Organic acid metabolism and the control of grape berry acidity in a warming climate

Abstract

The objective of this project was to identify potential targets for the manipulation of organic acid profiles in grapes, with a long-term goal of minimising the impact of climate change on grape must acidity. Transgenic grapevines were developed to better understand how acidity is regulated within berries and leaves. New metabolic models were generated from field- and chamber-based temperature experiments and from cultivars with inherently different acid profiles. These demonstrated correlative links between organic acid and amino acid metabolism. Therefore altering nitrogen supply may provide a relatively straightforward means for manipulating berry acid levels, warranting further investigation.

Summary

Two strategies could be used to combat low-acidity in grapes grown during hot seasons. The first is to identify a management tool to reduce the loss of malic acid upon exposure of the vine to elevated temperatures. The second is to increase levels of tartaric acid in the fruit, such that losses of acidity due to malic acid degradation are compensated by an abundance of heat-stable tartaric acid. This project aimed to advance progress on both of these strategies and could thus be divided into two general aims. The first was to pinpoint important regulatory junctions of malic acid metabolism that may be targeted for reducing acid losses during hot periods of the season. The second was to discover new genes involved in the largely uncharacterised tartaric acid biosynthesis pathway, such that tartaric acid production may be manipulated to control acidity in the berry regardless of seasonal temperature. To address the first aim, elevated temperature treatments in field and controlled-environment (chamber) conditions were used to explore the effects on various genes involved in malic acid metabolism, as well as the effects on other metabolite pools within the berry. Based on gene transcript levels, three distinct areas of malic acid metabolism seem to be affected by elevated temperature: some malic acid synthesis enzymes were down-regulated, some malic acid degradation enzymes were up-regulated, and some malic acid transporters were affected. Overall, improving the ability of a cell to compartmentalise malic acid such that it is protected from degradation, as well as improving the ratio of enzyme activities for malic acid synthesis relative to degradation, would decrease the likelihood that malic acid will encounter an enzyme capable of degrading it, and thus could help to retain higher levels of malic acid in response to elevated temperatures or during extended ripening periods. Based on metabolite levels, there was a negative correlation between malic acid and amino acid levels, which suggested that a change in the balance of carbon and nitrogen pools in the fruit could alter malic acid metabolism. This was consistent with some of the observed shifts in the expression of malic acid-metabolising enzymes, which can act as branch-points between organic acid and amino acid metabolism. It was also consistent with data from a grapevine cultivar comparison conducted within this study. This suggests that the levels of organic acids in the fruit at harvest may be malleable, through the management of nitrogen levels. In the literature there are some references to altered acidity of berries when nitrogen status is altered, but this has not been closely investigated as a management tool for malic acid levels. The effect of nitrogen supply on the transcript levels and activities of genes involved in malic acid metabolism seems to be an important way forward in this area. To address the second aim, candidate genes for uncharacterised steps of the tartaric acid biosynthesis pathway were explored to determine whether the corresponding enzymes could in fact carry out the expected reactions. For one of these genes, the expected activity could indeed be demonstrated, however the enzyme was far more active with some alternative substrates, and therefore the primary role of this gene product was unlikely to be tartaric acid biosynthesis. However, this may be an example of a biosynthetic pathway commandeering enzymes from other pathways and performing multiple functions within the cell. In addition, the already-known enzyme of the tartaric acid biosynthesis pathway in grapevine was recently shown to have diverged from an enzyme family involved in sorbitol metabolism in the V. vinifera genome. Therefore, when specific tartaric acid precursors are available in the grape berry cell, this new candidate enzyme could be capable of utilising them for tartaric acid biosynthesis. However, when the precursors are not available this enzyme may carry out other functions, as may be the case for more than one step in the tartaric acid biosynthetic pathway. In order to confirm whether this candidate enzyme contributes to tartaric acid biosynthesis, it is necessary to determine whether tartaric acid levels are affected by altering expression of this gene in grapevine. Transgenic plants for such an investigation have been generated during this project and await further analysis. Transgenic grapevines were also generated to target other genes thought to be involved in organic acid metabolism, in a regulatory (i.e. rate-limiting) capacity. A number of plants were generated, targeting different genes of the tartaric acid biosynthesis pathway, including the newly characterised gene mentioned above. These plants still need to be analysed comprehensively in order to determine the effects on metabolite levels in the leaves and/or fruit.