjugate (ID 285, FC HL/LL = 4.5) and the lignan-type hydroxycinnamic acid dimer isolariciresinol-β-4′- O -glucopyranoside (ID 613, FC = 2.2) (Figure 4.5). Hydroxycinnamic acids including lignans have been shown to possess antioxidant activity and are associated with the biosynthesis of lignins (Rodríguez-García et al., 2019), which are key in seed lignification (Lewis et al., 1998). In our experiments, the level of the lignan isolariciresinol increased from Green Véraison Mature 0.0 0.2 0.4 0.6 0.8 1.0 2.0 2.5 3.0 3.5 A,a A,b B,a C,b B,a B,a Total Monomers (mg of standard/g of samples DW) Green Véraison Mature 0 20 40 60 80 100 A,a A,b B,a C,b B,a B,a Total Procyanidins (ug of standard/g of samples DW) (a) LL HL (b) Total Flavan-3-ols Monomers Exocarp Total Procyanidin dimers Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 118 the green to véraison stage and then its abundance was maintained up to the mature stage (Figure 4.5b); this pattern is in agreement with the degree of lignification of grape seeds (Cadot et al., 2006). Coumaroyl conjugate Green Véraison Mature 0 4000 8000 30,000 40,000 A,a A,b B,a A,a B,a A,a (a) LL HL Coumaroyl conjugate (relative intensity) Green Véraison Mature 0 2000 4000 20,000 30,000 40,000 A,a A,b B,a B,a B,a B,a Lignan (b) Isolariciresinol--4'-O-glucopyranoside (Relative intensity) Seed Figure 4.5. Examples of seed hydroxycinnamic acids, (a) a coumaroyl conjugate (ID 285) and (b) a lignan type—isolariciresinol-β-4′- O -glucopyranoside (ID 613) detected by LCMS (mean values + SD, n = 8–12) significantly differing between canopy microclimates (LL and HL; independent of mitigation treatment) and at three developmental stages (green, véraison and mature). Statistical analysis (two-way ANOVA, p ≤ 0.05) was applied after data Log2 transformation. Statistical notation is the same as in Figure 4.3. The HL microclimate, as compared to LL, also led to a higher relative abundance of compounds from the flavonoid pathway itself, specifically flavan-3-ols in seeds at the green stage, including a procyanidin pentamer (ID 317, FC = 2.2), a (epi)gallocatechin-conjugate (ID 162, FC = 1.7), a pentahydroxyflavan dimer (ID 170, FC = 1.6) and the gallocatechin (ID 229, FC = 1.5) (Supplemental File S2, Table S3). In contrast, mature HL seeds accumulated less stilbene derivatives, such as (+)-alphaviniferin-hexoside (ID 990, FC = 0.8) and viniferin 3″-glucoside (ID 1027, FC = 0.7) (Figure S4.9). Stilbenes are an effective response against pathogen infection and abiotic stress and contribute to the final nutraceutical quality of both seeds and wine (Ananga et al., 2017; Duarte et al., 2020; Hasan and Bae, 2017). To our knowledge, the effect of light conditions on stilbenes in grape seeds has not been reported before. 4.2.1.2. Specific Effects of Irrigation The irrigation of the soil resulted in an up-regulation of thonningianin B (ID 521, putatively identified), a tannin-type of compound, in the exocarp at both the véraison and mature stage (Figure 4.6a) (Supplemental File S1, Tables S8 and S9). In seeds, at the véraison stage, the irrigation led to an accumulation of several flavan-3-ols such as gallocatechin (ID 229, Figure 4.6b) and an (epi)gallocatechinconjugate (ID 162) (Supplemental File S2, Table S5). Additionally, at the véraison stage, the irrigation resulted in a down-regulation of primary metabolites, including: D-fructose 1,6-bisphosphate (ID 79) and arginine (ID 77) in exocarps (Figure S4.10a,b, Supplemental File S1, Table S8) and both a hexose sugar Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 119 (ID 21) and the phenylpropanoid coutaric acid (ID 315) in seeds (Figure S4.10c,d, Supplemental File S2, Table S5). On the other hand, in exocarps at the mature stage, the irrigation treatment resulted in a lower accumulation of a hydroxy-procyanidin trimer (ID 151), a (epi)catechin-gallocatechin dimer (ID 122) and (+)-gallocatechin (ID 131) (Supplemental File S1, Tables S9). Véraison Mature 0 2.0104 4.0104 6.0104 A,a A,b Tannin (ID 521) A,a B,b (a) Tannin (Relative intensity) Véraison Mature 0 1.0105 2.0105 3.0105Gallocatechin (ID 229) A,a A,b B,a B,a (b) Gallocatechin (Relative Intensity) Non-Irrigation Irrigation Exocarp Seed Figure 4.6. Relative abundance of (a) a tannin (ID 521) in the exocarp and (b) for gallocatechin (ID 229) in the seed, as obtained by LCMS analysis (means + SD, n = 8–12). Grape berries were grown under non-irrigation (grey bars) and irrigation conditions (blue bars) and collected at two developmental stages ( véraison and mature). Data of HL and LL berries, combined. Statistical test: two-way ANOVA, p ≤ 0.05. Statistical notation above the bars: the capital letters refer to differences between developmental stages for the same treatment condition, while the lowercase letters refer to differences between the control and irrigation treatment, within each developmental stage. Genebra et al. (2014), likewise, showed that seeds from irrigated grapevines at full maturation had higher flavan-3-ols and tannins contents compared to non-irrigated ones, which was explained by a slower berry ripening upon irrigation. In fact, Castellarin et al., (2007) argued that water deficiency accelerates the ripening of grape berries. In another study, Koundouras et al. (2009) showed that the total amount of flavan-3-ols in the seed (per fresh weight) was higher in fully irrigated vines compared to non-irrigated ones, likely due to the effect of the more vigorous canopy growth on the berry microclimate. In addition, the skins and seeds of berries from fully irrigated plants of the red Syrah variety tasted more astringent than those from non-irrigated ones, which was attributed to the higher levels of various flavan-3-ol-type of polyphenols (Kyraleou et al., 2016). The present and previous studies thus indicate that irrigation can delay berry ripening and thereby result in the ripening-dependent decrease in flavan-3-ols (e.g., Figure 4.4 and Figure 4.6), which may have an effect on quality traits of both grape seed and wine. Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 126 mitigation effects in the grape exocarp, which is photosynthetically active, specifically. Figure 4.11 shows the levels of the three main individual sugars, as well as their summed values (i.e., here referred to as total sugar content) in the exocarp of the mature grapes. LL HL 0 2 4 6 8 10 a b a,b a,b Glucose (a) Concentration (mg of standard/g sample DW) LL HL 0 2 4 6 8 10 ab a,b a,b Fructose (b) LL HL 0 1 2 6 8 10 Sucrose (c) Concentration (mg of standard/g sample DW) LL HL 0 2 4 6 8 10 a b a,b a,b Total Sugar Content (d) Non-Kaolin, Non-Irrigation (NK, NI) Non-Kaolin, Irrigation (NK, I) Kaolin, Non-Irrigation (K, NI) Kaolin, Irrigation (K, I) Figure 4.11. Levels of main sugars (in mg/g sample DW, mean values n = 3–4, +SD) in the exocarp of mature berries grown in either low light (LL) or high light (HL) microclimate conditions in the canopy: (a) glucose, (b) fructose, (c) sucrose and (d) their summed values, in this paper referred to as total sugar content. Kaolin (K) or no kaolin (NK) was applied to the plant leaves before fruit set; plants were irrigated (I) or non-irrigated (NI) after the green stage. Statistical notations above the bars: the lowercase letters refer to differences between treatment combinations within the same light microclimate (values with a common letter or no letter at all, indicate no significant differences; two-way ANOVA, p ≤ 0.05). Under untreated control conditions (NK, NI), HL exocarps had sugar levels, similar to LL exocarps, while with kaolin only (K, NI), they contained significantly more glucose (Figure 4.11a; +37 %) and fructose (Figure 4.11b; +32 %), and consequently 30 % more total sugars (Figure 4.11d). Since the mature exocarps of HL and LL berries showed similar photosynthetic capacities (Garrido et al., 2019), we assume that this positive effect of kaolin on exocarp sugar content is related to the higher sugar biosynthesis in the grape leaves, leading to a higher import into the berries, including the exocarp, and/or to the higher biosynthesis from the accumulated malate that is used to produce sugars from the véraison stage, onwards. Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 127 4.3. Materials and Methods 4.3.1. Grapevine Field Conditions and Sampling Grape berry samples were collected from Alvarinho cultivar grapevines ( Vitis vinifera L.) grown in a field trial conducted in 2018 in the organic vineyard Quinta Cova da Raposa in the Demarcated Region of Vinho Verde, Braga, Portugal (41°34′16.4″ N 8°23′42.0″ W). Details concerning the vineyard and treatments are described in Garrido et al. (2019). Briefly, all treatments—kaolin (K) and non-kaolin (NK) application on leaves, and irrigation (I) and non-irrigation (NI) of grapevines—were applied in a complete factorial design in two blocks, each with three to four vines per treatment. The kaolin suspension (5 % w / v in water) was applied on both 6 July and 27 July 2018, corresponding to four and seven weeks after anthesis (WAA), respectively. Irrigation of half of the plants started on 26 July (seven WAA) by means of drip irrigation (one dripper per vine with an average capacity of 5.5 ± 1.6 L h−1) for 2 h every three days. Grape clusters with contrasting light exposures in the canopy, called low light (LL) and high light (HL) microclimates, were collected randomly at each experimental condition and at three distinct developmental stages; green (16 July, 6 WAA), véraison (29 August, 12 WAA), and mature (17 September) berries were immediately frozen in liquid nitrogen and stored at −80 °C. The exocarp (=skin) and seeds were isolated from the whole frozen grape berries. Firstly, the berry was broken with a slight impact of a pestle in a mortar (both pre-cooled with liquid nitrogen), which allowed us to isolate the seeds. Secondly, the exocarps were dissected from the remaining frozen berry fragments in a petri dish placed in a box with ice, and then quickly transferred to a liquid nitrogen cooled falcon tube. Finally, both seeds and exocarp pieces were ground to a fine powder, using a mortar, a pestle and liquid nitrogen, and freezedried for 48 h before metabolomic analyses. At each ripening stage, we sampled berries from 3 (both véraison and mature) or 4 (green) biological replicates for each condition, in which 1 replicate resembled a mix of 5 to 10 berries from 3 to 5 clusters from 6 to 8 plants, resulting in a total of 128 samples. All dried samples, conditioned in boxes with silica to maintain the dehydration, were transported to Wageningen, the Netherlands, in order to analyze them by complementary targeted and untargeted metabolomics platforms. 4.3.2. Untargeted Metabolomics by Liquid Chromatography Mass Spectrometry (LCMS) and Gas Chromatography Mass Spectrometry (GCMS) 4.3.2.1. LCMS Analysis All 128 samples were used and extracted according to De Vos et al. (2007). Quality control (QC) samples ( n = 5, per each batch analysis) were also prepared with a mix of the grape berry tissues in Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 128 order to estimate the overall technical variation per compound. In short, 20 mg dry weight (DW) grape berry tissue was transferred to 2 mL plastic safe-lock Eppendorf tubes and extracted with 600 μL of 75 % ( v / v ) methanol/water + 0.1 % formic acid (FA). After vortexing (10 s) and sonication (15 min) (these steps were performed twice), samples were centrifuged (16,100× g ) for 15 min and the supernatant was collected. Chromatographic separation (5 µL of in injection) was performed on an HPLC system (Waters Acquity, Milford, MA, USA) with a C18 column (Phenomenex Luna 150 × 2 mm i.d., 3 µm—Torrance, CA, USA) using ultra-pure water (eluent A) and acetonitrile (eluent B) both acidified with 0.1 % FA at a flow rate of 0.19 mL min−1, starting with 5% B and increasing linearly to 35 % B in 45 min, followed by 15 min of re-equilibration at 5 % B. The column was kept at 40 °C and detection was done with both a PDA detector (Waters) at 210–600 nm and an LTQ-Orbitrap FTMS hybrid mass spectrometer (Thermo Scientific, Bremen, Germany) in negative ionization mode. A mass resolution of 60,000 FWHM was employed for data acquisition. Eluting compounds were detected in full-scan mode in the m / z range of 90–1350. Separate LCMS/MS runs were performed by re-injecting a random set of extracts, using datadependent acquisition in discovery mode by selecting the 3 most intense ions per full scan for fragmentation up to MS3. Some selected phenolic compounds were identified or quantified using authentic standards: procyanidin B1, B2 (Extrasynthese®, Genay Cedex, France) and B3 (APIN Chemicals Ltd.®, Compton, United Kingdom), catechin (APIN Chemicals Ltd.®), epicatechin (Sigma®, Zwijndrecht, the Netherlands), epicatechin-3O -gallate (Extrasynthese®), quercetin-3-glucoside (Fluka®, Munich, Germany), piceid (APIN Chemicals Ltd.®) and resveratrol (Sigma®). 4.3.2.2. GCMS Analysis For the analysis of polar (primary) compounds, we used an untargeted GCMS platform. In view of limited sample amounts, we only analyzed the 24 exocarp samples from the mature stage. Extraction was according to the protocol described by Carreno-Quintero et al. (2012). Briefly, 10 mg of dry weight powder was extracted with 1.4 mL of methanol/water 75 % ( v / v ) containing 8 µg mL−1 of ribitol (Sigma®) as the internal standard. After sonication and centrifugation, 500 µL of the supernatant was mixed with 375 µL of chloroform (−20 °C) and 750 µL of distilled water (4 °C). After a new centrifugation, aliquots (50 µL) of the upper (polar) phase were transferred to an insert placed in a 2 mL vial. All samples were dried overnight (16 h) by vacuum centrifugation (Savant®, SPD121P, Thermo Scientific) at room temperature and the vials were closed under an argon atmosphere using magnetic crimp caps. Prior to analysis, dried samples were derivatized online using a TriPlusRSH autosampling/injection robot (Thermo Scientific) (Carreno-Quintero et al., 2012; Lisec et al., 2006). First, 12.5 µL of o-methylhydroxylamine Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 129 hydrochloride (20 mg mL−1 pyridine) was added to the samples and incubated for 30 min at 40 °C with agitation. Then, the samples were derivatized with 17.5 µL of N -methylN -trimethylsilyltrifluoroacetamide (MSTFA) for 60 min. An alkane mixture (C10-C32) was added to determine the retention indices of metabolites. The derivatized samples were analyzed by a GCMS system consisting of a Trace 1300 gas chromatograph (Thermo Scientific) with a PTV injector coupled to a TSQ8000 DUO-series triple quadrupole mass spectrometer (Thermo Scientific). One microliter of each sample was introduced to the injector at 70 °C using a split flow of 19 mL min−1. Chromatographic separation was performed using a VF-5ms capillary column (Varian, Palo Alto, CA, USA; 30 m × 0.25 mm × 0.25 mm) including a 10 m guardian column with helium as the carrier gas at a column flow rate of 1 mL min−1. The column effluent was ionized by electron impact at 70 eV. Mass spectra were acquired at a combined SRM and full scan mode with a m / z range of 50 to 600 at an ion source temperature of 290 °C. A solvent delay of 420 s was set. 4.3.2.3. Untargeted Data Processing and Multivariate Statistical Analysis Unbiased mass peak picking and alignment of the raw data sets from LCMS and GCMS were carried out separately for each tissue using MetAlign software (Lommen, 2009). Irreproducible individual mass signals (present in <3 samples) were filtered out using an in-house script called MetAlign Output Transformer (METOT) (Houshyani et al., 2012). The remaining mass peaks, including molecular ions, insource adducts (in case of LCMS), fragments and their natural isotopes, were subsequently clustered using MSClust software into so-called reconstructed metabolites (centrotypes) (Tikunov et al., 2012) according to their corresponding retention time and peak intensity pattern across samples. In the final LCMS dataset, the total number of non-detects, i.e., below the detection limit of 2500 ion counts per compound, was 12,154 and 14,308 for exocarp and seeds, respectively. These non-detects were subsequently filtered out when not present in all 3 or 4 biological replicates of at least one sample group. The values of the remaining non-detects (3394 and 2459 for exocarp and seeds, respectively) were randomized between 45 % and 55 % of the detection threshold, i.e., between 1125 and 1375. The resulting spreadsheets for exocarps (Supplemental File S1, Table S1, available on this link) and seeds (Supplemental File S2, Table S1, available on this link) with the relative intensity of each reconstructed metabolite in each sample were used for further statistical analyses. The on-line tool MetaboAnalyst was employed to compare the three developmental stages for each tissue (Xia et al., 2015). The MSClust output was uploaded into this platform and was Log10-transformed and scaled by the Pareto method (mean-centered and divided by the square root of standard deviation of Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 130 each variable). Principal component analysis (PCA) was used as an unsupervised approach. In addition, the heatmap plot was represented based on the 25 top-ranking metabolites according to Analysis of Variance (ANOVA) test. On this test, the p -values were adjusted using a false discovery rate (FDR) correction. A multivariate statistical analysis was carried out using MATLAB software. An ANOVA simultaneous component analysis (ASCA), a common tool for analysis of metabolomics data (Smilde et al., 2005), was applied to the Log-transformed data. The model comprised the following factors: HL vs. LL, K vs. NK, I vs. NI and their interactions. The significance of each factor was assessed by a permutation test using 1000 permutations, and Wilks Lambda as a test statistic (Engel et al., 2015). Separate ASCA models were fitted to the data from each developmental stage to study the influence of the factors of interest on the overall metabolome of grape berry tissues. In addition to the ASCA analysis, the effect of the factors mentioned above on each metabolite was studied in more detail by N-way ANOVA. For each factor of interest, the p -values were adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) approach. Adjusted p -values smaller than 0.05 were considered to be significant. For each factor, the significant metabolites showing the strongest effect (i.e., highest fold change (FC) values, estimated by the ANOVA) were considered for manual annotation. The mass of the molecular ion was manually verified within the clustered mass signals of selected, reconstructed metabolites. Metabolites were then annotated using an in-house metabolite database based on comparisons of retention time, accurate mass and UV spectra, if available. On-line available metabolite databases (e.g., KNApSAcK) and literature on grape analyses were also employed for annotation. In the case of GCMS metabolites, the mass spectrum of each ion cluster was compared with that in available EI-spectral libraries, such as the NIST2014 and the Golm spectral database (Hummel et al., 2010), as well as an in-house library of derivatized standards. In addition, the experimentally obtained RI was compared with reported RIs for verification of the automated spectra annotations. The level of annotation of compounds was performed following the rules described by Sumner et al. (2007), being classified at four levels: identified metabolites by comparison with standards (level 1), putatively annotated compounds (level 2), putatively characterized compound classes (level 3), and unknown compounds (level 4). Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 131 4.3.3. Targeted Analysis 4.3.3.1. Tocopherols The extraction procedure for tocopherols was the same as recently described for chlorophylls and carotenoids (Garrido et al., 2019). Briefly, 20 mg DW of all 128 samples of the grape berry tissues (exocarp or seed) were extracted in 1.8 mL of chloroform/methanol (1:1) with both 0.1 % ( w / v ) butylated hydroxytoluene (BHT) as an antioxidant and Sudan 1 (0.5 µg mL−1, Sigma®) as the internal standard (IS). The samples were vortexed, sonicated and centrifuged. The supernatant was dried for 1 h in a Speedvac and prior to analysis, the dried samples were dissolved in 200 µL ethylacetate containing 0.1 % ( w / v ) BHT, again sonicated and centrifuged, and the final supernatant (180 µL) was transferred to ambercolored 2 mL HPLC vials. Samples (20 μL for injection) were analyzed using an HPLC (Waters Alliance e2695 Separations Module, Milford, MA, USA) coupled to a fluorescence detector (Waters 2475) with excitation at 296 and emission at 340 nm. Separation was performed on a reverse-phase C30 column (250 × 4.6 mm i.d., S-5 μm—YMC Carotenoid, Komatsu, Japan) kept at 35 °C with a flow rate of 1 mL min−1. The three tocopherol species detected (i.e., α-, γand δ-tocopherol) were identified based on comparisons of retention times with authentic standards. Waters Empower 3 software (Waters, Milford, MA, USA) was used for data processing. The total tocopherol content (µg per g of DW tissue) was obtained by adding up the levels of the three detected tocopherols. 4.3.3.2. Sugars All 24 exocarp samples from the mature stage (10 mg DW) were extracted with methanol/water 75 % ( v / v ). After sonification (10 min), followed by centrifugation (10 min) at maximal speed (16,100× g ), the supernatant was transferred to new Eppendorf tubes and stored at −20 °C until use. For the sugar analysis, 20 µL was transferred to plastic vial and dried in a vacuum centrifugation without heating. The residue was resuspended in 0.2 mL MiliQ water and vortexed thoroughly. The residue dissolved in water was injected into a Dionex HPLC system (ICS 5000+DC) to analyze the sugar content, using a CarboPac PA 1, 4 × 250 mm column preceded by a guard column (CarboPac PA 1, 4 × 50 mm). Mono-, di-, and tri-saccharides were separated by elution in an increasing concentration of NaOH (20–350 mM) with a flow rate of 1 mL min−1. Peaks were identified by co-elution of standards. The sugar amount was expressed in mg of sugar per g of dry material. Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 132 4.3.3.3. Total Soluble Phenolics The Folin–Ciocalteu colorimetric method was used for total phenolics quantification in all 24 exocarp and 24 seed samples from the mature stage (Waterhouse, 2003). Ten mg DW were extracted in 300 μL of 75 % ( v / v ) methanol/water with 0.1 % formic acid (FA), and after vortexing (10 s) and sonication (15 min), samples were centrifuged (16,100× g ) for 15 min and the supernatant was collected. After that, 50 μL of extract was added to 300 μL of 10 % ( v / v ) Folin reagent and incubated for 5 min in the dark before adding 300 μL of 6 % ( w / v ) sodium carbonate. After 2 h of incubation in the dark, the absorbance was measured at 765 nm. The phenolic contents were determined using a gallic acid (Sigma®) calibration curve and expressed as mg of gallic acid equivalents [GAE]/g DW tissue. 4.3.3.4. Lipid Peroxidation Products The 16 green exocarp and 12 mature seed samples from the two distinct light microclimates were selected for the analysis of lipid peroxidation products. Ten mg DW were extracted in 800 µL of 0.5 % ( w / v ) 2-thiobarbituric acid (TBA) freshly dissolved in 20 % ( v / v ) trichloroacetic acid (TCA), and 800 µL of water was added. The mixture was vortexed, heated at 95 °C for 30 min in a water bath, cooled on ice and centrifuged at max speed for 10 min. The absorbance of the supernatant was measured at both 532 and 600 nm. Lipid peroxidation product levels were calculated as described by Hodges et al. (1999), and expressed in thiobarbituric acid-reactive-substances (TBARS) per g DW using malondialdehyde (MDA) as a standard. 4.3.3.5. Statistical Analysis Analysis of Variance tests (two-way ANOVA) were applied, followed by post hoc multiple comparisons using the Bonferroni test whenever the factors had significant effects (GraphPad Prism version 5.00 for Windows, GraphPad Software, La Jolla, CA, USA). 4.4. Conclusions We previously showed that both the exocarp and seed of berries from the white grape variety Alvarinho are photosynthetically active and more so in berries exposed to full sunlight (HL microclimate) than in those of shaded locations in the vines (LL microclimate), especially at the green stage of their development (Garrido et al., 2019). Here, we used unbiased, comprehensive LCMSand GCMS-based metabolomics approaches, as well as targeted analyses of selected key compounds for grape/wine quality traits, in order to get more insight into the effects of these contrasting canopy light microclimates, as well Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 133 as into the effects of soil irrigation and foliar kaolin spraying, on the metabolome composition of berry exocarps and seeds. Both strategies are regularly applied in viticulture as potential mitigation against abiotic stress. Our results indicate the significant influence of the microclimate in both photosynthetically active berry tissues, suggesting a potential role for in situ berry photosynthesis in contributing carbonskeletons and energy for the biosynthesis of berry components during development and ripening. More experimental research, e.g., by specifically applying artificial shading and exposing grapes to additional light of specific wavelengths, is needed to get a better understanding of the exact role of berry photosynthesis in the final grape quality. In addition, the foliar kaolin application and especially the irrigation treatment appeared to exert their own or combined additional modulating effects on the metabolome of these two berry tissues, possibly due to their direct or indirect influence on photosynthesis in both the leaves and berries. Several compounds affected by these mitigation treatments are also relevant to viticulture, e.g., modulation of a series of phenolic compounds including monoand polymers of flavan-3-ols, suggesting that good management of these treatments by farmers is necessary and may even further optimize their products by fine-tuning the berry metabolome. Moreover, it is also important to emphasize that this was an exploratory study which aimed to contribute new knowledge and generate hypotheses for future experiments. In addition, for more robust conclusions, more biological replicates and repeating campaigns are helpful in order to possibly link the grape berry metabolite composition to year-to-year variations in wine quality. 4.5. Supplementary Materials The following are available online at: https://www.mdpi.com/article/10.3390/metabo11040205/s1 Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 134 Figure S4.1. Liquid chromatography mass spectrometry (LCMS) chromatograms of exocarp at different berry developmental stages: green (a), véraison (b) and mature (c). Numbers above peaks represent, from top to bottom, the retention time and mass signals, respectively. The three chromatograms are in the same scale. Annotations of compounds, if known, are provided in (Supplemental File S1, Table S1, available on this link). Figure S4.2. LCMS chromatograms of seeds at different berry developmental stages: green (a), véraison (b) and mature (c). Numbers above peaks represent, from top to bottom, the retention time and mass signals, respectively. The three chromatograms are in the same scale. Annotations of compounds, if known, are provided in Supplemental File S2, Table S1, available on this link. RT: 0.00 - 45.00 0 5 10 15 20 25 30 35 40 45 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 Relative Abundance 0 20 40 60 80 100 2.58 133.0144 12.84 295.0461 11.59 289.0719 3.07 191.0199 23.76 477.0680 27.51 389.1248 18.04 577.1359 30.37 435.1093 37.79 493.2294 41.53 499.1403 2.59 133.0144 11.55 289.0719 23.88 449.1093 3.08 191.0199 14.11 435.1302 26.48 447.0936 18.05 577.1359 30.36 435.1091 34.35 161.0458 39.78 161.0457 2.30 133.0144 23.31 463.0886 12.83 295.0461 3.07 191.0200 26.49 447.0937 22.74 463.0888 17.68 366.1198 34.83 493.2299 36.81 161.0458 41.05 161.0458 NL: 1.90E7 Base Peak F: FTMS - c ESI Full ms [90.00-1350.00] MS F026181 NL: 1.90E7 Base Peak F: FTMS - c ESI Full ms [90.00-1350.00] MS f026151 NL: 1.90E7 Base Peak F: FTMS - c ESI Full ms [90.00-1350.00] MS f026172 (a) Green (b) Véraison (c) Mature RT: 0.00 - 45.00 0 5 10 15 20 25 30 35 40 45 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 Relative Abundance 0 20 40 60 80 100 2.30 133.0144 17.98 729.1462 22.02 441.0827 11.57 289.0718 9.84 577.1355 23.31 652.1336 27.41 729.1461 34.71 151.0401 37.71 151.0400 40.84 151.0400 11.55 289.0717 15.17 289.0717 2.10 387.1145 2.29 149.0092 21.99 441.0828 10.79 577.1353 17.98 729.1461 3.07 191.0196 23.29 652.1332 27.42 729.1455 30.58 439.1032 35.88 465.1187 40.00 629.2024 2.09 387.1145 11.57 289.0717 15.16 289.0718 2.29 149.0092 22.04 441.0829 10.83 577.1353 16.80 865.1985 27.40 729.1458 29.71 440.0749 35.90 465.1190 38.07 151.0400 NL: 1.90E7 Base Peak F: FTMS - c ESI Full ms [90.00-1350.00] MS f026301 NL: 1.90E7 Base Peak F: FTMS - c ESI Full ms [90.00-1350.00] MS f026304 NL: 1.90E7 Base Peak F: FTMS - c ESI Full ms [90.00-1350.00] MS f026310 (a) Green (b) Véraison (c) Mature Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 135 Figure S4.3. Heatmaps with the most significant LCMS-metabolites affected by developmental stages in exocarp (a) and seed (b). Heatmap plot with the 25 top-ranking metabolites from Analysis of Variance (ANOVA). Metabolites are represented as numbers on the right-hand side of the plot, using the metabolite number (metabolite ID) obtained from the data processing. Developmental stages are represented from left to right side of the plot by order, as well as, the replicates for low and high light (LL and HL, respectively) grape berries grown under the four combinations of the two treatments applied: irrigation (I)/ non-irrigation (NI) x kaolin (K)/non-kaolin (NK). Figure S4.4. Principal component analysis (PCA) score plots of the liquid chromatography mass spectrometry (LCMS) metabolite data for exocarp and seed at véraison stage, including all microclimates and treatments ( n = 3). Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 142 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—ref. POCI-01-0145-FEDER-028165 and ref. PTDC/BIAFBT/28165/2017, respectively. Artur Conde was supported by a post-doctoral researcher contract/position within the project “MitiVineDrought” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145FEDER-030341). This work also benefited from the networking activities within the European Unionfunded COST Action CA17111“INTEGRAPE—Data Integration to maximize the power of omics for grapevine improvement”. Authors acknowledge the owner from Quinta Cova da Raposa, Manuel Taxa, who provided the samples, and also all support given by the Biology Department of the School of Sciences from the University of Minho. The authors would also like to acknowledge Bert Schipper, Henriëtte van Eekelen and Jeroen van Arkel (all Plant Metabolomics group of WUR-Bioscience) for their help in the chemical analyses and data processing. 4.8. References Agati, G., & Tattini, M. (2010). Multiple functional roles of flavonoids in photoprotection. New Phytologist , 186 (4), 786–793. Ali, K., Maltese, F., Choi, Y. H., & Verpoorte, R. (2010). Metabolic constituents of grapevine and grapederived products. Phytochemistry Reviews , 9 (3), 357–378. Ananga, A., Obuya, J., & Ochieng, J. (2017). Grape Seed Nutraceuticals for Disease Prevention: Current and Future Prospects. In Phenolic Compounds-Biological Activity (pp. 119–137). Barbehenn, R. V., & Peter Constabel, C. (2011). Tannins in plant-herbivore interactions. Phytochemistry , 72 (13), 1551–1565. Bernardo, S., Dinis, L. T., Luzio, A., Pinto, G., Meijón, M., Valledor, L., Moutinho-Pereira, J. (2017). Kaolin particle film application lowers oxidative damage and DNA methylation on grapevine ( Vitis vinifera L.). Environmental and Experimental Botany , 139 , 39–47. Bernardo, Sara, Dinis, L. T., Machado, N., & Moutinho-Pereira, J. (2018). Grapevine abiotic stress assessment and search for sustainable adaptation strategies in Mediterranean-like climates. A review. Agronomy for Sustainable Development . Agronomy for Sustainable Development. Blancquaert, E. H., Oberholster, A., Ricardo-da-Silva, J. M., & Deloire, A. J. (2019). Effects of abiotic factors on phenolic compounds in the grape berry - A review. South African Journal of Enology and Viticulture , 40 (1), 1–14. Bouhamidi, R., Prévost, V., & Nouvelot, A. (1998). High protection by grape seed proanthocyanidins (GSPC) of polyunsaturated fatty acids against UV-C induced peroxidation. Comptes Rendus de l’Académie Des Sciences - Series III - Sciences de La Vie , 321 (1), 31–38. Brazel, A. J., & Ó’Maoileídigh, D. S. (2019). Photosynthetic activity of reproductive organs. Journal of Experimental Botany , 70 (6), 1737–1753. Breia, R., Vieira, S., Da Silva, J. M., Gerós, H., & Cunha, A. (2013). Mapping grape berry photosynthesis by chlorophyll fluorescence imaging: The effect of saturating pulse intensity in different tissues. Photochemistry and Photobiology , 89 (3), 579–585. Cadot, Y., Miñana-Castelló, M. T., & Chevalier, M. (2006). Anatomical, Histological, and Histochemical Changes in Grape Seeds from Vitis vinifera L. cv Cabernet franc during Fruit Development. Journal of Agricultural and Food Chemistry , 54 (24), 9206–9215. Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 143 Caravia, L., Collins, C., Petrie, P. R., & Tyerman, S. D. (2016). Application of shade treatments during Shiraz berry ripening to reduce the impact of high temperature. Australian Journal of Grape and Wine Research , 22 (3), 422–437. Carreno-Quintero, N., Acharjee, A., Maliepaard, C., Bachem, C. W. B., Mumm, R., Bouwmeester, H., Keurentjes, J. J. B. (2012). Untargeted metabolic quantitative trait loci analyses reveal a relationship between primary metabolism and potato tuber quality. Plant Physiology , 158 (3), 1306–1318. Castellarin, S. D., Matthews, M. A., Di Gaspero, G., & Gambetta, G. A. (2007). Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries. Planta , 227 (1), 101–112. Chidi, B. S., Bauer, F. F., & Rossouw, D. (2018). Organic acid metabolism and the impact of fermentation practices on wine acidity - A review. South African Journal of Enology and Viticulture , 39 (2), 1–15. Cholet, C., Claverol, S., Claisse, O., Rabot, A., Osowsky, A., Dumot, V., Gény, L. (2016). Tartaric acid pathways in Vitis vinifera L. (cv. Ugni blanc): A comparative study of two vintages with contrasted climatic conditions. BMC Plant Biology , 16 (1), 1–18. Cocaliadis, M. F., Fernández-Muñoz, R., Pons, C., Orzaez, D., & Granell, A. (2014). Increasing tomato fruit quality by enhancing fruit chloroplast function. A double-edged sword? Journal of Experimental Botany , 65 (16), 4589–4598. Conde, A., Neves, A., Breia, R., Pimentel, D., Dinis, L. T., Bernardo, S., Moutinho-Pereira, J. (2018). Kaolin particle film application stimulates photoassimilate synthesis and modifies the primary metabolome of grape leaves. Journal of Plant Physiology , 223 , 47–56. Conde, A., Pimentel, D., Neves, A., Dinis, L.-T., Bernardo, S., Correia, C. M., Moutinho-Pereira, J. (2016). Kaolin Foliar Application Has a Stimulatory Effect on Phenylpropanoid and Flavonoid Pathways in Grape Berries. Frontiers in Plant Science , 7 (August), 1–14. Conde, C., Agasse, A., Glissant, D., Tavares, R., Gerós, H., & Delrot, S. (2006). Pathways of glucose regulation of monosaccharide transport in grape cells. Plant Physiology , 141 (4), 1563–1577. Conde, C., Silva, P., Fontes, N., Dias, A. C. P., Tavares, R. M., Sousa, M. J., Gerós, H. (2007). Biochemical changes throughout grape berry development and fruit and wine quality. Food , 1 , 1–22. Cooley, N. M., Glenn, D. M., Clingeleffer, P. R., & Walker, R. R. (2008). The Effects of Water Deficit and Particle Film Technology Interactions on Cabernet Sauvignon Grape Composition. Acta Horticulturae , (792), 193–200. Dai, Z. W., Léon, C., Feil, R., Lunn, J. E., Delrot, S., & Gomès, E. (2013). Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry ( Vitis vinifera L.), a nonclimacteric fleshy fruit. Journal of Experimental Botany , 64 (5), 1345–1355. De Vos, R. C. H., Moco, S., Lommen, A., Keurentjes, J. J. B., Bino, R. J., & Hall, R. D. (2007). Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nature Protocols , 2 (4), 778. Degu, A., Hochberg, U., Sikron, N., Venturini, L., Buson, G., Ghan, R., Fait, A. (2014). Metabolite and transcript profiling of berry skin during fruit development elucidates differential regulation between Cabernet Sauvignon and Shiraz cultivars at branching points in the polyphenol pathway. BMC Plant Biology , 14 (1), 1–20. Deluc, L. G., Grimplet, J., Wheatley, M. D., Tillett, R. L., Quilici, D. R., Osborne, C., Cramer, G. R. (2007). Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development. BMC Genomics , 8 (1), 429. Deluc, L. G., Quilici, D. R., Decendit, A., Grimplet, J., Wheatley, M. D., Schlauch, K. A., Cramer, G. R. (2009). Water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay. BMC Genomics , 10 (1), 212. Dinis, L. T., Bernardo, S., Conde, A., Pimentel, D., Ferreira, H., Félix, L., Moutinho-Pereira, J. (2016). Kaolin exogenous application boosts antioxidant capacity and phenolic content in berries and leaves Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 144 of grapevine under summer stress. Journal of Plant Physiology , 191 , 45–53. Dinis, L. T., Ferreira, H., Pinto, G., Bernardo, S., Correia, C. M., & Moutinho-Pereira, J. (2016). Kaolinbased, foliar reflective film protects photosystem II structure and function in grapevine leaves exposed to heat and high solar radiation. Photosynthetica , 54 (1), 47–55. Dinis, L. T., Malheiro, A. C., Luzio, A., Fraga, H., Ferreira, H., Gonçalves, I., Moutinho-Pereira, J. (2018). Improvement of grapevine physiology and yield under summer stress by kaolin-foliar application: water relations, photosynthesis and oxidative damage. Photosynthetica , 56 (2), 641–651. Downey, M. O., Harvey, J. S., & Robinson, S. P. (2003). Synthesis of flavonols and expression of flavonol synthase genes in the developing grape berries of Shiraz and Chardonnay ( Vitis vinifera L.). Australian Journal of Grape and Wine Research , 9 (2), 110–121. du Plessis, K., Young, P. R., Eyéghé-Bickong, H. A., & Vivier, M. A. (2017). The transcriptional responses and metabolic consequences of acclimation to elevated light exposure in grapevine berries. Frontiers in Plant Science , 8 (July). Duarte, N., Ramalhete, C., Rijo, P., Reis, M. A., & Ferreira, M.-J. U. (2020). Stilbenoids in Grapes and Wine. In Handbook of Dietary Phytochemicals (pp. 1–28). Duchêne, E. (2016). How can grapevine genetics contribute to the adaptation to climate change? OENO One , 50 (3), 113–114. Engel, J., Blanchet, L., Bloemen, B., Van den Heuvel, L. P., Engelke, U. H. F., Wevers, R. A., & Buydens, L. M. C. (2015). Regularized MANOVA (rMANOVA) in untargeted metabolomics. Analytica Chimica Acta , 899 , 1–12. Estreicher, S. K. (2017). The beginning of wine and viticulture. Physica Status Solidi (C) Current Topics in Solid State Physics , 14 (7). Fraga, H., García de Cortázar Atauri, I., & Santos, J. A. (2018). Viticultural irrigation demands under climate change scenarios in Portugal. Agricultural Water Management , 196 , 66–74. Fraga, H., Malheiro, A. C., Moutinho-Pereira, J., Jones, G. V., Alves, F., Pinto, J. G., & Santos, J. A. (2014). Very high resolution bioclimatic zoning of Portuguese wine regions: Present and future scenarios. Regional Environmental Change , 14 , 295–306. Fraga, H., Malheiro, A. C., Moutinho-Pereira, J., & Santos, J. A. (2013). Future scenarios for viticultural zoning in Europe: ensemble projections and uncertainties. International Journal of Biometeorology , 57 (6), 909–925. Fraga, Helder, Pinto, J. G., & Santos, J. A. (2019). Climate change projections for chilling and heat forcing conditions in European vineyards and olive orchards: a multi-model assessment. Climatic Change , 152 (1), 179–193. Friedel, M., Stoll, M., Patz, C. D., Will, F., & Dietrich, H. (2015). Impact of light exposure on fruit composition of white “Riesling” grape berries ( Vitis vinifera L.). Vitis - Journal of Grapevine Research , 54 (3), 107–116. Fujita, A., Soma, N., Goto-Yamamoto, N., Mizuno, A., Kiso, K., & Hashizume, K. (2007). Effect of shading on proanthocyanidin biosynthesis in the grape berry. Journal of the Japanese Society for Horticultural Science , 72 (2), 112–119. Garrido, A., Breia, R., Serôdio, J., & Cunha, A. (2018). Impact of the Light Microclimate on Photosynthetic Activity of Grape Berry ( Vitis vinifera ): Insights for Radiation Absorption Mitigations’ Measures. In Theory and Practice of Climate Adaptation (pp. 419–441). Springer, Cham. Garrido, A., Serôdio, J., Vos, R. De, Conde, A., & Cunha, A. (2019). Influence of foliar kaolin application and irrigation on photosynthetic activity of grape berries. Agronomy , 9 (11), 685. Garrido, J., & Borges, F. (2013). Wine and grape polyphenols - A chemical perspective. Food Research International , 54 (2), 1844–1858. Genebra, T., Santos, R. R., Francisco, R., Pinto-Marijuan, M., Brossa, R., Serra, A. T., Zarrouk, O. (2014). Proanthocyanidin accumulation and biosynthesis are modulated by the irrigation regime in Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 145 tempranillo seeds. International Journal of Molecular Sciences , 15 (7), 11862–11877. Hasan, M. M., & Bae, H. (2017). An overview of stress-induced resveratrol synthesis in grapes: Perspectives for resveratrol-enriched grape products. Molecules , 22 (2), 294. Hassanein, M. M. M., & Abedel-Razek, A. G. (2009). Chromatographic quantitation of some bioactive minor components in oils of wheat germ and grape seeds produced as by-products. Journal of Oleo Science , 58 (5), 227–233. Hodges, D. M., DeLong, J. M., Forney, C. F., & Prange, R. K. (1999). Improving the thiobarbituric acidreactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta , 207 (4), 604–611. Houshyani, B., Kabouw, P., Muth, D., de Vos, R. C. H., Bino, R. J., & Bouwmeester, H. J. (2012). Characterization of the natural variation in Arabidopsis thaliana metabolome by the analysis of metabolic distance. Metabolomics , 8 (1), 131–145. Hummel, J. Strehmel, N., Selbig, J., Walther, D. and Kopka, J. (2010). Decision tree supported substructure prediction of metabolites from GC-MS profiles. Metabolomics , 6 (2), 322–333. Jeandet, P., Bessis, R., & Gautheron, B. (1991). The production of resveratrol (3, 5, 4’-trihydroxystilbene) by grape berries in different developmental stages. American Journal of Enology and Viticulture , 42 (1), 41–46. Jordão, A. M., Ricardo-da-Silva, J. M., & Laureano, O. (2001). Evolution of catechins and oligomeric procyanidins during grape maturation of Castelão Francês and Touriga Francesa. American Journal of Enology and Viticulture , 52 (3), 230–234. Joubert, C., Young, P. R., Eyéghé-Bickong, H. A., & Vivier, M. A. (2016). Field-grown grapevine berries use carotenoids and the associated xanthophyll cycles to acclimate to UV exposure differentially in high and low light (Shade) conditions. Frontiers in Plant Science , 7 , 786. Kadir, S., Von Weihe, M., & Al-Khatib, K. (2007). Photochemical Efficiency and Recovery of Photosystem II in Grapes After Exposure to Sudden and Gradual Heat Stress. Journal of the American Society for Horticultural Science , 132 (6), 764–769. Keller, M., Romero, P., Gohil, H., Smithyman, R. P., Riley, W. R., Casassa, L. F., & Harbertson, J. F. (2016). Deficit irrigation alters grapevine growth, physiology, and fruit microclimate. American Journal of Enology and Viticulture , 67 (4), 426–435. Kennedy, G. J. T., Pilbrow, J. R., Hutton, D. R., Hewitt, D., Hunter, C. R., Ristic, R., Jones, G. P., J. A. (2000). Development of seed polyphenols in berries from Vitis vinifera L. cv. Shiraz. Australian Journal of Grape and Wine Research , 6 (3), 244–254. Koundouras, S., Hatzidimitriou, E., Karamolegkou, M., Dimopoulou, E., Kallithraka, S., Tsialtas, J. T., Kotseridis, Y. (2009). Irrigation and rootstock effects on the phenolic concentration and aroma potential of Vitis vinifera L. cv. Cabernet Sauvignon grapes. Journal of Agricultural and Food Chemistry , 57 (17), 7805–7813. Koyama, K., Ikeda, H., Poudel, P. R., & Goto-Yamamoto, N. (2012). Light quality affects flavonoid biosynthesis in young berries of Cabernet Sauvignon grape. Phytochemistry , 78 , 54–64. Kyraleou, M., Kotseridis, Y., Koundouras, S., Chira, K., Teissedre, P. L., & Kallithraka, S. (2016). Effect of irrigation regime on perceived astringency and proanthocyanidin composition of skins and seeds of Vitis vinifera L. cv. Syrah grapes under semiarid conditions. Food Chemistry , 203 , 292–300. Leeuwen, V. C., & Seguin, G. (2006). The concept of terroir in viticulture. Journal of Wine Research , 17 (1), 1–10. Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998). Lignin and Lignan Biosynthesis. In ACS Symposium Series (pp. 1–27). American Chemical Society. Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., & Fernie, A. R. (2006). Gas chromatography mass spectrometry-based metabolite profiling in plants. Nature Protocols , 1 (1), 387–396. Lommen, A. (2009). Metalign: Interface-driven, versatile metabolomics tool for hyphenated full-scan mass Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 146 spectrometry data preprocessing. Analytical Chemistry , 81 (8), 3079–3086. Lytovchenko, A., Eickmeier, I., Pons, C., Osorio, S., Szecowka, M., Lehmberg, K., Fernie, a. R. (2011). Tomato Fruit Photosynthesis Is Seemingly Unimportant in Primary Metabolism and Ripening But Plays a Considerable Role in Seed Development. Plant Physiology , 157 (December), 1650–1663. Ma, W., Guo, A., Zhang, Y., Wang, H., Liu, Y., & Li, H. (2014). A review on astringency and bitterness perception of tannins in wine. Trends in Food Science and Technology , 40 (1), 6–19. Mira de Orduña, R. (2010). Climate change associated effects on grape and wine quality and production. Food Research International , 43 (7), 1844–1855. Obiadalla-Ali, H., Fernie, A. R., Lytovchenko, A., Kossmann, J., & Lloyd, J. R. (2004). Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size, but only small changes in carbohydrate metabolism. Planta , 219 (3), 533–540. OIV. (2018). International Organisation of Vine and Wine Statistical Report on World Vitiviniculture . France. Ollat, N., & Gaudillere, J. P. (2000). Carbon balance in developing grapevine berries. In V International Symposium on Grapevine Physiology (Vol. 526, pp. 345–350). Parpinello, G. P., Ricci, A., Rombolà, A. D., Nigro, G., & Versari, A. (2019). Comparison of Sangiovese wines obtained from stabilized organic and biodynamic vineyard management systems. Food Chemistry , 283 , 499–507. Pilati, S., Brazzale, D., Guella, G., Milli, A., Ruberti, C., Biasioli, F., Moser, C. (2014). The onset of grapevine berry ripening is characterized by ROS accumulation and lipoxygenase-mediated membrane peroxidation in the skin. BMC Plant Biology , 14 (1), 87. Reshef, N., Agam, N., & Fait, A. (2018). Grape Berry Acclimation to Excessive Solar Irradiance Leads to Repartitioning between Major Flavonoid Groups. Journal of Agricultural and Food Chemistry , 66 (14), 3624–3636. Reshef, Noam, Fait, A., & Agam, N. (2019). Grape berry position affects the diurnal dynamics of its metabolic profile. Plant Cell and Environment , 42 (6), 1897–1912. Reshef, Noam, Walbaum, N., Agam, N., & Fait, A. (2017). Sunlight modulates fruit metabolic profile and shapes the spatial pattern of compound accumulation within the grape cluster. Frontiers in Plant Science , 8 (70), 1–20. Reynolds, A. G. (2010). Viticultural and vineyard management practices and their effects on grape and wine quality. In Managing Wine Quality: Viticulture and Wine Quality (pp. 365–444). Woodhead Publishing. Rocchi, L. (2015). Physiological responses of white grape berries to sunlight exposure . Retrieved from https://air.unimi.it/bitstream/2434/336669/2/phd_unimi_R10017.pdf Rodríguez-García, C., Sánchez-Quesada, C., Toledo, E., Delgado-Rodríguez, M., & Gaforio, J. J. (2019). Naturally lignan-rich foods: A dietary tool for health promotion? Molecules . Sattler, S. E., Cahoon, E. B., Coughlan, S. J., & DellaPenna, D. (2003). Characterization of tocopherol cyclases from higher plants and cyanobacteria. Evolutionary implications for tocopherol synthesis and function. Plant Physiology , 132 (4), 2184–2195. Serrano, A., Espinoza, C., Armijo, G., Inostroza-blancheteau, C., Poblete, E., Meyer-regueiro, C., Arcejohnson, P. (2017). Omics Approaches for Understanding Grapevine Berry Development : Regulatory Networks Associated with Endogenous Processes and Environmental Responses. Frontiers in Plant Science , 8 , 1486. Smilde, A. K., Jansen, J. J., Hoefsloot, H. C. J., Lamers, R. J. A. N., van der Greef, J., & Timmerman, M. E. (2005). ANOVA-simultaneous component analysis (ASCA): A new tool for analyzing designed metabolomics data. Bioinformatics , 21 (13), 3043–3048. Song, J., Shellie, K. C., Wang, H., & Qian, M. C. (2012). Influence of deficit irrigation and kaolin particle film on grape composition and volatile compounds in Merlot grape ( Vitis vinifera L.). Food Chemistry , 134 (2), 841–850. Chapter 4 - Metabolomics of photosynthetically active tissues in white grapes __________________________________________________________________________________ 147 Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., Viant, M. R. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics , 3 (3), 211–221. Sweetman, C., Deluc, L. G., Cramer, G. R., Ford, C. M., & Soole, K. L. (2009). Regulation of malate metabolism in grape berry and other developing fruits. Phytochemistry , 70 (11–12), 1329–1344. Teixeira, Ana, Baenas, N., Dominguez-Perles, R., Barros, A., Rosa, E., Moreno, D. A., & Garcia-Viguera, C. (2014). Natural bioactive compounds from winery by-products as health promoters: A review. International Journal of Molecular Sciences , 15 (9), 15638–15678. Teixeira, António, Eiras-Dias, J., Castellarin, S. D., & Gerós, H. (2013). Berry phenolics of grapevine under challenging environments. International Journal of Molecular Sciences , 14 (9), 18711–18739. Tikunov, Y. M., Laptenok, S., Hall, R. D., Bovy, A., & de Vos, R. C. H. (2012). MSClust: A tool for unsupervised mass spectra extraction of chromatography-mass spectrometry ion-wise aligned data. Metabolomics , 8 (4), 714–718. Vallarino, J. G., & Osorio, S. (2018). Organic acids. In Postharvest Physiology and Biochemistry of Fruits and Vegetables (p. 207). Wang, L., Sun, X., Weiszmann, J., & Weckwerth, W. (2017). System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism. Frontiers in Plant Science , 8 , 1066. Waterhouse, A. L. (2003). Determination of Total Phenolics. In Current Protocols in Food Analytical Chemistry. (pp. 1–8). Wünsche, J.-N., Lombardini, L., Greer, D. H., & Palmer, J. W. (2004). “Surround” particle film applications - the effect on whole canopy physiology of apple. XXVI International Horticultural Congress: Key Processes in the Growth and Cropping of Deciduous Fruit and Nut Trees , 636 , 565–571. Xia, J., Sinelnikov, I. V., Han, B., & Wishart, D. S. (2015). MetaboAnalyst 3.0-making metabolomics more meaningful. Nucleic Acids Research , 43 (W1), W251–W257. Yamaki, S. (2010). Metabolism and accumulation of sugars translocated to fruit and their regulation. Journal of the Japanese Society for Horticultural Science , 79 (1), 1–15. Zamboni, A., di Carli, M., Guzzo, F., Stocchero, M., Zenoni, S., Ferrarini, A., Pezzotti, M. (2010). Identification of putative stage-specific grapevine berry biomarkers and omics data integration into networks. Plant Physiology , 154 (3), 1439–1459. Chapter 5 Light microclimate-driven changes at transcriptional level in photosynthetic grape berry tissues The work presented in this chapter was submitted (26th June 2021): Garrido, A., De Vos, R. C. H., Conde, A.* and Cunha, A.* (submitted). Light microclimate-driven changes at transcriptional level in photosynthetic grape berry tissues. Plants , 10 , x, https://doi.org/10.3390/xxxxx. *Equal senior authorship. Author Contributions: Conceptualization, A.G., R.C.H.D.V, A.C. (Artur Conde) and A.C. (Ana Cunha); methodology, A.G. and A.C. (Artur Conde); formal analysis, A.G. A.C. (Artur Conde) and A.C. (Ana Cunha); investigation, A.G. and A.C. (Ana Cunha); resources, A.C. (Artur Conde) and A.C. (Ana Cunha); writing— original draft preparation, A.G.; writing—review and editing, A.G., R.C.H.D.V, A.C. (Artur Conde) and A.C. (Ana Cunha); supervision, R.C.H.D.V, A.C. (Artur Conde) and A.C. (Ana Cunha); project administration, A.C. (Ana Cunha). Chapter 5 - Light microclimate-driven changes at transcriptional level __________________________________________________________________________________ 149 Abstract Viticulture practices that change the light distribution in grapevine canopy can interfere with several physiological mechanisms, like 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 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 metabolites 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 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 ( VvSuSy ), phenylpropanoid ( VvPAL1 ), stilbenoid ( VvSTS1 ) and flavan-3-ol synthesis ( VvDFR , VvLAR2 and VvANR ) were highest at green stage. In seeds, the expression of several genes associated with both phenylpropanoid ( VvCHS1 and VvCHS3 ) and flavan-3-ols synthesis ( VvDFR and VvLAR2 ) showed a peak at véraison stage, while that of RuBisCO was maintained up to mature stage. Overall, the HL microclimate, as compared to 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 of flavonoids, while 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 HL microclimate. This work suggests an interlink between berry photosynthesis and metabolism at the transcriptional level and provides relevant information for a smarter management of the light microenvironment at canopy level of the grapes. Keywords: light microclimate, exocarp, seed, gene expression, enzyme activity, grape berry photosynthesis, metabolic pathways. 5.1. Introduction Grapevine ( Vitis vinifera L.) is commonly cultivated across temperate to semi-dry areas, including the Mediterranean region (Estreicher, 2017; Santillán et al., 2020). Currently, grape berry and wine production are being affected by the escalation of environmental constraints, due to the intensification of climate changes, thus, adaptation and/or stress mitigation strategies are being implemented for a better management of vineyards (as reviewed by Santos et al., 2020). Chapter 5 - Light microclimate-driven changes at transcriptional level __________________________________________________________________________________ 150 Grape berry is composed by different tissues and cell layers, including the exocarp (skin), mesocarp (pulp) and seeds, which present distinct anatomical characteristics and biochemical profiles during development (Cadot et al., 2006; Famiani et al., 2000; Hardie et al., 1996). Different tissues of the grape berry have different functions, mainly anatomical/structural, physiological and ecological, but they are also important in viniculture because their composition has a direct impact on the wine organoleptic properties (e.g., color, aroma, flavor and texture) (Garrido and Borges, 2013; Niimi et al., 2020). Indeed, the exocarp contributes to the integrity of the whole berry by protecting inner tissues from mechanical damage or pathogen attack (Hardie et al., 1996), allowing timely seed dissemination (Martin and Rose, 2014), and also confers protection against ultraviolet light exposure, especially due to its flavonols content (Martínez-Lüscher et al., 2019). The seed is rich in flavan-3-ol monomers and procyanidins (tannins), which confer protection against herbivory but are also responsible for the bitterness and astringency to the wine (Rousserie et al., 2019). Both primary and secondary metabolites of grape berry tissues are extremely important for fruit nutritional and organoleptic characteristics (Pott et al., 2019). Complex regulatory mechanisms are involved in their synthesis, such as many transcriptional, translational and biochemical, that can be also modulated by biotic and abiotic factors (as reviewed by Serrano et al., 2017). It is well established that environmental conditions have a strong influence on metabolism of grape berry cells (Blancquaert et al., 2019). Light is an abiotic factor that influences the overall grapevine physiology but also grape berry composition (Poni et al., 2018). In this way, viticulture practices that involve canopy manipulations (e.g., leaf removal, shading covering, canopy conduction systems, and also irrigation) are directly related with levels of light reaching the leaves and grape berry clusters (Reynolds, 2010). Smart et al. (1985) introduced the concept of microclimate to define the specific environmental conditions in the vicinity of leaves and fruits. Several studies have addressed the effects of light conditions experienced by developing grape berries on their primary and secondary metabolites as well as on transcriptional changes related to key genes (Friedel et al., 2015; Koyama et al., 2012; Plessis et al., 2017; Reshef et al., 2017; Young et al., 2016). However, most of these studies focused on whole berries or just on the skin, and not specifically to other tissues/organs like seeds. To the best of our knowledge, no studies concerning the effects of light microclimate on grape seed metabolism and gene expression have been reported to date. Like leaves, fruits may present photosynthetic activity, at least at their early stage of development (Aschan and Pfanz, 2003; Brazel and Ó’Maoileídigh, 2019). In grape berries, both the exocarp and the seed outer integument exhibit photosynthetic activity (Breia et al., 2013) and their photosynthetic Chapter 5 - Light microclimate-driven changes at transcriptional level __________________________________________________________________________________ 151 competence is responsive to the light microclimate experienced by the grapes throughout their development (Garrido et al., 2018). More recently, we characterized the photosynthetic profiles of these two berry tissues collected from clusters growing in two contrasting light microclimates in the canopy: LL (low light) i.e., shaded inner zones of the canopy; and HL (high light) i.e., grape berry clusters are exposed to direct sunlight part of the day, receiving 3-fold more light intensity than LL clusters (Garrido et al., 2019). Moreover, these photosynthetic profiles of these two berry tissues were also assessed in vineyards under short-term mitigation treatments against climate adversities, i.e., foliar kaolin application and soil irrigation (Garrido et al., 2019). A metabolomics study showed that both light microclimate and irrigation were the main environmental factors influencing the metabolite composition of exocarp and seed (Garrido et al., 2021). Transcriptomics and genomics studies have disclosed the main elements involved in berry photosynthesis, especially in the skin (da Silva et al., 2005; Deluc et al., 2007; Grimplet et al., 2007; Terrier et al., 2005; Waters et al., 2005). In particular, Waters et al. (2005), using cDNA microarray analysis, verified that expressed sequence tag (ESTs) involved in photosynthesis and carbohydrate metabolism were co-regulated, suggesting that photosynthesis in the berry skin is a source of carbohydrate for the berry skin itself. However, much uncertainty still exists about the relation between grape berry photosynthesis and its primary and secondary metabolism, and whether this is regulated at the level of gene transcription or enzyme activity. The main objective of the present work was to evaluate the transcriptional changes in key genes involved in photosynthesis, sucrose metabolism and secondary metabolite pathways (carotenoids, phenolics), in both exocarp and seed of grape berries exposed to either HL or LL microclimate, aiming to establish a potential link between transcripts, metabolites (Garrido et al., 2021) and photosynthetic activity (Garrido et al., 2019) in these berry tissues.