Table_9_Proteomics of Heat-Stress and Ethylene-Mediated Thermotolerance Mechanisms in Tomato Pollen Grains.XLSX

Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.

热胁迫是包括蔬菜作物在内的多种作物产量损失的主要诱因。近年来愈发频发的短期高温事件，同样会对作物造成损害。花粉发育对高温最为敏感，这也是多种作物在热胁迫下生产力下降的核心原因。然而，花粉响应热胁迫及耐热性背后的分子机制尚未完全阐明。本研究团队此前已证实，气态植物激素乙烯（ethylene）参与番茄（Solanum lycopersicum）的花粉耐热性调控。本研究进一步验证了上述结论：在热胁迫处理前使用乙烯释放剂乙烯利（ethephon）提升乙烯水平，可改善花粉品质。为解析发育中花粉粒内花粉响应热胁迫及乙烯介导的花粉耐热性的潜在机制，本研究采用蛋白质组学方法。研究采用凝胶液相色谱-质谱（gel LC-MS）分级分离方案对蛋白质进行提取与分析，共鉴定得到1355种蛋白质。本研究所有分析均基于一组包含721种蛋白质的数据集：该数据集的蛋白质可在至少一种处理组的三次生物学重复中被检测到。分析采用基于肽段计数的定量方法。结果表明，热胁迫会影响花粉的发育程序，包括蛋白质稳态（翻译与降解系统组分）、碳水化合物及能量代谢相关过程。乙烯利预处理可使热胁迫下的花粉蛋白质组更接近非胁迫条件下的蛋白质组：具体表现为参与蛋白质合成、降解、三羧酸循环及RNA调控的蛋白质丰度更高。此外，经乙烯利处理后，花粉的抗氧化应激保护机制被上调：具体包括谷胱甘肽二硫键还原酶（glutathione-disulfide reductase）、谷氧还蛋白（glutaredoxin）及蛋白质二硫键异构酶（protein disulfide isomerase）的丰度提升。综上，本研究鉴定出花粉耐热性的系统性核心组分，可为后续研究提供极具价值的定量蛋白质组数据库。