Regulation of Grape Ripening: A Fine Balance Between Physiology and Gene Expression

Grape berry ripening in Vitis vinifera L. is a highly complex and finely regulated process influenced by genetic and environmental factors. Climate change is significantly affecting grapevine physiology, leading to variations in berry composition and harvest timing, with direct consequences on wine quality. Understanding the molecular mechanisms underlying fruit ripening is therefore crucial for developing innovative strategies to adapt viticulture to these emerging environmental challenges. This thesis explores the physiological and genetic regulation of grape berry ripening, emphasizing the hormonal and transcriptional dynamics governing this process. Additionally, it examines various biotechnological approaches as platforms for functional analysis of grapevine genes. The Chapter 1 provides a general introduction to the grapevine system, emphasizing its economic and agronomic significance on a global scale. It discusses the impact of climate change on grape quality and the mitigation strategies adopted within the viticulture sector. The ripening process is explored in depth, describing the key physiological, biochemical and molecular factors governing it. Additionally, innovative genetic improvement approaches, including modern breeding techniques and genetic modifications, are examined. Chapter 2 focuses on the characterization of a specific grapevine genotype, derived from a breeding population, which exhibits a delayed ripening phenotype compared to its normal-ripening siblings. The first section of the second chapter (Chapter 2.1 – draft of a paper ready to be submitted) describes the physiological dynamics of sugar accumulation, berry growth and berry firmness over three years, highlighting the differences between the slow-ripening (SR) mutant and a normal-ripening (NR) genotype within the same breeding population. Potential causes of ripening failure were investigated through targeted experiments. The second section (Chapter 2.2) presents transcriptomic and hormonal analyses of the SR, comparing it with the NR and evaluating its response to abscisic acid (ABA) treatments. The identification of differentially expressed genes (DEGs) provided insights into the key molecular regulators involved in ripening control and hormonal responses in this natural mutant. Chapter 3 (draft of a paper ready to be submitted) investigates the effects of auxin (NAA) treatments on berry ripening in Chardonnay and Corvina, with a specific focus on the interactions between hormones and gene expression of key-ripening genes. Physiological and molecular analyses revealed auxin-induced effects, providing insights into the potential application of these compounds for vineyard ripening management. While the previous chapters primarily focus on physiological and transcriptomic analyses as well as field-based mitigation strategies to either accelerate (Chapter 2) or delay (Chapter 3) the onset of berry ripening, Chapter 4 shifts attention toward functional gene analysis platforms for grapevine research. In Chapter 4.1 transcriptomic analysis through microarray technology was performed on stable overexpressing lines of VviAGL15a in Shiraz grapevines, providing a clearer understanding of its role in ripening regulation. These findings were further contextualized by comparing them with previous transcriptomic data obtained from transient overexpression of the same gene using the same microarray approach. Chapter 4.2 introduces the Microvine model, a natural grapevine mutant with a reduced life cycle that facilitates accelerated studies on ripening. The steps involved in obtaining embryogenic calli from immature inflorescences and subsequent whole-plant regeneration are detailed. Additionally, stable genetic transformation trials were performed on calli and embryos for GFP reporter gene overexpression, with the aim of optimizing a functional stable transformation protocol. This work sets the foundation for future functional analysis of two partially characterized genes, VviNAC33 and VviNAC60. Finally, Chapter 4.3 explores a modern DNA-free genome editing approach using protoplasts and subsequent whole-plant regeneration. The focus is on genome editing of VviMLO3 as a target gene in Sultana and VviAGL11 in Microvine. The studies conducted in Chapter 4 lay the groundwork for the implementation of next-generation genetic improvement techniques applicable to viticulture. Chapter 5 provides an overall conclusion, summarizing the main findings and discussing their implications. A particular focus is given to the advantages and limitations of modern genetic techniques for functional gene analysis in grapevine.