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.     

AM fungi might affect the root morphology of maize by increasing indole-3-butyric acid biosynthesis

Inoculation of maize ( Zea mays L.) with the arbuscular mycorrhizal (AM) fungus Glomus intraradices resulted in a distinct root phenotype ca 10 days after inoculation. Although the fresh weight of inoculated and control roots was about the same, the AM-inoculated roots showed a significant increase in the percentage of lateral fine roots. This increase coincided with an increase in free indole-3-butyric acid (IBA) as well as an increase in IBA synthesis. At later time points (31 days after inoculation), the free IBA content was not increased in infected roots; however, the fraction of bound IBA increased compared to controls. The phenotype of mycorrhizal maize roots could be mimicked by IBA applied exogenously to non-mycorrhizal roots. Addition of trifluoro-IBA (TFIBA), an inhibitor of IBA-induced root growth and lateral root induction, simultaneously with IBA resulted in a phenotype resembling that of untreated controls. In roots treated with TFIBA the inoculation with AM fungi did not increase the formation of fine roots. The TFIBA treatment also reduced endogenous free IBA and the AM infection rate in mycorrhizal roots. The results are discussed with respect to a possible role of IBA in the establishment of AM symbiosis.     

Arbuscular mycorrhiza enhances auxin levels and alters auxin biosynthesis in Tropaeolum majus during early stages of colonization

Plant hormones, including auxins, might be signals during the establishment of an arbuscular mycorrhizal (AM) symbiosis. Here, we report on the concentrations of three auxins native to nasturtium ( Tropaeolum majus L.) during early AM development. Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and phenylacetic acid (PAA) were previously identified as endogenous compounds in this species by full-scan gas chromatography–mass spectrometry. All auxinic compounds were influenced by AM colonization but showed completely different patterns. At very early stage, free IAA and IBA were lower in infected than in control roots, whereas PAA concentration was higher in infected roots than in controls. At later stages, PAA was reduced in colonized roots, whereas, especially, IBA was increased in colonized roots compared with controls. Measurement of total auxins confirmed a complex regulation pattern for the three compounds. In hyphae of Glomus intraradices , none of the auxins was detectable. Biosynthesis of the three auxins was measured using heavy labeled isotopes as precursors in control and AM-inoculated roots. While not much difference was found in the IAA labeling pattern between controls and AM-inoculated roots at both time points, IBA synthesis was slightly higher in AM-inoculated roots. Double labeling experiments showed that two distinct pathways, a tryptophan-dependent and a tryptophan-independent biosynthetic pathway contribute to the synthesis of IAA in T. majus roots. Because T. majus is difficult to genetically manipulate, we have used tobacco plants transformed with the auxin-inducible promoter GH3 fused to the β-glucuronidase (GUS) reporter gene to investigate whether AM structures would co-localize to cells harboring the auxin-inducible promoter. Although the GUS activity increased significantly in AM-inoculated roots, there was no obvious correlation between GH3::GUS expression and fungal structures.     

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.     

Auxin-conjugate hydrolysis in Chinese cabbage: Characterization of an amidohydrolase and its role during infection with clubroot disease

Auxin conjugates play a role in the regulation of free indole-3-acetic acid (IAA) content in plants. Not much is known about the enzymes involved in either conjugate synthesis or hydrolysis. In this study we have isolated and characterized an auxin conjugate hydrolase from Chinese cabbage seedlings and investigated it during the development of both the Chinese cabbage plants and the clubroot disease. The hydrolase isolated from light- and dark-grown seedlings accepted the amide conjugates indole-3-acetic acid-alanine (IAAla), IAA-phenylalanine (IAPhe), but not IAA-aspartate (IAAsp) as substrates. We also found a substantial amount of hydrolysis of an ester conjugate (IAA-glucose, IAGlu) in our enzyme preparation. The tentative reaction product IAA was identified by HPLC and subsequent GC-MS analysis. The pH optima for the different substrates were not identical, suggesting several hydrolase isoforms. After gel filtration chromatography we found at least two peaks containing different hydrolase isoforms. The isoform, which converted IAGlu to IAA, exhibited a molecular mass of ca 63 kDa, and an isoform of ca 21 kDa converted IAAla and IAPhe. The increased free IAA content in clubroot-diseased roots of Brassicaceae can be due to either de novo synthesis or release of IAA from conjugates. To answer this question free, ester- and amide-bound IAA was measured in 24- and 30-day-old leaves and roots of healthy and Plasmodiophora brassicae-infected Chinese cabbage, and the hydrolase activity with different substrates measured in the same tissues. The amide conjugates were dramatically enhanced in infected roots, whereas free IAA was only slightly enhanced compared to the control tissue. Hydrolase activity was also enhanced in clubbed roots, but the substrate specificity differed from that found in the seedlings. Especially, IAAsp hydrolysis was induced after inoculation with P. brassicae. We conclude that different auxin conjugates can be hydrolyzed at different developmental stages or under stress.     

Auxin conjugates: their role for plant development and in the evolution of land plants

Auxin conjugates are thought to play important roles as storage forms for the active plant hormone indole-3-acetic acid (IAA). In its free form, IAA comprises only up to 25% of the total amount of IAA, depending on the tissue and the plant species studied. The major forms of IAA conjugate are low molecular weight ester or amide forms, but there is increasing evidence of the occurrence of peptides and proteins modified by IAA. Since the discovery of genes and enzymes involved in synthesis and hydrolysis of auxin conjugates, much knowledge has been gained on the biochemistry and function of these compounds, but there is still much to discover. For example, recent work has shown that some auxin conjugate hydrolases prefer conjugates with longer-chain auxins such as indole-3-propionic acid and indole-3-butyric acid as substrate. Also, the compartmentation of these reactions in the cell or in tissues has not been resolved in great detail. The function of auxin conjugates has been mainly elucidated by mutant analysis in genes for synthesis or hydrolysis and a possible function for conjugates inferred from these results. In the evolution of land plants auxin conjugates seem to be connected with the development of certain traits such as embryo, shoot, and vasculature. Most likely, the synthesis of auxin conjugates was developed first, since it has been already detected in moss, whereas sequences typical of auxin conjugate hydrolases were found according to database entries first in moss ferns. The implications for the regulation of auxin levels in different species will be discussed.     

Hormonal Effects on Growth and Morphology of Normal and Hairy Roots of Hyoscyamus muticus

Treatment of normal and Agrobacterium rhizogenes-transformed root cultures of Hyoscyamus muticus with three different auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and naphthaleneacetic acid (NAA), revealed that the response varied considerably among auxins, between transformed and normal roots, and depending on the parameter. In normal roots all three auxins provoked abundant branching, with IBA and NAA being the most effective at 2.5 and 0.5 μm, respectively, whereas IAA was most effective at low concentrations (0.05 and 0.1 μm). In transformed roots exogenously supplied auxins were generally inhibitory or, at best, without effect on growth and branching. Only 0.01 μm IAA significantly enhanced lateral root number, whereas at the higher concentrations IBA, although inhibitory, was the least effective auxin. In both root types IBA had little effect on primary root growth, but normal roots were more sensitive to IAA and NAA. These results suggest a different sensitivity to auxins of normal and transformed roots since there was no significant difference in endogenous free and conjugated IAA content nor in IAA uptake capacity. Ethylene production and biosynthesis were approximately threefold higher in hairy roots, but production could be stimulated up to tenfold that of control levels in normal roots by supplying NAA or 1-aminocyclopropane-1-carboxylic acid (ACC). Treatment with 2.5 μm NAA, but not IAA or IBA, also enhanced ethylene biosynthesis in normal roots but not in transformed ones. ACC and malonyl-1-aminocyclopropane-1-carboxylic acid accumulated to detectable levels only after treatment with an auxin (NAA). Received March 3, 1997; accepted May 28, 1997     

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [³H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [³H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.     

Halogenated Auxins Affect Microtubules and Root Elongation in Lactuca sativa

We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.     

Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate Nα-(indol-3-ylacetyl)-glutamine and initiation of a mutant screen

The accumulation of conjugates of indole-3-acetic acid (IAA) in Arabidopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts. Using fluorescence detection, indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose were observed and quantitated in extracts of tissue after 24 h incubation with 500 μ M IAA. In addition, a new metabolite was detected and positively identified as indole-3-acetyl-glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between leaves, shoot axes and roots. In all three tissues, indole-3-acetyl-aspartate was the most abundant conjugate, the highest level being observed in roots. Highest levels of indole-3-acetyl-glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole-3-acetyl-glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA-inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole-3-acetyl-glutamine and less indole-3-acetyl-aspartate than wild-type, and is allelic to an existing class of photorespiration mutants ( gluS ) deficient in chloroplastic glutamate synthase.     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants,and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 [plus or minus] 7%, SD) than in old leaves (36 [plus or minus] 8%), whereas there was no difference between young (73 [plus or minus] 8%) and old internodes (70 [plus or minus] 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 [plus or minus] 10%) for both leaves and internodes, independent of the total IAA level or tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.     

The Effect of the Conversion of Indolebutyric Acid into Indoleacetic Acid on Root Formation on Microcuttings of Malus

The uptake and metabolism of tritiated indolebutyric acid (IBA)and indoleacetic acid (IAA) were related to root regenerationon stem bases of apple (Malus cv "Jork") shootlets culturedin vitro. The major part of the auxins taken up from the mediumwas located in the bottom 1 mm of the stem basis, the locationwhere the roots emerge. In this part of the shoot about 4% ofthe accumulated IBA-³H remained in the free acid. Analysis onnormal phase TLC followed by reversed phase HPLC revealed thatabout 1% of the IBA-metabolites co-chromatographed with standardIAA. Incubation of shoots on medium with IAA led also to anIAA^int content of about 1% of the amount absorbed. IAA was notconverted into IBA. A medium concentration of 3.2 µM IAAor IBA induced maximum root formation of 9 and 13 roots pershoot, respectively. The IAA^int content in the stem base was0.5 µmol per kg FW after 5 days regardless of the auxinsource. Incubation on medium with IBA led to an IBA^int concentrationof 3.4 µmol per kg FW. IBA may exert its action partlyvia conversion into IAA. However, the fact that IBA inducedmore roots than IAA suggests that IBA itself is also active,or modulates the activity of IAA. The partition of absorbed auxin over active free auxin acidand individual conjugates was not directly related to root formation.At inductive and non-inductive auxin concentrations no shiftin the ratio of free auxin acids to total absorbed auxin wasobserved during root formation. (Received March 4, 1992; Accepted May 25, 1992)     

Exogenous auxin effects on growth and phenotype of normal and hairy roots of Pueraria lobata (Willd.) Ohwi

Pueraria lobata hairy roots have faster elongationand more branches than normal roots. The responses of hairy roots and normalroots to treatment with three auxins, indole-3-acetic acid (IAA),indole-3-butyric acid (IBA), and naphthalene acetic acid (NAA) were different.In normal roots, all three auxins strongly stimulated lateral root formation atall tested concentrations. Responses to IAA and IBA in primary root growth andlateral root elongation were similar and depended on concentration; promotionat0.1 M, no effect at 1.0 M, and inhibition at2.5 M. In hairy roots, lateral root formation varied inresponseto the different auxins, i.e. depressed by NAA, unaffected by IAA, and promotedby IBA. Primary root growth was slightly inhibited by IBA and was unaffected byIAA. However, mean lateral root length was reduced in response to IAA and IBA.Only NAA exerted strong inhibition on primary and lateral root elongation inboth root types. The similar free IAA and conjugated IAA content but quitedifferent basal ethylene production and biosynthesis in hairy and normal rootssuggested different mechanisms of response to exogenous auxins in the two roottypes.     

Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis

Polar transport of the natural auxin indole-3-acetic acid (IAA) is important in a number of plant developmental processes. However, few studies have investigated the polar transport of other endogenous auxins, such as indole-3-butyric acid (IBA), in Arabidopsis. This study details the similarities and differences between IBA and IAA transport in several tissues of Arabidopsis. In the inflorescence axis, no significant IBA movement was detected, whereas IAA is transported in a basipetal direction from the meristem tip. In young seedlings, both IBA and IAA were transported only in a basipetal direction in the hypocotyl. In roots, both auxins moved in two distinct polarities and in specific tissues. The kinetics of IBA and IAA transport appear similar, with transport rates of 8 to 10 mm per hour. In addition, IBA transport, like IAA transport, is saturable at high concentrations of auxin, suggesting that IBA transport is protein mediated. Interestingly, IAA efflux inhibitors and mutations in genes encoding putative IAA transport proteins reduce IAA transport but do not alter IBA movement, suggesting that different auxin transport protein complexes are likely to mediate IBA and IAA transport. Finally, the physiological effects of IBA and IAA on hypocotyl elongation under several light conditions were examined and analyzed in the context of the differences in IBA and IAA transport. Together, these results present a detailed picture of IBA transport and provide the basis for a better understanding of the transport of these two endogenous auxins.     

The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis)

The effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H4]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled internal standards. [13C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [2H4]IAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. The presence of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.     

Dynamics of the concentration of IAA and some of its conjugates during the induction of somatic embryogenesis in Coffea canephora

Most of the somatic embryogenesis (SE) process requires the presence, either before or during the embryogenic process, of at least one exogenous auxin. This exogenous auxin induces the presence of endogenous auxins, which appears to be essential for SE induction. We found that during the preincubation period of SE in Coffea canephora, there is an important increase in both free and conjugated indole-3-acetic acid (IAA), as well as indole-3-butyric acid. This increase is accompanied by an increase in the expression of YUCCA (CcYUC), TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (CcTAA1), and GRETCHEN HAGEN 3 (GH3) genes. On the other hand, most of the IAA compounds decreased during the induction of SE. The results presented in this research suggest that a balance between free IAA and its amide conjugates is necessary to allow the expression of SE-related genes.     

Auxins and polyamines in relation to differential in vitro root induction on microcuttings of two pear cultivars

The internal levels of indole-3-acetic acid (IAA) and polyamines (PAs) and the metabolism of indole-3-butyric acid (IBA) were studied in relation to the in vitro rooting process of two pear cultivars, the easy-to-root Conference and the difficult-to-root Doyenne d'Hiver. Doyenne d'Hiver required about a 10 times higher concentration of IBA to achieve a rooting percentage similar to that of Conference. One- or two-day exposures to IBA were sufficient to stimulate rooting but with different efficiency for each cultivar. Longer exposure to auxin strongly increased the root number in Conference, whereas root elongation was inhibited in both cultivars. The metabolism of IBA in both cultivars was not significantly different when IBA was used at a high concentration to stimulate maximal rooting in Doyenne d'Hiver. IBA was mainly conjugated into IBA glucose, which was accumulated, and a small amount was converted into free IAA in both cultivars. However, in Doyenne d'Hiver this metabolic pathway appears to be active only at a higher exogenous IBA concentration. At a high IBA concentration more callus was formed by Doyenne d'Hiver, indicating that the cells of Doyenne d'Hiver are not capable of responding to the hormone in the same manner as Conference cells. Anatomic observations indicated that the capacity to induce initial dividing cells was more efficient in Doyenne d'Hiver, but subsequently the number of root primordia formed and root development were much reduced relative to Conference. A possible correlation between these processes and an early increase followed by a decrease of free IAA was seen in Conference. By day 4, a significant increase in IAA conjugates and free putrescine was observed in Doyenne d'Hiver. This higher putrescine content may be related to the lower amount of root development. Together with previous studies these results indicate that differences in the uptake and metabolism of applied auxins may affect rooting ability and the subsequent development of adventitious roots in microcuttings of pear.Abbreviations IBA indole-3-butyric acid - IAA indole-3-acetic acid - PA(s) polyamine(s) - HPLC high pressure liquid chromatography - GC-MS gas chromatography-mass spectrometry - TCA trichloroacetic acid dansyl, 1-dimethylaminonaphthalene-5-sulfonyl - TLC thin layer chromatography - TBA terbutilic alcohol - IBAGluc IBA glucose - IAAGluc IAA glucose - IAAsp IAA aspartate     

Differential responses of primary and lateral roots to indole-3-acetic acid,indole-3-butyric acid,and 1-naphthaleneacetic acid in maize seedlings

The role of auxins on root system architecture was studied by applying indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA) to maize roots and analysing the main processes involved in root development: primary root (PR) elongation, lateral root (LR) formation, and LR root elongation. We found that these effects were not dependent only on concentration, but also on the type of auxin applied. We also studied temporal changes in auxin inhibition of PR elongation. These temporal changes were analysed calculating the elongation ratio between two consecutive one day periods after auxin application. It was observed that a reduction in root elongation was also dependent on the type of auxin applied and its concentration. The inhibitory effect of IBA and IAA decreased on the second day, and the ratio also increased with the concentration. In contrast, NAA increased root elongation inhibition with time. Indeed, the ratio decreased as the NAA concentration increased. Regarding LR formation, we observed that external auxin increased only LR formation in certain zones of the PR. Finally, comparison of inhibition elongation associated with auxin in the LR and PR clearly demonstrates that PR elongation was more sensitive to auxin than LR elongation.     

Identification and quantification of three active auxins in different tissues of Tropaeolum majus

Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and phenylacetic acid (PAA) were identified as endogenous compounds with auxin activity in nasturtium ( Tropaeolum majus L.) by full scan gas chromatography-mass spectrometry. The endogenous concentrations of the three auxins were measured by GC-selected ion monitoring-MS and isotope dilution analysis using stable labelled isotopes. PAA was present at concentrations about 10- to 100-fold lower than IAA, whereas IBA was found to be in the same concentration range as IAA. Free IAA was highest in roots followed by young leaves. IBA was also highest in the roots, and relatively high concentrations were found in young leaves and flowers. The distribution of PAA was quite different from that found for IBA. No PAA could be detected in young leaves and flowers, and in all other tissues studied the concentrations were well below those of the other two auxin compounds. The presence of a nitrilase gene family and nitrilase activity in extracts from T. majus suggests that PAA might be synthesized by the nitrilase pathway using benzylglucosinolate as precursor.