Quantifying new rhizospheric roles of JA in shaping root architecture response in compacted soil

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Soil compaction represents a major challenge facing modern farming due to changes in agricultural management practices. Over half of Europe's farmed soils are prone to compaction, costing billions of pounds of losses. Despite its importance, little was known about why roots stop growing in compacted soils. A series of (literally) ground-breaking experiments by our team (Pandey et al, 2021, Science) recently revealed that roots can penetrate highly compacted soil after disrupting their sensitivity to a plant hormone signal called ethylene. However, the current understanding of the key rhizosphere signals a lacking to fully understand the soil compaction response in crop roots.Aim: In this project, we aim to discover the new roles of a key rhizosphere signal JA in shaping root system architecture in compacted soil using state-of-art imaging and molecular biology techniques.Work plan:3D imaging of tomato JA biosynthetic and JA signalling mutants' roots growing in compacted and noncompacted soil (Year1)Jasmonic Acid (JA) biosynthetic mutant (def1 in Castlemart background) and JA signalling mutant (jai1-1 in Alisa craig background) will he grown in non-compacted and compacted soil for 10- 20 days.X-ray CT imaging will quantify root system architecture (e.g. 3D root branch structure, length, lateral number etc) in compacted soil. Root tips of def1 and jai1-1 mutants will be harvested to image their root anatomical responses such as cortical cell expansion, epidermal cell elongation and vasculature anatomy. Transcriptomic and JA profiling of tomato roots from compacted and non-compacted soil (Year1-2)The expression level of key candidate genes (highly upregulated and downregulated) from transcriptome analysis will be used generating CRISPR ca9 mutants. Based on the temporal dynamics of key JA signalling repressor gene expression, we will select the best JA responsive as well as compaction responsive gene for designing new JA reporter in tomato. We will also analyse the quantification of JA in tomato root tips grown in compacted and noncompacted soil to corroborate the expression level of JA signalling and biosynthetic genes with the Actual JA level.Generating novel JA-response reporters and CRISPR based JA signalling mutants in tomato (Year2-4)The jas motif of selected JAZ repressor from expression datasets will be used to create a new JA reporter (SlJAZx:3xVENUS) which will provide the spatial and temporal quantification of JA level in various cells of tomato roots. CRISPR mutants will be created from the selected key candidate genes then imaged using CT and LAT imaging.Dissecting the nexus of Ethylene and JA signalling networks to regulate soil compaction responses (Year4)We will use our tomato intogression lines (highly ethylene sensitive and insensitive) and never ripe 2 tomato ethylene signalling mutants to cross them with def1, jai1-1 and newly created CRISPR mutants to understand whether JA acts downstream or upstream to ethylene signalling in compaction. Output: Discovering a new rhizospheric signal and underlying mechanism of JA which shape root system architecture in compacted soil.