Decarboxylation Of Indole-3-acetic Acid Research Articles

Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice

Clinical efficacy of current therapies for hepatocellular carcinoma (HCC) treatment is limited. Indole-3-acetic acid (IAA) is non-toxic for mammalian cells. Oxidative decarboxylation of IAA by horseradish peroxidase (HRP) leads to toxic effects of IAA. The purpose of this study was to investigate the effects of a novel gene-targeted enzyme prodrug therapy with IAA on hepatoma growth in vitro and in vivo mouse hepatoma models. We generated a plasmid using adenovirus to express HRP isoenzyme C (HRPC) with the HCC marker, alpha-fetoprotein (AFP), as the promoter (pAdv-AFP-HRPC). Hepatocellular cells were infected with pAdv-AFP-HRPC and treated with IAA. Cell death was detected using MTT assay. Hepatoma xenografts were developed in mice by injection of mouse hepatoma cells. The size and weight of tumors and organs were evaluated. Cell death in tumors was assessed using hematoxylin and eosin-stained tissue sections. HRPC expression in tissues was detected using Reverse Transcriptase-Polymerase Chain Reaction. IAA stimulated death of hepatocellular cells infected with pAdv-AFP-HRPC, in a dose- and time-dependent manner, but not in control cells. Growth of hepatoma xenografts, including the size and weight, was inhibited in mice treated with pAdv-AFP-HRPC and IAA, compared with that in control group. pAdv-AFP-HRPC/IAA treatment induced cell death in hepatoma xenografts in mice. HRPC gene expressed only in hepatoma, but not in other normal organs of mice. pAdv-AFP-HRPC/IAA treatment did not cause any side effects on normal organs. These findings suggest that pAdv-AFP-HRPC/IAA enzyme/prodrug system may serve as a strategy for HCC therapy.

Effects of phenolic compounds on adventitious root formation and oxidative decarboxylation of applied indoleacetic acid in Malus ‘Jork 9’

Stem slices (1-mm thick) cut from apple microshoots were cultured on a modified Murashige-Skoog medium with indole-3-acetic acid (IAA) or α-naphthaleneacetic acid (NAA), and increasing concentrations of various phenolic compounds. Both auxins were added at a concentration suboptimal for rooting. Indole-3-acetic acid is metabolized through oxidation and conjugation but NAA through conjugation only; which might have affected the results. With IAA, all tested orthodiphenols, paradiphenols and triphenols promoted adventitious root formation from the stem slices. Ferulic acid (FA, a methylated orthodiphenol) had the largest effect and increased the number of adventitious roots from 0.9 to 5.8. With NAA there was little or no promotion after addition of phenolics. Phloroglucinol (a triphenol) and FA were examined in detail. Their effects on the dose–response curve of IAA and the timing of their action indicated that both acted as antioxidants protecting IAA from decarboxylation and the tissue from oxidative stress. Experiments with carboxyl-labelled IAA showed that IAA was massively decarboxylated by the slices and that decarboxylation was strongly reduced by phenolics. Decarboxylation was to a great extent attributable to the wound response and did not occur to such an extent in non-wounded plant tissues. In shoots, FA promoted little rooting. Slices were cultured on top of the medium and shoots were stuck into the medium. Possibly, the anaerobic conditions in the medium near the basal part of the stem of shoots reduced the wound response and consequently decarboxylation of IAA. The monophenolic compound salicylic acid (SA) promoted IAA decarboxylation. Accordingly, SA reduced rooting when added during the initial days of the rooting process (the period during which auxin enhances rooting), and promoted outgrowth of root primordia later on (the period during which auxin inhibits rooting).

Stimulation of the oxidative decarboxylation of indole-3-acetic acid in citrus tissues by ethylene

Ethylene has been shown to stimulate the degradation of indole-3-acetic acid (IAA) in citrus leaf tissues via the oxidative decarboxylation pathway, resulting in the accumulation of indole-3-carboxylic acid (ICA). Preliminary data indicated that ethylene stimulates only the first step of this pathway, i.e. the decarboxylation of IAA which leads to the formation of indole-3-methanol. The effect of ethylene seems to be a specific one since 2,5-norbornadiene, an ethylene action inhibitor, significantly inhibited the stimulation of IAA decarboxylation by ethylene. It has long been suggested that peroxidase or a specific form of the peroxidase complex (`IAA oxidase') catalyse this step. However, we did not observe a clear effect of ethylene on the peroxidase system. An alternative possibility, that the stimulatory effect of ethylene on IAA catabolism results from increased formation of hydrogen peroxide (H2O2), a co-factor for peroxidase activity, was verified by direct measurements of H2O2 in the tissues or by assaying the activity of gluthathione reductase, which has been shown to be induced by oxygen species. This possibility is further supported by the observations showing that IAA decarboxylation in control tissues was enhanced to the level detected in ethylene-treated tissues by application of H2O2.

Decarboxylation of lndole‐3‐Acetic Acid and Inhibition of Growth in Avena sativa Seedlings by Plant‐Derived Photosensitizers*

Abstract— A number of plant phototoxins, when supplemented with UVA (320–400 nm) radiation, are capable of sensitizing the decomposition of indole‐3‐acetic acid (IAA), as measured by release of 14CO2 from carboxyl‐labeled IAA. Alpha‐terthienyl (αT) and harmine caused significant rates of IAA decarboxylation at concentrations as low as 1 nM and were approximately 80% as effective as riboflavin and flavin mononucleotide. Partial inhibition by sodium azide indicates that the aT‐induced decarboxylation of IAA is predominately, but not entirely, a type II reaction mediated by singlet oxygen. Based on changes in UV ab sorption spectra, it appears that the hormones gibberellic acid, abscisic acid and 6‐benzylaminopurine (a cytokinin) are less susceptible to photosensitized decomposition than is IAA. Alpha‐terthienyl plus UVA also inhibited elongation growth and reduced endogenous IAA levels in Avena sativa L. coleoptile sections and promoted senescence in intact Avena seedlings. These results confirm the allelopathic potential of plant photosensitizers such as αT and indicate that the phytohormone IAA may represent an additional target for the action of photosensitizers.

Mechanism of indole-3-acetic acid oxidation by plant peroxidases: anaerobic stopped-flow spectrophotometric studies on horseradish and tobacco peroxidases

Indole-3-acetic acid (IAA) is a powerful plant growth regulator. The oxidative decarboxylation of IAA by plant peroxidases is thought to be a major degradation reaction involved in controlling the in vivo level of IAA. Horseradish peroxidase isoenzyme C and an anionic tobacco peroxidase isolated from transgenic Nicotiana sylvestris have been used in experiments in vitro designed to determine the mechanism of IAA oxidation. In particular, the initial reduction of ferric to ferrous enzyme, a key step in previously proposed mechanisms, has been investigated by rapid-scan stopped-flow spectrophotometry under strictly anaerobic conditions and at defined oxygen concentrations. The data provide the first evidence for a ternary complex comprising peroxidase, IAA and oxygen that is kinetically competent both at the initiation stage and during the catalytic cycle of IAA oxidation. A general scheme describing the oxidative cycles of both anionic and cationic peroxidases is proposed that includes native ferric enzyme and compound II as kinetically competent intermediates. For anionic peroxidases, addition of hydrogen peroxide switches on the oxidative cycle thereby promoting IAA oxidation. 2-Methyl-IAA is not a substrate of the oxidase reaction, suggesting a specific interaction between plant peroxidases and IAA.

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

The rate of decarboxylation of [1′-14C]indole-3-acetic acid (IAA) infiltrated into tomato (Lycopersicon esculentum Mill.) pericarp discs was much more rapid in green than in breaker and pink tissues. Studies were carried out in order to determine whether the decarboxylative catabolism occurring in the green pericarp discs was associated with ripening or was a consequence of wound-induced peroxidase activity and/or ethylene production. After a 2-h lag, the decarboxylative capacity of the green pericarp discs increased exponentially during a 24-h incubation period. This increase was accompanied by increases in IAA-oxidase activity in cell-free preparations from the intercellular space and cut surface of the discs. Although higher IAA-oxidase activity was detected in extracts from the tissue residue, which comprises mainly intracellular peroxidases, this activity did not increase during the 24-h incubation period. Analysis of the cell-free preparations by isoelectric focusing revealed the major component in all samples was a highly anionic peroxidase (pI=3.5) the levels of which did not increase during incubation. However, the intercellular and cut-surface preparations contained additional anionic and cationic peroxidases which increased in parallel with the increases in both the IAA-oxidase activity of the preparations and the decarboxylative capacity of the green pericarp discs from which they were derived. Treatment of green discs with the ethylene-biosynthesis inhibitors aminooxyacetic acid and CoCl2, inhibited the development of an enhanced capacity to decarboxylate [1′-14C]IAA but the inhibition was not counteracted by exogenous ethylene. Another ethylene-biosynthesis inhibitor, aminoethoxyvinyl glycine, also reduced ethylene levels but did not affect IAA decarboxylation, indicating that the decarboxylation was not a consequence of wound-induced ethylene production. The data obtained thus demonstrate that the enhanced capacity to decarboxylate [1′-14C]IAA that develops in green tomato pericarp discs following excision is not associated with ripening but instead is attributable to a wound-induced increase in anionic and cationic peroxidase activity in the intercellular fluid and at the cut surface of the excised tissues.

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

The products of indole-3-acetic acid (IAA) metabolism by incubating hypocotyl sections and decapitated seedlings of Lupinus albus were investigated. Single treatments using [1-14C]-IAA, [2-14C]-IAA or [5-3H]-IAA and double treatments using [1-14C]-IAA+[5-3H]-IAA were carried out. Extracts from treated plant material were analyzed by paper chromatography (PC), Thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). When hypocotyl sections were incubated in [2-14C]-IAA, several IAA decarboxylation products including indole-3-aldehyde (IA1), indole-3-methanol (IM), 3-hydroxymethyloxindole (HMOx), methyleneoxindole (MOx) and 3,3′-bisindolylmethane (BIM) were detected in the 95% ethanol extract; a latter extraction with 1M NaOH rendered IAA, IM and BIM, suggesting that conjugated auxins were formed in addition to conjugated IM. In sections incubated with [1-14C]-IAA, the 1M NaOH extraction also produced IAA so confirming the formation of conjugated auxins. The same decarboxylation products and two conjugated auxins, indole-3-acetylaspartic acid (IAAsp) and 1-O-(indole-3-acetyl)-β-D-glucose (IAGlu), were detected in the acetonitrile extracts from decapitated seedlings treated with [5-3H]-IAA. After a double isotope treatment ([1-14C]-IAA+[5-3H]-IAA) of decapitated seedlings, the ratio 14C/3H measured in the HPLC fractions of the acetonitrile extracts confirmed the presence of decarboxylation products as well as conjugated auxins.

Evaluation of Indole-3-acetic Acid Decarboxylating Activity in Hypocotyl Sections of Etiolated Lupinus albus Seedlings

Summary It has been suggested that the endogenous Indole-3-acetic acid (IAA) level in plant tissues is regulated, in part, by its decarboxylation. However, in vivo decarboxylation assays do not permit one to attribute a definitive role to this metabolic pathway in the control of the normal plant growth. Therefore a method was developed in which 14 CO 2 is collected during incubation in [1- 14 C]-IAA of excised hypocotyl sections of etiolated lupin ( Lupinus albus ) seedlings to measure the in vivo IAA decarboxylation. IAA decarboxylation by purified peroxidase (HRP) was utilized as a model system to calibrate the C0 2 measurements as well as a reference system to evaluate the relative incidence of decarboxylation in the IAA metabolism by lupin sections. The 14 C0 2 evolved per mg tissue was linear for 1.5 h. In this period 6-15 % of total 14 CO 2 was produced. After a temporary fall in the rate (from 1.5 to 3 h), decarboxylation increased again and evolution of 14 C02 was detected until 20-24 h. It is suggested that an inactivation of the decarboxylating activity occurs during the enzymatic IAA breakdown, and new activity is synthesized to explain the restoration of the IAA decarboxylation. The percentage of initial IAA decarboxylated by lupin hypocotyl sections was higher in rapidly-growing hypocotyls of 7 day old seedlings than in non-growing hypocotyls of 18 days. Localization of the section along the hypocotyl influenced decarboxylation mainly at an intermediate age, decarboxylation being higher in apical (growing) than in middle (non growing) sections. Thus a correlation between decarboxylation and cell elongation is suggested.

In vivo metabolism of labelled indole-3-acetic acid during polar transport in etiolated hypocotyls of Lupinus albus: Relationship with growth

The in vivo metabolism of indole-3-acetic acid (IAA) in etiolated hypocotyls of lupin (Lupinus albus L., from Bari, Italy) was investigated by appliying IAA labelled with two radioisotopes ([1-14C]-IAA+[5-3H]-IAA) to the apical end of decapitated seedlings, followed by extraction of the radioactivity in the different regions along the hypocotyl. This method allowed detection of IAA decarboxylation in zones distant from the cut surface and, therefore, containing intact cells. When IAA was added directly in solution to the cut surface, decarboxylation was high especially in those hypocotyl regions where transient accumulations characteristic of the polar transport of IAA occurred. In 10-day-old seedlings such accumulations were observed both in the elongation zone (2nd, 3rd, and 4th cm) and in the non elongating basal zone (8th, 9th and 10th cm). When the IAA, instead, was applied with an agar block deposited on the cut surface, IAA metabolism (decarboxylation as well as conjugation) was increased but almost exclusively in tissues within 10 mm of the cut surface. In both kinds of experiment, the increase in IAA decarboxylation seemed to coincide with a decrease in the transport of IAA, since in the assay without agar the transient accumulations of radioactivity were probably due to a decrease in the transport velocity, while in the assay with agar the transport intensity was much lower than in the assay without agar. These results point to a competitive relationship between IAA metabolism and transport. Consequently, it is suggested that hypocotyl regions that probably use auxin for development processes (e.g., cell elongation and differentiation) may have a more intense IAA metabolism in parallel with their higher IAA concentrations.

Nonenzymatic Oxidation of Indole-3-Acetic Acid by H2O2and Fe2+Ions

Indole oxidation products detected by dimethylaminocinnamaldehyde were formed nonenzymatically from indole-3-acetic acid (IAA) in the presence of low concentrations of H2O2 Decarboxylation of IAA-[1-14C] was promoted by H2O2 concentrations as low as 5 μM, and this effect was greatly increased in the presence of Fe2+ ions Incubation of IAA-[1-14C] with H2O2 did not yield any radioactive compounds that were separated from free IAA by paper or thin-layer chromatography in four solvent systems These results indicate that IAA is nonenzymatically decomposed by H2O2 and do not support the hypothesis that the nonenzymatic inactivation of IAA by H2O2 is caused by the reversible formation of an IAA-H2O2 complex

Destruction of indole-3-acetic acid during the aerobic oxidation of sulfite

Indole-3-acetic acid (IAA) was rapidly destroyed in the presence of Mn 2+, oxygen and sulfite ion. The optimal pH for the reaction was between 5 and 6. The destruction was dependent on the aerobic oxidation of sulfite, but was not inhibited by superoxide dismutase. Tracer studies indicate that IAA was converted into at least 3 compounds. Decarboxylation of IAA was not involved in the destruction.

Decarboxylation and transport of auxin in segments of sunflower and cabbage roots

The movement of (14)C from indole-3-acetic acid (IAA) (14)C has been examined in 5 mm root segments of dark-grown seedlings of Helianthus annuus and Brassica oleracea. Contaminants from distilled water, phosphate buffer and the razor-blade cutter increase the decarboxylation of IAA-(14)C, and cutting of root segments results in an activation of IAA-destroying enzymes at the cut surfaces. When these sources of errors were eliminated the following was shown: a) Both in sunflower and cabbage there is a slight acropetal flux of (14)C through the root segments into the agar receiver blocks. The amount of (14)C found in the receiver blocks increases with the lenght of the transport period. b) When the root segments, after the transport period, are cut in two equal parts and these assayed separately, the amounts of (14)C in the two parts indicate a greater acropetal than basipetal transport. c) The total radioactivity of the receiver blocks is in part due to IAA-(14)C and in part to (14)CO2, the latter being a result of enzymatic destruction of auxin. d) Addition of ferulic acid, an inhibitor of IAA oxidases, to the receiver blocks markedly inhibits the decarboxylation of IAA-(14)C and thus increases the amount transported. This effect is more pronounced after a 20 hr than after a 6 hr transport period.