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Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves

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Abstract

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.

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Abbreviations

IgG:

Immunoglobulin

LAP:

Leucine aminopeptidase

MeJA:

Methyl jasmonate

RbcS:

Small subunit of ribulose bisphosphate carboxylase

TEM:

Transmission electron microscope

2D-PAGE:

Two-dimensional polyacrylamide gel electrophoresis

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Acknowledgments

The authors thank the CFAMM staff for their helpful suggestions and Dr. Patricia Springer for the use of her Leica DMR light microscope. This research was supported by a National Science Foundation grant # IBN-9974612 to L.L.W.

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Author notes

  1. Chao-Jung Tu

    Present address: Department of Plant Biology, Carnegie Institute of Washington, Stanford, CA, 94305-4101, USA

Authors and Affiliations

  1. Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California-Riverside, Riverside, CA, 92521-0124, USA

    Javier Narváez-Vásquez, Chao-Jung Tu, Sang-Youl Park & Linda L. Walling

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  1. Javier Narváez-Vásquez
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  2. Chao-Jung Tu
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  3. Sang-Youl Park
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  4. Linda L. Walling
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Correspondence to Linda L. Walling.

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Narváez-Vásquez, J., Tu, CJ., Park, SY. et al. Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves. Planta 227, 341–351 (2008). https://doi.org/10.1007/s00425-007-0621-0

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