Prohormone processing and regulation of the wound response by plant death proteases

虽然肽激素信号的重要性在动物系统中得到了很好的认识，但肽作为信号分子在植物中长期被忽视。然而，近年来，大约有十几种植物分泌的信号肽被发现并认识到在细胞通讯中起重要作用。此外，生物信息学已经预测了数百种潜在的信号肽。大多数这些信号肽需要翻译后修饰才能成熟，包括蛋白水解处理以从其较大的前体蛋白中释放活性肽。这也适用于系统素，它于1991年被确定为植物中第一个激素样信号肽，是茄科植物诱导对草食性昆虫防御反应的伤口信号通路的重要组成部分。然而，对于系统蛋白和大多数其他信号肽一样，它们如何从前体蛋白中释放出来以及加工蛋白酶的身份问题仍然没有解决。为了鉴定原系统加工酶，我们从番茄中纯化了一种天冬氨酸（Asp）特异性蛋白酶。发现该蛋白酶在系统蛋白肽两侧的两个Asp残基上切割原系统蛋白。裂解产物在诱导伤口反应标志物的生物测定中具有活性。这些位点上的切割也被发现是激活所必需的，因为在位点导向的原系统蛋白双突变体中，两个Asp残基被Ala取代，蛋白质水解过程和生物活性丧失。通过质谱分析，我们可以确定该蛋白酶为SlPhyt-1或SlPhyt-2，这是植酸酶的两个同源物，植酸酶是一种与细胞死亡相关的特异性蛋白酶，之前在烟草和水稻中被发现。这些数据表明，在基因复制后，番茄中的植酸酶同源物可能在原系统蛋白加工和伤口反应调节中获得了新的作用，除了植酸酶在程序性细胞死亡中的已知功能外。这一假设将在拟议的研究项目中得到解决。通过功能增益和功能损失分析，我们将在体内描述SlPhyt-1和SlPhyt-2的功能，并确定它们一方面对细胞死亡，另一方面对原系统蛋白加工，伤口信号传导和昆虫抗性的具体贡献。通过提出的研究，我们解决了长期存在的问题，即伤害如何导致信号肽系统的形成，作为对昆虫食草动物的系统防御反应的一部分。总的来说，这项研究将为了解肽激素成熟的机制和蛋白酶作为细胞死亡和植物防御的调节剂的功能提供见解。

While the importance of peptide hormone signaling is well recognized in animal systems, peptides as signaling molecules have long been neglected in plants. In recent years, however, about a dozen of secreted plant signaling peptides have been identified and recognized to play important roles in cellular communication. In addition, hundreds of potential signaling peptides have been predicted by bioinformatics. Most of these signaling peptides require post-translational modifications for maturation including proteolytic processing to release the active peptides from their larger precursor proteins. This also applies to systemin, which was identified in 1991 as the first hormone-like signaling peptide in plants, and as an essential component of the wound signaling pathway for the induction of defense responses against herbivorous insects in plants of the nightshade family. However, for systemin and most other signaling peptides alike, the question of how they are released from their precursor proteins and the identity of the processing proteases remain unresolved. In an attempt to identify the prosystemin-processing enzyme, we purified an aspartate (Asp)-specific protease from tomato. The protease was found to cleave prosystemin at two Asp residues flanking the systemin peptide. The cleavage product was active in bioassays for the induction of wound response markers. Cleavage at these sites was also found to be necessary for activation, since proteolytic processing and bioactivity were lost in a site-directed prosystemin double mutant, in which the two Asp residues had been substituted by Ala. Using mass spectrometry we could identify the protease as either SlPhyt-1 or SlPhyt-2, two homologs of phytaspase, a cell death-related Asp-specific protease previously characterized in tobacco and rice. The data suggest that after gene duplication, phytaspase homologs in tomato may have acquired novel roles in prosystemin processing and the regulation of the wound response, in addition to the recognized function of phytaspase in programmed cell death. This hypothesis will be addressed in the proposed research project. Using gain- and loss-of-function analyses we will characterize the function of SlPhyt-1 and SlPhyt-2 in vivo and identify their specific contributions to cell death on the one hand, and prosystemin processing, wound signaling and insect resistance on the other. With the proposed research we address the long-standing question of how wounding leads to the formation of the signaling peptide systemin as part of the systemic defense response against insect herbivores. On a more general note, the study will provide insight into the mechanisms of peptide hormone maturation and the function of proteases as regulators of cell death and plant defense.