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In most New Zealand vineyards planted in Sauvignon blanc, cane pruning is an essential management practice with direct consequences on yield, ripeness, and balance. In Marlborough, the largest wine grape producing region of New Zealand, Sauvignon blanc is generally pruned to three or four canes contrary to the traditional two-cane pruning system. Not only is four-cane pruning more labour demanding and more challenging for the selection of fruiting canes, but also it results in the production of more above-ground biomass as the vine is loaded with more nodes compared with two-cane pruning. Cane pruning requires skilled labour, which has become a rare commodity to find and retain. Artificial Intelligence (AI) technologies can support least skilled human pruners and guide automated pruning systems, thus alleviating this labour shortage. However, training an AI system in cane pruning requires learning from the decisions of expert human pruners. Besides being a specialised task, cane pruning also ensures that grapevines are kept in balance, and node number retention is one of the initial and cost-effective ways of achieving it. At the pre-flowering growth stage, the vine budburst percentage is used to assess capacity and infer balance, with values around 100% being regarded as optimal. However, this metric does not accurately account for other underlying physiological conditions such as number, distribution and location of double shoots, non-count shoots and blind nodes, which are also key indicators of potential balance. From veraison to harvest, ratios such as LA/FM – leaf area to fruit mass, ELA/FM – exposed leaf area to fruit mass, FM/PM – fruit mass to pruning mass, FM/CM – fruit mass to cane mass as well as fruit composition through ripening provide measures of the vine performance and balance. The interpretation of these metrics requires their comparison with optimal ranges, which in most cases, are often non-existent for many grapevine cultivars and vineyard conditions. When such metrics exist, their wide range makes their use impractical for specific growing conditions warranting research to find optimal values that are specific to cultivars, locations, and growing conditions. Therefore, to train an AI system in cane pruning and to enhance the monitoring and assessment of vine balance, retained dormant shoot attributes were characterised and the effect of retained node numbers on the vegetative and reproductive growth of Sauvignon blanc were analysed. To characterise winter cane pruning decisions for the selection of fruiting canes and renewal spurs, eight dormant shoot attributes – diameter, length, vertical distance from the bottom fruiting wire (VD), horizontal distance from the vine trunk (HD), node count, internode length, origin, and position relative to bottom fruiting wire (PFW) – were measured before and after pruning. This first experiment was conducted in two vineyard sites located in the Awatere Valley, Marlborough (New Zealand) over two growing seasons (2019/20-2020/21). In one site (Site 1), vine canopies were modified according to a 5 [total node numbers on canes: 10, 20, 30, 40, 50] x 3 [total node numbers on spurs: 1, 2, 3] factorial design and in the other site (Site 2) there was no canopy modification. The second experiment was conducted to investigate the physiological response of Sauvignon blanc to retained node numbers at budburst, pre-flowering, veraison, harvest, and winter dormancy in three different sites over two growing seasons (2019/20-2020/21). For this second experiment, the three sites used were coded Site 1, Site 2 and Site 3. Site 1 was the same as in the first experiment (site with vine canopies modified), Site 2 was located within the same vineyard row as Site 1, and Site 3 was in a vineyard block in Waipara, Canterbury. At Site 1, vine node numbers were set according to the same factorial design as in the first experiment (i.e. 5 [total node numbers on canes: 10, 20, 30, 40, 50] x 3 [total node numbers on spurs: 1, 2, 3]). At Site 2 and Site 3 vine node numbers were set according to a 5 [total node numbers on canes: 10, 20, 30, 40, 50] x 2 [total node numbers on spurs: 1, 2] factorial design. Cane pruning stripped the vine of 82% dormant shoots, the remaining 18% formed the next season’s canes and spurs. Retained and non-retained dormant shoots significantly differed in all their attributes except for node count. On average, retained dormant shoots at Site 1 were 9.2±0.07 mm diameter, 104.7±0.93 cm long, 11.4±0.65 cm HD and 83.2±0.54 cm VD. Retained dormant shoots that formed spurs had the shortest HD (9.8±0.93 cm) and VD (77.8±0.7 cm) and originated by order of preference from the vine head, basal nodes or first nodes on old spurs and old canes. Still at Site 1, retained dormant shoots that formed fruiting canes were 9.1±0.08 mm diameter, 106.4±0.89 cm long, 85.7±0.69 cm VD, 12.2±0.85 cm HD and originated by order of preference from old canes, old spurs and vine head. Increasing the vine node load in 2019 resulted the following winter (2020) in more dormant shoots for spurs being selected from old canes (49%), less from the vine head (25% reduction) and old spurs (14% reduction). Moreover, the number of spurs retained on old canes in winter 2020 increased with node load. Retained dormant shoots on 50-node vines compared with 10-node vines increased in VD (from 83.8 to 87.8 cm; p100%), a cane budburst close to 100%, the growth of numerous non-count shoots on the vine head (29.5±3.0 shoots, p

Grape pomace, the residue obtained after making wine, contains large quantities of polyphenols, dietary fibre, and has a high antioxidant capacity. The contents of the valuable compositions in grape pomace are believed to be much higher than the ones extracted from the grape to wine. The main polyphenols detected in grape pomace include hydroxycinnamic acids, hydroxybenzoic acids, flavan-3-ols, flavonols, and stilbenes. Cereal and cereal-based products are popular foods all over the world. However, they have a high carbohydrate content and a low polyphenol and antioxidant content. In addition, too high an intake of these highly digestible starch foods is believed to be involved in global issues of obesity and high blood sugar levels. Consumers’ demand for healthier and natural origin products has been increasing in recent decades, along with the awareness of the importance of “fresh” products. Hence, the use of plant parts to fortify cereal-based products has attracted much research, resulting in proven benefits to human health. Grape pomace has been added to many types of product, including starchy food and dairy products. Overall, cereal-based products have been confirmed to be an apparopriate food matrix to apply grape pomace based on its nutritional and physical characteristics. In this study, three white grape pomaces (Sauvignon Blanc, Pinot Gris, and Gewürztraminer) and two red grape pomace (Merlot and Pinot Noir) obtained from a New Zealand winery were the main objects. Analysis of compositions of these grape pomaces showed a high content of total phenolic compounds, in which the red grape pomaces had higher concentrations than the white ones. Between varieties, Pinot Noir had the highest content of phenolic compounds, followed by Merlot, Pinot Gris, and Gewürztraminer, while Sauvignon Blanc possessed the lowest value. The results also showed that the concentration ranges of total phenolic content found in New Zealand grape pomaces were similar to the values found in grape pomaces obtained elsewhere such as Brazil, France or Italy. Further analysis by HPLC found that catechin and epicatechin were the most abundant phenolics in all pomaces. A significant concentration of malvidin-3-o-glucoside was found in Merlot and Pinot Noir pomaces, but was not detected in white pomaces. The antioxidant capacity was reflected in the total phenolic content of each pomace with the red pomaces showed a better antioxidant capacity than the white pomaces.The red pomaces had a significantly higher content of both soluble and insoluble dietary fibre content. In contrast, white pomaces had a higher sugar content than the red pomaces. Replacement of wheat flour with different levels (5 %, 10 %, and 15 %) of grape pomace powder was performed to investigate the change in physicochemical and nutritional properties of cookies. Inclusion of pomace powder caused a significant reduction (p < 0.05) of both the thickness and diameter of the cookies. The cookies hardness decreased significantly (p < 0.05) with increased supplement levels, but there was no difference (p > 0.05) in the reduction between grape varieties. The colour of cookies also changed significantly (p < 0.05), they became darker with higher levels of addition (L* value decreased), the increase of a* value and decrease of b* value indicated that the final products got redder and bluer. The total phenolic content of cookies increased with increasing of pomace addition. The results of scavenging capacity determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric ion reducing antioxidant power (FRAP) were in agreement with the phenolic content of cookies. The amount of sugar released during the in vitro digestion of cookies was attenuated due to the fortification with grape pomaces. Samples with the addition of 15% Pinot Noir and Merlot showed the greatest reduction in reducing sugar release. All other samples reduced the content of sugar in comparison with the control sample. Fortification of rice starch using grape pomace powder was conducted by using a rapid visco-analyser (RVA) to investigate their impacts in another type of food matrix, namely a paste. Pasting properties of paste significantly changed (p < 0.05) with the addition of pomace powder. The peak viscosity, breakdown, final viscosity, and setback of the pastes decreased significantly (p < 0.05), and the pasting temperature increased. The pasting profile of starch is defined by the apparent ratio of amylose to amylopectin; hence the inclusion of grape pomace powder changed this ratio and the final pasting profile. In an in vitro digestion, the general trend of reducing sugar released was similar to the control paste and pastes with pomace powder, but the actual amounts of reducing sugars released were lower than the control. A higher reduction was observed in samples with Pinot Noir powder. Such reductions were attributed to the content of polyphenols and dietary fibre. The influences of polyphenols from grape pomaces were tested in the mean of extracts instead of traditional powder. The changes in pasting profile were similar to the changes observed with grape pomace powder, means that peak viscosity, breakdown, final viscosity and setback decreased with the increase of added level of polyphenol extracts. The temperature increased but the range of variation was much lower than the ones with pomace powder inclusion. The extracts showed the similar impacts to reduce sugar released during in vitro digestion process in comparison with pomace powder enrichment. In addition, the total phenolic content and antioxidant capacity before and after digestion were in similar trend with pomace powder samples did. The α-amylase inhibitory was also tested on these samples, with the highest inhibition rate was attributed to Pinot Noir (64.3%)

New Zealand Pinot noir has seen impressive growth in export sales in recent years. It is now second only to Sauvignon Blanc in production volume. There are four main Pinot noir regions in New Zealand: Central Otago, Waipara, Marlborough and Wairarapa. In these regions, the soil, climate and other conditions are suitable for Pinot noir growth. However, some environmental issues challenge Pinot noir growth in New Zealand, such as water deficit and UV-B. The mean annual rainfall in regions where Pinot noir is grown, is low, and long dry spells can occur, especially in summer. UV-B radiation in New Zealand is 30-40% higher than at similar latitudes in the Northern Hemisphere. In this research, the aim was to determine effects of the separate and combined UV-B and water deficit on vine physiology and chemical composition of Pinot noir fruit. In the 2015-2016 and 2016-2017 vintages, two rows of Pinot noir grapevines in the West Vineyard at Lincoln University were chosen for the study. Treatments were a combination of leaf removal with plastic screens or shade cloth around the fruiting zone, and/or restricted irrigation. Grapevines and the fruit both responded to UV-B and water deficit. In comparison to exposure to natural UV-B in the vineyard, the potted vines were moved into a glasshouse for preparation for the experiments in September, prior to budbreak. From October (fruit-set) to December (veraison), the grapevines were uniformly irrigated on a regular basis to soil capacity and were exposed to normal daylength hours in the glasshouse. From veraison, vines in treatments were exposed to supplemental UV-B interaction with restricted irrigation in a glasshouse. The physiology of Vitis vinifera L. var. Pinot noir vines were altered by water deficit and UV-B. The combination of UV-B and water deficit changed the vine water status and leaf greenness in the glasshouse, but there was only a slight effect on berry parameters. However, UV-B exposure/exclusion interaction with water deficit did not affect vine water status and leaf greenness, even with no significant changes in fruit production capacity and °Brix in the vineyard. Amino acids did not show consistent results in the two-year trial. In the glasshouse, amino acids were decreased by UV-B and water deficit in 2015-2016. However, there was no change in amino acids in response to UV-B in 2016-2017. The concentration of amino acids under water deficit was enhanced by its combination with UV-B. In the vineyard, there were no changes in fruit amino acids with UV-B exposure or exclusion over the two years, but the interaction of UV-B with water deficit significantly changed His, Val, Thr and Lys in 2016-2017. Under UV-B and water deficit, vines were found to respond in both the glasshouse and vineyard trials. When berries were directly exposed to UV-B or water deficit, phenolic compounds accumulated to a greater degree in the berry skins. The combined stresses caused larger increases in phenolic composition than the individual stresses alone, such as skin anthocyanin contents in +UV-W (0.578 mg/berry) vs. -UV+W (0.398 mg/berry). A similar pattern was shown in the volatile compounds. For example, the concentration of hexanol was 1028.6 µg/L in +UV-W compared with 683.3 µg/L in -UV+W. Overall, this study clearly demonstrates that UV-B exposure can interact with water deficit to increase the effects on physiological parameters of the vine and fruit composition to UV-B or water deficit alone. Water deficit can, potentially, increase additional responses to UV-B in both glasshouse and vineyard situations.

Sauvignon blanc wine currently comprises about 86% of total NZ wine exports. Its success has been ascribed to its distinctive flavour and aroma, which has been the subject of much research. However, less attention has been paid to the role of palate structure (e.g. mouthfeel) in defining the style, and to the effects of specific winemaking techniques such as lees contact during wine aging. In this study, the effects of different lees management techniques (lees type, stirring frequency, and addition of β-glucanse) on the release of chemical compounds (i.e. nitrogenous compounds, polysaccharides, mannoprotein, β-glucan and glycerol) in model wine were investigated. Because yeast autolysis is a slow process at low temperatures and at the low pH values existing during winemaking, the study also investigated the release of chemical compounds from pulsed electric field (PEF) induced yeast autolysis using rehydrated active yeast with/without addition of β-glucanse. In addition to limited information on lees management appropriate to New Zealand styles of Sauvignon blanc, the lexicon to describe Sauvignon blanc wine mouthfeel is poorly investigated. In this study, napping® was used in order to try and capture terms for the description of mouthfeel in Sauvignon blanc wine, and to determine how these words relate to the chemical composition of wines. Time and lees materials had a significant impact on the concentrations of total protein, primary amino acids nitrogen (PAN), neutral and acidic polysaccharides, mannoprotein, β-glucan and glycerol in model wines (P < 0.001). In addition, inactivated yeast was a good source of amino acids, especially Pro, Arg, Glu and Gln. Inactivated yeasts was a better source of polysaccharides than rehydrated yeasts and collected lees. The majority of the polysaccharide released was in the neutral form and reached concentrations ranging from 600 to 800 mg galactose/L on day 160 in model wine. Both addition of β-glucanases and stirring frequency were minor factors that affected the release of chemical compounds from lees material. Stirring frequency had a significant impact on the concentrations of protein, PAN and neutral polysaccharide (P < 0.05). In addition, stirring frequency showed a statistical impact on protein, PAN and polysaccharide in model wines, but differences were small compared with the overall changes that were achieved at the end of the aging experiment. The study also showed it was feasible to accelerate on-lees aging with pulsed electric field. The experiment demonstrated that the application of PEF (5.5 and 10.0 kV/cm field strength) to lees (5% w/v, rehydrated yeasts) provoked a release of intracellular materials, such as nitrogenous compounds, polysaccharides, and mannoprotein. In addition, time and lees materials also had a significant impact on the concentrations of total protein, primary amino acids nitrogen (PAN), neutral and acidic polysaccharides, mannoprotein, β-glucan and glycerol in model wines (P < 0.001). In the current study, perceived sweetness and acidity seemed to be the major criteria for sorting the commercial examples of Sauvignon blanc wines. In total, 253 descriptors were collected but more than half of these descriptors were used no more than twice on different occasions, and a combination of synonyms was not able to reduce this number significantly. Descriptors that had a frequency of more than 20 were too few to create a comprehensive lexicon. There was no precise correlation between chemical composition and collected descriptors (especially for the ones with low frequency). However, the distribution of wines on the consensus map seemed to be associated with the vinification technique, namely, on-lees aging and vessels used during fermentation and post fermentation. Panellists were able to distinguish the standard and on-lees aged Sauvignon blanc wines.

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.

Winter pruning is the highest yearly expenditure in the typical New Zealand vineyard budget, yet few attempts have been made to bring quantitative measurement tools into its management. The research presented here constitutes first steps towards this end, in tandem with University of Canterbury researchers working towards an artificially intelligent pruning robot. In pursuit of information regarding cane pruning preferences and decision-making criteria, a two-part survey was conducted in the regions of Marlborough, Hawke’s Bay, Waipara and Central Otago. Part One of this survey asked participants to rate a set of already-made cane pruning decisions for one (cv.) Sauvignon Blanc vine. Participants rated these decisions on 24 individual pruning criteria and also provided two overall assessments. One of these overall assessments was recorded before participants rated the decisions on the 24 individual criteria, and a second overall assessment was recorded after such time. All ratings were collected via Qualtrics software, either online or via the Qualtrics offline survey application. Part Two of the survey asked participants to indicate, with highlighter pens on paper, their own preferred pruning decisions for the same vine. Linear Models, based on the relationship between the individual criteria and overall assessments (Part One), have revealed spur and cane position to be the dominant influencing factors in the pruning of the subject vine. Participant first impressions, as measured by the first overall assessment (before the individual criteria ratings), were almost exclusively reflective of participant attitude towards spur and cane position. The dominance of position was corroborated by Correspondence Analysis of the preferred pruning decisions (Part Two), which suggested that the decision to modify the vine’s cane or spur position was a fundamental point of divide within participant responses. In a related finding, results from Principal Component Analysis (Part One) have suggested that overall impressions were a heavy influence throughout the course of participant responses to Part One of this study. By extension, this finding suggested that attitudes towards position, which were strongly linked to participant overall assessments, permeated into participant attitudes towards other aspects of the presented decision set (Part One). Generally speaking, the dominance of a single group of decision-making criteria calls for further investigation into how pruning is conceptualised as a task. Results from this study suggest that there exists a somewhat broad, non-specific, view of whether or not a particular set of spur and cane selections are acceptable. This finding, while perhaps not immediately impactful for practitioners, has considerable implication for the design of future pruning research, as well as for the evaluation of artificially intelligent pruning. This research also reports the detection of pruning preference (Part One and Part Two) groups, based on region and organisational role. Correspondence Analysis and Multiple Correspondence Analysis (Part Two) have revealed that participants from Hawke’s Bay and, particularly, Central Otago tended towards a decision to restructure the subject vine by not leaving a spur from its existing right half. This was in contrast to those participants from Marlborough and Waipara who tended towards a maintaining of the current vine configuration. Aside from these differing propensities to restructure the vine, several regions were associated with unique spur and cane selections. It is unclear at present whether regional differences are due to social influences, regional viticulture conditions, cultivar familiarity, or some unknown factor. Participants also differed in their preferences when grouped based upon their organisational role. Those participants identifying exclusively as labourers were considerably less likely to restructure the vine, compared to those participants identifying as supervisors, managers, or proprietors. Managerial implications of this finding are discussed, with several potential remedies explored.

Pathogen infection or plant disease cause major losses in crop production across many species. In grapevine, in particular, there is an ongoing need to decrease dependence on chemical agents as a method to control or manage pathogen infection. Therefore, new approaches need to be explored to provide effective methodologies or approaches to minimise the impacts of pathogen infections. Jasmonic acid is known to be an important compound in plants that orchestrates both wound and plant defence responsiveness against a range of plant herbivores and pathogens. Jasmonic acid, via complex signalling cascades, induces plant defence genes such as those encoding proteinase inhibitors (involved in the protection of plant from insect damage), defensins and thionin (involved in the production of antimicrobials), and a raft of biosynthetic genes that lead to the accumulation of antimicrobial secondary metabolite such as alkaloids, terpenoids, flavonoids, and glucosinolates. Furthermore, jasmonic acid also facilitates the interaction between other defence signalling pathways such as those mediated by salicylic acid and ethylene to acquire the most effective ways to combat herbivore and pathogen attacks. Allene oxide synthase is the first committed biosynthetic step in the formation of jasmonic acid. Previous studies indicate that genetic variation within allene oxide synthase that alter its biosynthetic capacity have the potential to confer to the host plant increased resistance to attack from fungal pathogens. Therefore characterisation of grapevine allene oxide synthase function and genetic variation is an important step in ascertaining the potential this enzyme to contribute to increased tolerance to a wide range of fungal pathogens. Allene oxide synthase (hydroperoxide dehydratase; EC 4.2.1.92) is an enzyme belonging to the cytochrome P-450 (CYP74A) that known to catalyse the first step in the biosynthesis of jasmonic acid from lipoxygenase-derived hydroperoxides. A functional study of grapevine allene oxide synthase has not been previously reported. Therefore in this study we focused on the identification and functional characterization of the putative allene oxide synthase from Vitis vinifera L. Sauvignon blanc via complementation of an Arabidopsis allene oxide synthase null mutant. We investigated the relationships between allene oxide synthase and the other members of the CYP74 family in grapevine, in terms of sequence similarities, subcellular localisations and transcriptional regulation, both spatially and in response to mechanical wounding. We also determined the range of genetic variation of the grapevine allene oxide synthase within a commercial grapevine population. Our findings clearly demonstrate that there is a single allene oxide synthase gene in grapevine and that this gene is able to function in a heterologous system (Arabidopsis) to compliment a null mutation in allene oxide synthase. We show that grapevine allene oxide synthase is localised within the chloroplast and likely associated with chloroplast membranes. In addition the remaining members of the grapevine CYP74 family are found to be localised in varying cellular locations, not necessarily those predicted by in silico sequence analysis. The members of the CYP74 family show differential spatial and developmental transcript accumulation in grapevine. In order to assess the potential for increasing allene oxide synthase levels to increase biochemical flux through to jasmonic acid we overexpressed both the grapevine and Arabidopsis allene oxide synthases in a wild type Arabidopsis background. Our findings suggest that grapevine AOS might not be the only limitation in production of enhanced levels of jasmonic acid in response to wounding or pathogen attack. While we obtained increased levels of allene oxide synthase transcription, this did not result in a concomitant increase in jasmonic acid and consequently increases in the transcription of jasmonate regulated genes. However, while the alterations in jasmonate levels in the transgenic lines was below expectations, we did note that increased levels of jasmonate as a result of overexpression of allene oxide synthase did result in a limited and transient increase in tolerance to Botrytis infection. Investigation of the potential levels of genetic diversity of allene oxide synthase locus in grapevine indicated that this locus is highly conserved with no variation being evident among 100 vines in a commercial vineyard. While the levels of genetic variation strongly suggest that identification of suitable genetic variation in allene oxide synthase that would contribute to increased jasmonate accumulation from within existing grapevine populations is uneconomically practical or efficient. In conclusion our data suggests that to increase jasmonate mediated resistance against fungal disease in grapevine would likely require a coordinated alteration in allene oxide synthase as well as downstream genes in the biosynthetic pathway such as allene oxide cyclase and 12-oxophytodienoic acid reductase. To achieve such an alteration without resorting to transgenic approaches would require the use of a hybridization/breeding approach (which is currently unpalatable to industry) or identification of a suitable gain-of-function mutation from the native transposon mutation population that our group is currently producing.

Leaf removal is a normal commercial practice in viticulture to lower disease pressure through reduction in humidity and increased light exposure. However, New Zealand has 30-40% higher levels of UV radiation compared to similar latitudes in the Northern hemisphere, an increase of light exposure may therefore have a profound influence on berry composition. The aim of this research was to determine the effects of leaf removal and UV-B on flavonoid biosynthesis and regulation of gene activity in Vitis vinifera L. var. Sauvignon blanc grapes, particularly those genes associated with the low and high fluence UV-B responses. During the 2010 and 2011 seasons, three north-south oriented rows of Sauvignon blanc grapevines in the Lincoln University research vineyard were chosen to study the specific effects of UV-B on flavonoid accumulation and gene expression. This was achieved by using a combination of leaf removal and plastic screening techniques, which alter the light environment that the fruits are exposed to. UV-B exposure had a dramatic effect on the physical appearance of the berries causing specific pigmentation post-veraison. However, the accumulation of the total soluble solids and berry tissue development were not affected either by leaf removal around the fruiting zone or increased exposure to UV-B. In contrast, flavonols, particularly quercetin and kaempferol glycosides, increased substantially on fruit exposure due to UV-B. Spatial analysis of these flavonols located the changes to the berry skin. To understand the role of UV-B in regulating flavonoid biosynthesis, the transcript abundance for genes and transcription factors that are involved in the flavonoid biosynthetic pathway were analysed by qRT-PCR. These genes include five flavonol synthase genes (VvFLS1-5), three chalcone synthase genes (VvCHS1-3), and genes for the MYB-bHLH-WD40 transcription factor complex (VvMYB12, VvMYCA1 and VvWDRs). Of the five identified VvFLS, only two (VvFLS4 and VvFLS5) were found to be transcriptionally active in Sauvignon blanc berries and responsive to UV-B exposure. Of three VvCHS, only VvCHS1 and VvCHS2 showed a significant UV-B induction. Of the transcription factor genes, only VvMYB12 was found to be responsive to UV-B radiation and its expression pattern was consistent with VvFLS. The relative involvement of genes in the high and low fluence UV-B transduction pathways (VvUVR8, VvHY5 and VvCOP1; PR genes and VvMAPK3; respectively) were also studied. VvUVR8 did not response to UV-B but showed a developmental regulation. A similar lack of a UV-B response was found for VvCOP1. In contrast, VvHY5 showed a UV-B induction and higher levels of transcript abundance took place at harvest. All of PR genes showed a significant developmental regulation, with very low or no detectable transcript abundance pre-veraison and high transcript abundance post-veraison. These PR genes were not responsive to the natural fluence UV-B in the vineyard environment, but some showed a significant UV-B induction to a relatively high fluence UV-B under the controlled environment. Overall, this study clearly demonstrates the effect of UV-B exposure on the flavonoid biosynthesis in Sauvignon blanc grapes is achieved through a complex regulation of genes and transcription factors involved in the flavonoid biosynthetic pathway. This research makes a substantial contribution, not only in improvement of our scientific understanding of UV-B responses in important commercial species, but also provides valuable information for vineyard management to ultimately improve wine quality.

Protein stabilization of white wine is a process whereby proteins which may later give rise to a haze are removed prior to bottling. This is normally achieved by fining with bentonite, a clay material that has a strong affinity for proteins and other larger molecules. It is now well-established that pathogenesis-related (PR) proteins, which are originally derived from grape berries, are mostly responsible for haze formation. In this study, PR proteins in specific Sauvignon Blanc grape tissues were identified and quantified. Changes in the PR protein composition of grape skin and pulp during ripening and in response to environmental stresses (UV exclusion and powdery mildew infection) were determined, followed by evaluation of the impact of these changes on the extraction of PR proteins into juice. In addition, as grape harvesting and processing conditions can influence the extraction of proteins and other grape components that may interact with them, the effects of harvesting and grape processing on juice composition were investigated and protein heat instability in corresponding wines determined. Finally, the effect of skin contact on juice composition was examined in an experiment designed to investigate how the interactions between proteins and phenolics affect the extraction of grape components. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology, the main soluble PR proteins in wine, thaumatin-like proteins (TLPs) and chitinases, were identified as present in both Sauvignon Blanc grape skin and pulp, but not in the seed. The majority of identified proteins were involved in metabolism and energy. Proteins identified in grape seed were less diverse than those identified in grape skin and pulp. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fractions of protein extracts separated by high performance liquid chromatography (HPLC) indicated the individual elution time of TLPs and chitinases. Thus, the area of these peaks was accordingly used for relative quantification of TLPs and chitinases in protein extracts of grape tissues. Ripening of Sauvignon Blanc grapes coincided with the increase in phenolic content on a per berry basis in both grape skin and pulp, but the phenolic concentration on per kg fresh weight basis showed a decreasing trend in grape skin during ripening. This suggested that the accumulation of phenolics in skin during ripening was less than the dilution effect caused by berry growth. Tannin was only detected in grape skin and the changes in tannin content was similar to total phenolics. Accumulation of proteins along with the increase of °Brix during ripening was observed in both grape skin and pulp. PR proteins were synthesized and accumulated in both skin and pulp from véraison until harvest. This increase of PR proteins in grape berries was also reflected in the corresponding juice. In addition, the concentration of PR proteins in grape berries showed a positive correlation with the concentration of total proteins. UV exclusion and powdery mildew infection resulted in decreased phenolic concentration and increased PR protein concentration, respectively, which was in agreement with previous studies. Furthermore, the results showed that the concentration of PR proteins in the resultant juice was predominantly determined by the concentration of PR proteins in grape pulp, but the concentration of PR proteins in the skin and interactions of proteins with phenolics/tannin during the juicing process may modulate the final concentration of PR proteins in juice. The UV exclusion treatment compared to the control treatment resulted in a lower concentration of PR proteins in grape skin, but not in the pulp. However, the corresponding juice showed a higher concentration of PR proteins from UV exclusion treatment suggesting the loss of PR proteins as a result of phenolic-protein interactions in UV exclusion treatment was less than control as the concentration of phenolics in grapes was significantly reduced by UV exclusion. Significantly higher concentration of PR proteins in grapes from highly scarred berries with powdery mildew infection was reflected in the resultant juice. In addition, the lower concentration of tannin in juice from these berries also suggested the loss of tannin as a result of interactions between tannin and proteins. The harvesting and grape processing experiment showed that the year and vineyard site had a major impact on juice and wine composition. For grapes harvested in the same vineyard and vintage, machine harvesting resulted in a lower concentration of proteins, particularly PR proteins, than hand harvesting with destem, crush and 3 h skin contact. These juices and wines therefore required less bentonite addition for protein stabilization as bentonite requirement had been observed to be positively correlated with concentration of chitinases in juice and wine. The results suggested that the phenolic-protein interaction and juice oxidation, which was greater in mechanical harvesting than hand harvesting may have resulted in the loss of PR proteins. Furthermore, the potential increase in PR proteins as a result of 3 h skin contact was likely having little impact on final concentration of PR proteins in juice. Pressing treatments also affected juice composition since desteming, crushing and 3 h skin contact resulted in greater juice yield and higher concentration of phenolics and proteins (including PR proteins) than whole bunch pressing. Greater extraction of phenolics was also found in juice obtained at higher pressing pressure. Investigation of the effects of skin contact showed that juice treated with 24 h skin contact had the highest concentration of total phenolics and chitinases using both fresh berries and berries that were chilled for 24 h. Thus, longer skin contact will increase the protein concentration in juice, in particular the haze forming PR proteins, but the degree of this increase might be reduced along with more phenolic compounds co-extracted from skin as a result of phenolic-protein interactions. The effect of such interaction on reducing protein concentration was reflected in decreased concentration of bovine serum albumin (BSA) in extractants after skin extraction. The concentration of phenolic and tannin in skin extracts by using extractants added with BSA was lower than by using extractants without addition of BSA, also indicating the occurrence of phenolic-protein interactions. In addition, compared to mechanically pressed juice in which no tannin was observed, hand-squeezed juice had considerable amount of tannin but lower protein concentration. This suggested that hand squeezing was likely more effective in extracting grape skin components (mainly phenolics) through repeatedly squeezing and rubbing the mixture of skin and pulp, while mechanical pressing was likely more effective to extract grape pulp components (mainly proteins) through continuously applying high pressing pressure. This PhD project has revealed the distribution of haze forming TLPs and chitinases in Sauvignon Blanc grape skin and pulp. The concentration of PR proteins in juice is predominantly determined by their concentration in grape pulp, but the extraction of phenolics from grape skin might modulate the final concentration of PR proteins in juice through the phenolic-protein interaction during juicing process. The wine protein heat instability has been found positively correlated with the concentration of chitinases. Thus, any practices that can reduce extraction of chitinases into juice are important to improve the protein stabilization in final wine products.

The formation of inflorescence primordia (IP) marks an important step in the reproductive cycle of grapevines. The development of an IP to a grape bunch occurs over the course of two growing seasons. During season one, an IP initiates from a group of uncommitted cells, termed an anlage, where initiation is thought to occur around the time of flowering. Further branching and development of IP continues until the onset of winter dormancy. During spring of the following season, branching of IP resumes and each branch terminates in a floral identity, where each floral identity has the potential to form a berry. Inflorescence primordia initiation and development is sensitive to carbohydrate (CHO) availability and temperature. This thesis explores the influence of CHO availability and temperature during IP initiation and their continued development the following season. An inflorescence or bunch has two main components: the inner and outer arm. For the purpose of this thesis, inflorescence or bunch architecture is defined by the type of structure occurring at the outer arm position: and outer arm with floral identities; a tendril; or no structure. While the inner arm is required for an inflorescence structure to occur, development of the outer arm to a floral bearing structure does not always occur. Although initiation of the inner and outer arm likely occurs at the same time, the development of the outer arm into a floral bearing structure is frequently delayed compared to its inner arm structure. The causes and consequences of delayed outer arm formation on yield and grape composition at harvest are also addressed in this thesis. The effect of restricting CHO availability to IP during their second season of development was achieved by pre-budbreak (BB) cane girdling. First, it was established that pre-BB cane girdling alters the CHO availability to developing shoots and IP structures (Chapter 3.1). Canes were girdled 5, 10 or 20 cm from the terminal bud of the cane and shoot growth of the terminal bud was monitored over a single growing season. A linear relationship was found between the initial rate of shoot growth and the amount of cane isolated by the girdle. A decrease in available CHOs during initial shoot growth appeared to inhibit the shoot’s ability to produce new vegetative nodes past the point of discontinuity. This resulted in a decrease in total leaf area due to incomplete leaf expansion. The transition of the vine’s dependence on reserve CHOs to a net positive state appeared to occur when shoot growth reached a steady state. In the case of severe CHO restriction, no lateral growth occurred, suggesting the CHO status of the vine may play a role in lateral bud growth. The cross sectional area of canes or shoots were shown to have a linear relationship to their CHO content, which allowed for an estimation of the amount of CHOs required to obtain growth similar to the Control treatment. Additionally, main shoot leaf area can be used to predict total CHO content in the shoot at harvest. In the same experiment, the dates of flowering, flower number, berry number and grape berry soluble solids (SS) were measured for the inner and outer arm components of the basal and apical inflorescence and bunches separately (Chapter 3.2). Restricting pre-BB CHOs resulted in the abortion of some pre-formed inflorescences and reduced branching of the inflorescences that did develop. In general, berry SS were greatest for the basal inner arm, followed by those of the apical bunch inner arm, then those of the basal bunch outer arm, then lastly by those of the apical bunch outer arm. However, this was influenced by the relative berry numbers between the inner and outer arm. Bunches with more similar berry numbers on the inner and outer arms had more synchronous flowering and uniform SS. The differences in SS were largely a reflection of the timing of flowering of the various inflorescence components and may be an important source of variation in SS within a vine at harvest. The effects of girdling shoots and / or periodic leaf removal post fruit set on the initiation and development of IP was studied (Chapter 4). Dormant latent buds from treatment shoots (shoot node positions one to ten) were grown as single node cuttings (SNCs). Inflorescence number per SNC and their architecture were scored for every SNC. Girdling increased the proportion of SNC basal bunches with an outer arm, but had no effect on the number if inflorescence structures per bud (fruitfulness). However, there was a decrease in fruitfulness per bud and in the proportion of SNC basal inflorescences with an outer arm when girdled shoots had their leaves removed at zero and four weeks post fruit set respectively. This chapter presents novel information regarding the timing of IP initiation, including the initiation and development of the outer arm. As well, for the first time, the formation of an outer arm is shown to be sensitive to girdling and / or leaf removal. The influence of temperature during IP initiation on the resulting fruitfulness, distribution and architecture over the course of two consecutive growing seasons, at a single vineyard (2-Cane, 4-Cane and Spur pruning) was studied (Chapter 5). The pruning system had no effect on the fruitfulness per bud, or on the resulting bunch architecture. Warmer temperatures during IP initiation were correlated to: an increase in fruitfulness; an increase in the occurrence of an outer arm; and a decrease in the basal bunch insertion point on a shoot. As well, an increase in cane cross sectional area correlated to an increase in the average fruitfulness per shoot along a cane, where the influence of cane cross sectional area on fruitfulness was consistent between seasons. Additionally, an increase in cane cross sectional area correlated to an increase in the average occurrence of an outer arm per shoot along a cane, where the influence of cane cross sectional area was greater when temperatures during IP initiation were cooler. Temperatures during winter dormancy and BB were altered to determine its influence on the timing of BB and the branching of IP structures (Chapter 6). Winter dormant buds were passively heated using plastic heating chambers for different periods during dormancy to BB. Heating buds from either July or August to BB advanced the date of BB and the start date of flowering for the basal inner arm component by 14 and 16 days respectively compared to the Control treatment. Heating buds during winter dormancy had no effect on the fruitfulness of buds, the distribution of inflorescence structures on shoots or on the identity of the structure occurring at the outer arm position. However, elevated bud temperatures 12 days pre-BB caused a statistically significant decrease in the flower number per shoot (P < 0.05). Additionally, it was found that any delay in the start of flowering (which can be influenced by the date of BB, the position of the inflorescence on the shoot and / or the inflorescence component in question) is reflected in the accumulation of SS at harvest. The influence of temperature during IP initiation and CHO availability during BB were combined in a final experiment to improve the ability to predict when IP is occurring (Chapter 7). In this experiment, the influence of temperatures during IP initiation on fruitfulness and inflorescence architecture was studied between vineyards from separate growing regions throughout New Zealand. Winter dormant canes were harvested from each vineyard, and nodes three and ten were grown as SNCs. The volume of the SNC, inflorescence number, the position of the bunch on the SNC shoot and the occurrence of an outer arm were recorded. Inflorescence number and the occurrence of an outer arm increased as the volume of the SNC increased. The timing of IP initiation was predicted using stepwise regressions from 80 days pre to 90 days post 50% flowering with a variable window of time. Regressions of average daily temperature during IP initiation versus bunch number and versus the occurrence of an outer arm resulted in a linear continuum between node three and node ten for both bunch number (R²=0.73) and the occurrence of an outer arm (R²=0.47). The results also indicate that the use of 50% flowering as a reference point to calculate IP initiation is a valid alternative to destructively sampling buds to determine bud fruitfulness and IP architecture. This thesis provides an understanding of two of the major factors affecting bunch architecture, specifically outer arm development, which are temperature and CHO availability. The results indicate that temperature is likely the major factor influencing bunch architecture. Whereas CHO availability is a modifying factor, and is likely not to be an issue in New Zealand where grapevines are rarely CHO stressed.

Variety, temperature and viticulture practices (e.g. pruning, trimming, cluster thinning, irrigation etc.) all have an influence on grapevine (Vitis vinifera L.) phenology and berry maturation. Process-based phenological modelling which assumes temperature as the driving factor can be used to predict differences in development among varieties. Management practices including those that alter the Leaf Area to Fruit Weight (LA:FW) ratio may also influence the timing of key phenological stages and berry maturation. Regression modelling the relationship between LA:FW ratio and phenology or maturation parameters will enable a better understanding of these responses for different varieties. The aims of this work were 1) to characterise the differences between varieties for three key phases of development: flowering, veraison and the time to reach a target sugar concentration set at 200g/L using process-based phenological models; 2) to investigate different methods of characterising veraison as a phenological event to define the value of alternate objective measures; 3) to investigate the influence of altering the LA:FW ratio via trimming shoots (reducing leaf area) or removing crop (reducing the fruit weight) on two occasions (shortly after fruit set or at veraison) on the time of veraison and berry maturation parameters (soluble solids, titratable acidity, pH and berry weight). 2) and 3) were considered for two different varieties, Pinot noir and Sauvignon blanc. Linear and non-linear regression modelling was used to explore the relationships in 2) and 3). For the first aim, the Grapevine Flowering Veraison (GFV) model that uses a linear thermal summation of average daily temperatures from the 60th day of the year (in the Northern hemisphere) and a base temperature of 0°C was used to classify 95 varieties for the parameter F* (the accumulation of forcing units) for the time of 50% flowering and 105 varieties for 50% veraison. For maturity, data corresponding to a threshold sugar concentration of 200g/L were calibrated for seven different process-based models and calibrations calculated from either daily mean, daily maximum, average or hourly temperatures. The best fit model was the Sigmoid model in terms of efficiency and parsimony - assessed by the lowest Akaike Information Criterion (AIC), model efficiency, (EF), and Root Mean Squared Error (RMSE) - and the GFV model in terms of parsimony using daily mean temperatures. 35 varieties were parameterised and classified using both models for the day of the year to reach a sugar concentration of 200g/L. Seven different ways of assessing veraison were used to determine the relationship between soluble solids concentration and the measurement considered. Soluble solids ranged from 7 to 9 °Brix for the different methods. 8 °Brix was found to be a simple robust measure that allows comparison between different varieties, seasons and sites and was used in subsequent modelling to investigate the influence of LA:FW ratio on phenology and berry composition. Altering the LA:FW ratios of two varieties (Pinot noir and Sauvignon blanc) by trimming main shoots (leaf removal) delayed veraison, and both leaf removal and crop thinning influenced the dynamics of soluble solids accumulation; leaf removal slowed rates of soluble solids accumulation and resulted in lower concentrations of soluble solids at harvest whereas crop removal had the opposite effect. Titratable acidity, berry weight and pH were unaffected by the LA:FW ratio changes. The method of removing crop (removing all bunches on alternate shoots versus removing alternating bunches-apical then basal- from each shoot) did not influence the response but the presence of laterals advanced soluble solids accumulation. Non-linear exponential regression models across a wide range of LA:FW ratios best described the following different parameters for both varieties: Day of year to 8 °Brix, duration from 8 to 14 °Brix and soluble solids at harvest. The classification of flowering, veraison and the day to reach a sugar concentration of 200g/L are the most extensive to date within the literature. The methodology developed for these classifications and to assess the quality of prediction could also be applied to other crops. LA:FW ratio manipulations can shift the timing of keys events, such as veraison and soluble solids accumulation; the regression results indicated that it is possible to delay timing by the same magnitude of time as observed for between varietal differences in timing. This represents a potential tool to manipulate changes in development under future climate scenarios.

Aims: Sauvignon blanc is the flagship wine of Marlborough, with its style described as the definitive benchmark of the varietal. The majority of Marlborough’s vineyard plantings are in the Wairau plain; where the young alluvial soils were formed by sedimentation from the Wairau River. The braided nature of the river and frequent flood events has created significant vertical and horizontal soil texture variation. This soil texture variation has been shown to reflect changes in trunk circumference, which lead to the use of trunk circumference as an indicator of soil texture. The aim of this study was to investigate the impact of soil texture and yield on vine performance and fruit composition, within a single vineyard. Methods: Trunk circumference measurements were grouped to create five vine size classes, while two pruning methods (two and four-cane VSP) were applied to create two crop load treatments with two different canopy types. Vine vigour, canopy density, vine phenology, yield and fruit composition measurements were taken from each vine size and crop load treatment throughout the season. Results: Vine vigour and canopy density increased with increasing vine size while increases in crop load resulted in decreased vigour and increased canopy density. Vine phenology was delayed with both increasing vine size and increasing crop load. Vine size did not influence yield but did influence fruit composition; increases in vine size delayed ripening with lower soluble solids and higher titratable acidity levels measured at harvest. Crop load influenced both yield and fruit composition with increases in crop load leading to higher yields and delayed ripening. Lower soluble solids and higher titratable acidity levels were measured at harvest as crop load increased. Conclusions: The variation in soil texture found in the Squire Vineyard lead to variation in vine vigour, canopy density and phenology. These differences in vine growth had no influence on vine yield but did have a significant impact on fruit composition at harvest. Variation in vine growth and fruit composition within a single vineyard creates challenges with vineyard management; particularly with canopy management, irrigation, nutrition, and harvest decisions.

Green leaf volatiles (GLVs) are short-chain acylic aldehydes, alcohols and esters produced by plants via enzymatic metabolism of polyunsaturated fatty acids (PUFAs). GLVs are known to affect flavour and aroma of fruits and vegetables, including grapes. It has also been suggested that C6 and C5 GLVs are the precursors of volatile thiols, the aroma compounds that are important in Sauvignon blanc wine. GLVs are produced during grape berry development and released in high quantities upon berry crush. GLV aldehydes are formed from PUFAs by the action of two enzymes, lipoxygenase (LOX) and hydroperoxide lyase (HPL). This biochemical pathway is well characterised in other plant species and is known as the GLV pathway. To date, the GLV pathway has not been characterised in grapes. This thesis focuses on identification and initial characterisation of LOX and HPL genes and enzymes involved in the GLV-pathway. LOXs are a group of non-haem iron-containing dioxygenases that catalyse oxygenation of PUFAs producing PUFA-hydroperoxides. The most common PUFA substrates in plants are linoleic acid (LA, 18:2) and α-linolenic acid (LnA, 18:3). Depending on the position of oxygenation of the 18-carbon chain PUFAs, all plant LOXs are classified as 13(S) - and 9(S)-LOXs. 13(S)-LOXs are further regarded as type II and type I enzymes, depending on the presence or absence of transit peptides in the amino acid sequences of these enzymes respectively. HPLs belong to the CYP74 enzyme family, which is represented by atypical members of cytochrome P450 oxidases superfamily. HPLs cleave PUFA-hydroperoxides, producing aldehydes and oxoacids. Depending on the substrate specificity, HPLs are classified as 13-HPLs, 9/13-HPLs or 9-HPLs. The research reveals the complexity of the genetic makeup of the GLV metabolic pathway in grapes. Eighteen putative LOX genes and six putative HPL genes were identified in the Pinot noir grape genome. Phylogenetic analysis of the identified grape LOXs classified them as members of two groups, type II 13-LOXs and 9-LOXs, whereas all identified grape HPLs were classified as 13-HPLs (CYP-74B) and 9/13-HPLs (CYP-74C). Several LOX and HPL genes were expressed at different levels in Sauvignon blanc berry. Study of selected LOX and HPL gene expression revealed different levels of expression and differential tissue distribution of individual LOX and HPL genes within the berry. The studied genes also displayed different patterns of expression across different stages of berry development, upon wounding and in berries infected with Botrytis cinerea. Amongst the four LOX gene studied, transcripts of VvLOXA were the most abundant at all stages during berry development. VvLOXO was induced transiently upon berry damage and was a clear candidate involved in berry response to wounding. Expression levels of VvLOXC and VvLOXO were significantly increased in berries infected with Botrytis cinerea compared to the uninfected berries. In vitro biochemical analysis of the reaction products of recombinant VvLOXA (LOXA-TP) and VvLOXO (LOXO-TP) confirmed that these two enzymes are 13-LOXs. Both enzymes preferred LnA as a substrate. Both enzymes had the same temperature optima of 25°C, but preferred different pH conditions. Recombinant LOXA-TP preferred acidic environment and had pH optimum of pH 5.5, while LOXO-TP preferred neutral-to-basic conditions and had pH optimum of pH 7.5. Preliminary experiments with recombinant VvHPLA showed its ability to metabolise 13(S)-hydroperoxides, releasing C6 volatile aldehydes. Recombinant VvHPLA exhibited maximum activity with 13(S)-hydroperoxides of LnA as substrate at pH 5.0.

The influence of soil texture on canopy growth, vine yield, and fruit composition of Sauvignon blanc were assessed on a mature vineyard from the Rapaura area in Marlborough. The subject vines were mature Sauvignon blanc in a commercial vineyard trained to four cane VSP and planted with a north south orientation. Row and vine spacings were typical of the area at 3m x 1.8m. Four areas of different soil texture were identified using trunk circumference measurements, visual assessment of the surface soil and aerial photographs to identify changes in vine growth. Soil pits were excavated at a later date to determine root numbers and record the soil texture in the different areas. Two crop treatments were imposed on half of the plots at approximately 50% veraison: unthinned crop and 50% crop. A shading treatment was also imposed at 50% veraison where three sets of tagged shoots had bunches that were exposed to sunlight, bunches that had some natural shading from leaves or bunches that had paper bags fastened over each to provide a completely shaded environment. Vine vegetative vigour for each plot was assessed during the growing season using the Point Quadrat method and at pruning using bud counts and pruning weights. Thirty berry samples were collected from the 32 plots and analysed weekly for soluble solids and berry weight. Harvest of bunches from the tagged shoots occurred on two different dates with the first harvest picked when fruit from one soil and crop treatment had reached 21 °Brix. The second picking took place just before the commercial harvest, which coincided with last soil and crop treatment reaching 21 °Brix. Bunches from each tagged shoot were weighed, frozen and later analysed for soluble solids, pH, organic acids, and methoxypyrazines. Vines on very stony textured soils had small trunk circumferences with a high root density and yield to pruning weight ratio compared to those growing on deep silt soils. Vines on deep silt soils had more vigorous canopies with large shoots and a higher leaf layer number. The target soluble solids was reached first by the vines on the very stony plots despite the higher crop load with soil texture appearing to be a dominant factor by influencing the time of flowering. Cluster shading decreased soluble solids, consistent with other studies, whilst crop thinning resulted in an earlier harvest. This was contrary to popular opinion that crop thinning at veraison would have no impact on sugar accumulation. pH and organic acids were unaffected by shading or bunch thinning. Vines growing on deep silt soils had a significantly higher level of total acidity and malic acid than those on the stony soils. At harvest, methoxypyrazine levels were very low compared to previously reported figures for Marlborough, which may have been a result of sample preparation. IBMP was significantly higher on deep silt soils, however, with no impact from the shading or crop treatment. The findings are consistent with the hypothesis that IBMP synthesis may be increased by the number of leaves surrounding bunches (Roujou de Boubee, 2003). The results suggest that soil texture plays a dominant role in influencing Sauvignon blanc flavour and aroma and due consideration should be given to vineyard layout and site selection prior to new plantings taking place.

The effects of crop load and berry exposure on the composition of Marlborough Sauvignon blanc grapes and wine from the Brancott vineyard, Blenheim, were explored. Commercially grown, 2-cane and 4-cane Sauvignon blanc vines were used with a row orientation of north-south. Two exposure treatments were imposed in the following manner: complete leaf removal was undertaken in the fruit zone and 50% shade cloth was erected to give a uniform shading treatment to half the trial vines. Weekly thirty-berry and whole bunch samples were taken from each of the 32 plots with the exception of the veraison period when two samples per week were taken. Vine vigour was assessed using pruning and leaf area per vine data. Harvest occurred on different dates for 2-cane and 4-cane pruned vines so that fruit attained from both treatments had similar °Brix. Fruit was processed at the Lincoln University winery. Must analysis and wine analysis were undertaken. As expected, 4-cane vines had almost double the yield of 2-cane vines. Higher crop load significantly reduced leaf area per shoot and shoot thickness. Lower leaf area to fruit ratio for 4-cane berries resulted in delayed onset of veraison and slowed the rate of sugar accumulation. Crop load, which limited leaf area to fruit ratio, appeared to be the dominant factor in determining timing of grape physiological ripeness as expressed by °Brix over other factors such as fruit exposure. Malic acid, tartaric acid, IPMP (iso-propylmethoxypyrazine) and IBMP (iso-butyl-methoxypyrazine) were lower at equivalent °Brix in 4-cane compared with 2-cane berries. Significantly higher concentrations of quercetin were found in exposed compared to shaded berries. Must analysis showed a significant influence of crop load on berry titratable acidity and pH, reflecting berry ripening results. Exposure significantly increased the concentrations of nitrogenous compounds in 4-cane must yet showed no influence on 2-cane must. After wine processing lower malic acid concentrations in wines made from 100% exposed fruit became evident in lower wine titratable acidity but showed no influence on wine pH. Bentonite addition to wines had a small but statistically significant influence on wine by reducing pH, titratable acidity and alcohol. Bound sulphur concentrations were significantly higher in 4-cane versus 2-cane wines. At harvest, methoxypyrazine levels in grapes and wines were very low; IBMP concentrations where significantly lower than those normally found in Sauvignon blanc wines from Marlborough. This was attributed to the absence of basal leaves from the shoots of ripening berries. The results suggest that leaf area to fruit ratio is a powerful determinant of grape and wine quality.

Grape composition is important in determining the flavour and aroma characteristics of the resultant wine. Samples of the juice composition are described by their average value, yet the variability in fruit composition around the mean may also have a large impact on wine quality. The objective of this study was to identify the variance contribution of row orientation and trellis type within the vineyard, and bunch and shoot position within the vine. For two years primary and secondary bunches were sampled from basal, mid-cane and apical shoots of five year old Sauvignon blanc vines on the Wairau Plains of Marlborough, New Zealand (41º 29' south, 173º 54' East). Vines were trained on either the Scott-Henry (S-H) or vertically shoot positioned (VSP) trellis and located in either east/west (E/W) or north/south (N/S) oriented rows. The bunch position contributed 30% to 50% to juice composition variance in 1999 and 50% to 90% in 2000. Whilst trellis type and row orientation contributed 42%, 50% and 40% to the brix, TA and pH variance respectively in 1999, they contributed only 26%, 16% and 4% in 2000. This was attributed to a change in canopy management which increased the fruit and leaf exposure, therefore reducing the effects of trellis and row orientation on juice composition variance. Despite bunch position accounting for most of the data variance in 2000, differences between apical and basal shoots were the largest single differences in brix (0.9°), TA (0.9 g/L) and pH (0.07). The least mature bunches on the vine were secondary bunches on basal shoots regardless of trellis type or year. They were from 1.3° - 2.2° brix lower, and 1.0 - 2.7 g/L TA higher than primary bunches on mid cane or apical shoots. In the first year of the trial differences in fruit exposure caused maturity differences between trellis types. Fruit exposure levels were improved in the second year, and maturity differences were similar to differences in phenology at flowering. The phenology of apical shoots was advanced by 2 to 3 days compared to basal shoots in both years, whilst primary bunches were advanced 1 to 2 days relative to secondary bunches. Variation in the leaf area or leaf area: fruit weight ratio of shoots was not correlated to variation in berry weight or soluble solids. This was probably because of low vine crop loads and remobilisation of carbohydrate reserves. The results indicated that to reduce variability in the grape crop the viticulturist must promote uniform fruit exposure and try to reduce phenological differences between shoot positions. Further study should consider the relationship between vine crop load and the leaf area or leaf area: fruit weight ratio of individual shoots on the vine. A better understanding of how light exposure on the fruit and leaves contributes to the weighted average juice composition of a vine would also be useful. Whilst the effects of variable fruit exposure on the variance of the data were not clear, biologically significant differences in maturity were not reflected in the data variance, so this line of research is of less concern to the viticulturist/wine maker.