Author: Crystal Sweetman
Sweetman, Crystal, 2012 An examination of malate metabolism in Vitis vinifera during fruit ripening and in response to elevated vineyard temperature, Flinders University, School of Biological Sciences
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Acidity is an important characteristic for many fruits, particularly in aspects of fruit biochemistry and sensory quality. In grapes (Vitis vinifera), one of the most important agricultural fruiting crops, the use of fruit with low acidity and high pH in winemaking can increase risk of microbial spoilage and undesirable fermentative outcomes. Levels of the organic acid malate can determine acidity and pH in many fruits, through species- and cultivar-specific regulation of developmental and environmental responses. Malate is also extensively involved in primary and secondary metabolic pathways that can be critical to fruit development and ripening. The grape berry demonstrates particularly striking changes in malate accumulation and degradation during development and in response to vine temperature, likely driven by changes in metabolic pathways involving the acid. If harvested late in the season, or if ripening berries are exposed to unusually warm temperatures, the fruit are likely to contain less-than-optimal acid due to reduced levels of malate. Despite the considerable influence that berry acidity imparts on berry and wine quality, the mechanisms of regulation for malate metabolism in response to changes in grape berry development and vine temperature are still largely unknown. This thesis contains an evaluation of the activities and transcripts of enzymes in V. vinifera fruit that are involved in processes such as glycolysis, gluconeogenesis, CO2 assimilation, respiration and fermentation, each involving malate either directly or indirectly. Enzymes from purified grape berry mitochondrial preparations were also explored, providing evidence for two activities that were, until now, undetermined in grapes: NAD-dependent malic enzyme and alternative oxidase. A developmental series of grapes from fruit set until harvest maturity displayed the expected pattern of pre-veraison malate accumulation, and post-veraison malate loss. Activities or transcripts that were differentially regulated with development included NADP-dependent malic enzyme, PEP carboxylase, PEP carboxykinase, pyruvate, orthophosphate dikinase, alcohol dehydrogenase and the terminal oxidases of the mitochondrial transport chain. To test the effect of elevated vine temperature on grape berry malate, field-based heat trials were established at numerous developmental stages. Temperature experiments were set up as mild, long-term heating trials to mimic a climatic shift, as well as shorter, more intense trials to represent heat-wave events. Effects of raising bunch temperature during the day and night were also investigated. Heating of grapevines at the post-set developmental stage (i.e. malate accumulation phase) led to higher berry malate, particularly when bunches were heated at night. Heating of grapevines around the veraison and pre-harvest stages (i.e. malate degradation phase) generally led to lower berry malate, unless bunches were also heated at night, or if the majority of malate had already been lost from the fruit before the time of treatment. Data indicated that the temperature-sensitive changes in grape berry malate during the day and/or night at different developmental stages could be linked to alterations in PEP carboxylase, PEP carboxykinase, pyruvate, orthophosphate dikinase and NAD-dependent malic enzyme. Implications of such shifts in metabolism are discussed in detail within the text.
Keywords: grape,organic acid,temperature,gene transcript,enzyme
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Kathleen Soole