Abstract
The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.
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Abbreviations
- ACC:
-
1-aminocyclopropane-carboxylic acid
- FP:
-
fusion protein
- IgG:
-
immunoglobulin G
- PEPCase:
-
phosphoenolpyruvate carboxylase
- SP:
-
synthetic peptide
- TBS:
-
Tris-buffered saline
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This work represents some of the research submitted by C. Rombaldi in partial fulfillment of the requirements for the Doctorate degree. It was partly carried out at the Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes (CNRS/IN-RA, Toulouse-Auzeville, France) with generous assistance and advice from Dr. G. Truchet and F. de Billy. The authors are grateful to Professor D. Grierson and Dr. A. Hamilton (University of Nottingham, UK) for providing us with pTOM13 antisense tomatoes and the latter for critical reviewing of the manuscript. The PEPCase antibodies were kindly provided by Dr. J.Vidal (CNRS, Orsay, France). This work was supported by the Ministère de l'Education Nationale (JE 179), the EEC (ECLAIR Grant AGRE 015) and by CAPES-COFECUB (doctoral fellowship to C. Rombaldi).
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Rombaldi, C., Lelièvre, JM., Latché, A. et al. Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit. Planta 192, 453–460 (1994). https://doi.org/10.1007/BF00203582
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DOI: https://doi.org/10.1007/BF00203582