Graduate Thesis Or Dissertation


Spatiotemporal Phytohormone Distributions in Developing Pinot Noir Berries and the Effects of Two Viticulture Practices Thereon Public Deposited

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  • The Effects of Cluster-thinning and Cluster-zone Leaf Removal on the Distributions of Phytohormones in a Spatiotemporal Context
  • In recent years, many studies have focused on the molecular and biochemical mechanisms regulating the development of wine grapes. The course of grape berry development is directed by genetic design and is mediated by phytohormones, which regulate grape berry growth and development by orchestrating a complex network of interacting genes, proteins, and other metabolites while simultaneously facilitating adaptive responses to external growing conditions. Hormones are bioactive at very low concentrations, and their accumulation is tightly regulated by transcription of genes that encode enzymes responsible for the biosynthesis, inactivation, and degradation of the bioactive forms. Understanding how the bioactive hormones are regulated is integral to understanding how the environment influences their accumulation and response. Because bioactive hormones mediate developmental transitions, their accumulation in the berry tissues is temporally regulated, and because hormone function is specific to tissue or organ, their accumulation is spatially-regulated. It is necessary, therefore, to identify the dynamic hormone profiles in a tissue and stage-specific context. For these reasons, we have used an extraction protocol optimized in our lab to extract 34 hormone-related analytes from separated berry tissues sampled from eight time points from early growth through post-ripening. Liquid chromatography coupled to tandem mass spectrometry was then used to analyze the extracted hormones and then quantify their concentration in the lyophilized tissues at each developmental stage. Included in this study are the bioactive forms, the precursors, inactivated storage conjugates, and inactive catabolites of auxin, abscisic acid, cytokinin, and gibberellin, as well as jasmonic acid, salicylic acid, and castasterone (bioactive brassinosteroid). An important aspect of phytohormone activity is cross-talk and other forms of hormone interactions. Some plant hormones are known to act in synergy to facilitate and coordinate various plant processes (e.g. auxin and gibberellin in cell expansion). Others are believed to work antagonistically. Therefore, the concomitant study of phytohormones is key to identifying potentially-novel interactions. The environmental conditions and nutrient status of the plants have a major impact on the dynamics of plant hormones. We proposed in this thesis to determine the effects of cultural practices that are known to influence cues during the pre-ripening and ripening phases. We used the same protocol to extract, analyze, and quantify ripening-related hormone compounds (auxins, abscisic acid-related compounds, and castasterone) at five developmental stages in the separated tissues of berries following two common practices: cluster-thinning (CT) and cluster-zone leaf removal (LR). Our results indicate that both practices have significant effects on ripening-related hormone accumulation and their regulation through the biosynthetic, conjugation, and catabolic pathways. Bioactive castasterone was especially interesting and could open the gate for meaningful research in determining the efficacy of exogenous castasterone application in mitigating deleterious effects of thermal stress and UV damage to the photosynthetic machinery following defoliation in the cluster zone, especially if the climate projections of significant warming in wine-grape growing regions are realized.
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Table of Contents
  • Chapter 1. General Introduction 1 Chapter 2. The spatial and temporal distribution of phytohormones in Vitis Vinifera cv. Pinot noir berries 7 2.1 Introduction 7 2.2 Materials & Methods 13 2.2.1 Berry sample selection 14 2.2.2 Hormone extraction 15 2.2.3 Liquid chromatography and mass spectrometry conditions 17 2.2.4 Chemicals and reagents 17 2.2.5 Statistical analyses 18 2.3 Results 19 2.3.1 Phytohormone profiles 21 Auxin-related compounds 21 Abscisic acid-related compounds 27 Gibberellin-related compounds 30 Cytokinin-related compounds 34 Jasmonate 36 Salicylate 37 Brassinosteroid 38 2.3.2 Pearson hierarchical clustering of hormone profiles 38 Phytohormone profile clustering in seed tissue 38 Phytohormone profile clustering in pulp tissue 41 Phytohormone profile clustering in skin tissue 41 TABLE OF CONTENTS (Continued) Page Phytohormone profile clustering common in all tissues 42 2.4 Discussion 42 Chapter 3. The impacts of two viticulture practices on the dynamic hormone accumulation in the tissues of Pinot noir berries during ripening. 72 3.1 Introduction 72 3.2 Materials & Methods 74 3.2.1 Field experiments 74 3.2.2 Sample selection 76 3.2.3 Hormone extraction 77 3.2.4 Hormone analysis 77 3.2.5 Chemicals and reagents 78 3.2.6 Statistical analysis 78 3.3 Results & Discussion 79 3.3.1 Effects of cluster-thinning on the hormone profiles 79 Auxins 80 Brassinosteroid 81 Abscisic acid-related compounds 82 3.3.2. Effects of cluster-zone leaf removal on the hormone profiles 84 Auxins 84 Brassinosteroid 85 Abscisic acid-related compounds 86 3.4 Conclusions 88 Chapter 4. Conclusion 100 Bibliography 104
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