Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses. Arabidopsis thaliana

Arabidopsis microRNA expression regulation was studied in a wide array of abiotic stresses such as drought, heat, salinity, copper excess/deficiency, cadmium excess and sulphur deficiency. A home-built RT-qPCR mirEX platform for the amplification of 289 Arabidopsis microRNA transcripts was used to study their response to abiotic stresses. Small RNA sequencing and Northern hybridization were performed to study the expression of mature microRNAs. In the case of common climate change related stresses such as drought, heat and salinity we observed broad induction of the level of primary miRNAs that was not observed at the level of microRNAs.. In the case of local soil pollution stresses, that are represented by heavy metal contaminations or deprivation of a specific micro- or macroelement, the transcriptional response of pri-miRNAs was quite limited but also not predictive to the level of the mature microRNA. This points to an essential role of posttranscriptional regulation of microRNAs expression. We found that the level of several microRNAs can be differentially regulated in early and late response to stress. New Arabidopsis microRNAs responsive to abiotic stresses were discovered. Three microRNAs: miR319a/b, miR319b.2, and miR400 have been found to be responsive in several abiotic stresses and thus can be regarded as a general stress-responsive microRNA species. Additionally, a new target for miR319b.2 – TBL10 has been experimentally confirmed. However, its level under different abiotic stresses is unchanged in comparison to control conditions. In the promoter region of the TBL10 gene we found the presence of many stress-responsive elements. We suggest that transcriptional induction resulting in the increase of transcript levels is downregulated by the increase of the miR319b.2 ultimately resulting in a stable level of TBL10 mRNA. Our experiments show the existence of a complex regulatory network involved in the microRNA level control that is necessary to fine-tune plant response to environmental cues. Overall design: Arabidopsis thaliana abiotic stress responses miRNAs: 30%SWC drought, 20%SWC drought, 0.5h heat (37˚C), 6h heat (37˚C), 24h NaCl [250mM] excess, 24h copper deficiency, 24h copper [10μM] excess, 24h cadmium [10μM] excess, 24h sulfur deficiency

本研究围绕拟南芥（Arabidopsis）在多种非生物胁迫（abiotic stresses）下的微小RNA（microRNA, miRNA）表达调控机制展开探究，所涉胁迫类型涵盖干旱、高温、盐胁迫、铜过量/缺乏、镉过量及硫缺乏。本研究采用自研的实时荧光定量逆转录聚合酶链反应（RT-qPCR）mirEX扩增平台，对289条拟南芥miRNA转录本进行扩增，以分析其对非生物胁迫的响应情况；同时通过小RNA测序（small RNA sequencing）与Northern印迹杂交（Northern hybridization）技术，检测成熟miRNA的表达水平。针对干旱、高温、盐胁迫这类与气候变化相关的典型胁迫，研究观察到初级microRNA（pri-miRNA）的表达水平出现广泛诱导，但该现象并未在成熟miRNA层面得到体现。而针对重金属污染或特定微量/常量元素缺乏所代表的本土土壤污染胁迫，pri-miRNA的转录响应相当有限，且同样无法预测成熟miRNA的表达水平，这表明miRNA表达的转录后调控（posttranscriptional regulation）发挥着关键作用。本研究发现，部分miRNA在胁迫的早期与晚期响应过程中呈现差异调控模式；同时鉴定出一批响应非生物胁迫的新型拟南芥miRNA。其中miR319a/b、miR319b.2及miR400在多种非生物胁迫中均表现出响应特性，可被视为通用胁迫响应miRNA家族成员。此外，本研究通过实验证实了miR319b.2的一个全新靶基因——TBL10，但在不同非生物胁迫条件下，其转录本水平与对照组相比并无显著变化。对TBL10基因启动子区域（promoter region）的分析显示，该区域存在大量胁迫响应元件（stress-responsive elements）。据此推测，转录诱导所带来的转录本水平提升，会因miR319b.2的表达上调而被负调控，最终使TBL10 mRNA的水平维持稳定。本实验证实，存在一套复杂的调控网络参与miRNA水平的调控，这对于植物精准响应环境信号至关重要。实验设计概述：以拟南芥（Arabidopsis thaliana）为研究对象，探究其非生物胁迫响应相关miRNA的表达模式，处理组设置如下：30%土壤含水量（soil water capacity, SWC）干旱胁迫、20%SWC干旱胁迫、0.5小时高温（37℃）处理、6小时高温（37℃）处理、24小时250mM氯化钠盐胁迫、24小时铜缺乏处理、24小时10μM铜过量处理、24小时10μM镉过量处理、24小时硫缺乏处理。