Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase during Leaf Ontogeny in White Clover1

Abstract

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 1-aminocyclopropane-1-carboxylic 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.The plant hormone ethylene is an important regulator of several physiological processes in higher plants (Abeles et al., 1992) and also functions as a mediator of responses to external stimuli, such as wounding, flooding, and pathogen invasion (Kende, 1993). The biosynthetic pathway of the hormone in higher plants has now been characterized (Adams and Yang, 1979) and two committed enzymes in the pathway, ACC synthase (EC 4.4.1.14) and ACC oxidase (EC 1.4.3), have been identified (Yang and Hoffman, 1984;Kende, 1993).ACC synthase is recognized as the rate-determining step in the ethylene-biosynthesis pathway, and many inducers are proposed to act by stimulation of this enzyme (Yang and Hoffman, 1984;Theologis, 1992;Kende, 1993). The enzyme is known to be coded for by a multigene family in several plant species, with many of these genes cloned from a wide variety of tissues and in response to a variety of stimuli (Fluhr and Mattoo, 1996).In contrast, the ability of most plant tissues to convert ACC to ethylene was interpreted originally as evidence that the regulation of ACC oxidase is not a major control point of ethylene biosynthesis (Yang and Hoffman, 1984). More recently, two significant advances have provided the foundation for more detailed biochemical analysis of the enzyme. The first was the expression of a cDNA, designated pTOM13, coding for the putative ethylene-forming enzyme (Hamilton et al., 1990) in yeast (Hamilton et al., 1991), and another hi...

The role of PnACO1 in light- and IAA-regulated flower inhibition in Pharbitis nil

“…(PnACO1) and determined its sequence. The PnACO1 gene encodes for a protein whose expected molecular mass (34.82 kDa) is similar to that of ACC oxidases identified in other plant species (Hudgins et al 2006;Hunter et al 1999;Nie et al 2001;Vriezen et al 1999;Zanetti et al 2002). The largest similarity with respect to the PnACO1 amino acid sequence As an internal control, we used a constitutively expressed actin gene (83 %) is shown in ACO1 proteins from N. attenuata, N. glutinosa, S. tuberosum, as well as A. thaliana ACO2-69 % (Fig.…”

Regulation of ethylene biosynthesis at the level of 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene

“…An expression study in white clover indicated that ACO plays a regulatory role in leaf ontogeny (Hunter et al 1999). In the apical hook of A. thaliana, AT-ACO2 expression is positively regulated by ethylene and restricted to the time period that the cells are ethylene sensitive (Raz and Ecker 1999).…”

The proteomics of senescence in leaves of white clover, Trifolium repens (L.)

“…Side branches, formed by the outgrowth of axillary buds, were removed as these can affect the developmental sequence. Stolons grown in this way produce the full sequence of leaf development from initiation at the apex, through maturity to senescence and necrosis allowing the comparison of stage-specific physiological and biochemical differences within the same plant [5]. Developmental stages were characterised by measuring the chlorophyll content of leaves although it is known that some senescence related genes show altered expression prior to loss of chlorophyll [6].…”