Leucine Aminopeptidases: Role in Wounding and Defense

对植物发育过程中蛋白质的降解以及对胁迫的反应知之甚少。特别是，有一个空白的理解一类蛋白酶称为氨肽酶。番茄中的两类亮氨酸氨肽酶基因（LapA和LapN）已被鉴定。LapA基因表达响应于创伤、毛虫进食以及一些细菌和真菌病原体。相反，LapN基因在所有时间和所有组织中表达。对表达LapA反义基因（35S：asLap）的基因工程番茄植物的分析表明，Lap基因的下调损害了番茄产生伤口反应的能力。该提案建立在对前一个授予期内建立的番茄LAPs的强大知识基础之上。这项综合研究的重点是了解LAPs的位置及其在创伤，病原体，缺水和盐度反应中的作用。 细胞分级分离研究表明，LAPs存在于两个细胞隔室：细胞质和叶绿体。显然，可用于抑制作用的底物由LAPs在番茄细胞内的位置决定。因此，LAP-A和LAP-N特异性抗体将用于确定这两类氨肽酶在细胞内的位置。将测试控制LAP-A和LAP-N在两个细胞位置中蓄积的机制。LapA反义植物（35S：asLap）不能有效地诱导伤口反应基因。这些数据表明LAP-A可能是调节植物信号传导途径的第一种肽酶之一。由于这一结论是新的植物群落，这些数据应确认使用其他策略。将评价三组具有改变的Lap基因表达模式的转基因番茄植物。首先，制备表达两个Lap反义基因的植物。这些植物表达来自创伤诱导的LapA启动子（LapA：asLap）和组成型35S启动子（35S：asLap）的LapA反义RNA。这些植物应该提供最严格的伤口反应调节。第二，将表征共抑制植物。与下调LapA和LapN的反义植物不同，共抑制的植物仅下调LapA。第三，将表征组成型表达高水平LAP-A的转基因番茄。LapA和/或LapN下调与伤口反应的抑制的紧密相关性将暗示LAP作为调节植物胁迫反应的蛋白酶。如果LAP显示调节伤口反应，则通过饲喂LAP下调的植物系统素、脱落酸、茉莉酸和十八烷酸途径的中间体来确定抑制作用的位点。如果涉及系统素活化，则将表征LAP-A和LAP-N对推定的系统素前体的活性。 Lap基因表达上调和下调的转基因番茄将用于确定Lap是否对控制细菌、病毒和真菌感染以及昆虫侵染和对缺水和盐度的耐受性很重要。 对LAPs在植物发育和胁迫响应中的表达和影响的理解大大增加。然而，持续的努力是必不可少的，以阐明在应激反应的LAPs的确切作用。这些研究将增强我们对有利于最佳生长和发育的机制的理解。这些因素将直接影响作物生产力的提高，并可能产生显着的见解，为发展有说服力的战略，以提高植物的内源性保护机制，以一系列的压力。

Little is known about the degradation of proteins during plant development and in response to stress. In particular, there is a gap in the understanding of the class of proteases called aminopeptidases. Two classes of leucine aminopeptidase genes (LapA and LapN) from tomato have been characterized. LapA genes are expressed in response to wounding, caterpillar feeding, and some bacterial and fungal pathogens. In contrast, LapN genes are expressed at all times and in all tissues. The analysis of genetically engineered tomato plants that express a LapA antisense gene (35S:asLap) showed that down-regulation of Lap genes impairs the ability of tomatoes to mount a wound response. This proposal builds on a strong foundation of knowledge about the tomato LAPs built during the previous granting period. This proposed integrated research effort is focused on understanding the location of LAPs and their role in responses wounding, pathogens, water deficit, and salinity. Cell fractionation studies show that LAPs reside in two cellular compartments: the cytoplasm and the chloroplast. Obviously, the substrates available for LAP action are determined by the location of LAPs within the tomato cell. Therefore, LAP-A- and LAP-N-specific antibodies will be used to determine the location of these two classes of aminopeptidases within the cell. The mechanisms that control the accumulation of LAP-A and LAP-N in two cellular locations will be tested. The LapA antisense plants (35S:asLap) do not effectively induce wound-response genes. These data suggest that LAP-A may be one of the first peptidases to regulate a plant-signaling pathway. Since this conclusion is novel for the plant community, these data should be confirmed using additional strategies. Three sets of transgenic tomato plants that have altered patterns of Lap gene expression will be evaluated. First, plants expressing two Lap antisense genes will be made. These plants express the LapA antisense RNA from both the wound-induced LapA promoter (LapA:asLap) and the constitutive 35S promoter (35S:asLap). These plants should afford the tightest regulation of the wound response. Second, co-suppressed plants will be characterized. Unlike the antisense plants that down regulate both LapA and LapN, the co-suppressed plants only down regulate LapA. Third, transgenic tomatoes that express high levels of LAP-A constitutively will be characterized. A tight correlation of LapA and/or LapN down regulation with the suppression of the wound response will implicate LAPs as protease that regulates plant stress responses. If LAPs are shown to modulate the wound response, the site of LAP action will be determined by feeding Lap down-regulated plants systemin, abscisic acid, jasmonic acid and intermediates of the octadecanoid pathway. If systemin activation is implicated, the activity of LAP-A and LAP-N on putative systemin precursors will be characterized. Transgenic tomatoes that are up- and down-regulated for Lap gene expression will be used to determine if LAPs are important for controlling bacterial, viral, and fungal infections, as well as insect infestations and tolerance to water-deficit and salinity. The understanding of the expression and impact of LAPs in plant development and in responses to stress has increased greatly. However, continued efforts are essential to elucidate the exact roles of LAPs in stress responses. These studies will enhance our understanding of the mechanisms that favor optimal growth and development. These factors will directly impact on increased crop productivity and may yield significant insights for the development of cogent strategies to enhance a plant's endogenous protection mechanisms to an array of stresses.