Relationship of indole-3-butyric acid and adventitious rooting in M.26 apple (Malus Pumila mill.) shoots cultured in vitro

The role of indole-3-butyric acid (IBA) in adventitious root formation was studied by analyzing the uptake and subsequent metabolism of IBA in shoots of M.26 apple (Malus pumila Mill.) rootstock grown in vitro. Roots were induced by exposing shoots to 4 M IBA and [³H]IBA for 5 days in the dark and then transferring them to plant growth regulator (PGR)-free medium in the light until roots formed. Approximately 50% of the total radioactivity applied was taken up from the agar medium by the shoots during the 5-day incubation period in IBA. Indole-3-butyric acid metabolism was studied by extraction and high-performance liquid chromatographic (HPLC) separation of [³H]IBA and metabolites from the basal sections of treated shoots. The major [³H]IBA metabolite co-eluted with authentic [¹⁴C]indole-3-acetic acid (IAA) suggesting that IBA was converted to IAA in the shoots. The proportion of newly synthesized IAA present as conjugates was higher at the end of the 5-day IBA treatment period than after 13 days in PGR-free medium. There appeared to be no conjugation of IBA at any time.     

Indole-3-butyric acid in Arabidopsis thaliana III. In vivo biosynthesis

Indole-3-butyric acid (IBA) was identified by HPLC and GC-MS as one of the reaction products after incubation of sterile cultures of Arabidopsis thaliana seedlings with labeled indole-3-acetic acid (IAA). This is the first demonstration of IBA biosynthesis in a dicotyledonous plant. After 1 h of incubation most of the IBA was found in the free form, while after longer periods of incubation most of it was detected in conjugated forms. Formation of IBA conjugates was inhibited by the addition of unlabeled IBA. The biosynthesis of IBA and its conjugates was followed throughout the development of the seedlings and at different pH values. All parts of the plant (isolated roots, leaves, shoots and flowers) were able to convert IAA to IBA to the same extent.IAA was more readily transported than IBA in mature Arabidopsis plants. Feeding of labeled phenylacetic acid (PAA) and -naphthylacetic acid (NAA) to Arabidopsis seedlings resulted in a new small peak which was hydrolyzed by 7N NaOH, but the formation of compounds with longer side chains (analogous to IBA) could not be detected.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthylacetic acid - PAA phenylacetic acid     

Effect of ethylene treatment on transport and metabolism of indole-3-butyric acid in citrus leaf midribs

Transport and metabolism of radiolabeled indole-3-butyric acid (IBA) were studied in midrib sections of Cleopatra mandarin (Citrus reticulata Blanco) and compared to that of indole-3-acetic acid (IAA). Exogenous IBA was metabolized by the midribs to a polar compound, probably an ester conjugate. Ethylene pretreatment of the midribs reduced their capacity to metabolize IBA by ca. 70% as compared to air pretreatment. IBA transport capacity in the leaf midribs was ca. two times greater in the basipetal direction than the acropetal. The basipetal transport capacity of ³H-IBA was lower than that of ¹⁴C-IAA (ca. 24% and 39% of the uptake, respectively). While ethylene treatment reduced basipetal transport of IAA by ca. 70% it did not affect the transport of IBA. Most of the transported label was found as free IBA, but the reduction of IBA conjugation by ethylene treatment did not affect the transport capacity.     

Indole-3-butyric acid in plants: occurrence, synthesis, metabolism and transport

Indole-3-butyric acid (IBA) was recently identified by GC/MS analysis as an endogenous constituent of various plants. Plant tissues contained 9 ng g^?1 fresh weight of free IBA and 37 ng g^?1 fresh weight of total IBA, compared to 26 ng g^?1 and 52 ng g^?1 fresh weight of free and total indole-3-acetic acid (IAA), respectively. IBA level was found to increase during plant development, but never reached the level of IAA. It is generally assumed that the greater ability of IBA as compared with IAA to promote rooting is due to its relatively higher stability. Indeed, the concentrations of IAA and IBA in autoclaved medium were reduced by 40% and 20%, respectively, compared with filter sterilized controls. In liquid medium, IAA was more sensitive than IBA to non-biological degradation. However, in all plant tissues tested, both auxins were found to be metabolized rapidly and conjugated at the same rate with amino acids or sugar. Studies of auxin transport showed that IAA was transported faster than IBA. The velocities of some of the auxins tested were 7. 5 mm h^?1 for IAA, 6. 7 mm h^?1 for naphthaleneacetic acid (NAA) and only 3. 2 mm h^?1 for IBA. Like IAA, IBA was transported predominantly in a basipetal direction (polar transport). After application of ³H-IBA to cuttings of various plants, most of the label remained in the bases of the cuttings. Easy-to-root cultivars were found to absorb more of the auxin and transport more of it to the leaves. It has been postulated that easy-to-root, as opposed to the difficult-to-root cultivars, have the ability to hydrolyze auxin conjugates at the appropriate time to release free auxin which may promote root initiation. This theory is supported by reports on increased levels of free auxin in the bases of cuttings prior to rooting. The auxin conjugate probably acts as a ‘slow-release’ hormone in the tissues. Easy-to-root cultivars were also able to convert IBA to IAA which accumulated in the cutting bases prior to rooting. IAA conjugates, but not IBA conjugates, were subject to oxidation, and thus deactivation. The efficiency of the two auxins in root induction therefore seems to depend on the stability of their conjugates. The higher rooting promotion of IBA was also ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. IAA was converted to IBA by seedlings of corn and Arabidopsis. The K_m value for IBA formation was low (approximately 20 μM), indicating high affinity for the substrate. That means that small amounts of IAA (only a fraction of the total IAA in the plant tissues) can be converted to IBA. It was suggested that IBA is formed by the acetylation of IAA with acetyl-CoA in the carboxyl position via a biosynthetic pathway analogous to the primary steps of fatty acid biosynthesis, where acetyl moieties are transferred to an acceptor molecule. Incubation of the soluble enzyme fraction from Arabidopsis with ³H-IBA, IBA and UDP-glucose resulted in a product that was identified tentatively as IBA glucose (IBGIc). IBGIc was detected only during the first 30 min of incubation, showing that it might be converted rapidly to another conjugate.     

Transport and metabolism of indole-3-butyric acid in cuttings of Leucadendron discolor

Indole-3-butyric acid (IBA) greatly enhanced the rooting of an early-flowering variety of protea, Leucadendron discolor, but had very little effect on a late-flowering variety. IBA transport and metabolism were studied in both varieties after incubating the cuttings in ³H-IBA. More of the radio-label was transported to the leaves of the easy-to-root variety than the difficult-to-root (35–45% and 10%, respectively). IBA was metabolized rapidly by the cuttings of both varieties and after 24 h most of the label was in the new metabolite. However, free IBA (about 10%) was present in the cuttings during the whole period up to the time of root emergence (4 weeks). More free IBA was accumulated in the base of easy-to-root cuttings, while in the difficult-to-root variety most of the IBA was found in the leaves. The metabolite was identified tentatively as an ester conjugate with a glucose. It is possible that IBA-glucose serves as a source for free IBA, and the difference between the varieties is a consequence of the free IBA which is released, transported and accumulated in the site of a root formation.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Endogenous indole-3-acetic acid and abscisic acid in apple microcuttings in relation to adventitious root formation

The effects of applying indole-3-butyric acid (IBA) for periods up to 48 h were examined in difficult-to-root microcuttings (from newly-established cultures) and in easy-to-root microcuttings (from long-term subcultures) of Jonathan apple (Malus X domestica Borkh). In easy-to-root material, 20% of the microcuttings produced roots in the absence of IBA, while 6 h exposure to 10 M IBA gave 100% rooting of microcuttings. In contrast, root formation in difficult-to-root material was IBA-dependent. Maximum rooting of these microcuttings (50%) required 24 h exposure to 10 M IBA.Variation in the endogenous levels of free indole-3-acetic acid (IAA) during the course of root induction was similar in microcuttings of both types but there were marked differences in endogenous abscisic acid (ABA) levels. In easy-to-root microcuttings ABA remained at a constant low level, but in difficult-to-root material ABA exhibited marked fluctuations and was present at higher concentrations than in easy-to-root microcuttings.     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

Degradation of exogenous indole-3-butyric acid and riboflavin and their influence on rooting response of papaya in vitro

Varying concentrations of riboflavin were added to a De Fossard et al. (1974) basal medium containing 10 µM IBA and the effect on adventitious root initiation on shoots of Carica papaya L. was studied. Ninety percent root initiation occurred in 11 days when 1 µM riboflavin was added to the culture medium. Smaller rooting percentages were observed and roots emerged more slowly with riboflavin concentrations greater and less than 1 µM. Tissue culture media were maintained at 27°±1°C in either darkness or 12-h photoperiods for 28 days, and concentrations of riboflavin and IBA were measured at regular intervals using HPLC analysis. In a De Fossard et al. (1974) basal medium, riboflavin concentrations (0.1, 1.0, 10.0 µM) decreased rapidly in light and were independent of the presence of IBA. IBA concentration steadily decreased when media was placed in light, and increasing riboflavin concentrations accelerated the reduction of IBA levels. Concentrations of IBA and riboflavin were stable with dark incubation.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Transport of indole-3-butyric acid and indole-3-acetic acid in Arabidopsis hypocotyls using stable isotope labeling

The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling.     

Endogenous levels of abscisic acid and indole-3-acetic acid during in vitro rooting of Wild Cherry explants produced by micropropagation

Levels of endogenous ABA and IAA were quantified during the first week of in vitro rooting of Wild Cherry (Prunus avium L.) using IBA in the culture medium. Hormones were measured in the apical, median and basal parts of the explants using an avidin-biotin based enzyme linked immunosorbent assay (ELISA), after a purification of the methanolic extracts by high-performance liquid chromatography (HPLC).Root primordia started to differentiate from day 5 at the basal part of the explants. ABA and IAA showed considerable changes and high levels were detected during the first week of culture. ABA levels increased transiently mainly in the apical part during root formation. Exogenous IBA was possibly transformed into IAA mainly in the basal part of the explants.     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

Lateral root formation in rice (Oryza Sativa L.): differential effects of indole-3-acetic acid and indole-3-butyric acid

Comparative effects of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on lateral root (LR) formation were studied using 2-day-old seedlings of IR8 rice (Oryza sativa L.). Results showed that IBA at all concentrations (0.8–500 nmol/L) increased the number of LRs in the seminal root. However exogenous IAA, failed to increase the number of LRs. On the other hand, both IBA and IAA caused inhibition of seminal root elongation and promotion of LR elongation, but IAA can only reach to the same degree of that of IBA at a more than 20-fold concentration. Exogenous IBA had no effect on endogenous IAA content. We conclude from the results that IBA could act directly as a distinct auxin, promoting LR formation in rice, and that the signal transduction pathway for IBA is at least partially different from that for IAA.     

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

Effects of phenolic substances on metabolism of exogenous indole-3-acetic acid in maize stems

Four-day-old stem segments of Zea mays L. cv. Seneca 60 were treated sequentially with phenolic substances and indole-3-acetic [2-¹⁴C] acid ([2-¹⁴C]IAA). Formation of bound IAA was rapid, but a pretreatment with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. The decrease is not likely related to the effect of the phenolics on enzymic oxidation of IAA, since the level of free IAA was not limiting and the activity of ferulic acid in enzymic oxidation of IAA is different from that of p-coumaric acid and 4-methylum-belliferone. Apparently these compounds inhibited the formation of bound IAA and consequently caused an accumulation of free IAA. In contrast, caffeic acid, protocatechuic acid and 2,3-dihydro-2, 2-dimethyl-7-benzofuranol had little effect. After the uptake of IAA there was a slow but steady incorporation of the radioactivity into the 80% ethanol-insoluble, 1 M NaOH-soluble fraction. Phenolic substances also affected this process. The compounds which are cofactors of IAA-oxidase increased the incorporation while those which are inhibitors of IAA-oxidase decreased the incorporation. Results suggest that the phenolics also affected the enzymic oxidation of IAA in vivo in the same way as in vitro.     

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

Interaction of free indole-3-acetic acid and its amino acid conjugates in tomato hypocotyl cultures

The interaction of free IAA and its amino acid conjugates on growth and development of cultured tomato hypocotyl tissue (Lycopersicon esculentum Mill. cv. Marglobe) was studied. In a nutrient medium containing 10 mol/L of benzyladenine, free IAA stimulated shoot and root development with little callus proliferation. In contrast, all IAA-amino acid conjugates tested supported mostly callus growth. Simultaneous application of free IAA and its conjugates resulted in the expression of mixed morphogenetic responses (i.e., both vigorous callus growth and organogenesis resulted). Growth kinetics and the effect of temporal exposure of the tissues to the bound and the free auxin suggest that some IAA-amino acid conjugates may specifically influence plant morphogenesis in ways that cannot be easily explained as simply a function of their slow hydrolysis to release free IAA.Abbreviations IAA indole-3-acetic acid - IAA-Ala N-(indol-3-ylacetyl)-l-alanine - IAA-Asp N-(indol-3-ylacetyl)-dl-aspartic acid - IAA-Lys N -(indol-3-ylacetyl)-l-lysine - IAA-Orn N -(indol-3-ylacetyl)-l-ornithine - IAA-Thr N-(indol-3-ylaetyl)-l-threonine     

Identification of indole-3-butyric acid as an endogenous constituent of maize kernels and leaves

Indole-3-butyric acid (IBA) was identified by thin layer chromatography, gas-liquid chromatography and gas chromatography-mass spectrometry in kernels and leaves of corn (Zea mays) var. Hazera 224. Free and ester conjugated IBA were present in dry and germinating corn kernels and leaves. This is the first report of IBA in a monocotyledonous plant and, as far as we know, the first evidence for the presence of conjugated IBA.