Phytohormones,Rhizobium mutants, and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of³H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.     

Synthesis and biological activities of 4-chloroindole-3-acetic acid and its esters

4-Chloroindole-3-acetic acid (4-Cl-IAA) and its esters were synthesized from 2-chloro-6-nitrotoluene as the starting material. The biological activities of 4-CI-IAA and its esters were determined by four bioassays. Except for the tert-butyl ester, 4-Cl-IAA and its esters had stronger elongation activity toward Avena coleoptiles than had indole-3-acetic acid. The biological activities of the methyl, ethyl and allyl esters were as strong as the activity of the free acid. All the esters, except for the tert-butyl, inhibited Chinese cabbage hypocotyl growth more than the free acid did, and all the esters induced severe swelling and formation of numerous lateral roots in black gram seedlings even at a low concentration. Furthermore, adventitious root formation was strongly promoted in Serissa japonica cuttings by all the esters. The root formation-promoting activities of the ethyl and allyl esters were about three times the value for indole-3-butyric acid which is used to promote and accelerate root formation in plant cuttings.     

Structure of the inclusion complexes of heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin with indole-3-butyric acid and 2,4-dichlorophenoxyacetic acid

The crystal structures of the complexes of heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin with indole-3-butyric acid and with 2,4-dichlorophenoxyacetic acid were studied by X-ray diffraction. The complexes crystallize in the monoclinic P2(1) space group. The host molecules are elliptically puckered and stacked along the a crystal axis, in a head-to-tail fashion, forming columns. One primary methoxy group of the host molecule of the complex with indole-3-butyric acid has the unusual trans-gauche conformation for permethylated CDs. All the secondary O-3-CH(3) methoxy groups, some secondary O-2-CH(3) and some primary methoxy groups pointing inwards the cavity enclose the indole or the 2,4-dichlorophenoxy moieties of the guest molecules inside the cavity, while the chains of the guests protrude between two adjacent host molecules of the columns. The mean planes of the indole and 2,4-dichlorophenoxy moieties of the guests are nearly perpendicular to the mean planes of the elliptical heptagons defined by the O-4n atoms of the hosts. The carboxyl group of the guests form hydrogen bonds with oxygen atoms of the host molecules or with the water molecules found in the space between the complexes of the same column.     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Phytohormones,Rhizobium mutants,and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of³H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.     

The influence of phytohormones on growth,organ differentiation and fructan production in callus of Symphytum officinale L.

Summary Callus derived from Symphytum officinale L. regenerants was cultured in the presence of various phytohormones. The growth rate of callus was stimulated by all phytohormones at various concentrations. With 1-naphthaleneacetic acid no organ differentiation could be observed. With indole-3-butyric acid at low concentrations only roots were formed, whereas 6-benzylaminopurine, kinetin and zeatin at various concentrations induced either root or shoot formation or the simultaneous regeneration of both. Minor amounts of fructans were formed at high 6-benzylaminopurine-, zeatin- and at all indole-3-acetic acid-concentrations. The concentration of 1-naphthaleneacetic acid had no influence on the fructan content. Highest rates of fructan synthesis occurred at low zeatin-concentrations up to 1.5 mg/l. Only zeatin at all concentrations induced the synthesis of polyfructans, whereas appreciable amounts of oligofructans were formed under the influence of all other phytohormones.Abbreviations NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - DP degree of polymerisation - GLC gas liquid chromatography - TLC thin layer chromatography     

Double-standard isotope dilution assay: I. Quantitative assay of indole-3-acetic acid

Isotope dilution analysis for the quantitation of labile compounds has been limited in applicability by the amount of sample necessary to determine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determination of specific activity by gas chromatography. It requires the availability of the radiolabeled internal standard, as is customarily used in an isotope dilution assay, and also requires a chemically related radiolabeled compound to serve as a second internal standard. It is this second internal standard, added in known amounts, that permits quantitation of the gas chromatography. The method is illustrated by assaying indole-3-acetic acid in plant extracts using [¹⁴C]indole-3-acetic acid as the internal standard and adding [¹⁴C]indole-3-butyric acid as the second internal standard for quantitation of the gas chromatographic procedures. Used with a nitrogen-specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of [2-ring-¹⁴C]indole-3-butyric acid is also described.     

Mutagen formation on nitrite treatment of indole compounds derived from indole-glucosinolate

The mutagenicities of 8 indole compounds (indole-3-acetonitrile, indole-3-carbinol, indole-3-acetamide, indole-3-acetic acid, 3-methylindole, indole-3-aldehyde, indole-3-carboxylic acid and indole) derived from indole glucosinolate were studied by mutation tests on Salmonella typhimurium TA98 and TA100 and Escherichia coli WP2 uvrA/pKM101 with and without S9 mix. None of the 8 indole compounds were mutagenic, but they became mutagenic on these 3 tester strains when treated with nitrite at pH 3. The nitrite-treated indole compounds were mutagenic without metabolic activation system (S9 mix), and their mutagenicities were decreased by the addition of S9 mix.     

Endogenous auxins in plant growth-promoting Cyanobacteria—Anabaena vaginicola and Nostoc calcicola

Three isolates of heterocystous cyanobacteria, belonging to the genera Anabaena and Nostoc, gathered from Iranian terrestrial and aquatic ecosystems exhibited considerable growth promotion effect on several vegetables and herbaceous plants. To study the ability of these three isolates to produce auxins, three endogenous auxins, including indole-3-acetic acid (IAA), and two of its main homologues, indole-3-propionic acid and indole-3-butyric acid, were extracted and analyzed with high-performance liquid chromatography equipped with diode array detector and fluorescence detector, and the results were further confirmed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the negative-ion mode. The dominant auxin observed in all isolates was indole-3-butyric acid (IBA) in the range of 140.10–2146.96 ng g^?1 fresh weight (FW), and only small amounts of IAA (2.19–9.93 ng g^?1 FW) were detected. The predominance of IBA in these strains is reported for the first time which is different from the previously reported auxin profiles in microalgae and algae with the predominance of IAA.     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl^–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl^–1 indole-3-acetic acid or 0.1 mgl^–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl^–1) with indole-3-butyric acid (0.1 mgl^–1) was added to NN medium supplemented with casein hydrolysate (250 mgl^–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NN Nitsch and Nitsch (1969) - NOA 2-naphthoxyacetic acid     

Several known indole compounds are not important precursors of direct mutagenic N-nitroso compounds in green cabbage

In this study we investigated the role of indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan in the formation of N-nitroso compounds in green cabbage extracts. Green cabbage extracts were separated by gel permeation chromatography. Fractions were treated with nitrite, tested for mutagenicity and analysed for total N-nitroso content. Fractions in which spiked indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan eluted appeared to be low in mutagenic activity and contained relatively small amounts of N-nitroso compounds. To detect indole compounds other than the ones used in the gel permeation chromatography experiments, high-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed of green cabbage extracts. Indole-3-carboxaldehyde was found to be the most commonly occurring indole compound, but it did not show direct mutagenic activity upon nitrite treatment. Indole-3-acetonitrile was the second most common compound; although it was mutagenic after nitrite treatment, its contribution to the mutagenicity of nitrite-treated green cabbage was roughly estimated to be only 2%. No other indole compounds were detected. From this study we conclude that neither the tested indole compounds nor indole-3-carboxaldehyde play a significant role in the formation of direct mutagenic N-nitroso compounds in nitrite-treated green cabbage extracts.     

Endogenous levels of auxin and tumorigenesis in a Nicotiana amphiploid

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown aseptically on nutrient medium in a controlled environment chamber. At regular intervals after the seeds had been sown, the incidence of tumor formation was scored and plants were harvested. Indole-3-acetic acid was extracted, purified, and assayed spectrofluorometrically. A close correlation in time between onset of tumor formation and decline in endogenous level of indole-3-acetic acid was demonstrated.     

Embryo culture of Costus speciosus (Koen.) Sm. to regenerate variable diosgenin yielding clones

Mature embryos of Costus speciosus were excised and cultured on Schenk and Hildebrandt's (1972) nutrient medium containing auxins and cytokinins either alone or in combination. Multiple shoots were obtained when kinetin and indole-3-butyric acid were supplemented each at 0.1 mg 1^–1 concentration. Embryo-derived plantlets were multiplied through propagation of rhizomes and the propagules derived from a single embryo were designated as an embryoclone. Twenty such embryo-clones were maintained in the field. Variations in rhizome biomass yield and diosgenin contents of these embryoclones were noted. Thirty-six percent of the embryo-clones studied were high diosgenin yielding types. Diosgenin contents at the intraclonal level were uniform. The in vitro raised plants were morphologically uniform and indistinguishable from their parent.Abbreviations SH Schenk and Hildebrandt (1972) medium - Kn kinetin - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - CA casaminoacids (vitamin free) - TLC thin layer chromatography     

Influence of Aryl Esters of Indole-3-acetic and Indole-3-butyric Acids on Adventitious Root Primordium Initiation and Development

Synthetic aryl esters of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) greatly enhanced adventitious root primordium initiation in bean (Phaseolus vulgaris L. cv. Top Crop) and jack pine (Pinus banksiana Lamb.) cuttings, respectively. Bean cuttings produced 95 to 154% more macroscopically visible root primordia in 2 days when treated with phenyl indole-3-acetate (P-IAA), in comparison with an equal concentration of IAA. Substantial but lesser increases occurred when treatment was done with 3-hydroxyphenyl indole-3-acetate (3HP-IAA). On a molar basis, either P-IAA or 3HP-IAA were 10 or more times as efficient as IAA in inducing adventitious root primordium initiation in bean cuttings. Methyl indole-3-acetate was no more effective than IAA in these tests. Phenyl indole-3-butyrate (P-IBA) consistently enhanced the number of rooted jack pine seedling cuttings by 11 to 12% in comparison with a 27% higher concentration of IBA. The number of elongated roots (2 mm or more) after 5 days was 165 to 276% greater for P-IAA than for IAA-treated bean cuttings. Similar but lesser increases occurred as a result of 3HP-IAA treatment. P-IBA in comparison with IBA treatment did not influence either the number of roots or length of the longest root per rooted jack pine cutting. Enzymes in bean and jack pine cuttings hydrolyzed the aryl esters. However, check experiments showed that initial integrity of the esters was required for enhanced activity in inducing root primordium initiation. Treatment of bean cuttings with hydrolysates of P-IAA, or with IAA and phenol, alone or combined, did not influence root primordium initiation or development in a manner different from treatment with IAA alone.     

Plant regeneration from mesophyll protoplasts in Isatis indigotica

Protoplasts of an accession of Isatis indigotica Fort. were isolated from mesophyll tissue by enzymatic digestion and cultured using a feeder cell system. Shoot regeneration efficiency was 100% via organogenesis among 627 isolated calluses within 30–37 days. Among these shoot initiating calluses, 162 (22.6%) developed normal shoots with multiple (2–5) shoots per callus. The remaining calluses developed only vitreous shoots. High concentration (5 μM) of indole-3-butyric acid had a positive effect on rooting compared to low concentration (0.5 μM) of indole-3-butyric acid and α-naphthalene-acetic acid. The average rooting efficiency of regenerated shoots of two experiments was higher on LS medium with 5 μM indole-3-butyric acid than on LS medium without growth regulators. Twenty-nine plantlets, with 2–3 expanded leaves and roots were potted in soil and 22 developed normally to maturity in the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of indole-3-Acetic acid on ethylene formation in wheat seedlings

Isoperoxidase B 1 isolated from winter wheat (Triticum aestivum L., cv. Jubilar) seedlings was shown to catalyze ethylene formation from α-keto, γ-methylmercaptobutyric acid (KMBA). In the presence of Mn²⁺, indole-3-acetic acid (IAA), andp-coumaric acid, the kinetics by isoperoxidase B 1 catalyzed conversion of KMBA into ethylene and other products was similar to that of IAA oxidation. The reaction rate was therefore controlled by IAA through its electrondonating properties. Exogenous IAA induced ethylene formation in the segments of etiolated wheat coleoptiles. IAA-induced ethylene production was enhanced by L-methionine and mitomycin C. Aminoethoxy-analogue of rhizobitoxine, ferulic acid, sodium benzoate, cycloheximide and actinomyoin D exhibited significant inhibitory effects. These data indicate that the overall reaction mechanism in coleoptile segments involves RNA and protein synthesis. The site of IAA action is not specific; 2,4-dichlorophenoxyacetic, α-naphthylacetic and indole-3-butyric acids, respectively, possessed comparable inductive effect as IAA. Indole-3-propionic acid, indole, L-tryptophan and glucobrassicin had only low inductive efficiency, and moreover indole and L-tryptophan slowed down IAA-induced ethylene formation.     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus

Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter of about 1cm were transferred to shoot induction media, containing different concentrations (1–50µM) of thidiazuron. Shoot formation was observed after 16 weeks up to 12% efficiency. Thidiazuron (10µM) was the most effective concentration in all experiments. Shoot buds elongated in medium supplemented with N-isopentenyl adenine and indole-3-butyric acid. Since rooting was almost impossible in these thidiazuron-induced shoots, shoots were grafted onto young pea seedlings and regenerated to fertile plants.Abbreviations 2ip N-isopentenyl adenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MES 2-(Nmorpholino)ethane acid - NAA naphthaleneacetic acid - TDZ thidiazuron - BAP 6-benzylaminopurine - PEG polyethylene glycol     

Measurement of indolebutyric Acid in plant tissues by isotope dilution gas chromatography-mass spectrometry analysis

An internal standard, [¹³C][indole-2]-indole-3-butyric acid, was synthesized from indole-2[¹³C] and was shown to be effective for the quantitative determination of indole-3-butyric acid from plant tissue. When this standard was used along with [¹³C₆]indole-3-acetic acid, both indolic auxins could be quantified from the same tobacco (Nicotiana tabacam) leaf sample by isotope dilution analysis using selected ion monitoring gas chromatography-mass spectrometry for detection.