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

由于农业管理实践的变化，土壤压实是现代农业面临的主要挑战。欧洲一半以上的耕地土壤容易压实，造成数十亿英镑的损失。尽管它的重要性，很少有人知道为什么根停止在压实的土壤中生长。我们团队（Pandey et al，2021，Science）最近进行的一系列（字面上）突破性实验表明，根系在破坏了对植物激素信号（称为乙烯）的敏感性后，可以穿透高度压实的土壤。然而，目前对关键根际信号的理解还不足以完全理解作物根系的土壤压实响应。目的：在本项目中，我们旨在利用最先进的成像和分子生物学技术，发现关键根际信号JA在压实土壤中塑造根系构型的新作用。工作计划：在压实和非压实土壤中生长的番茄JA生物合成和JA信号传导突变体的根的3D成像（Year 1）茉莉酸（JA）生物合成突变体（Castlemart背景中的def 1）和JA信号传导突变体（Alisa克雷格背景中的jai 1 -1）将在非压实和压实土壤中生长10- 20天。X射线CT成像将量化压实土壤中的根系结构（例如，3D根分支结构、长度、横向数量等）。将收获def 1和jai 1 -1突变体的根尖，以成像它们的根解剖学响应，例如皮层细胞扩张、表皮细胞伸长和脉管系统解剖学。来自压实和非压实土壤的番茄根的转录组学和JA谱分析（第1 -2年）来自转录组分析的关键候选基因（高度上调和下调）的表达水平将用于产生CRISPR ca 9突变体。基于JA信号抑制基因表达的时间动态，我们将选择最佳JA响应以及压缩响应基因，用于设计番茄中的新JA报告基因。我们还将分析在压实和非压实土壤中生长的番茄根尖中JA的定量，以证实JA信号传导和生物合成基因的表达水平与实际JA水平。在番茄中产生新的JA响应报告基因和基于CRISPR的JA信号传导突变体（Year 2 -4）（SlJAZx：3xVENUS），其将提供番茄根的各种细胞中JA水平的空间和时间定量。CRISPR突变体将从选定的关键候选基因中产生，然后使用CT和LAT成像进行成像。解剖乙烯和JA信号网络的联系以调节土壤压实反应（Year 4）我们将使用我们的番茄导入系（高乙烯敏感性和不敏感性）和从未成熟的2个番茄乙烯信号传导突变体与def 1杂交，jai 1 -1和新创建的CRISPR突变体，以了解JA在压实中是在乙烯信号传导的下游还是上游起作用。输出：发现JA在紧实土壤中塑造根系结构的新根际信号和潜在机制。

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.