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

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 ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Inhibition of Polar Indole-3-acetic Acid Transport by Cycloheximide

Cycloheximide inhibits polar indoleacetic acid transport in midrib tissues of leaves of citrus (Citrus sinensis [L.] Osbeck) and poplar (Populus deltoides Bartr.) as measured by the donor-receiver agar cylinder technique. It appears that the mechanism of auxin transport inhibition by cycloheximide consists in arresting protein synthesis and not in the disruption of energy flow. The interpretation of the data takes into account the involvement of either a carrier protein or auxin-induced proton excretion in auxin transport.     

Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays

A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth.     

Indole-3-acetic Acid Levels and the Role of Indole-3-acetic Acid Oxidase in the Normal Root and Club-root of Cabbage

The auxin content of club-root (Plasmodiophora brassicae Wor.) is 50–100 times higher than that of normal cabbage root. The importance of this difference in the disease development is discussed. Both normal root and club-root of cabbage contain allosteric IAA oxidase and IAA oxidase with ordinary kinetic properties. In normal cabbage root the allosteric one is associated with cell fractions sedimenting at 20,000 × g and 105,000 × g, in club-root it remains in the supernatant after 105,000 × g centrifugation. IAA oxidase with conventional kinetic properties is present in both these tissues in the cell fraction sedimenting at 10,000 × g, which contains mainly cell wall fragments. It is concluded that IAA oxidase is not primarily involved in regulation of the endogenous IAA level.     

Movement of Indole-3-acetic Acid and Tryptophan-derived Indole-3-acetic Acid from the Endosperm to the Shoot of Zea mays L

The structures and the concentrations of all of the indolylic compounds that occur in the endosperm of the seeds of corn (Zea mays L.) are known. Thus, it should be possible to determine which, if any, of the indolylic compounds of the endosperm can be transported to the seedling in significant amounts and thus help identify the seed-auxin precursor of Cholodny (1935. Planta 23:289-312) and Skoog (1937. J. Gen. Physiol. 20:311-334). Of interest is the transport of tryptophan, indole-3-acetic acid (IAA), and the esters of IAA, which comprise 95% of the IAA compounds of the seed. We have shown that: (a) IAA can move from the endosperm to the shoot; (b) the rate of movement of IAA from endosperm to shoot is that of simple diffusion; (c) 98% of the transported IAA is converted into compounds other than IAA, or IAA esters, en route; (d) some of the IAA that has moved into the shoot has been esterified; (e) labeled tryptophan applied to the endosperm can be found as labeled IAA in the shoot; and (f) with certain assumptions concerning IAA turnover, the rate of movement of IAA and tryptophan-derived IAA from the endosperm to shoot is inadequate for shoot growth or to maintain IAA levels in the shoot.     

Concentration of Indole-3-acetic Acid and Its Derivatives in Plants

Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed.     

Polar Indole-3-acetic Acid Diffusion in Nonliving and Model Systems

Polar indole-3-acetic acid movement was observed in killed plant segments and in artificial model systems. The polar diffusion of indole-3-acetic acid was observed in tissue killed by chemical or physical means in an agar-plant system and in a multicelled Plexiglas dialysis chamber containing hypocotyl tissue gradients or gradients of anion exchange material.     

Arabidopsis Seedlings Over-Accumulated Indole-3-acetic Acid in Response to Aminooxyacetic Acid

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.     

Polarity of Indoleacetic Acid in young Coleus Stems

Young internodes of Coleus blumei Benth. have long been known for their sizable amount of acropetal indoleacetic acid movement. However, plants of the same clone, under improved growing conditions, now show almost absolute basipetal polarity of ¹⁴C-indoleacetic acid, as measured by liquid scintillation counting of ¹⁴C in the receiver cylinders of agar. The ratio of basipetal to acropetal movement is now as much as 85:1, instead of the 3:1 ratio found years ago under conditions providing slower growth.     

Studies on the Destruction of Indole-3-acetic Acid by a Species of Arthrobacter. V. Indole-3-acetic Acid Production from Tryptophan

Tryptophan (Try) metabolism of Arthrobacter sp. was examined. The inducibility of the Try oxidizing enzyme system seems to be correlated with that of the indole-3-acetic acid (IAA) oxidizing enzyme system. Try is metabolized to IAA via indole-3-pyruvic acid (Ip) and indole-3-acetaldehyde (IAAId). Indole-3-acetamide (IAm) is formed as a product of Try oxidation. Exogenous IAm, indole-3-acetonitrile (IAN) and tryptamine are not oxidized by Try-induced cells.     

Enzymatic Esterification of Indole-3-acetic Acid to myo-Inositol and Glucose

Incubation of mature sweet corn kernels of Zea mays in dilute solutions of ¹⁴C-labeled indole-3-acetic acid leads to the formation of ¹⁴C-labeled esters of myo-inositol, glucose, and glucans. Utilizing this knowledge it was found that an enzyme preparation from immature sweet corn kernels of Zea mays catalyzed the CoA- and ATP-dependent esterification of indole-3-acetic acid to myo-inositol and glucose. The esters formed were 2-O-(indole-3-acetyl)-myo-inositol, 1-dl-1-O-(indole-3-acetyl)-myo-inositol, di-O-(indole-3-acetyl)-myo-inositol, tri-O-(indole-3-acetyl)-myo-inositol, 2-O-(indole-3-acetyl)-d-glucopyranose, 4-O-(indole-3-acetyl)-d-glucopyranose and 6-O-(indole-3-acetyl)-d-glycopyranose. An assay system was developed for measuring esterification of ¹⁴C-labeled indole-3-acetic acid by ammonolysis of the esters followed by isolation and counting the radioactive indole-3-acetamide.     

Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid.

In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [¹⁴C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices.

It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants.

The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined.

     

Indole-3-acetic Acid oxidation and crocin bleaching by horseradish peroxidase

During indoleacetic acid (IAA) oxidation by horseradish peroxidase the water soluble model polyene, crocin, is bleached. IAA-oxidation and crocin bleaching are stimulated at acidic pH as well as by the monophenol p-hydroxyacetophenone. IAA oxidation and crocin bleaching are neither influenced by catalase or superoxide dismutase nor by different OH-radical scavengers, whereas both ascorbate and propylgallate are inhibitory.     

Concentrations of Indole-3-acetic Acid and Its Esters in Avena and Zea

An isotope-dilution method has been developed for the assay of free indole-3-acetic acid and ester indole-3-acetic acid as measured by indole-3-acetic acid liberated by mild alkaline hydrolysis. Application of this method to seedlings of Avena sativa and Zea mays indicates the upper limit of free indole-3-acetic acid in Avena to be about 16 μg per kg and in Zea, about 24 μg. The amount of 1 n alkali-labile indole-3-acetic acid in Zea is about 330 μg per kg and there is very little 1 n alkali-labile IAA in Avena. A chemical characterization of the indole-3-acetic acid of Avena and a confirmation of the chemical characterization of the indole-3-acetic acid of Zea is presented.     

Involvement of Abscisic Acid and Indole-3-acetic Acid in the Flowering of Pharbitis nil

The involvement of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the regulation of flowering of Pharbitis nil was investigated through exogenous applications and analyses of endogenous levels. Both hormones inhibited the flowering of P. nil when they were applied before or after a single 15-h dark treatment. The inhibitory effect of ABA and IAA was significant when they were applied before the dark treatment, and the application to plumules was more effective than that to cotyledons. In all applications, the inhibitory effect of IAA was stronger than that of ABA. Endogenous levels of ABA and IAA in the plumules were compared between flower-inductive (15-h dark treatment) and noninductive (continuous light) light conditions. There was no significant difference in the ABA level between light and dark conditions, whereas the level of IAA was decreased by the dark treatment. These results suggest that biosynthesis and/or catabolism of IAA is affected by the light treatment and therefore may be involved in the regulation of early flowering processes in the apex. The inhibitory effects of ABA and IAA were reversed by an application of gibberellin A₃, indicating that gibberellin A₃ counteracts the flowering processes affected by ABA and IAA. Application of aminoethoxyvinylglycine restored the flowering response inhibited by IAA, which suggests the possibility that the inhibitory effect of IAA is the result of enhanced ethylene biosynthesis. Received November 22, 1996; accepted February 17, 1997