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容易压实，造成数十亿英镑的损失。尽管它很重要，但人们对为什么根在压实的土壤中停止生长知之甚少。我们的团队（Pandey et al ., 2021, Science）最近进行了一系列（字面上）突破性的实验，揭示了根系在破坏了对一种叫做乙烯的植物激素信号的敏感性后，可以穿透高度密实的土壤。然而，目前对关键根际的认识表明缺乏对作物根系土壤压实反应的充分了解。目的：在本项目中，我们的目标是利用最先进的成像和分子生物学技术发现根际信号JA在夯实土壤中形成根系结构中的新作用。工作计划：番茄JA生物合成和JA信号突变体在夯实和非夯实土壤中的根系三维成像（Year1）茉莉酸（Jasmonic Acid， JA）生物合成突变体（Castlemart背景中的def1）和JA信号突变体（Alisa craig背景中的jai1-1）将在非夯实和夯实土壤中生长10- 20天。x射线CT成像将量化夯实土壤中的根系结构（如三维根枝结构、长度、横向数等）。将采集def1和jai1-1突变体的根尖，对其根解剖反应进行成像，如皮层细胞扩增、表皮细胞伸长和脉管系统解剖。来自夯实和非夯实土壤的番茄根系转录组学和JA谱分析（Year1-2）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 ca9突变体。基于关键JA信号抑制基因表达的时间动态，我们将选择最佳的JA响应基因和压实响应基因来设计新的番茄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JA的定量，以证实JA信号和生物合成基因的表达水平与实际JA水平。利用从表达数据集中选择的JAZ抑制因子的jas基元，将创建一个新的JA报告基因（SlJAZx:3xVENUS），该基因将提供番茄根系不同细胞中JA水平的时空量化。将从选定的关键候选基因中创建CRISPR突变体，然后使用CT和LAT成像进行成像。我们将使用我们的番茄侵入系（高度乙烯敏感和不敏感）和未成熟的2个番茄乙烯信号突变体与def1， jai1-1和新创建的CRISPR突变体进行杂交，以了解JA在压实过程中是下游还是上游作用于乙烯信号。成果：发现了一种新的根际信号和JA在夯实土壤中形成根系结构的潜在机制。

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.