Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves.
The constitutive and wound-inducible leucine aminopeptidases (LAP-N and LAP-A, respectively) of tomato encode 60-kDa proteins with 5-kDa presequences that resemble chloroplast-targeting peptides. Cell fractionation studies and immunoblot analyses of chloroplast and total proteins have suggested a dual location of the mature LAP-A proteins in the cytosol and the plastids. In this study, the subcellular localization of tomato LAPs was further investigated using in vitro chloroplast-targeting assays and immunocytochemical techniques at the light and TEM levels. In vitro-translated LAP-A1 and LAP-N preproteins were readily transported into pea chloroplasts and processed into mature proteins of 55 kDa indicating the presence of a functional chloroplast-targeting signal in the LAP-A1 and LAP-N protein precursors. In addition, a LAP polyclonal and a LAP-A-specific antisera were used to immunolocalize LAP proteins in leaves from healthy, wounded and methyl jasmonate (MeJA)-treated plants. Low levels of LAPs and/or LAP-like proteins were detected in leaves from unwounded plants. The LAP polyclonal antiserum, which detected LAP-A, LAP-N and LAP-like proteins, and the LAP-A specific antibodies, which detected only LAP-A, showed that LAP levels increased in leaf sections after wounding and MeJA treatments. LAP-A proteins were primarily detected within the chloroplasts of spongy and palisade mesophyll cells. The localization of LAP-A was distinct from the location of early wound-response proteins that are important in the biosynthesis of jasmonic acid or systemin and more similar to the late wound-response proteins with primary roles in defense. The importance of these findings relative to the potential roles of LAP-A in defense is discussed.
Narváez-Vásquez
Javier
J
Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California-Riverside, Riverside, CA 92521-0124, USA.
Tu
Chao-Jung
CJ
Park
Sang-Youl
SY
Walling
Linda L
LL
D000595:Amino Acid Sequence; D018506:Gene Expression Regulation, Plant; D007150:Immunohistochemistry; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000201:enzymology*; D018515:Plant Leaves / Q000201:enzymology*; D021381:Protein Transport
D007931:Leucyl Aminopeptidase