Leucine Aminopeptidases: Role in Wounding and Defense

人们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，人们对一类被称为氨基肽酶的酶的理解存在差距。番茄中的两类亮氨酸氨基肽酶基因(LapA和LapN)已被鉴定。LapA基因在创伤、毛虫取食以及一些细菌和真菌病原体的作用下表达。相反，LapN基因在任何时候和所有组织中都有表达。对表达LapA反义基因(35S：ASLAP)的转基因番茄植株的分析表明，LAP基因的下调削弱了番茄对伤口的反应能力。这项提议建立在对前一个授权期内建立的番茄圈的坚实知识基础上。这项拟议的综合研究工作侧重于了解LAPS的位置及其在伤害、病原体、水分亏缺和盐度反应中的作用。细胞分离研究表明，LAPS存在于两个细胞室：细胞质和叶绿体。显然，可用于LAP作用的底物由LAP在番茄细胞内的位置决定。因此，将使用LAP-A和LAP-N特异性抗体来确定这两类氨基肽酶在细胞内的位置。将测试控制LAP-A和LAP-N在两个细胞位置积累的机制。LAPA反义植物(35S：ASLAP)不能有效地诱导创伤反应基因。这些数据表明，LAP-A可能是最早调节植物信号通路的多肽酶之一。由于这一结论对植物群落来说是新的，这些数据应该用其他策略来证实。将对三组改变了Lap基因表达模式的转基因番茄植株进行评估。首先，将获得表达两个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)和构成35S启动子(35S：ASLAP)的LapA反义RNA。这些植物应该能够对伤口反应进行最严格的调控。其次，共抑制的植物将被定性。与同时下调LapA和LapN的反义植物不同，共抑制的植物只下调Lapa。第三，结构性地表达高水平LAP-A的转基因西红柿将被鉴定。LapA和/或LapN的下调与伤口反应的抑制密切相关，这将意味着LAPS可能是调节植物逆境反应的蛋白酶。如果LAP被证明可以调节伤口反应，LAP的作用部位将通过饲喂LAP下调的植物系统素、脱落酸、茉莉酸和十八烷类途径的中间体来确定。如果牵涉到系统蛋白的激活，LAP-A和LAP-N在可能的系统蛋白前体上的活性将被表征。LAP基因表达上调和下调的转基因番茄将被用来确定LAPS是否对控制细菌、病毒和真菌感染以及昆虫侵袭和对缺水和盐碱的耐受性至关重要。对LAPS在植物发育和逆境反应中的表达和影响的了解大大增加。然而，继续努力对于阐明LAPS在应激反应中的确切作用是至关重要的。这些研究将加深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，并可能为制定有效的战略以增强植物的内源保护机制以应对一系列压力提供重要的见解。

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.