Leucine Aminopeptidases: Role in Wounding and Defense

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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.