Effects and Modes od Action of Canopy Management Practices on Vine Physiology and Berry Composition in Organically-Cultivated cv. Sangiovese (Vitis Vinifera L.)

In organic and biodynamic vineyards, canopy management practices should be carefully and timely modulated, particularly in a context of climate change, for successfully achieving balanced plants, ventilated and exposed berries, elevated grape and wine quality. In 2013 and 2014, characterized by contrasting climatic conditions, the implications of post-veraison (late) or pea-size trimming, post-veraison or pre-harvest late defoliations and shoot-positioning (post-veraison) were assessed against long-shoots non treated controls, under field conditions on organically-cultivated cv. Sangiovese. The key agronomic and enological relevance of late trimming and defoliations clearly emerged in both seasons. Berry skin phenolics (e.g. anthocyanins, flavonols) increased markedly, without changes in technological parameters. In case of early trimming, such positive effects were observed only in 2013. Maintaining long shoots for shading decreased anthocyanins, flavonols and total phenolics concentrations and promoted the production of compact bunches. Experimental data strongly designated late trimming, a practice proved to contain yield and bunch compactness, as a valuable alternative to cluster thinning. Late trimming, defoliations and shoot positioning reduced the severity of Botrytis cluster rot. The highest levels of berry skins phenolic compounds in late trimmed and defoliated plants could have contributed control the severity of this pathogen. The enological benefits induced by late trimming and defoliations and shoot positioning emerged in both young and aged wines. For the first time, cell cultures from cv. Sangiovese berry tissues were obtained and enabled to investigate, in controlled conditions, the relations between mechanisms regulating secondary metabolism in grapevine cells and changes induced by environmental and agronomic factors. The Doctoral Dissertation strongly highlights the need to consider, for a proper interpretation of the multiple modifications induced by canopy management strategies, physiological mechanisms other than the canonic source-sink relationships, in particular their impact on the vine hormonal status

Uso de fitorreguladores en el viñedo como herramienta para mitigar los efectos del cambio climático en la calidad de la uva

El cambio climático afecta a todas las regiones del mundo en las que se incluyen las vitivinícolas, y muchos de los viticultores se están viendo en la necesidad de adoptar ciertas medidas que puedan contrarrestar los efectos que este fenómeno produce sobre la vid. El impacto del calentamiento global repercute en todo el proceso de maduración de la uva, puesto que adelanta las etapas fenológicas, y como consecuencia se produce una alteración en la composición de la uva y el vino. El aumento de temperatura produce un desacoplamiento entre la madurez tecnológica y la madurez fenólica, de la uva lleva a un aumento en el grado alcohólico de los vinos y a pérdidas de calidad en parámetros sensoriales. Una de las estrategias posibles para minimizar estos efectos es aplicar técnicas que retrasen la maduración de la uva para que esta tenga lugar de una forma más equilibrada a temperaturas más frescas. En esta revisión bibliográfica se recopila la información actualizada sobre la aplicación de reguladores de crecimiento, análogos a las hormonas vegetales, como herramienta para inducir una maduración del fruto más retrasada en la vid. Se repasa el potencial uso de auxinas, citoquininas, giberelinas, ácido abscísico, etileno, brasinoesteroides y ácido salicílico con este fin.Grado en Enologí

Genetic Improvement of Grapevine: Tailoring Grape Varieties for The Third Millennium - A Review

The remarkable propagative aptitute of grapevine is one of the key factors contributing to its success as a cultivated species and to the spread of the domesticated grapevine, establishing it as one of the most important fruit species worldwide. Today there are some 8 million hectares of vineyards across the world. It is therefore titting that the successful implementation of the powerful technology of gene manipulation in grapevine is to a large extent reliant on this regenerative ability. Currently, several varieties of grapevines have been successfully genetically transformed, largely by employing somatic embryogenesis to generate highly regenerative target material. Especially attractive in the wine industry is the possibility of improving grapevine varieties by the addition of genes that confer usefultraits, such as resistances against biotic and abiotic factors and manipulation of certain metabolic functions. In principle, gene transfer technology allows for the directed manipulation of a specific trait without altering the characteristic nature of the cultivar, permitting the improvement of the traditional cultivars while maintaining their established varietal characteristics. For the most part, targeted traits currently include disease resistance andimproved berry quality. The promise of this technology is threatened by worldwide resistance to genetically modified organisms, and in the wine industry by complications surrounding the property rights and naming of transgenic vines. If it is not possible to maintain the varietal name when a transgenic vine has the same properties as the original well known variety, the significant advantages of gene technology over traditional breeding programmes are to a large extent lost. If these and other complications can be overcome, the integration of this powerful technology with traditional breeding programmes, and with other initiatives such as the study of the grapevine genome, will ensure a new era in the cultivation of this ancient species

Late pruning to delay maturity and preserve wine identity in Barossa Shiraz

Warming is shifting vine phenology, compressing harvests, and altering the balance of fruit traits relevant to wine. The aim of this thesis was to test late pruning as a tool to delay maturity of Shiraz in the Barossa Valley of Australia, and its impact on vine yield, and wine chemical and sensory attributes. Pruning at three phenological stages were compared: winter (control), budburst and 2-3 leaves emerged. Two trials were established. First, three pruning treatments were carried out during four consecutive seasons on the same vines, to evaluate carry-over effects. Second, two thermal regimes (heating with open-top chambers vs unheated control) was combined with three pruning times during three seasons. In general, late pruning treatments delayed maturity with neutral or positive effects for yield and berry traits without carry-over effects on phenology, yield, leaf area and berry traits. Further, late pruning shifted the onset of berry anthocyanin in relation to sugars and increased the anthocyanin to sugar ratio, improved wine phenolics and altered sensory attributes. In a context of warming, delaying pruning until 2-3 leaves have emerged can effectively spread the harvest and partially restore anthocyanin : sugar ratio, improve wine phenolics and preserve wine sensory attributes with no yield penalties.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

The study of the reprogramming of metabolism of Trincadeira grapes upon infection with Botrytis cinerea

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017As uvas (espécie Vitis) são, a nível económico, o fruto mais importante à escala mundial. No entanto, as videiras são suscetíveis a várias doenças, sendo que os fungos são a principal causa para a redução de qualidade em uvas e do seu respetivo rendimento. A Trincadeira é uma variedade de videira muito importante em Portugal, pois dá origem a excelentes vinhos em alguns anos. No entanto, é também extremamente suscetível a doenças derivadas de fungos como o bolor cinzento causado por Botrytis cinerea, que é uma das doenças mais perigosas para as uvas. Esta tese de mestrado tem como objetivo aumentar o conhecimento relativo à forma como o metabolismo das uvas, bem como a qualidade do vinho, é afetado pela infeção com B. cinerea, complementando estudos recentes. A iniciação dos mecanismos de defesa contra patógenos necrotróficos, biotróficos e hemibiotróficos está já bem documentada no estudo de tecidos vegetativos, enquanto outros órgãos como os frutos (neste caso, a uva) ainda não foram bem estudados nesse aspeto. Aquando a infeção, o metabolismo hormonal das uvas é reprogramado graças ao envolvimento putativo de jasmonatos, ácido abcísico, auxinas e outras fito-hormonas, ao passo que o ácido salicílico não aparenta estar envolvido neste processo. Como tal, estamos interessados em estudar a forma como o metabolismo hormonal é regulado em uvas infetadas com B. cinerea, especialmente tendo em conta o papel desempenhado por estas hormonas tanto no amadurecimento do fruto como na resposta a stress biótico. Com este trabalho, temos como objetivo revelar nova informação relativamente às respostas das uvas durante interações patógeno-hospedeiro. Isto será feito através de uma análise combinada de perfis metabólicos de bagos infetados com análise da expressão de alguns genes envolvidos no metabolismo de hormonas. Pretendemos comparar as mudanças observadas no metabolismo hormonal aquando infeção com B. cinerea entre uma variedade suscetível (Trincadeira) e uma variedade resistente (Syrah). Por outro lado, o estudo da forma de como o aroma das uvas é influenciado por este tipo de infeção (em particular, o estudo do metabolismo de terpenos), e quais as consequências na qualidade do vinho são aspetos importantes neste contexto. De modo a conseguir estes resultados, bagos de uva controlo e infetados em três estágios de amadurecimento (green, EL32; veraison, EL35; harvest, EL38) foram colhidos para podermos efetuar quantificação de hormonas, análise de expressão de genes envolvidos no metabolismo hormonal e identificação de voláteis. Para fazer a análise de voláteis, foram usadas amostras liofilizadas a -40ºC. No entanto, foi concluído que, através do uso desta abordagem, muito poucos compostos conseguem ser detetados no processo e que uma grande quantidade inicial de material é necessária, de preferência proveniente de amostras frescas. Relativamente ao metabolismo de hormonas, os resultados obtidos mostram que os jasmonatos (OPDA e JA-Ile) estão envolvidos na resposta das uvas contra stress biótico proveniente da infeção. Estas hormonas já tinham sido caracterizadas como estando envolvidas no amadurecimento através do crescimento e do desenvolvimento de coloração dos bagos. Estudos prévios indicam que os jasmonatos são também responsáveis pela regulação de respostas de defesa como respostas contra stress oxidativo e stress por dessecação. Neste caso, as uvas da variedade Syrah apresentaram um elevado conteúdo basal de jasmonatos, ao passo que as uvas da variedade Trincadeira apresentaram um grande aumento na concentração de JA-Ile após estarem infetadas. As mudanças observadas na expressão dos genes envolvidos na biossíntese de JA-Ile (genes que codificam para as proteínas allene oxide synthase e 12-oxophytodienoate reductase 1) apoiam este aumento observado na quantidade de jasmonatos, já que uma baixa expressão destes genes a nível basal significa que não são necessários nas primeiras fases de crescimento devido ao já elevado conteúdo em jasmonatos. O metabolismo relativamente ao ácido abcísico apoia o já conhecido papel desta hormona no amadurecimento. É sabido de estudos anteriores que o ácido abcísico tem um papel importante no amadurecimento das uvas, sendo responsável por processos importantes na acumulação de açúcares e aumento da coloração dos bagos. Estes dados foram apoiados pelo aumento de ABA que observámos durante o início do amadurecimento. Para além do amadurecimento, os resultados que obtivémos sugeriram ainda que esta hormona possa estar também envolvida na defesa das uvas contra a infeção por Botrytis cinerea. Esta observação advém do facto de que o teor em ácido abcísico tende a aumentar após a infeção e de que se verificou uma maior expressão dos genes envolvidos na síntese e na sinalização desta hormona (genes que codificam para a 9-cis-epoxycarotenoid dioxygenase e para o ABA receptor PYL4 RCAR10, respetivamente). No entanto, observou-se também um conteúdo basal em ABA baixo na variedade Syrah, que é resistente contra a infeção. Como tal, futuras investigações terão de ser feitas relativamente ao papel do ácido abcísico aquando a infeção. Relativamente ao papel das auxinas, sabe-se que estas hormonas estão presentes em baixas concentrações no início do amadurecimento, pois níveis elevados de auxinas atrasam a acumulação de açúcares que é essencial nesta fase. Estes dados estão de acordo com os resultados que obtivemos relativamente à expressão génica. No que toca ao seu papel na defesa, foram observados níveis basais elevados em uvas da variedade Syrah (que podem sugerir a existência de uma resposta acelerada quando infetadas) e aumentos de concentração em uvas da variedade Trincadeira após estas serem infetadas. Ambos estes fatores podem ser interpretados como indicadores de que as auxinas estão envolvidas na resposta das uvas contra o patógeno. Foram ainda observadas mudanças na expressão de genes envolvidos na síntese, sinalização e transporte de auxinas (genes que codificam para a IAA-amido synthetase e para o auxin-responsive SAUR29), em especial durante o início do amadurecimento. Estas mudanças sugeriram que as auxinas têm um papel importante tanto no crescimento como no amadurecimento das uvas. No que toca à importância do ácido salicílico, esta hormona tinha sido, devido a estudos anteriores, associada apenas à resposta contra fungos biotróficos. Estudos prévios estudaram o seu envolvimento na resposta contra fungos necrotróficos e foi colocada a hipótese de que, aquando a infeção, um fungo como Botrytis cinerea ativa alguns dos mecanismos de defesa da uva mas inibe as vias relacionadas com o ácido salicílico. No entanto, ao contrário do sugerido, também aparenta estar envolvida na resposta à infeção por fungos deste tipo, devido aos resultados obtidos em que a variedade de vinha resistente apresentou um alto conteúdo basal de ácido salicílico, o que pode significar uma resposta rápida contra a infeção. A expressão de genes envolvidos na sinalização mediada por ácido salicílico (genes que codificam para a proteína enhanced disease susceptibility 1 e para o seu co-regulador, phytoalexine deficient 4) mostrou ter uma progressão que, inicialmente, apoiava estes resultados. No entanto, foi observado que esta expressão decresceu em uvas da variedade Syrah durante os primeiros estágios de crescimento, o que não está de acordo com os resultados obtidos pela quantificação de hormonas. Desta forma, serão precisos estudos mais desenvolvidos no que toca ao papel do ácido salicílico na defesa contra patógenos necrotróficos. Em conclusão, este trabalho permitiu efetuar uma análise de um modo mais detalhado do papel desempenhado por algumas fito-hormonas tanto no crescimento e amadurecimento de uvas como na defesa relativamente a stress biótico. Permitiu também a sugestão de mecanismos, tanto moleculares como metabólicos, que podem estar envolvidos na regulação de ambos os processos.Grapes (Vitis species) are economically the most important fruit crop worldwide. However, grapevines are prone to several diseases, with fungi being the major cause of reduction in grape quality and yield. Trincadeira is a very important Portuguese grapevine cultivar, giving rise to unique and excellent wines in certain years. However, it is extremely susceptible to fungal diseases such as grey mold caused by Botrytis cinerea, which is one of the most dramatic diseases for grapes. This master thesis aims at further elucidating how the metabolism of grapes, as well as wine quality, is affected upon infection with Botrytis cinerea, complementing recent studies. The initiation of defense mechanisms against necrotrophic, biotrophic and hemibiotrophic pathogens has been documented mostly for vegetative tissues, while organs like the fruits have not been well studied in that regard. Upon infection, hormonal metabolism is reprogrammed with the putative involvement of jasmonates, abscisic acid, auxins and other phytohormones, whereas salicylic acid does not seem to play a role in the process as assessed by microarray. As such, we are interested in studying how hormonal metabolism is regulated in infected grapes given the role of hormones in both fruit ripening and biotic stress response. With this work, we aim at bringing novel insights into the responses of grapes during a pathogen-host interaction by a combined metabolic profiling analysis of infected fruits together with an analysis of the expression of genes involved in hormonal metabolism. We want to compare the changes in hormonal metabolism in a susceptible (Trincadeira) and a resistant (Syrah) variety upon infection with Botrytis cinerea. Furthermore, we want to study how the aroma of grapes is influenced by the infection, in particular the metabolism of terpenes, with consequences on wine quality. To this end, control and infected berries at the green, veraison and harvest stages were collected for hormonal quantification, analysis of gene expression regarding the metabolism of hormones and volatile identification. Regarding volatile analysis using samples lyophilized at -40ºC, it was concluded that few compounds can be detected using this approach and that a great quantity of starting material is needed and preferably fresh samples. In what concerns hormonal metabolism, our results showed that jasmonates were heavily involved in biotic stress response, as not only did Syrah grapes have a high basal content in jasmonates, but Trincadeira grapes displayed a significant increase in JA-Ile concentration upon infection. Changes in the expression of genes involved in JA-Ile biosynthesis accompanied this increase in quantity. Abscisic acid metabolism, while mostly cementing its role in ripening, also suggested an involvement in grape defense against B. cinerea, mainly due to increases in ABA content upon infection, as well as heightened expression of genes involved in its synthesis and signaling. However, due to the low basal content in ABA in the Syrah variety, further investigation will be pursued. Regarding auxins, the high basal levels in Syrah grapes and increased concentration in infected Trincadeira grapes were indicators of the hormone’s involvement in response to the pathogen. Changes in the expression of genes involved in IAA synthesis, signaling and transport, especially during the onset of ripening, also suggested an important role of auxin regulation during grape growth and ripening. Salicylic acid, which had only been previously suggested to be involved in response against biotrophic fungi, also appears to play a part during Botrytis cinerea infection due to elevated basal SA content in the resistant cultivar. Expression of genes involved in SA-mediated signaling, although in accordance with these results at first, decreases in Syrah grapes during earlier stages, which does not support the obtained results from hormonal quantification. As such, further studies might be needed concerning the role of salicylic acid in response to necrotrophic pathogens. In conclusion, this work allowed a more detailed analysis of the role played by hormones in both grape ripening and defense and suggested molecular and metabolic mechanisms that might regulate both processes

Influence of the ethylene on the grape berry development and related-gene expression

Le raisin est un fruit non climactérique dont la maturation semble ne pas nécessiter l'éthylène. Ici, il est confirmé que l’accumulation d’anthocyanes est liée à l’accumulation d’une glucoslyltransferase (UFGT), dont le promoteur a été cloné. Il a été trouvé 7 cis -éléments éthylène-dépendants. Cette étude a montré la stimulation d'expression de l'ufgt par l’éthylène n'est pas dépendant de MybA, un des régulateurs de la transcription de l'ufgt. Des expériences ont été conçues pour étudier l’ensemble des gènes affecté par éthylène au début de la maturation du raisin. Parmi eux certains sont en relation avec les variations de diamètre de la baie. Ces gènes sont impliqués dans la circulation de l'eau : plusieurs aquaporines, et dans la structure de la paroi cellulaire : polygalactoronases, xyloglucan endotransglucosylases, pectine méthylesterase, cellulose synthase et expansines. L’éthylène stimule l'accumulation de la plupart de leurs transcrits entre 1 heure et 24 heures d’incubation. ABSTRACT : The grape is a non-climacteric fruit which maturation apparently does not require ethylene. Here, it is confirmed that the accumulation of anthocyanins is linked to the accumulation of a glucoslyltransferase (UFGT), whose promoter was cloned. We found 7 cis-elements ethylene-dependent. This study showed the stimulation of ufgt expression by ethylene is not dependent to MybA, transcription regulators of the ufgt. Experiments were designed to investigate all genes affected by ethylene in early ripening grapes. Among them some are in relation to variations in berry diameter. These genes are involved in the movement of water : several aquaporins, and the structure of the cell wall : polygalactoronases, xyloglucan endotransglucosylases, méthylesterase pectin, cellulose synthase and expansines. Ethylene stimulates the accumulation of most of their transcripts between 1 hour and 24 hours of incubation

Characterisation of the Auxin Signalling Pathway in Vitis vinifera L. cv. Shiraz

Auxin is a key regulator of plant growth, mechanistically controlled by a finely tuned transcriptional system. The auxin signalling pathway consists of the transcription factors, auxin response factors (ARFs), the transcriptional repressors, auxin/indole-3-acetic acid (Aux/IAAs), and the transport inhibitor response 1/auxin signalling F-box (TIR1/AFB) proteins that form a co-receptor complex with the Aux/IAA proteins. These proteins modulate the plant’s response to auxin, mediating a myriad of downstream responses. Auxin is involved in most, or all, developmental processes and in responses to environmental cues. It has been found to be a repressor of ripening in grapes, and a decrease in berry auxin concentration is required to allow the initiation of ripening. To investigate the role of the auxin signalling pathway in grape, 19 VviARF transcription factors, 23 VviIAA repressors and six VviAFB receptor family members were identified in Vitis vinifera sp. The conserved domains were analysed in each group of proteins and their presence or absence related to possible function. Phylogenetic trees demonstrated the relationship of the Vvi genes with apple, Arabidopsis, poplar, and tomato family members, some of which have proven function in fruit development. Expression analysis across a 16 week V. vinifera L. cv. Shiraz berry developmental series suggested that 39 of the 48 auxin signalling transcripts were highly expressed pre-veraison (before the onset of ripening) and were down-regulated from veraison and throughout berry ripening, correlating with the high concentration of auxin pre-veraison. The varied expression patterns of these genes suggest participation in a range of developmental processes at different stages during development. The change in expression of a large proportion (39 of 48) of auxin signalling genes at veraison indicates that it is a key change-point in berry development. Those genes expressed early in development may play roles in cell division and cell expansion. Two VviARF transcripts, VviARF27 and 2b, and five VviIAA transcripts, VviIAA15b, 19, 31, 38 and 40, were highly up-regulated post-veraison suggesting that they may play roles in fruit ripening. ARF proteins have also been found to play roles in fruit ripening through interactions with other proteins such as MYBs and bHLHs. The expression patterns of the 48 auxin signalling genes in a nine stage leaf developmental series could be clustered into 12 groups. Interestingly, very few auxin signalling pathway genes were expressed in leaves with a pattern that correlated to the pattern of IAA or IAA-Asp accumulation. Various transcripts had high transcript expression in flowers, roots and/or tendrils again suggesting a diversity in the roles these genes play. Yeast 2-hybrid and bimolecular fluorescence techniques showed that VviARF4-VviIAA19, VviARF27- VviIAA19 and VviARF27-VviIAA27 protein pairs interact and have nuclear localisation. The ARF activator, VviARF27, and VviIAA19 have overlapping expression patterns, in post-veraison berries and flowers, suggesting the interaction between these two proteins may occur in planta and play a role in flowering and berry ripening. From their expression patterns, VviARF4 and VviIAA19 may interact in tendrils, and VvARF27 and VviIAA27 may interact in flowers. Ex planta berry treatments suggested that auxin and ethylene/abscisic acid (ABA) have antagonistic effects on the auxin signalling pathway in grape berries. Auxin treatment up-regulated VviIAA transcripts pre- and post-veraison whilst the enhancers of ripening, ethylene, in the form of Ethrel, and ABA, down-regulated some auxin signalling transcripts, both pre- and post-veraison. ABA may play an important role in ripening by switching off the vegetative pathways pre-veraison, such as photosynthesis, while ethylene enhances ripening factors post-veraison. A model explaining the role of auxin signalling during berry development is proposed where the majority of VviARF and VviIAA proteins function during pre-veraison berry development, when the levels of IAA are high. These are switched off towards veraison, and allow the transition to ripening. During fruit ripening, a select number of VviARF and VviIAA proteins may interact to affect the ripening process – potentially through interactions with other protein families. ABA may down-regulate the auxin signalling pathway pre-veraison, and ethylene down-regulates some members of the pathway post-veraison. These findings support the existence of a fruit specific, complex hormonal network that works in concert to modulate and ensure grape berry growth and ripening. This research represents the most in-depth analysis of the auxin signalling pathway components in V. vinifera to date and highlights the pleiotropic roles the candidates play throughout plant development. These findings may aid in the development of strategies to manipulate berry ripening and identifies areas for future research.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201