The role of Gamma-aminobutyric acid and Hydrogen peroxide in cell death in grape berry development

Loss of cell vitality (i.e., cell death) in the mesocarp of grape berries occurs during ripening. However, cell death is genotype-dependent and modulated by increasing temperatures and drought. Cell death and associated berry shrivel negatively affect the grape quality and wine chemistry. The causes of cell death are not yet completely understood, and research suggests that cell death may be correlated to hydrogen peroxide (H2O2) production in berries. Another metabolite of interest is Gamma-aminobutyric acid (GABA) which might have a role in cell death in berries because the concentration of H2O2 and GABA in cells tends to increase under stress. Chapter 1 analyses the literature on H2O2, and GABA, a four-carbon non-protein amino acid and their role in berry development and Chapter 2 discusses the materials and methods employed in the current study. This study aims to understand the interactions between GABA and H2O2 in three grape cultivars, Grenache, Shiraz and Chardonnay, which show contrasting characteristics for cell death. Physiological and biochemical techniques were used to analyse berry samples collected from field-grown (2018-2019, and 2019-2020 seasons) and potted grapevines (2019-2020). Hormones, phytochemicals, and reactive oxygen species (ROS), primarily H2O2, are involved in plant responses to stresses, such as higher temperatures, hypoxia, salinity and water deficit. Many developmental processes, including budburst, flowering, and grape berry ripening, are controlled by H2O2; there is always a balance between the production and dissociation of H2O2. plant adaptation mechanisms under stress influence the development of fruits; one of the strategies include an increase in internal GABA, which enhances antioxidant enzymes mitigating H2O2 accumulation and oxidative damage; GABA accumulation and the production of ethanol marks the beginning of hypoxic stress in plants. In order to understand the physiological and biochemical changes during the development of grape berries of the three cultivars chapter 3 focuses on physical changes such as berry mass, total soluble solids (TSS), cell death, and biochemical changes such as GABA, H2O2, and antioxidant enzyme (ascorbic peroxidase and catalase) concentrations in grape berries during development. Berries from Grenache, Shiraz and Chardonnay cultivars were sampled from veraison (70-80 days after flowering) for two seasons (2018-2019, and 2019-2020). Weather changes can cause changes in berry biochemistry and, as a result impact wine quality. Previous research suggests that GABA treatment could extend fruit shelf life and delay senescence (cell death) by regulating antioxidant enzymes and ROS (H2O2) metabolism. Since GABA is one of the globally recognised safe (GRAS) molecules approved by the FDA to be safe at 100 mg per serving level equivalent to 3.9 mM (0.041%)–64 mM (0.66%) (CFSAN/Office of Food Additive Safety, 2015), it is safe to spray on berries. The current study aims to provide an overview of changes in physiology and biochemistry that occur in grape berries in response to exogenous GABA application. Chapter 4 assesses the effects of exogenous GABA treatment on the Shiraz grape berries during two seasons (2018-19, and 2019-20) and chapter 5 investigates the effects of exogenous GABA application on Shiraz berries under imposed soil water deficit in potted vine experiments in the glass house. Shiraz grape bunches were sprayed with 5 mM GABA once a week from the post-veraison stage to the post-harvest stage to investigate the effect of GABA on cell death (field experiment). The results show that GABA-treated berries had lower berry mass, TSS and CD and that exogenous GABA application increases antioxidant activity, reduces H2O2 accumulation, which may delay cell death. Imposed soil water deficit has a negative impact on berry growth and development (chapter 5), resulting in a significant decrease in berry mass and an increase in total soluble solids (TSS) and cell death. Higher cell death can be explained by increased H2O2 and decreased antioxidant enzymes observed in water-stressed Shiraz berries. Climate change poses challenges to grape berry development, with the accelerated rate of ripening leading to a significant strain on wineries' ability to process fruit in a timely manner. The wine industry may benefit from delaying ripening in order to control harvest dates and berry composition. The observations thatH2O2 acts as a signalling molecule for berry ripening initiation and exogenous GABA application mitigates H2O2 by enhancing antioxidant enzymes lead to the hypothesis that exogenous GABA application may play a key role in delaying ripening in grape berries by enhancing antioxidants and reducing H2O2 accumulation. Chapter 6 discusses the GABA treatment (5 mM) applied weekly from pea to veraison stage of grape berries to understand the concept of delay in ripening in Chardonnay, Shiraz and Grenache cultivars. GABA treatment in all three cultivars delayed the onset of ripening. This delay in ripening manifested as slowed berry growth (delay in the increase in berry mass), total soluble solids, and cell death during development. Since H2O2 acts as a signalling molecule in grape berry ripening initiation, berries treated with GABA showed lower H2O2 concentration and higher antioxidant activity (catalase, APX), thus causing a delay in ripening. These findings support the hypothesis that GABA may play a role in modulating grape berry ripening by influencing the H2O2 and antioxidant systems involved in the ripening process. My doctoral research focuses on the interactions of GABA and H2O2 in grape berry development. The results indicate that GABA might have a role in preventing oxidative damage caused by increased accumulation of H2O2 during berry development. Exogenous GABA application on berries has promising potential for use in drought-prone agricultural areas. The trials conducted in this study also attempt to understand the interactions between GABA and H2O2 in berries of vines grown in the field and under imposed soil water deficit in the glasshouse. Understanding the influence of GABA on cell death at different phenological stages will help grape growers to mitigate or reduce berry shrivel in specific cultivars prone to increased cell death and shrivel. GABA can be applied exogenously as part of their routine spray. These trials have the potential to delay ripening so that grape ripening can occur in the cooler months of the season to preserve quality and thus play a role in protecting the grape crop under adverse weather conditions.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 202