In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

Abstract

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which '4CO2 is collected following uptake of IAA is usually found in highest concentration in the juvenile parts of the plant, and at lower levels in regions removed from the growing points (10) and in the older leaves (4). In some cases, the senescence process may, in fact, be regulated by the changing levels of this hormone. This is perhaps best illustrated by the process of leaf abscission in Coleus (5). Based upon current evidence, it appears that under natural conditions a depletion of IAA in the older petioles is a prerequisite for abscission to occur, although some interaction with other hormones may be involved as well (5) It was first proposed by Galston and Dalberg (3) that IAA oxidase activity increases with the age of the tissue, and thereby reduces the IAA concentration. This concept was supported by several subsequent papers which showed higher rates of IAA oxidase (7), peroxidase (12), and decarboxylating (6) activities in older tissues of cotton, cherry, and tobacco, respectively. However, most of these findings were based on work done in vitro using tissue homogenates or partially purified enzymes, and caution must be used in attempting to extrapolate these results to the situation in the intact plant, where additional enzymes, cofactors, inhibitors, and nonenzymic processes may be involved.In the experiments reported here, we have attempted to devise a method which will enable us to accurately measure total IAA decarboxylation rates in vivo. This paper describes the procedure using excised Coleus petiole sections, and presents data which indicate that in this system the IAA decarboxylating activity of the tissue decreases with age. Benth. were grown in the greenhouse under natural day lengths that were supplemented by continuous light from incandescent bulbs. All plants in a given experiment were of identical age, as measured by the time since the cuttings were started. The petioles were numbered in a basipetal direction, with the No. 2 petioles defined as the youngest ones whose leaf blades were between 60 and 100 mm long. MATERIALS AND METHODS Plants of the Princeton clone of Coleus blumeiIn the decarboxylation experiments, 10-mm segments were cut from the center of the petioles being tested. The petiole segments were weighed and the cut ends dipped in collodion (Mallinckrodt Chemical Co., St. Louis) in order to minimize cut surface effects (13). For ease of handling, the segments within each group were then strung together into a loop using a needle and thread. The segments were then rinsed with sterile distilled H20 (surface sterilization had no effect) and placed in a 25-ml Erlenmeyer flask containing 5 ml 0.05 M sodium acetate buffer (pH 5.0) and 0.2 ,Ci [1-'4C]IAA (57 mCi/mmol), t...

Uptake and decarboxylation of indole-3-acetic acid during auxin-induced growth in lupin hypocotyl segments

“…Thus, the idea proposed by Galston and Dalberg in 1954 that increased IAA-oxidase might account for the decreased IAA level in older, non-growing tissues has long been accepted. However, several studies have reported higher 1AA-oxidase in younger growing tissues which also exhibited higher IAA concentrations (Rajagopal and Larsen 1974;Brennan and Jacobs 1983;Valpuesta et al 1989;Sfinchez-Bravo et al 1989). Though a compartmentalization of IAA and IAA-oxidase might explain these results, the presence of a high IAA concentration in tissues with a high capacity to destroy IAA would agree with the proposed hypothesis that IAA must be destroyed following "the growth promoting act" (Cohen and Bandurski 1978).…”

Decarboxylative metabolism of [1′-14C]indole-3-acetic acid by tomato pericarp discs during ripening

“…In contrast, after a 2-h lag, the decarboxylative capacity of green discs increased in a linear manner and 24h after excision > 60% of the [I'-14C]IAA absorbed by the discs was subjected to decarboxylation during the subsequent 1-h incubation. The development of the IAA-decarboxylative capacity after excision of the immature green pericarp discs is suggestive of the involvement of wound-induced increases in peroxidases that are associated with repair processes in a number of tissues (Brennan and Jacobs 1983;Lagrimini and Rothstein 1987;Svalheim and Robertsen 1990). The lack of a similar increase in the IAA-decarboxylation capacity of the more-mature breaker and pink discs is in keeping with the lower increases in peroxidase activity that are reported to occur after the wounding of older tissues (Birecka et al 1976;Macheix et al 1986).…”

Identification of the metabolites of Indole-3-acetic acid in growing hypocotyls of Lupinus albus

“…For the HPLC analysis the seedlings were treated with a double isotope solution containing 1,539 Bq (0.19pM) of [5-3H]-IAA + 781 Bq (42.2pM) of [l-l4 Cl-IAA. At different times the apical first cm of the hypocotyls was divided into sections of 0.5cm length (for better extraction they were cut in half longitudinally) and extracted according to Brennan and Jacobs [3] with the following modifications: 0.5 ml acetonitrile/l cm section, 48 h at 4°C in darkness. Treated plants were individually analyzed either by TLC or HPLC.…”

“…Frequently, oxidative degradation of IAA implies decarboxylation [3,141, IAI, HMOx, IM and BIM being the major products of this route [2,9,11,12,15,19,24,30,331. Alternatively, a non-decarboxylative pathway producing oxindole-3-acetic acid-like compounds has been reported [IO, 16,201.…”