ENHANCING DROUGHT TOLERANCE IN TOMATO THROUGH CRISPR-CAS9 EDITING OF ABA SIGNALING AND HIGH-THROUGHPUT PHENOTYPING

Improving agricultural resilience is now more important than ever due to the escalation of climate change impacts. In recent years, the European Union has experienced a decline in tomato production, with a 14% decrease in 2022 attributed to the increasingly arid landscapes of Spain and Italy. The vulnerability of the agricultural sector underscores the pressing need for innovative solutions to mitigate the impacts of water scarcity. The core strategy of this study is the CRISPR-Cas9-mediated manipulation of the signaling pathway of the stress-hormone abscisic acid (ABA). Upon increased ABA production (i.e., under water deficit), the PYR/PYL/RCAR receptors bind ABA, sequester the phosphatases proteins PP2Cs, and therefore release the kinases SnRK2s from the PP2Cs inhibition. This process will lead SnRK2s proteins to induce stomatal closure as well as the activation of the ABAstress responsive genes. The SlPP2Cs are structurally similar and functionally redundant in tomato. We reasoned that the combinatorial alterations of the SlPP2Cs-encoding genes could generate a favorable drought resistance phenotype with limited impact on biomass accumulation under optimal growing conditions. We have generated 8 Cas9-free T1 families of 4 PP2Cs-encoding genes, and through sequencing we verified the presence of different mutations (InDels) in all four targets, in a heterozygous, homozygous (homo/hetero allelic) state. We used high throughuput phenotyping and RNAseq to explore the impact of this genetic variability (allelic and dosage heterogeneity) under control or water deficit conditions. As anticipated, phenotypic analysis under drought conditions revealed strong drought-resistance traits resulting from the CRISPR-Cas9-mediated editing of various SlPP2C genes. These traits include reduced plant growth, likely due to decreased transpiration rates. Under optimal watering conditions, different SlPP2C alleles (and their combinations) exhibited varying degrees of growth reduction, with some alleles causing only mild phenotypic deviations compared to the wild type. Ongoing RNA-seq analysis is being used to uncover the molecular signatures underlying these phenotypic differences. Collectively, our findings suggest that a more favorable trade-off between growth and drought tolerance can be achieved by leveraging the quantitative effects of functionally conserved and expanded gene families.