RNA-seq GLV6 overexpression during lateral root initiation in Arabidopsis thaliana

The goal of this experiment was to identify the downstream targets of the GOLVEN6 peptide signaling pathway in Arabidopsis thaliana, specifically during lateral root initiation. Using an estradiol inducible GLV6 overexpression construct in wildtype and rgi1rgi5 double mutant (mutant in receptors for the GLV6 peptide) backgrounds, in combination with gravistimulation induced lateral root formation, the RGI receptor dependent transcriptional effects of GLV6 overexpression were characterized. An estradiol inducible GLV6 overexpression line in a wildtype (iGLV6) and in an rgi1rgi5 double receptor mutant background (rgi1rgi5/iGLV6) were used. 4-day old seedlings of both lines were gravistimulated (vertically grown seedlings were turned counterclockwise by 90°) to induce lateral root initiation in the resulting root bends. 8h after gravistimulation, seedlings of both lines were treated with 2µM of estradiol to induce GLV6 overexpression, or DMSO as a mock treatment. 3h and 6h after treatment, root bends were dissected and collected for RNA-sequencing. This yielded a total of 8 samples per replicate; 3h mock treated iGLV6 (IM3), 3h estradiol treated iGLV6 (IE3), 3h mock treated rgi1rgi5/iGLV6 (RM3), 3h estradiol treated rgi1rgi5/iGLV6 (RE3), 6h mock treated iGLV6 (IM6), 6h estradiol treated iGLV6 (IE6), 6h mock treated rgi1rgi5/iGLV6 (RM6), 6h estradiol treated rgi1rgi5/iGLV6 (RE6). For each sample, 4 replicates were obtained. This setup enabled the comparison of the GLV6 induced transcriptional effects between wildtype and rgi1rgi5 mutants at 2 time points after treatment, in samples that are strongly enriched for lateral root initiation events.

本实验旨在鉴定拟南芥（Arabidopsis thaliana）中GOLVEN6（GLV6）肽信号通路的下游靶标，尤其聚焦侧根起始阶段。本研究采用雌二醇（estradiol）诱导型GLV6过表达载体，分别在野生型背景与GLV6肽受体突变体rgi1rgi5双突变体背景中构建实验体系，并结合重力刺激诱导侧根形成，以此表征GLV6过表达的RGI受体（RGI receptor）依赖性转录调控效应。本实验分别构建了两种株系：野生型背景下的雌二醇诱导型GLV6过表达株系（iGLV6），以及rgi1rgi5双受体突变体背景下的该过表达株系（rgi1rgi5/iGLV6）。将两种株系的4日龄幼苗进行重力刺激处理：将垂直生长的幼苗逆时针旋转90°，以在根尖弯曲部位诱导侧根起始。重力刺激8小时后，对两种株系的幼苗分别施加2μM雌二醇以诱导GLV6过表达，同时以二甲基亚砜（DMSO）作为空白对照处理。分别在处理后3小时与6小时，收集根尖弯曲部位的组织进行RNA测序（RNA-sequencing）。每组生物学重复共包含8组样本，具体为：处理3小时的野生型iGLV6 mock组（IM3）、处理3小时的野生型iGLV6雌二醇处理组（IE3）、处理3小时的rgi1rgi5/iGLV6 mock组（RM3）、处理3小时的rgi1rgi5/iGLV6雌二醇处理组（RE3）、处理6小时的野生型iGLV6 mock组（IM6）、处理6小时的野生型iGLV6雌二醇处理组（IE6）、处理6小时的rgi1rgi5/iGLV6 mock组（RM6）、处理6小时的rgi1rgi5/iGLV6雌二醇处理组（RE6）。每组样本设置4个生物学重复。该实验体系可实现在侧根起始事件高度富集的样本中，对比处理后两个时间点下野生型与rgi1rgi5突变体中GLV6诱导的转录调控差异。