La vigne (Vitis vinifera) est sensible à un grand nombre de maladies. Les politiques de limitation des traitements phytosanitaires font qu'aujourd'hui, les viticulteurs ne disposent d'aucun moyen de lutte contre certains bioagresseurs. C'est le cas des maladies du bois causées par des complexes fongiques nécrosant les ceps et du court noué, maladie virale transmise par des nématodes. La vigne est une espèce végétale connue pour sa production particulière de métabolites secondaires en réponse à des infections. Dans le cadre de cette thèse, nous nous sommes intéressés aux phytoanticipines, préformées dans les plantes et dont le potentiel de toxicité envers des agents pathogènes est intéressant, c’est le cas des triterpénoïdes. Les objectifs de cette thèse ont donc été dans un premier temps d'estimer la capacité de la vigne à produire de tels composés. Pour cela, la composition en triterpénoïdes de différents organes et de différents cépages a été analysée. Nous avons pu montrer que la composition générale en triterpénoïdes est caractéristique de chaque cépage et de chaque organe. Dans une deuxième partie, 9 gènes impliqués dans la synthèse de triterpènes chez la vigne ont été identifiés et leur expression a été évaluée dans différents organes, chez différents cépages et sous différentes conditions de stress biotique et abiotique. Cette étude exploratoire nous donne des pistes pour mettre en corrélation l'expression de certaines triterpène synthases avec la production différentielle de certains triterpènes à la surface des feuilles de différents cépages. Enfin, nous nous sommes intéressés aux triterpènes glycosylés, les saponines, afin d'évaluer leur potentiel dans la lutte contre certaines maladies majeures de la vigne pour lesquelles aucun traitement n'est actuellement disponible. Pour cela, l'efficacité de saponines issues de la gypsophile et du quillaja a été testée contre certains champignons associés aux maladies du bois ainsi que contre les nématodes vecteurs des virus du court noué. Nous avons pu mettre en évidence que les souches de champignons testées étaient capables de contourner la toxicité des saponines, tandis qu'un tel traitement était rapidement efficace pour lutter contre les nématodes. Afin de vérifier l'innocuité de ce traitement pour l'environnement, les doses efficaces ont été testées et n'ont pas eu d'impact significatif sur différents bioindicateurs. / Vitis vinifera is susceptible to many pathogens. These past few years, treatment policies led to the withdrawal of many pesticides. Renee, no chemical treatments are available to treat some grapevine diseases such as the grapevine trunk diseases caused by fungi complexes and the grapevine fanleaf degeneration, a viral disease transmitted from grapevine to grapevine by vector nematodes. Grapevine is known for the production of secondary metabolites as a response to pathogen infections. In this work, we focused on phytoanticipins such as triterpenoids, that are found as preformed compounds and that confer a basal resistance level to plants. First, a chemical analysis was made on the triterpenoid composition of some grapevine cultivars and organs. This study revealed that the triterpenoid composition is specific to the V. vinifera cultivar and the organ. In a genomic approach, 9 candidate genes involved in the triterpene biosynthesis were identified and their expression was studied in different organs, varieties and biotic or abiotic stress conditions. This explorative study shows correlations between gene expression and differential triterpene production at the leaf surface of the different varieties. In the last part of this study, the use of glycosylated triterpenes, also called saponins, as a substitution solution to withdrawed treattnents against major grapevine diseases was tested. Therefore, the efficiency of saponins extracted from gypsophila and quillaja was tested against fungi associated to grapevine trunk diseases and some nematodes vector of the grapevine fanleaf degeneration. These tests evidenced that the fungi were able to avoid saponins toxicity, whereas such treatment was efficient to kill nematodes. In order to evaluate the effect of the treatment on the environment, the efficient doses were tested and bad no significant impact on some bioindicators.

A antracnose da videira tem como agente causal o fungo Elsinoë ampelina Shear, o qual ataca tecidos tenros da parte aérea causando lesões necróticas e comprometendo o desenvolvimento da planta com consequente queda na quantidade e qualidade dos frutos produzidos. Cultivares de Vitis apresentam diferentes níveis de susceptibilidade à antracnose. Uma vez que características anatômicas e químicas podem atuar como mecanismos de resistência a patógenos, o presente estudo propõe comparar caracteres histológicos e bioquímicos em folhas jovens de Vitis labrusca \'Niagara Rosada\' (NR, susceptível), Vitis vinifera \'Thompson Seedless\' (TS, susceptível) e V. vinifera \'Pinot Noir\' (PN, resistente). As análises anatômicas foram realizadas em folhas sadias aos 4 e 11 dias após o brotamento (DAB) e em folhas lesionadas com 11 DAB, enquanto os compostos bioquímicos foram quantificados em folhas sadias aos 4 e 11 DAB. Os caracteres analisados foram: índice estomático, densidade de idioblastos cristalíferos, espessura da cutícula da face adaxial, espessura da parede periclinal externa e altura das células epidérmicas de ambas as faces, espessura do mesofilo total, do parênquima paliçádico e do parênquima lacunoso, o conteúdo de compostos fenólicos totais, flavonoides e de clorofila a, b e total, bem como a atividade de peroxidase e de polifenoloxidase. Os dados obtidos foram submetidos à análise estatística. A cultivar TS apresentou índice estomático maior nas duas idades avaliadas em comparação com o genótipo resistente PN, a qual apresentou maior espessura de cutícula nas duas idades e de mesofilo total aos 11 DAB. Os parâmetros quantitativos das amostras inoculadas apresentaram redução significativa em relação às não inoculadas, com exceção da área foliar de PN, a qual não foi significativamente menor. Para NR e TS, 100% das amostras inoculadas apresentaram várias lesões expandidas, enquanto PN apenas 60% das folhas analisadas apresentaram poucas lesões bem delimitadas e de formato circular. A cultivar resistente PN apresentou forte reação positiva a compostos fenólicos bem como o maior conteúdo desses compostos na idade 11 DAB, enquanto a cultivar susceptível TS apresentou reação muito fraca e a menor concentração. O conteúdo de flavonoides foi maior em PN em relação à TS nas duas idades avaliadas, e NR apresentou o valor mais baixo aos 4 DAB, quando é altamente susceptível. A atividade de polifenoloxidase aos 11 DAB foi maior na cultivar PN, intermediária em NR e mais baixa em TS. A análise de todos os parâmetros anatômicos e bioquímicos avaliados indica que a maior resistência da cultivar PN esteja relacionada ao menor índice estomático, à cutícula mais espessa, ao conteúdo mais elevado de compostos fenólicos totais e de flavonoides, bem como a maior atividade enzimática de polifenoloxidase. / Grapevine anthracnose is caused by the fungus Elsinoë ampelina Shear, which attacks tissues of the aerial part causing necrotic lesions, compromising plant growth with consequent decrease in quantity and quality of the fruits produced. Vitis cultivars show different levels of susceptibility to anthracnose. Anatomical and genetic characteristics can act as a resistance mechanism against pathogens, therefore, this study proposes to compare histological and biochemical characteristics in young leaves of Vitis labrusca \'Niagara Rosada\' (NR, susceptible), Vitis vinifera \'Thompson Seedless\' (TS, susceptible) and V. vinifera \'Pinot Noir\' (PN, resistant). The anatomical analyses were performed on healthy leaves at 4 and 11 days after budding (DAB) and at 11 DAB on lesioned leaves, while biochemical compounds were quantified on healthy leaves at 4 and 11 DAB. The features analyzed were: stomatal index, idioblasts density, adaxial face cuticle thickness, external periclinal wall thickness, epidermal cell height of both faces, total mesophyll thickness, palisade parenchyma and spongy parenchyma, total phenolic compounds content, flavonoids, chlorophyll a, b, and total, and peroxidase and polyphenoloxidase activity. The data were submitted to the statistical analysis. The TS cultivar had a higher stomatal index at both ages evaluated compared to the PN resistant genotype, which presented greater cuticle thickness at leaf ages and total mesophyll at 11 DAB. The quantitative parameters of the inoculated samples presented a significant reduction in relation to the non- inoculated, except for the PN leaf area, which was not significantly lower. For NR and TS, 100% of the inoculated samples presented several expanded lesions, whereas PN only 60% of the analyzed leaves showed few lesions well delimited and circular in shape. The resistant cultivar PN showed strong positive reaction to phenolic compounds as well as the highest content of these compounds at 11 DAB, while the TS susceptible cultivar presented very weak reaction and the lowest concentration. The content of flavonoids was higher in PN compared to TS at both ages evaluated, and NR presented the lowest flavonoid content at 4 DAB, when it was highly susceptible. The polyphenoloxidase activity at 11 DAB was higher in the PN cultivar, intermediate in NR, and lower in TS. The analyses of all the anatomical and biochemical parameters indicate that the higher resistance of the PN cultivar is related to the lower stomatal index, thicker cuticle, higher content of total phenolic compounds and flavonoids, as well as higher enzymatic activity of polyphenoloxidase.

The grape flavonoids include anthocyanins, tannins and flavonols, all of which contribute to grape and wine quality by influencing the colour and mouthfeel of red wine. These compounds are synthesized in different parts of the berry and during different stages of berry development. In addition, environmental and viticultural factors such as light exposure can also alter the flavonoid composition of grapes. An understanding of how synthesis of these compounds is coordinated, their relationship to wine quality and the influence of bunch light exposure on the flavonoid composition of grapes, could be used to improve fruit quality by enhanced viticultural management. The first part of this study sought to investigate the relationship between the different products of the flavonoid biosynthetic pathway (anthocyanins, flavonols and tannins), from two climatic regions (warm and cool) and determine their role in grape and wine quality. In collaboration with a major winery, whole Shiraz grapes were sampled at the weighbridge from a range of different vineyards from two climatic regions; warm (Riverland) and cool (McLaren Vale) in 2003 and 2004. A total of 80 grape samples were collected in each season and processed (i.e. 100 berries, separated into skin, seeds and juice, weighed and frozen). Anthocyanins and flavonols were measured, in triplicate, in skins by HPLC. Tannins were determined in the skins and seeds by two methods; phloroglucinol hydrolysis (HPLC) and protein precipitation (UV-VIS spectrophotometer). A comprehensive comparison of the two methods is discussed. In both years, the grapes from warm and cool climates formed two distinct data sets based on flavonoid composition. There was a correlation between anthocyanins and flavonols for both the warm and cool climate samples in both years, however those from the warm region had lower anthocyanin for a given level of flavonol. As expected, the level of tannin in the seeds was greater than in skin for all samples. In both years, there was a weak correlation between anthocyanin levels in the skin and skin tannins, but no relationship with seed tannins. These results suggest there is some co-ordination in the synthesis of anthocyanins, flavonols and skin tannins. Also, the two regions clearly separated based on yield and despite the weak correlations in both regions, the levels of total anthocyanins were inversely related to yield. In addition, there was no relationship with any of the flavonoids and grape quality, indicating the need for improvement in streaming fruit for quality using these flavonoid compounds. The second part of the study was to investigate the effect of bunch light exposure on flavonol synthesis and accumulation in Shiraz and Chardonnay grapes during development. Light-excluding boxes were applied to bunches at budburst. Boxes were removed at four sampling times; flowering, pre-veraison, veraison and harvest. At each sampling time, berry skins were sampled when the boxes were removed and then every second day (light induced), along with exposed controls for one week. Flavonol accumulation and flavonol synthase (VvFLS1) gene expression was determined by HPLC and Real Time-PCR (RT-PCR) respectively. As expected, for all four sampling times, flavonol accumulation and VvFLS1 expression in the boxed fruit was significantly less than bunches exposed to light. On removal of boxes at flowering, pre-veraison and veraison, flavonols accumulated to levels similar to that of the exposed control fruit over a period of 4-6 days. There was a significant increase in VvFLS1 expression 2 days after exposure to light in parallel with the accumulation of flavonols. At harvest, in Chardonnay, VvFLS1 expression peaked by day 4, while in Shiraz VvFLS1 expression increased linearly and was highest at day 6. In contrast to the results for the earlier sampling times, the total amount of flavonols accumulated at harvest was less than 50% of exposed controls in Chardonnay and Shiraz grapes. These results show that flavonols are able to be induced by bunch light exposure at different times during berry development, including times when flavonols are not normally being synthesised. This suggests bunch light exposure can override the developmental control of flavonol accumulation. To further investigate the light induced expression of VvFLS1 in grapevines the molecular mechanism of transcriptional control was explored. Using genomic walking PCR techniques, two Shiraz VvFLS1 promoter sequences were cloned and their sequences were analysed. These promoter sequences were ~800bp in length and were 99% identical. A putative MYB responsive element (MRE) and several light responsive elements (LRE) were identified in the promoter region of these genes. To functionally test the VvFLS1 promoter(s), a transient assay was developed in Chardonnay suspension cells. Cells were bombarded with constructs containing potential transcription factors and the VvFLS1 promoter(s), fused to a luciferase reporter vector. After 48hrs incubation in the dark, cells were harvested and luciferase activity measured as an indicator of VvFLS1 promoter activity. Of the different transcription factors tested with the VvFLS1 promoter(s) the highest luciferase activity was observed using AtMYB12 (a flavonol-specific regulator of AtFLS1 in Arabidopsis (Mehrtens et al. 2005). While this result shows activation of the VvFLS1 promoters by AtMYB12 and the development of a transient reporter assay for testing the VvFLS1 promoter(s) a grapevine transcription factor specific for VvFLS1 was sought. Two techniques were employed to identify potential transcription factor regulators of the VvFLS1 promoter(s). The first involved BLAST sequence search analysis in a grapevine expression (EST) database with AtMYB12 and the second involved using DNA microarray technology to identify candidate transcription factors that were up-regulated in light exposed Chardonnay cell suspension cultures. Thirteen potential transcription factors were identified and after correlative RT-PCR analysis (with VvFLS1 expression patterns) two candidates were selected for further isolation and characterisation. These results have made significant progress in unravelling the molecular mechanisms of regulation of the flavonol biosynthetic, however additional experiments are required to unravel the transcriptional control of flavonol biosynthesis. This investigation contributes to our knowledge of flavonoid synthesis in grapes; how it is coordinated, the relationship with wine quality, and the influence of light particularly on synthesis of flavonols. It also explores the molecular mechanisms of VvFLS1 control, through isolation of the VvFLS1 promoter and identification of potential transcription factors, which may regulate it. An understanding of the synthesis of flavonoids and how they may be coordinated, particularly in response to light, could be used to improve fruit quality by enhanced viticultural management. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326767 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

La peronospora della vite è causata dall’Oomicete eterotallico Plasmopara viticola ed è ampiamente diffusa in tutte le aree caratterizzate da primavere miti e piovose. Gli organi svernanti di questo patogeno sono le oospore, che rappresentano l’unica fonte di inoculo per le infezioni primarie. Recenti studi molecolari hanno evidenziato l’importanza delle infezioni primarie e suggerito una revisione dell’epidemiologia di P. viticola. Questa tesi è costituita dagli studi epidemiologici realizzati per indagare il processo di maturazione e germinazione delle oospore mirati all’elaborazione di un modello dinamico per la simulazione delle infezioni primarie di P. viticola. Il modello è stato successivamente validato in diverse aree viticole italiane e, inoltre, utilizzato come base per un warning system in tempo reale per la gestione dei trattamenti in vigneto. / The heterothallic Oomycete Plasmopara viticola represents the causal agent of downy mildew of grapevine (Vitis spp.). The unique source of inoculum is represented by the overwintering sexual spores, originated by the fusion between anteridium and oogonium, so called oospores. Despite their critical impact on the epidemiology of this disease, knowledge about oospores presents some inconsistencies that are engaged in the present dissertation. Initially, the effect of water moistening the grape leaf litter holding overwintering P. viticola oospores was investigated. A close relationship was found between vapour pressure deficit (VPD in hPa) and aW (water activity) of the leaf litter, so that when VPD is lower than 2.13 hPa there is sufficient water for oospores to develop. Results showed that moisture of the leaf litter due to the water flow from the atmosphere makes the oospore development possible also during non rainy periods. Then, the effects of environmental conditions on the variability in germination dynamics of Plasmopara viticola oospores were studied over five years. The germination course was determined indirectly as the relative infection incidence (RII) occurring on grape leaf discs kept in contact with oospores sampled from a vineyard between March and July. The time elapsed between the 1st of January and the infection occurrence was expressed as physiological time, using sums of hourly rates from a temperature-dependent function only in hours when VPD was not a limiting factor (hydro-thermal time, HT). The Gompertz equation calculated over hydro-thermal time produced a consistent modelling of the general relationships between the germination dynamics of a population of P. viticola oospores and weather conditions. It represents the relative density of the seasonal oospores that should have produced sporangia when they have experienced favourable conditions for germination. Finally, a dynamic model for Plasmopara viticola primary infections on grapevine was elaborated according to a mechanistic approach. Development of the sexual stage of the pathogen was split into different state variables, in which changes from one state to another were regulated by rates depending on environmental conditions. The conceptual model was based on the definition of a primary inoculum season, a seasonal oospore (inoculum) dose, and its division into many coeval cohorts. Each cohort progresses along the primary infection cycle (production and survival of sporangia, release, survival and dispersal of zoospores, infection, appearance of disease symptoms) simultaneously, with a time step of one hour. The model was evaluated by comparing model predictions with disease onset in: i) 100 vineyards of Northern, Southern and Insular Italy (1995 to 2007); ii) 42 groups of potted grapevine plants exposed to inoculum (2006 to 2008). Most of the wrong positive predictions occurred in early season, when the host was in the earlier growth stages, or when the oospore germination was triggered by isolated weak rain events. Considering that neither calibration nor empirical adjustment of model parameters were necessary to obtain accurate simulation, it was concluded that this model produces a reasonable approximation of the primary infection processes underlying oospore development. A warning system based on such a model and on short-term weather forecasts was developed and its use was evaluated in experimental vineyards over a 3-year period in North Italy. An unsprayed control was compared with a “warning” treatment (fungicides were applied when the warning system predicted an infection), a “threshold” treatment (fungicides were applied as in the warning treatment, but only for the oospore cohorts higher than a fixed threshold), and the grower’s schedule. Average efficacy in decreasing disease incidence on leaves compared to the unsprayed control was > 90% for all treatments. On the average, 6.8 sprays were applied following the grower’s schedule; use of the warning system reduced applications by about one half (warning treatment) or two third (threshold treatment). The grower’s schedule was the most expensive control strategy, with average of 337 €/ha; the average saving was 174 and 224 €/ha for the warning and the threshold treatments, respectively. The value of this dissertation consists in two relevant and connected aspects. From one side the studies performed on the oospore maturation and germination allowed to better understand and clarify a key point of the downy mildew epidemics still wrapped by a lack of information. From the other side the model elaborated during this thesis represents a practical and efficient tool that leads to the reduction both of growers’ costs and chemical input in the environment.

Molybdenum (Mo) is an essential micronutrient required by plants. It is biologically inactive until bound in a pterin compound named the molybdenum cofactor (Moco) that binds to apoproteins used in both reductive and oxidative reactions such as nitrate reductase (NR), xanthine dehydrogenase (XDH), aldehyde oxidase (AO) and sulfite oxidase (SOX). In Vitis vinifera cv. Merlot, molybdenum deficiency is common amongst vines grown on own roots in acidic soils often resulting in yield reductions. Foliar application of molybdenum sprays increases yield and remedies deficiency indicating that Merlot grown on own roots has a reduced capacity for molybdenum uptake from the soil. Molybdenum generally occurs as molybdate (MoO₄ ²⁻) within the soil solution. The mechanism(s) involved in molybdenum transport have recently been discovered in plants, although are well characterised within prokaryotic systems. Unfortunately, no homologues of prokaryotic genes involved in molybdate transport exist within eukaryotes. It has been suggested that molybdenum transport in plants may occur through other systems including sulfate transporters due to chemical similarities between sulfate and molybdate. A yeast functional complementation approach using a sulfate transport mutant was initially used to identify novel putative plant molybdenum transport proteins. A cDNA library derived from Pinot noir roots starved of molybdenum was screened for transporters. Unfortunately, no cDNAs were identified that met the requirements of a molybdenum transporter when screened on media containing low amounts of molybdenum. However, a number of putative cDNA’s partially complemented the yeast mutant YSD1, however none of these could be validated in second round screens. A candidate gene approach was then initiated to identify pre-characterised genes that may also have capacity to transport molybdenum. The plant sulfate transporter, SHST1, restored growth of YSD1 on media containing low amounts of molybdenum. Kinetic analysis using ⁹⁹MoO₄²⁻ to trace molybdenum transport in yeast cells demonstrated that SHST1 enhanced the uptake of molybdenum at nM concentrations. The uptake was not inhibited by sulfate, but the transport of sulfate was reduced with molybdenum. Further analysis demonstrated that SHST1 did prefer sulfate as the substrate but molybdenum could compete at higher concentrations. This result is the first measurement of molybdenum being transported through a pre characterised sulfate transport protein. Whole plant experiments using rooted grapevine cuttings and ⁹⁹MoO₄²⁻ to trace molybdenum movement into plants indicated that Merlot did not have reduced capacity to uptake molybdenum compared to other varieties that do not suffer from molybdenum deficiencies such as Chardonnay. When plants were grown with molybdenum, Merlot accumulated more molybdenum than Chardonnay, with the reverse being true when plants were grown without molybdenum. Similar experiments were performed on symbiosomes isolated from Glycine max grown with and without molybdenum. Symbiosomes absorbed more molybdenum when plants were grown without molybdenum. A field site was established to look at the molybdenum profiles within petioles against yield responses over a 3-year period. Molybdenum application did not increase the yield amongst vines despite all vines initially being deficient in molybdenum. There were no cumulative effects of molybdenum application over the trial, however, molybdenum did have limited translocation ability within the vine system. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1318770 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Molybdenum (Mo) is an essential micronutrient required by plants. It is biologically inactive until bound in a pterin compound named the molybdenum cofactor (Moco) that binds to apoproteins used in both reductive and oxidative reactions such as nitrate reductase (NR), xanthine dehydrogenase (XDH), aldehyde oxidase (AO) and sulfite oxidase (SOX). In Vitis vinifera cv. Merlot, molybdenum deficiency is common amongst vines grown on own roots in acidic soils often resulting in yield reductions. Foliar application of molybdenum sprays increases yield and remedies deficiency indicating that Merlot grown on own roots has a reduced capacity for molybdenum uptake from the soil. Molybdenum generally occurs as molybdate (MoO₄ ²⁻) within the soil solution. The mechanism(s) involved in molybdenum transport have recently been discovered in plants, although are well characterised within prokaryotic systems. Unfortunately, no homologues of prokaryotic genes involved in molybdate transport exist within eukaryotes. It has been suggested that molybdenum transport in plants may occur through other systems including sulfate transporters due to chemical similarities between sulfate and molybdate. A yeast functional complementation approach using a sulfate transport mutant was initially used to identify novel putative plant molybdenum transport proteins. A cDNA library derived from Pinot noir roots starved of molybdenum was screened for transporters. Unfortunately, no cDNAs were identified that met the requirements of a molybdenum transporter when screened on media containing low amounts of molybdenum. However, a number of putative cDNA’s partially complemented the yeast mutant YSD1, however none of these could be validated in second round screens. A candidate gene approach was then initiated to identify pre-characterised genes that may also have capacity to transport molybdenum. The plant sulfate transporter, SHST1, restored growth of YSD1 on media containing low amounts of molybdenum. Kinetic analysis using ⁹⁹MoO₄²⁻ to trace molybdenum transport in yeast cells demonstrated that SHST1 enhanced the uptake of molybdenum at nM concentrations. The uptake was not inhibited by sulfate, but the transport of sulfate was reduced with molybdenum. Further analysis demonstrated that SHST1 did prefer sulfate as the substrate but molybdenum could compete at higher concentrations. This result is the first measurement of molybdenum being transported through a pre characterised sulfate transport protein. Whole plant experiments using rooted grapevine cuttings and ⁹⁹MoO₄²⁻ to trace molybdenum movement into plants indicated that Merlot did not have reduced capacity to uptake molybdenum compared to other varieties that do not suffer from molybdenum deficiencies such as Chardonnay. When plants were grown with molybdenum, Merlot accumulated more molybdenum than Chardonnay, with the reverse being true when plants were grown without molybdenum. Similar experiments were performed on symbiosomes isolated from Glycine max grown with and without molybdenum. Symbiosomes absorbed more molybdenum when plants were grown without molybdenum. A field site was established to look at the molybdenum profiles within petioles against yield responses over a 3-year period. Molybdenum application did not increase the yield amongst vines despite all vines initially being deficient in molybdenum. There were no cumulative effects of molybdenum application over the trial, however, molybdenum did have limited translocation ability within the vine system. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1318770 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

The grape flavonoids include anthocyanins, tannins and flavonols, all of which contribute to grape and wine quality by influencing the colour and mouthfeel of red wine. These compounds are synthesized in different parts of the berry and during different stages of berry development. In addition, environmental and viticultural factors such as light exposure can also alter the flavonoid composition of grapes. An understanding of how synthesis of these compounds is coordinated, their relationship to wine quality and the influence of bunch light exposure on the flavonoid composition of grapes, could be used to improve fruit quality by enhanced viticultural management. The first part of this study sought to investigate the relationship between the different products of the flavonoid biosynthetic pathway (anthocyanins, flavonols and tannins), from two climatic regions (warm and cool) and determine their role in grape and wine quality. In collaboration with a major winery, whole Shiraz grapes were sampled at the weighbridge from a range of different vineyards from two climatic regions; warm (Riverland) and cool (McLaren Vale) in 2003 and 2004. A total of 80 grape samples were collected in each season and processed (i.e. 100 berries, separated into skin, seeds and juice, weighed and frozen). Anthocyanins and flavonols were measured, in triplicate, in skins by HPLC. Tannins were determined in the skins and seeds by two methods; phloroglucinol hydrolysis (HPLC) and protein precipitation (UV-VIS spectrophotometer). A comprehensive comparison of the two methods is discussed. In both years, the grapes from warm and cool climates formed two distinct data sets based on flavonoid composition. There was a correlation between anthocyanins and flavonols for both the warm and cool climate samples in both years, however those from the warm region had lower anthocyanin for a given level of flavonol. As expected, the level of tannin in the seeds was greater than in skin for all samples. In both years, there was a weak correlation between anthocyanin levels in the skin and skin tannins, but no relationship with seed tannins. These results suggest there is some co-ordination in the synthesis of anthocyanins, flavonols and skin tannins. Also, the two regions clearly separated based on yield and despite the weak correlations in both regions, the levels of total anthocyanins were inversely related to yield. In addition, there was no relationship with any of the flavonoids and grape quality, indicating the need for improvement in streaming fruit for quality using these flavonoid compounds. The second part of the study was to investigate the effect of bunch light exposure on flavonol synthesis and accumulation in Shiraz and Chardonnay grapes during development. Light-excluding boxes were applied to bunches at budburst. Boxes were removed at four sampling times; flowering, pre-veraison, veraison and harvest. At each sampling time, berry skins were sampled when the boxes were removed and then every second day (light induced), along with exposed controls for one week. Flavonol accumulation and flavonol synthase (VvFLS1) gene expression was determined by HPLC and Real Time-PCR (RT-PCR) respectively. As expected, for all four sampling times, flavonol accumulation and VvFLS1 expression in the boxed fruit was significantly less than bunches exposed to light. On removal of boxes at flowering, pre-veraison and veraison, flavonols accumulated to levels similar to that of the exposed control fruit over a period of 4-6 days. There was a significant increase in VvFLS1 expression 2 days after exposure to light in parallel with the accumulation of flavonols. At harvest, in Chardonnay, VvFLS1 expression peaked by day 4, while in Shiraz VvFLS1 expression increased linearly and was highest at day 6. In contrast to the results for the earlier sampling times, the total amount of flavonols accumulated at harvest was less than 50% of exposed controls in Chardonnay and Shiraz grapes. These results show that flavonols are able to be induced by bunch light exposure at different times during berry development, including times when flavonols are not normally being synthesised. This suggests bunch light exposure can override the developmental control of flavonol accumulation. To further investigate the light induced expression of VvFLS1 in grapevines the molecular mechanism of transcriptional control was explored. Using genomic walking PCR techniques, two Shiraz VvFLS1 promoter sequences were cloned and their sequences were analysed. These promoter sequences were ~800bp in length and were 99% identical. A putative MYB responsive element (MRE) and several light responsive elements (LRE) were identified in the promoter region of these genes. To functionally test the VvFLS1 promoter(s), a transient assay was developed in Chardonnay suspension cells. Cells were bombarded with constructs containing potential transcription factors and the VvFLS1 promoter(s), fused to a luciferase reporter vector. After 48hrs incubation in the dark, cells were harvested and luciferase activity measured as an indicator of VvFLS1 promoter activity. Of the different transcription factors tested with the VvFLS1 promoter(s) the highest luciferase activity was observed using AtMYB12 (a flavonol-specific regulator of AtFLS1 in Arabidopsis (Mehrtens et al. 2005). While this result shows activation of the VvFLS1 promoters by AtMYB12 and the development of a transient reporter assay for testing the VvFLS1 promoter(s) a grapevine transcription factor specific for VvFLS1 was sought. Two techniques were employed to identify potential transcription factor regulators of the VvFLS1 promoter(s). The first involved BLAST sequence search analysis in a grapevine expression (EST) database with AtMYB12 and the second involved using DNA microarray technology to identify candidate transcription factors that were up-regulated in light exposed Chardonnay cell suspension cultures. Thirteen potential transcription factors were identified and after correlative RT-PCR analysis (with VvFLS1 expression patterns) two candidates were selected for further isolation and characterisation. These results have made significant progress in unravelling the molecular mechanisms of regulation of the flavonol biosynthetic, however additional experiments are required to unravel the transcriptional control of flavonol biosynthesis. This investigation contributes to our knowledge of flavonoid synthesis in grapes; how it is coordinated, the relationship with wine quality, and the influence of light particularly on synthesis of flavonols. It also explores the molecular mechanisms of VvFLS1 control, through isolation of the VvFLS1 promoter and identification of potential transcription factors, which may regulate it. An understanding of the synthesis of flavonoids and how they may be coordinated, particularly in response to light, could be used to improve fruit quality by enhanced viticultural management. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326767 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Molybdenum (Mo) is an essential micronutrient required by plants. It is biologically inactive until bound in a pterin compound named the molybdenum cofactor (Moco) that binds to apoproteins used in both reductive and oxidative reactions such as nitrate reductase (NR), xanthine dehydrogenase (XDH), aldehyde oxidase (AO) and sulfite oxidase (SOX). In Vitis vinifera cv. Merlot, molybdenum deficiency is common amongst vines grown on own roots in acidic soils often resulting in yield reductions. Foliar application of molybdenum sprays increases yield and remedies deficiency indicating that Merlot grown on own roots has a reduced capacity for molybdenum uptake from the soil. Molybdenum generally occurs as molybdate (MoO₄ ²⁻) within the soil solution. The mechanism(s) involved in molybdenum transport have recently been discovered in plants, although are well characterised within prokaryotic systems. Unfortunately, no homologues of prokaryotic genes involved in molybdate transport exist within eukaryotes. It has been suggested that molybdenum transport in plants may occur through other systems including sulfate transporters due to chemical similarities between sulfate and molybdate. A yeast functional complementation approach using a sulfate transport mutant was initially used to identify novel putative plant molybdenum transport proteins. A cDNA library derived from Pinot noir roots starved of molybdenum was screened for transporters. Unfortunately, no cDNAs were identified that met the requirements of a molybdenum transporter when screened on media containing low amounts of molybdenum. However, a number of putative cDNA’s partially complemented the yeast mutant YSD1, however none of these could be validated in second round screens. A candidate gene approach was then initiated to identify pre-characterised genes that may also have capacity to transport molybdenum. The plant sulfate transporter, SHST1, restored growth of YSD1 on media containing low amounts of molybdenum. Kinetic analysis using ⁹⁹MoO₄²⁻ to trace molybdenum transport in yeast cells demonstrated that SHST1 enhanced the uptake of molybdenum at nM concentrations. The uptake was not inhibited by sulfate, but the transport of sulfate was reduced with molybdenum. Further analysis demonstrated that SHST1 did prefer sulfate as the substrate but molybdenum could compete at higher concentrations. This result is the first measurement of molybdenum being transported through a pre characterised sulfate transport protein. Whole plant experiments using rooted grapevine cuttings and ⁹⁹MoO₄²⁻ to trace molybdenum movement into plants indicated that Merlot did not have reduced capacity to uptake molybdenum compared to other varieties that do not suffer from molybdenum deficiencies such as Chardonnay. When plants were grown with molybdenum, Merlot accumulated more molybdenum than Chardonnay, with the reverse being true when plants were grown without molybdenum. Similar experiments were performed on symbiosomes isolated from Glycine max grown with and without molybdenum. Symbiosomes absorbed more molybdenum when plants were grown without molybdenum. A field site was established to look at the molybdenum profiles within petioles against yield responses over a 3-year period. Molybdenum application did not increase the yield amongst vines despite all vines initially being deficient in molybdenum. There were no cumulative effects of molybdenum application over the trial, however, molybdenum did have limited translocation ability within the vine system. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1318770 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Soil salinity is one of the major abiotic stresses that decreases agricultural crop production through imposition of both ionic and osmotic stresses. The accumulation of Na⁺ and Cl⁻ in the cytosol to toxic levels inhibits metabolism. Unlike Na⁺, less is known about Cl⁻ uptake and transport in plants. Grapevine is moderately sensitive to salinity and accumulation of toxic levels of Cl⁻ in leaves is the major reason for salt-induced symptoms. In this study Cl⁻ uptake and transport mechanism(s) were investigated in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance: 1103 Paulsen (salt tolerant) and K 51-40 (salt sensitive). Increased external salinity caused high Cl⁻ accumulation in shoots of the salt sensitive K 51-40 in comparison to Paulsen. Measurement of ¹ ⁵ NO₃⁻ net fluxes under high salinity showed that by increasing external Cl⁻ concentrations K 51-40 roots showed reduced NO₃⁻ accumulation. This was associated with increased accumulation of Cl⁻. In comparison to Paulsen, K 51-40 showed reduced NO₃⁻ / Cl⁻ root selectivity with increased salinity, but Paulsen had lower selectivity over the whole salinity range (0-45 mM). In order to examine if root hydraulic and permeability characterisations accounted for differences between varieties, the root pressure probe was used on excised roots. This showed that the osmotic Lpr was significantly smaller than hydrostatic Lpr, but no obvious difference was observed between the rootstocks. The reflection coefficient (σ) values (0.48-0.59) were the same for both rootstocks, and root anatomical studies showed no obvious difference in apoplastic barriers of the main and lateral roots. Comparing the uptake of Cl⁻ with an apoplastic tracer, PTS (3-hydroxy-5, 8, 10-pyrentrisulphonic acid), showed that there was no correlation between Cl⁻ and PTS transport. These results indicated that by-pass flow of salts to the xylem is the same for both rootstocks (10.01±3.03 % and 12.1±1.21 %) and hence pointed to differences in membrane transport to explain difference in Cl⁻ transport to the shoot. ³ ⁶Cl⁻ fluxes across plasma membrane and tonoplast of K 51-40 and Paulsen roots showed that ³ ⁶Cl⁻ influx in root segments of Paulsen was greater than K 51-40 over the first 10 minutes. Unidirectional influx within 10 min loading time showed increases with increases in the external concentrations in both rootstocks but Paulsen had higher influx rate when compared to K 51-40. This appeared to be due to a greater Vmax. There was no significant difference in Km. It was shown that ³ ⁶Cl⁻ accumulation and transport rate to the shoot of K 51-40 was higher than that of Paulsen. Compartmental analysis of ³ ⁶Cl⁻ efflux from intact roots confirmed that the difference in influx observed between the rootstocks was consistent with the results obtained for excised roots, although the values were not exactly the same. It was also shown that the main root of Paulsen had greater contribution to ³ ⁶Cl⁻ uptake than lateral roots. ³ ⁶Cl⁻ fluxes by lateral roots were not significantly different between the rootstocks. Cl⁻ and Na⁺ distribution patterns in different root cell types were determined using the X-ray microanalysis technique. It was shown that Cl⁻ content in the hypodermis and cortical cells was higher than the other cell types in both rootstocks, but overall Cl⁻ content in the root of Paulsen was higher than K 51-40. The pericycle of the main root of Paulsen accumulated more Cl⁻ than K 51-40. It was concluded that Cl⁻ loading to the xylem was different in the rootstocks and Paulsen tended to prevent the xylem Cl⁻ loading process. Lateral roots also displayed opposite behaviour consistent with flux analysis. Membrane potential difference (PD) of the cortical cells showed a rapid and transient depolarization by adding 30 mM NaCl in both rootstocks that was followed by a gradual hyperpolarization. Depolarizations caused by 30 mM Choline-Cl, Na-MES and NaCl measured by the root surface potential method showed that Choline-Cl in K 51-40 and Na-MES in Paulsen caused greater depolarization than that of Na-MES in K 51-40 and Choline-Cl in Paulsen respectively. Assuming that PD measured in this method was the trans-root potential (TRP), it was concluded that the higher depolarization by Choline-Cl in K 51-40 can be due to higher Cl⁻ efflux rate to the xylem. Two different mechanisms were also detected for Cl⁻ transport: HATS which was observed in the range of 0.5-5 mM and a LATS in the range of 10-30 mM of the external NaCl concentration. This was consistent with the concentration dependence of Cl⁻ influx. In conclusion, evidence obtained from different experiments of this study indicated that in the grapevine rootstocks (Paulsen and K 51-40) Cl⁻ was mostly transported through the symplastic pathway. From ECl values determined for the rootstocks by the Nernst equation, a proton-driven transport system was responsible for Cl⁻ transport in both the HATS and LATS range of external NaCl concentrations. The rate of Cl⁻ transport from the root to shoot (xylem loading) was the major difference in Cl⁻ transport between the rootstocks in terms of salinity tolerance. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339051 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Soil salinity is one of the major abiotic stresses that decreases agricultural crop production through imposition of both ionic and osmotic stresses. The accumulation of Na⁺ and Cl⁻ in the cytosol to toxic levels inhibits metabolism. Unlike Na⁺, less is known about Cl⁻ uptake and transport in plants. Grapevine is moderately sensitive to salinity and accumulation of toxic levels of Cl⁻ in leaves is the major reason for salt-induced symptoms. In this study Cl⁻ uptake and transport mechanism(s) were investigated in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance: 1103 Paulsen (salt tolerant) and K 51-40 (salt sensitive). Increased external salinity caused high Cl⁻ accumulation in shoots of the salt sensitive K 51-40 in comparison to Paulsen. Measurement of ¹ ⁵ NO₃⁻ net fluxes under high salinity showed that by increasing external Cl⁻ concentrations K 51-40 roots showed reduced NO₃⁻ accumulation. This was associated with increased accumulation of Cl⁻. In comparison to Paulsen, K 51-40 showed reduced NO₃⁻ / Cl⁻ root selectivity with increased salinity, but Paulsen had lower selectivity over the whole salinity range (0-45 mM). In order to examine if root hydraulic and permeability characterisations accounted for differences between varieties, the root pressure probe was used on excised roots. This showed that the osmotic Lpr was significantly smaller than hydrostatic Lpr, but no obvious difference was observed between the rootstocks. The reflection coefficient (σ) values (0.48-0.59) were the same for both rootstocks, and root anatomical studies showed no obvious difference in apoplastic barriers of the main and lateral roots. Comparing the uptake of Cl⁻ with an apoplastic tracer, PTS (3-hydroxy-5, 8, 10-pyrentrisulphonic acid), showed that there was no correlation between Cl⁻ and PTS transport. These results indicated that by-pass flow of salts to the xylem is the same for both rootstocks (10.01±3.03 % and 12.1±1.21 %) and hence pointed to differences in membrane transport to explain difference in Cl⁻ transport to the shoot. ³ ⁶Cl⁻ fluxes across plasma membrane and tonoplast of K 51-40 and Paulsen roots showed that ³ ⁶Cl⁻ influx in root segments of Paulsen was greater than K 51-40 over the first 10 minutes. Unidirectional influx within 10 min loading time showed increases with increases in the external concentrations in both rootstocks but Paulsen had higher influx rate when compared to K 51-40. This appeared to be due to a greater Vmax. There was no significant difference in Km. It was shown that ³ ⁶Cl⁻ accumulation and transport rate to the shoot of K 51-40 was higher than that of Paulsen. Compartmental analysis of ³ ⁶Cl⁻ efflux from intact roots confirmed that the difference in influx observed between the rootstocks was consistent with the results obtained for excised roots, although the values were not exactly the same. It was also shown that the main root of Paulsen had greater contribution to ³ ⁶Cl⁻ uptake than lateral roots. ³ ⁶Cl⁻ fluxes by lateral roots were not significantly different between the rootstocks. Cl⁻ and Na⁺ distribution patterns in different root cell types were determined using the X-ray microanalysis technique. It was shown that Cl⁻ content in the hypodermis and cortical cells was higher than the other cell types in both rootstocks, but overall Cl⁻ content in the root of Paulsen was higher than K 51-40. The pericycle of the main root of Paulsen accumulated more Cl⁻ than K 51-40. It was concluded that Cl⁻ loading to the xylem was different in the rootstocks and Paulsen tended to prevent the xylem Cl⁻ loading process. Lateral roots also displayed opposite behaviour consistent with flux analysis. Membrane potential difference (PD) of the cortical cells showed a rapid and transient depolarization by adding 30 mM NaCl in both rootstocks that was followed by a gradual hyperpolarization. Depolarizations caused by 30 mM Choline-Cl, Na-MES and NaCl measured by the root surface potential method showed that Choline-Cl in K 51-40 and Na-MES in Paulsen caused greater depolarization than that of Na-MES in K 51-40 and Choline-Cl in Paulsen respectively. Assuming that PD measured in this method was the trans-root potential (TRP), it was concluded that the higher depolarization by Choline-Cl in K 51-40 can be due to higher Cl⁻ efflux rate to the xylem. Two different mechanisms were also detected for Cl⁻ transport: HATS which was observed in the range of 0.5-5 mM and a LATS in the range of 10-30 mM of the external NaCl concentration. This was consistent with the concentration dependence of Cl⁻ influx. In conclusion, evidence obtained from different experiments of this study indicated that in the grapevine rootstocks (Paulsen and K 51-40) Cl⁻ was mostly transported through the symplastic pathway. From ECl values determined for the rootstocks by the Nernst equation, a proton-driven transport system was responsible for Cl⁻ transport in both the HATS and LATS range of external NaCl concentrations. The rate of Cl⁻ transport from the root to shoot (xylem loading) was the major difference in Cl⁻ transport between the rootstocks in terms of salinity tolerance. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339051 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008