Castaldo, Carmine (2024) Comprehensive analysis of stress response and fruit quality mechanisms in tomato: insights into the role of glutathione s-transferases and source-to-sink dynamics. [Tesi di dottorato]

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Abstract

Climate change is dramatically affecting cultivable areas and diminishing natural resources, posing significant challenges to food security worldwide. As a critical horticultural crop and a key model for genetic studies, tomato (Solanum lycopersicum) is at the forefront of agricultural research due to its relatively short biological cycle and comprehensive genetic and genomic resources, including a fully and well-sequenced genome. The primary aim of this doctoral thesis was to develop a comprehensive understanding of stress response mechanisms and fruit quality in tomato, focusing on the role of glutathione S-transferase (gst) genes and the intricacies of source-to-sink dynamics under drought stress. We started with a thorough bioinformatics analysis of the gst loci in the S. lycopersicum according to the latest version of the tomato genome annotation (ITAG 4.1) and for the first time, we have identified and characterized the GST genes in its wild relatives: Solanum pennellii, Solanum lycopersicoides, and Solanum pimpinellifolium. This analysis included domains, structural features, motifs, phylogenetic relationships, expression patterns, orthologs, and synteny, offering a deep dive into the evolutionary history and functional diversification of the gst gene family. This foundational work highlighted the potential of these genes in environmental stress adaptation and improving fruit quality through a comparative expression analysis, covered different tissues (i.e. leaf, fruit, root) and a wide spectrum of stresses (heat, drought, salt, cold), enabling a detailed elucidation of GST tissue-specific gene expression dynamics in response to abiotic stressors. Furthemore, our study employed polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) to verify the enhanced accuracy of the Glutathione S-transferase (GST) gene family annotations between Solanum lycopersicum ITAG 4.1 and ITAG 2.4, underscoring the necessity of ongoing updates and experimental validation for the reliability of genomic data. The thesis also delved into the functional roles of the S. lycopersicum tau Glutathione S-Transferase gene (Solyc075056420), which was functionally characterized through its stable over-expression in Nicotiana tabacum transgenic plants. The genetic transformation was facilitated using the Agrobacterium tumefaciens strain LBA4404, with the coding sequence regulated by the 35S2 promoter. T2 offspring transgenic plants with different levels of the gst mRNA expression underwent eco-physiological characterization under drought and salinity as well as molecular and metabolic characterization adding insights on the effect of the target genes on the plant response to environmental stresses through the control of the cellular redox potential. The expression of the gst gene appeared to regulate the antioxidant defense system by enhancing the efficiency of some antioxidants while reducing dependence on others. The modulation of the expression of other genes, particularly within the glutathione-ascorbate (GSH-AsA) cycle, seemed to help optimize the plant's antioxidant response. This may have allowed to manage the accumulation of reactive oxygen species (ROS) more effectively under conditions of both drought and salt stress. A non-redundant reference S. lycopersicum core collection was selected from a global collection using genome-wide SNP data. Plants were grown under semi-controlled conditions with full water reintegration (FWR) and halved water reintegration (HWR) and changes in evapotranspiration, CO2 assimilation, water use efficiency, leaf colorimetric parameters and leaf electrolyte leakage were measured. The most drought-resistant and susceptible phenotypes were selected for a deeper analysis of the plant response to drought in a second experiment. Upon the administration of the differential watering treatments (FWR and HWR), different tissues (leaves, stems, and mature green fruits) were sampled for metabolic and transcriptomic profiling by RNAseq analysis. Numerous differentially expressed genes (DEGs) were identified, indicating significant changes in gene expression that respond to drought stress. Specifically, the Red Setter tomato variety exhibited high shifts in gene expression across all plant tissues between control and stress condition, with 1,018 genes up-regulated and 646 down-regulated in leaves, 328 up-regulated and 835 down-regulated in fruits, and 320 up-regulated and 365 down-regulated in stems. Conversely, the Severianin variety showed a more moderate response, with 288 genes up-regulated and 242 down-regulated in leaves, 578 up-regulated and 545 down-regulated in fruits, and 231 up-regulated and 394 down-regulated in stems. These differentially expressed genes (DEGs) were then analyzed across various pathways to identify genes involved in key biological processes such as photosynthesis, hormone signaling, redox homeostasis, carbohydrate metabolism, solute transport, and cell wall organization. Our study has identified key genes that could better clarify the source-to-sink mechanism in tomato plants, particularly under drought stress. This investigation enhances our understanding of how these plants manage resource allocation and maintain fruit quality in adverse conditions.

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