Quantitative distribution of gibberellins and indole-acetic acid in pea (Pisum sativum L.) seedlings

The objective was to determine the distribution in pea seedling of GA (by thin layer chromatography) and IAA (by HPLC). Three samples of 30 7/day-old seedlings were sectioned into: apical meristem + plumular hook; stem elongation zone; stem non-elongation zone; cotyledons; root non-elongation zone; root tip. The material was frozen in liquid N and stored at -80 degrees C. The stem elongation zone, the apical meristem+plumular hook and the root tip had low IAA, whereas the stem non-elongation zone and root non-elongation zone had high IAA content. The stem elongation zone and apical meristem plumular hook had high GA, while root non-elongation zone had the lowest GA content.

Interactions of indole acetic acid with EGF and FSH in the culture of ovine preantral follicles

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. The objective of this study was to evaluate the effects of adding indole acetic acid (IAA), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) to the media for in vitro culture of ovine ovarian fragments and determine their effects on growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2 or 6 days in culture plates with: minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, bovine serum albumine and antibiotics (MEM+); MEM+ plus IAA (40 ng/mL); MEM+ plus EGF (100 ng/mL); MEM+ plus FSH (100 ng/mL); MEM+ plus IAA + EGF; MEM+ plus IAA + FSH; MEM+ plus EGF + FSH; or MEM+ plus IAA + EGF + FSH. After 2 or 6 days of culture in each treatment, the pieces of ovarian cortex were fixed in Bonin for histological examination. Follicles were classified as primordial or developing (primary and secondary) follicles. Compared to the control, in all media tested, the percentages of primordial follicles were reduced (P < 0.05) and the percentages of developing follicles were increased (P < 0.05) after 2 or 6 days of culture. Furthermore, culture of ovarian cortex for 6 days reduced the percentages of healthy, viable follicles when compared with the control (P < 0.05), except for cultures supplemented with IAA + EGF and EGF + FSH. In conclusion, the addition of IAA and EGF or EGF and FSH to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture. (c) 2005 Elsevier B.V. All rights reserved.

Indole acetic acid and ACC deaminase fromendophytic bacteria improves the growth of Solanum lycopersicum

Background: Endophytic bacteria are ubiquitous in all plant species contributing in host plant\'s nutrient uptake and helping the host to improve its growth. Moringa peregrina which is a medicinal plant, growing in arid region of Arabia, was assessed for the presence of endophytic bacterial strains. Results: PCR amplification and sequencing of 16S rRNA of bacterial endophytes revealed the 5 endophytic bacteria, in which 2 strains were from Sphingomonas sp.; 2 strains from Bacillus sp. and 1 from Methylobacterium genus. Among the endophytic bacterial strains, a strain of Bacillus subtilis LK14 has shown significant prospects in phosphate solubilization (clearing zone of 56.71 mm after 5 d), ACC deaminase (448.3 ± 2.91 nM α-ketobutyrate mg-1 h-1) and acid phosphatase activity (8.4 ± 1.2 nM mg-1 min-1). The endophytic bacteria were also assessed for their potential to produce indole-3-acetic acid (IAA). Among isolated strains, the initial spectrophotometry analysis showed significantly higher IAA production by Bacillus subtilis LK14. The diurnal production of IAA was quantified using multiple reactions monitoring method in UPLC/MS–MS. The analysis showed that LK14 produced the highest (8.7 μM) IAA on 14th d of growth. Looking at LK14 potentials, it was applied to Solanum lycopersicum , where it significantly increased the shoot and root biomass and chlorophyll (a and b) contents as compared to control plants. Conclusion: The study concludes that using endophytic bacterial strains can be bio-prospective for plant growth promotion, which might be an ideal strategy for improving growth of crops in marginal lands.

Microbicidal Action of Indole-3-Acetic Acid Combined with Horseradish Peroxidase on Prototheca zopfii from Bovine Mastitis

We investigated the toxic effect of indole-3-acetic acid (IAA) combined with horseradish peroxidase (HRP) on Prototheca zopfii from bovine mastitis. P. zopfii isolates were identified and characterized by morpho-physiological parameters; presences of P. zopfii genotype 2 were also investigated. Subsequently, P. zopfii was incubated in the absence (control) or presence of IAA/HRP and examined for: (i) cell viability; (ii) colonies number formation; (iii) antioxidant enzyme activity; and (iv) DNA integrity. Significance of differences was calculated using ANOVA and Tukey`s test (P a parts per thousand currency sign 0.05). As evidenced by Trypan blue exclusion and colony formation in Sabouraud dextrose agar, IAA/HRP addition to the culture reduced respective P. zopfii viability and P. zopfii colony formation in a concentration- and time-dependent manner. IAA/HRP specifically reduced cell viability in 10, 15, 20, 25, and 32% after 4, 6, 8, 10, and 12 h of incubation, respectively, compared with the control at the same time. The number of colony formation was inhibited (45, 82, and 88%) by IAA/HRP after 4, 6, and 9 h of incubation, respectively, compared with the control at the same time. In addition, P. zopfii antioxidant activity increased measurably in the presence of IAA/HRP (6 h); superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase increased by 90, 120, 150% and 3.4 times, compared with the controls. IAA/HRP did not appear to effect P. zopfii DNA integrity when examined by electrophoresis. In conclusion, IAA/HRP appears to function as a microbicidal mechanism on P. zopfii genotype 2 from bovine mastitis.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interaction of Gibberellic Acid and Indole-3-acetic Acid in the Growth of Excised Cuscuta Shoot Tips in Vitro1

Gibberellic acid (GA3) induced a marked elongation of 2.5-centimeter shoot tips of Cuscuta chinensis Lamk. cultured in vitro. In terms of the absolute amount of elongation, this growth may be the largest reported for an isolated plant system. The response to hormone was dependent on an exogenous carbohydrate supply. The hormone-stimulated growth was due to both cell division and cell elongation. The growth response progressively decreased if GA3 was given at increasingly later times after culturing, but the decreased growth response could be restored by the application of indole-3-acetic acid (IAA) to the apex. Explants deprived of GA3 gradually lost their ability to transport IAA basipetally, but this ability was also restored by auxin application. The observations are explained on the basis that: (a) the growth of Cuscuta shoot tip in vitro requires, at least, both an auxin and a gibberellin; and (b) in the absence of gibberellin the cultured shoot tip explants lose the ability to produce and/or transport auxin.

Hormones andCuscuta development: interaction of cytokinin and indole-3-acetic acid in the growth and curvature of subapical stem segments

Cuscuta stem (vines) exhibits two modes of growth—longitudinal elongation forming free-hanging vines, or coiling growth to twine around the host. The elongation zone of free-hanging vine extended up to 160 mm from the stem apex and in vivo growth rate (during 8 h of growth) was maximal in the 20-to-40-mm region. While gibberellic acid (GA3) or fusicoccin (FC) could maintain (GA3) or enhance (FC) the growth rate of apical (10 or 25 mm) segments, indole-3-acetic acid (IAA) (10 mgrM) induced growth only in subapical (5–160 mm) segments. In vitro growth rate induced by IAA (10 mgrM) was similar to the in vivo growth rate up to 40 mm. Thereafter, up to 100 mm, IAA induced growth rate exceeded in vivo growth. p ]Subapical segments (sim13 mm) from 5- to 40-mm regions responded to a cytokinin (BA, Z, or iP) or to low IAA (0.1 mgrM) with curved growth, whereas the segments grew straight in the presence of high IAA (10 mgrM). Curvature (measured as the angle subtended at the center of the circle of which the segment formed an arc) induced by BA and low (0.1 mgrM) IAA was greater than either added separately. Besides, segments induced to curve in BA + low-IAA solution could be made to straighten out by transferring to a solution containing high IAA (10 mgrM) with or without BA. Thus in vivo patterns of straight and coiling growth could be mimicked reversibly in vitro by adjusting the relative concentrations of cytokinin and auxin; low auxin and cytokinin induced coiling growth, whereas high auxin and cytokinin induced straight growth. p ]Beyond 40 mm, BA had no growth-promoting or curvative-inducing effect.Cuscuta vine segments thus showed sequential sensitivity to applied hormones, the apical region (0–25 mm) to GA3, the subapical (5–40 mm) region to BA and IAA and the region beyond (40–160 mm) to IAA alone.

The triad indole-3-acetic acid ethyl ester/esterase/horseradish peroxidase as a new cytotoxic prodrug/enzyme combination

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against l-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.

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 24h 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 pentosephosphate pathway

An in Vitro System from Maize Seedlings for Tryptophan-Independent Indole-3-Acetic Acid Biosynthesis1

The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [14C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [14C]Trp nor [14C]serine substituted for [14C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.

Metabolism of Indole-3-Acetic Acid in Arabidopsis1

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 μm, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 μm drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.

Indole-3-Acetic Acid Controls Cambial Growth in Scots Pine by Positional Signaling1

The vascular cambium produces secondary xylem and phloem in plants and is responsible for wood formation in forest trees. In this study we used a microscale mass-spectrometry technique coupled with cryosectioning to visualize the radial concentration gradient of endogenous indole-3-acetic acid (IAA) across the cambial meristem and the differentiating derivatives in Scots pine (Pinus sylvestris L.) trees that had different rates of cambial growth. This approach allowed us to investigate the relationship between growth rate and the concentration of endogenous IAA in the dividing cells. We also tested the hypothesis that IAA is a positional signal in xylem development (C. Uggla, T. Moritz, G. Sandberg, B. Sundberg [1996] Proc Natl Acad Sci USA 93: 9282–9286). This idea postulates that the width of the radial concentration gradient of IAA regulates the radial number of dividing cells in the cambial meristem, which is an important component for determining cambial growth rate. The relationship between IAA concentration in the dividing cells and growth rate was poor, although the highest IAA concentration was observed in the fastest-growing cambia. The radial width of the IAA concentration gradient showed a strong correlation with cambial growth rate. The results indicate that IAA gives positional information in plants.

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.