Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase during Leaf Ontogeny in White Clover
We examined the expression of three distinct 1-aminocyclopropane-1-carboxylic acid oxidase genes during leaf ontogeny in white clover (Trifolium repens). Significant production of ethylene occurs at the apex, in newly initiated leaves, and in senescent leaf tissue. We used a combination of reverse transcriptase-polymerase chain reaction and 3-rapid amplification of cDNA ends to identify three distinct DNA sequences designated TRACO1, TRACO2, and TRACO3, each with homology to
... 1carboxylic acid oxidase. Southern analysis confirmed that these sequences represent three distinct genes. Northern analysis revealed that TRACO1 is expressed specifically in the apex and TRACO2 is expressed in the apex and in developing and mature green leaves, with maximum expression in developing leaf tissue. The third gene, TRACO3, is expressed in senescent leaf tissue. Antibodies were raised to each gene product expressed in Escherichia coli, and western analysis showed that the TRACO1 antibody recognizes a protein of approximately 205 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed preferentially in apical tissue. The TRACO2 antibody recognizes a protein of approximately 36.4 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed in the apex and in developing and mature green leaves, with maximum expression in mature green tissue. No protein recognition by the TRACO3 antibody could be detected in senescent tissue or at any other stage of leaf development. Abbreviations: RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; UTR, untranslated region. Abeles FB, Morgan PW, Saltveit ME (1992) Ethylene in Plant Biology, Ed 2. Academic Press, London Adams DO, Yang SF (1979) Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc Natl Acad Sci USA 76: 170-174 Altschul SF, Gish W, Millar W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410 Barry CS, Blume B, Bouzayen M, Cooper W, Hamilton AJ, Grierson D (1996) Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato. Plant J 9: 525-535 Bouquin T, Lasserre E, Pradier J, Pech J-C, Balague C (1997) Wound and ethylene induction of the ACC oxidase melon gene CM-ACO1 occurs via two direct and independent transduction pathways. Plant Mol Biol 35: 1029-1035 Bradford MM (1976) A rapid and sensitive assay for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 Chomczynski P (1992) One hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal Biochem 201: 134-139 Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81: 1991-1995 Clark DG, Richards C, Hiloti Z, Lind-Iversen S, Brown K (1997) Effect of pollination on accumulation of ACC synthase and ACC oxidase transcripts, ethylene production and flower petal abscission in geranium (Pelargonium ϫ hortorum L.H. Bailey). Plant Mol Biol 34: 855-865 Dong JG, Fernandez-Maculet JC, Yang SF (1992) Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit. Proc Natl Acad Sci USA 89: 9789-9793 Dupille E, Rombaldi C, Lelievre J-M, Cleyet-Marel J-C, Pech J-C, Latche A (1993) Purification, properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits. Planta 190: 65-70 Dupille E, Zacarias L (1996) Extraction and biochemical characterization of wound-induced ACC oxidase from citrus peel. Plant Sci 114: 53-60 Fernandez-Maculet JC, Yang SF (1992) Extraction and partial characterisation of the ethylene-forming enzyme from apple fruit. Plant Physiol 99: 751-754 Finlayson SA, Reid DM, Morgan PW (1997) Root and leaf specific ACC oxidase activity in corn and sunflower seedlings. Phytochemistry 45: 869-877 Fluhr R, Mattoo AK (1996) Ethylene: biosynthesis and perception. Crit Rev Plant Sci 15: 479-523 140 Hunter et al.