A study of amino acid metabolism in grape berries (Vitis vinifera L.Sauvignon blanc): A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Lincoln University

Amino acids are important primary metabolites and one of the major sources of nitrogen in grapes. Amino acids also occupy a central junction in grapevine biochemistry and are important for vine metabolism and berry homeostasis. The majority of a grapevines photosynthesis and nitrogen assimilation occurs in the leaves, the products of which (sugars and amino acids) are exported to the grape bunches (fruit). In the grape berries themselves, amino acids are the precursors to secondary compounds supporting grapevine growth and physiology, while additionally having an important role in wine quality outcomes. Yet, despite the importance of amino acids in grapevine, their regulation and accumulation in grapes is poorly understood and usually inferred through research in other plant species. Leaf and shoot removal are common practice in commercial vineyards, with any such canopy management interventions having the potential to influence berry biochemical composition. This well-established viticultural intervention was used as an experimental technique to investigate the effect on amino acid biochemistry in Sauvignon blanc grapes. Basal leaf removal had a significant effect on amino acid accumulation in Sauvignon blanc grape berries. This was reflected in reductions of total amino acid concentrations in the berry, differential accumulation at the level of amino acid families and within families, individual amino acids. Individual amino acids also had differential responses to leaf removal. Aside from the quantitative effect of basal leaf removal on amino acid concentrations, leaf removal also had a qualitative effect on some individual amino acids, modifying their proportions of accumulation in the grape berries. The α-ketoglutarate family of amino acids (glutamine, glutamate, arginine and proline) was further studied, being the family that contains the predominant concentrations of amino acids through berry development and having both quantitative and qualitative aspects in their response to basal leaf removal. Glutamine was the dominant amino acid early in development and decreased throughout. Proline and arginine both increased steadily through development to become most abundant amino acids in grape berries at harvest. Glutamate levels stay relatively consistent through development despite being the direct precursor of the two most abundant amino acids at harvest (arginine and proline) and contributing as a substrate / co-factor for other amino acids via other metabolic reactions (pathways). The significant reduction in amino acid concentrations in grape berries due to leaf removal, was hypothesised to be predominantly due to the effect of an altered source/sink balance and partitioning impacts. The accumulation of sugars in the berry was less impacted by the same leaf removal treatments. A model was proposed, whereby the younger leaves further up the shoot can compensate with an increased carbohydrate export to the bunches, but this same compensation is not achieved with respect to amino acids. The mechanisms of amino acid accumulation in the grape berries was further examined, by investigating a number of genes involved in different aspects of α-ketoglutarate amino acid biochemistry. Investigating transcriptional changes of genes involved in these amino acid metabolic pathways, demonstrated differential expression of a range of transcripts involved in metabolism and regulation of glutamine, glutamate, arginine and proline. These experiments also present novel information, regarding the up-regulation of expression of genes involved in arginine and proline metabolism through berry development. In other plant species, proline is induced as a stress response, but in grapevine, the proline metabolic pathways are not well characterised and the accumulation of proline is poorly understood. An increase in expression of proline biosynthetic genes through veraison was demonstrated with a maintenance of transcript counts at postveraison time points, overlapping the period in berry development when proline accumulation in the berry is increasing and the berry is starting to accumulate larger amounts of sugars. An increase in expression of genes related to proline and arginine degradation pathways was also demonstrated through berry development. This implied that even as the berry is accumulating arginine and proline, there appeared to be the potential for interconversion and turnover of these amino acids through the upregulation of enzymes involved in their catabolism. The relative activity of an associated enzyme (ornithine aminotransferase) which was transcriptionally up-regulated, was also studied. Enzyme assays performed on treatment samples indicated that ornithine aminotransferase specific activity increased through Sauvignon blanc berry development and ripening. Additionally, the maintenance of a leaf canopy was important in regulating ornithine aminotransferase enzyme activity, as relative specific activity was increased by basal leaf removal compared to control (maintained leaf canopy) samples. This indicated the potential for allosteric regulation of key amino acid pathway enzymes in grape berries. Overall, the results presented in this thesis substantially improve the understanding of the mechanisms influencing amino acid biochemistry in Vitis vinifera L. var. Sauvignon blanc grapes.