Root hair elongation is inhibited by hypaphorine, the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by indole-3-acetic acid

Hypaphorine, the major indolic compound isolated from the ectomycorrhizal fungus Pisolithus tinctorius, controls the elongation rate of root hairs. At inhibitory concentrations (100 μM), hypaphorine induced a transitory swelling of root hair tips of Eucalyptus globulus Labill. ssp. bicostata. When the polar tip growth resumed, a characteristic deformation was still visible on elongating hairs. At higher hypaphorine concentrations (500 μM and greater), root hair elongation stopped, only 15 min after application. However, root hair initiation from trichoblasts was not affected by hypaphorine. Hypaphorine activity could not be mimicked by related molecules such as indole-3-acetic acid (IAA) or tryptophan. While IAA had no activity on root hair elongation, IAA was able to restore the tip growth of root hairs following inhibition by hypaphorine. These results suggest that hypaphorine and endogenous IAA counteract in controlling root hair elongation. During ectomycorrhiza development, the absence of root hairs might be due in part to fungal release of molecules, such as hypaphorine, that inhibit the elongation of root hairs. Received: 27 October 1999 / Accepted: 14 March 2000     

Morphological and anatomical changes in soybean roots subjected to indole-3-acetic acid and tryptophol: indole compounds present in plant auxin metabolism

The auxin metabolism is practically elucidated, and the compounds that are part of the biosynthesis are well characterized, but the indole-3-ethanol or tryptophol, a molecule that has a regulatory position in the indole-3-acetic acid biosynthesis, still represents a gap in the understanding of this pathway. We examined the hypothesis that tryptophol present the function of plant growth regulation on soybean root development. We evaluated two doses of auxin and two doses of tryptophol (100 e 200 mg L^??1), respectively, beside a control treatment (water), via leaf application, in soybean plants under V1–V2 phonological stages. After 18 days of application, the roots were collected for their volume and area measurement, thereafter small segments (0.5 cm of length), were collected at 1 cm below the root-collar, for anatomical analysis. We observed that the control showed greater area and root volume, but using 200 mg L^??1 auxin and 100 mg L^??1 tryptophol led to a radial increase of roots with significant increases in width radius vascular and cortical parenchyma. These results suggest that the application of both compounds had a potential of modify the vascular and ground tissues in soybean roots, which may be beneficial for the development of plants.     

The transport of indole-3-acetic Acid in boron- and calcium-deficient sunflower hypocotyl segments

Transfer of sunflower (Helianthus annuus L. cv Russian Mammoth) seedlings from complete nutrient solution to solutions deficient in either boron or calcium resulted in a steady decline in the rate of auxin transport, compared to seedlings that remained in the complete solution. In seedlings transferred to solutions deficient in both B and Ca, the decline in auxin transport was greater than seedlings deficient in only one element. The transfer of B- or Ca-deficient seedlings back to the complete solution prevented further decline in auxin transport, but auxin transport did not increase to the same level as seedlings maintained in complete solution. The significant reduction in auxin transport during the early stages of B or Ca deficiency was not related to (a) reduced growth rate of the hypocotyl, (b) increased acropetal movement of auxin, or (c) lack of respiratory substrates in the hypocotyl. In addition, no difference was found in the water-extractable total and ionic Ca in B-deficient and control nondeficient hypocotyls, indicating a direct effect of B on auxin transport, rather than indirectly by affecting Ca absorption. The rate of auxin transport in hypocotyls deficient in either B or Ca, was inversely correlated with K⁺ leakage and rate of respiration. The data presented strongly support the view that there are separate sites for B and Ca in the basipetal transport of the plant hormone indoleacetic acid.     

Growth, auxin requirement, and indole-3-acetic acid content of cultured crown-gall and habituated tissues of tobacco

Abstract. We used radioimmunoassay to compare the indole-3-acetic acid (IAA) concentration in cultured auxinautonomous tissues of Nicotiana tabacum L. cv.'Havana 425'that differ in growth rate, auxin requirement, capacity for organogenesis, and proximal cause of transformation. Lines HT37 and HB6 were crown-gall tissues transformed by the T37 and B6 strains of Agrobacterium tumefaciens . The tissue line 156AH, like the crown-gall lines, was auxin and cytokinin autotrophic, but arose spontaneously in culture and did not result from crown-gall transformation. The IAA content of the lines was variable and between 10⁻⁷ and 10⁻⁶ moles kg⁻¹ fresh weight. This same range of IAA concentration was found in growing leaf tissues on the plant. IAA concentrations were 2–5 fold higher in HB6 than HT37 during the first few days of culture, and exogenous auxin induced the teratomatous HT37 tissue to grow in an unorganized fashion suggesting that auxin plays a role in regulating tumor morphology. The major difference between genetically transformed crown-gall tissues and the auxin-cytokinin autotrophic tissue line was that net IAA production continued to rise for several weeks of culture in tumor tissues but only for the first few days of culture in the autotrophic tissue.     

Effects of indole-3-acetic acid and auxin transport inhibitors on the style curvature of three Alpinia species (Zingiberaceae)

The effects of indole-3-acetic acid (IAA) and the auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA) and 1-N-naphthylphthalamic acid (NPA) on the style curvature of Alpinia platychilus, A. blepharocalyx, and A. mutica were studied. Exogenous IAA stimulated the style curvature movement of the anaflexistylous morph (ana-morph) and cataflexistylous morph (cata-morph) of three Alpinia species in light, but had no effect in the dark. Treatment with auxin efflux inhibitors (NPA and TIBA) before flower opening did not affect the first curvature of the two morphs in darkness; however, the subsequent second movement of the ana-morph was enhanced by NPA or TIBA, while the second movement of the cata-morph was completely inhibited. After the first curvature, NPA and TIBA treatments at 06:00?hours (before significant illumination) and 11:00?hours (after the styles were illuminated for 4?h) increased the second curvature of the ana-morph, but significantly decreased that of the cata-morph. The effect at 06:00?hours was more significant than the effect at 11:00?hours. These results suggested that auxin and auxin transport affected the style curvature in a different way in the two morphs, and two morphs had distinct mechanisms for style movement at different times.     

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.     

Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone, indole-3-acetic acid.

From an Arabidopsis thaliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiella ozaenae bromoxynil nitrilase encoded by the bxn gene. The cDNA contained a long open reading frame, starting from two possible neighbouring ATG codons and capable of encoding 340 or 346 amino acids with calculated molecular masses of 37526 Da or 38176 Da, respectively. The sequence similarity between the deduced polypeptides from the Arabidopsis cDNA and bxn was clustered in three domains, one at the C-terminus, one in the center and one near the N-terminus of the two proteins, suggesting important functional elements in these parts of the proteins. The cDNA was cloned into different vectors under the control of the lacZ promotor and was functionally expressed by induction with isopropyl-beta-D-thiogalactoside. Using a combination of high-performance liquid chromatography, monoclonal-antibody based enzyme-linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole-3-acetonitrile to the plant growth hormone, indole-3-acetic-acid.     

The decrease in auxin polar transport down the lupin hypocotyl could produce the indole-3-acetic Acid distribution responsible for the elongation growth pattern

The variation of indole-3-acetic acid (IAA) transport along Lupinus albus L. hypocotyls was studied using decapitated seedlings and excised sections. To confirm that the mobile species was IAA and not IAA metabolites, dual isotope-labeled IAAs, [5-³H]IAA + [1-¹⁴C]IAA, were used. After apical application to decapitated seedlings, the longitudinal distribution of both isotopes at different transport periods showed that the velocity of IAA transport was higher in the apical, elongating region than in the basal, non-growing region. This variation in velocity was not a traumatic consequence of decapitation because after application of IAA to the basal region of decapitated seedlings, both the velocity and intensity of IAA transport were lower than in the apical treatment. The variation in IAA transport down the hypocotyl was confirmed when it was measured in excised sections located at different positions along the hypocotyl. The velocity and, to a greater extent, the intensity of IAA transport decreased from the apical to the basal sections. Consequently, if the amount of IAA reaching the apical zones of lupin hypocotyl were higher than the IAA transport capacity in the basal zones, accumulation of mobile IAA might be expected in zones located above the basal region. In fact, an IAA accumulation occurred in the elongating region during the first 4-h period of transport after apical treatment with IAA. It is proposed that the fall in IAA transport along the hypocotyl might be responsible for the IAA distribution and, consequently, for the growth distribution reported in this organ. An indirect proof of this was obtained from experiments that showed that the excision of the slowly transporting basal zones strongly reduced the growth in the remaining part of the organ, whereas excision of the root caused no significant modification in growth during a 20-h period.     

Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin

Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 μg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.     

Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3-acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid

Glucosinolates are natural plant products known as flavor compounds, cancer-preventing agents, and biopesticides. We report cloning and characterization of the cytochrome P450 CYP79B2 from Arabidopsis. Heterologous expression of CYP79B2 in Escherichia coli shows that CYP79B2 catalyzes the conversion of tryptophan to indole-3-acetaldoxime. Recombinant CYP79B2 has a K(m) of 21 microm and a V(max) of 7.78 nmol/h/ml culture. Inhibitor studies show that CYP79B2 is different from a previously described enzyme activity that converts tryptophan to indole-3-acetaldoxime (Ludwig-Müller, J. , and Hilgenberg, W. (1990) Phytochemistry, 29, 1397-1400). CYP79B2 is wound-inducible and expressed in leaves, stem, flowers, and roots, with the highest expression in roots. Arabidopsis overexpressing CYP79B2 has increased levels of indole glucosinolates, which strongly indicates that CYP79B2 is involved in indole glucosinolate biosynthesis. Our data show that oxime production by CYP79s is not restricted to those amino acids that are precursors for cyanogenic glucosides. Our data are consistent with the hypothesis that indole glucosinolates have evolved from cyanogenesis. Indole-3-acetaldoxime is a precursor of the plant hormone indole-3-acetic acid, which suggests that CYP79B2 might function in biosynthesis of indole-3-acetic acid. Identification of CYP79B2 provides an important tool for modification of the indole glucosinolate content to improve nutritional value and pest resistance.     

Comparison of growth,exogenous auxin sensitivity,and endogenous indole-3-acetic acid content in roots ofHordeum vulgare L. and an agravitropic mutant

The chemically induced barley (Hordeum vulgare L.) mutation, agr, was found to be a simple recessive trait resulting in agravitropic roots and normal gravitropic shoots. The total seedling root growth was similar for mutant and wild-type roots, although the mutant had fewer roots per seed and greater elongation per root. Although the concentration of exogenous indole-3-acetic acid (IAA) required to reduce root growth by 50% (GR50) was 12 times greater for the agravitropic mutant, agravitropic and gravitropic roots were equally sensitive to exogenous applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA). Root IAA contents, determined by high-pressure liquid chromatography (HPLC), were not different for gravitropes and agravitropes. The greater root elongation rates, lack of sensitivity to exogenous IAA, and normal endogenous IAA levels indicate that auxin-controlled growth regulation may be altered in the mutant.     

The effect of shoot-bending on the amount of diffusible indole-3-acetic acid and its transport in shoots of Japanese pear

The amount of diffusible indole-3-acetic acid (IAA) in shoots ofJapanese pear (Pyrus pyrifolia) decreased when vertical shootswere bent at an angle of 45°. A significant decrease of diffusibleIAA was observed one day after shoot bending (DAB), and the degree ofthis decrease was larger in the apical region of the shoot than in thebasal region. The decrease caused by the shoot bending increased withthe duration of the treatment. The IAA amounts in the bent shoot in theapical, central, and basal segments on 1 DAB were58.2±6.4%, 92.6±7.6%, and79.1±7.1% of the control, while 43.7±4.1%,30.8±2.9%, and 39.4±2.5% on 14 DAB.Radiolabelled IAA transport velocity was also examined, but it was notinfluenced by the shoot angle in the apical region of the shoot.However, the IAA transport velocity in the basal region decreased. Itdropped first on 1 DAB, but it recovered to the control level 3 DAB,then it decreased again on 14 DAB. A large increase in ethyleneproduction was observed in the bent shoot, but it seemed transient anddid not continue for 14 days. These results suggest that the decrease ofdiffusible IAA amounts may be induced not by the decrease of IAAtransport velocity but by the production/supply of IAA in the apicalregion.     

Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.     

The effect of CCC on the levels of endogenous indole-3-acetic acid,abscisic acid,gibberellins and cytokinins inVicia faba plants

Vicia faba plants cv. ‘Erfordia’ were treated with single application of CCC at 250 mg l^?1, 7 days before extraction. Such a concentration resulted in a 10.4, 14, 5 and 3.3 fold, respectively, increase in the levels of endogenous IAA, ABA, gibberellins and cytokinins relative to the controls. The results obtained indicate that a single application of CCC at a low concentration was sufficient to enhance the endogenous growth hormones in the treated plants. The results were obtained using GLC analyses for IAA, ABA and cytokinins, and the lettuce hypocotyl and soybean callus bioassay for gibberellins and cytokinins, respectively.     

WIN-55,212-2 and SR-141716A alter nicotine-induced changes in locomotor activity, but do not alter nicotine-evoked [3H]dopamine release

Nicotine, the main psychoactive ingredient in tobacco, plays a key role in the development of cigarette smoking addiction. The endocannabinoid system has been demonstrated to have an important role in the motivational and reinforcing effects of drugs. The present study used behavioral and neurochemical techniques to study the interaction of cannabinoid receptors and nicotine pharmacology. In a locomotor activity experiment in rats, the CB(1)/CB(2) cannabinoid receptor agonist WIN-55,212-2 (0.28-2.8 mg/kg) attenuated nicotine (0.4 mg/kg)-induced hyperactivity, but did not alter nicotine (1.0 mg/kg)-induced hypoactivity. In contrast, the selective CB(1) cannabinoid receptor antagonist SR-141716A (1.0 mg/kg) diminished nicotine-induced hypoactivity, but did not alter nicotine-induced hyperactivity. In a neurochemical experiment, rat striatal slices preloaded with [(3)H]dopamine were superfused with WIN-55,212-2 or SR-141716A. A high concentration (100 microM) of WIN-55,212-2 evoked [(3)H]overflow, but this effect was not blocked by the cannabinoid receptor antagonist AM-251. SR-141716A did not evoke [(3)H]overflow, and neither WIN-55,212-2 nor SR-141716A altered nicotine-evoked [(3)H]overflow. Overall, these results indicate a behavioral interaction between cannabinoid receptors and nicotine pharmacology. Likely, WIN-55,212-2 and SR-141716A block nicotine-induced changes in behavior through an indirect mechanism, such as alteration in endocannabinoid regulation of motor circuits, rather than directly through blockade of nicotinic acetylcholine receptors.     

Basipetally polarised transport of [3H]gibberellin A1 and [14C]gibberellin A3, and acropetal polarity of [14C]indole-3-acetic acid transport,in stelar tissues of Phaseolus coccineus roots

Summary Movement of both [³H]GA₁ and [¹⁴C]GA₃ through root segments from P. coccineus seedlings was basipetally polarised. The basipetal/acropetal ratio of radioactivity from [³H]GA₁ in agar receiver blocks was 9.2 for apical, elongating segments, and 4.0 for more basal, non-elongating segments. Polarity of gibberellin transport was restricted to the stele, and absent from cortical tissues. Transport of [¹⁴C]IAA through root segments to agar receivers was preferentially acropetal, particularly so in the stele. Despite the existence of basipetal polarity of gibberellin transport in the root, [³H]GA₁ injected into cotyledons moved into and acropetally along the seedling root.     

The relationship between oxidase activity,peroxidase activity,hydrogen peroxide,and phenolic compounds in the degradation of indole-3-acetic acid in vitro

The peroxidase catalyzed degradation of indole-3-acetic acid (IAA) results in the formation of indole-3-methanol (IM) in the presence of phenolic compounds or in 3-hydroxymethyloxindole (HMOx) in their absence. Apparently the phenols compote with a methyleneindolenine intermediate for H₂O₂ which is produced by oxidase action preceding the peroxidase action in the course of IAA degradation. The substitution pattern of various phenolic compounds tested strongly effects the rate of the reaction. However, this substitution pattern does not appear to effect the type of the reaction or the products formed. We suggest that the function of the “oxindole pathway” is to detoxify excess H₂O₂ in the absence of phenolic cosubstrates. The results lead to a number of interesting aspects of IAA biochemistry and to the proposal of a new reaction scheme for the peroxidase catalyzed degradation of IAA.     

The effect of dinitrophenol,sodium arsenate,sodium phosphate and indole-3-acetic acid on the heat of respiration of germinating wheat seeds

The rate of carbon dioxide evolution, respiration quotient, and the rate of liberation of heat were determined on the 3rd day of germination of wheat seeds. The seeds were germinated with pure water (control) or they were transferred for a 5-hour period to a solution of dinitrophenol, sodium arsenate, sodium phosphate or indole-3-acetic acid. The heat liberated was compared with the total energy released in respiratory processes of the seedlings, calculated from the gas exchange measurements. The amount of heat liberated for 1mm of carbon dioxide evolved and for 1mm of oxygen absorbed was definitely changed in the presence of the compounds used. However, the ratio of heat liberated to the total energy released in respiration of the seedlings changed only slightly.