Tef (Eragrostis tef) iTRAQ-labelled protein fractions

The orphan crop, Eragrostis tef, was subjected to controlled drought conditions to observe the physiological parameters and proteins changing in response to dehydration stress. Physiological measurements involving electrolyte leakage, chlorophyll fluorescence and ultra-structural analysis showed tef plants to tolerate water loss to 50% RWC before adverse effects in leaf tissues were observed. Proteomic analysis using iTRAQ mass spectrometry and appropriate database searching enabled the detection of 5727 proteins, of which 211 proteins, including a number of spliced variants, were found to be differentially regulated with imposed stress conditions. Validation of the stress-related proteins, fructose-bisphosphate aldolase (FBA), glutamine synthetase (GLN) and the protective antioxidant proteins, monodehydroascorbate reductase (MDHAR) and peroxidase (POX), using immunodetection and enzymatic assay confirmed protein presence according to iTRAQ findings and showed increased protein abundance levels and enzymatic activity in response to water-deficit. GO-term enrichment and analysis revealed terms involved in biotic and abiotic stress response, signaling, transport, cellular homeostasis and pentose metabolic processes, to be enriched in tef up-regulated proteins, while terms linked to ROS-producing processes under water-deficit, such as photosynthesis and associated light harvesting reactions, manganese transport and homeostasis, the synthesis of sugars and cell wall catabolism and modification, to be enriched in tef down-regulated proteins.

本研究针对孤儿作物苔麸（Eragrostis tef）设置可控干旱条件，以观测其在脱水胁迫下的生理参数与蛋白质表达变化。生理测定涵盖电解质渗漏、叶绿素荧光检测与超微结构分析，结果显示苔麸植株可耐受水分流失至相对含水量（Relative Water Content, RWC）的50%，此时其叶片组织尚未出现不良效应。本研究采用同位素标记相对和绝对定量（isobaric Tags for Relative and Absolute Quantitation, iTRAQ）质谱技术结合适配数据库检索开展蛋白质组分析，共检测到5727种蛋白质，其中211种蛋白质（含多种剪接变体）在胁迫处理下呈现差异表达调控。通过免疫检测与酶活性测定，对胁迫相关蛋白果糖二磷酸醛缩酶（fructose-bisphosphate aldolase, FBA）、谷氨酰胺合成酶（glutamine synthetase, GLN）以及保护性抗氧化蛋白单脱氢抗坏血酸还原酶（monodehydroascorbate reductase, MDHAR）与过氧化物酶（peroxidase, POX）进行验证，结果不仅证实了iTRAQ质谱分析的蛋白质检出结果，同时发现水分亏缺胁迫下这些蛋白的丰度水平与酶活性均显著升高。基因本体（Gene Ontology, GO）术语富集分析结果显示，苔麸上调表达蛋白显著富集于生物与非生物胁迫响应、信号转导、物质转运、细胞稳态以及戊糖代谢过程等相关术语；而水分亏缺下产活性氧（Reactive Oxygen Species, ROS）相关过程（如光合作用及其关联光捕获反应、锰转运与稳态、糖类合成以及细胞壁分解代谢与修饰）相关的术语，则在苔麸下调表达蛋白中显著富集。