Differential identification of natural forms of indolyl-3-acetic acid

An original micromethod ELISA for differential quantitative determination of free (indolyl-3-acetic acid) and bound (indolyl-3-acetyl-L-aspartic acid) forms of auxins is suggested. Our immunoassay approach is basic and may be applied to problems in biology, medicine, agriculture, and environmental protection for the identification of low-molecular compounds related in chemical structure and characterized by different biological activity.     

A rapid,sensitive and accurate determination of indolyl-3-acetic acid

A method is described for the routine determination of absolute amounts of free indolyl-3-acetic acid (IAA) in plant material. The time required for a determination is in the order of 2 hr. Several samples can be handled at the same time. Depending on the contents of IAA, the amount of fresh plant material varies from 0·05 to 5 g. Losses are corrected for by applying ¹⁴C-labeled IAA.     

Gravitropic plant growth regulation and ethylene: an unsought cardinal coordinate for a disused model

According to the Cholodny-Went hypothesis, gravitropic differential growth is brought about by the redistribution of auxin (indolyl-3-acetic acid, IAA). We reinvestigated the relevance of different auxins and studied the role of ethylene in hypocotyls of sunflower and shoots and roots of rye and maize seedlings. Incubation of coleoptiles and of sunflower hypocotyls in solutions of IAA and dichlorophenoxyacetic acid as well as naphthylacetic acid resulted in a two- to threefold length increase compared to water controls. In spite of this pronounced general effect on elongation growth, gravi-curvature was similar to water controls. In contrast to this, inhibition of ethylene synthesis by aminoethoxyvinylglycine prevented differential growth of both hypocotyls and coleoptiles and of roots of maize. In horizontally stimulated maize roots growing on surfaces, inhibition of ethylene perception by methylcyclopropene inhibited roots to adapt growth to the surface, resulting in a lasting vertical orientation of the root tips. This effect is accompanied by up- and down-regulation of a number of proteins as detected by two-dimensional matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Together the data query the regulatory relevance of IAA redistribution for gravitropic differential growth. They corroborate the crucial regulatory role of ethylene for gravitropic differential growth, both in roots and coleoptiles of maize as well as in hypocotyls.     

Responsiveness to abscisic acid of embryos of dormant oat (Avena sativa) seeds. Involvement of ABA-inducible proteins

An in vitro bioassay system for floral bud formation has been established using Nicotiana tabacum L. cv. MC, explants excised from floral stalks cultured on modified Murashige-Skoog medium containing excess auxin and antiauxin. Three auxins. indolyl-3-acetic acid (IAA), 4-chloroindolyl-3-acetic acid and 5,6-dichloroindolyl-3-acetic acid, were tested for floral bud-forming activity; IAA was most efficient. Three antiauxins. 5,7-dichlorindolyl-3-isobutyric acid (5,7-Cl₂-HBA), p -chlorophenoxyiso butyric acid and 2,3,5-triiodobenzoic acid, were tested for the ability to reverse the inhibition of floral bud formation caused by excess IAA. Only 5,7-Cl₂-HBA was very effective. Leaf exudates from short day- and long day-treated tobacco plants were added to the bioassay system which contained 1 µ M 6-benzylaminopurine, 5 µ M IAA and 5 µ M 5, 7-Cl₂-HBA, Interestingly, only the leaf exudate from short day-treated plants stimulated floral bud formation. Moreover, the buds produced grew into large. well-developed ones with shapes similar to those of the natural floral buds of intact tobacco plants.     

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.     

Poly-N-vinylcaprolactam gel: A novel matrix for entrapment of microorganisms

Summary A new gel-type support poly-N-vinylcaprolactam for microbial cell immobilization is presented. The method allows one to obtain beads of biocatalyst in a single step. The properties of beads obtained using different types of gel stabilizers were compared; the best stabilizer was found to be tannin. The method developed was used for entrapment of viable bacterial cells and fungal spores. The biocatalysts obtained were used for transformations of both hydrophilic (sorbitol, indolyl-3-acetic acid) and lipophilic (cortexolone, hydrocortisone) substrates.Abbreviations PVC poly-N-vinylcaprolactam - ImC immobilized cells - IAA indolyl-3-acetic acid - TLC thin layer chromatography     

Three-phase extraction and partitioning with the aid of dialysis – a new method for purification of indolyI-3-acetic and abscisic acids in plant materials

A three-phase partitioning method to separate indolyl-3-acetic acid (IAA) and abscisic acid (ABA) from a crude plant extract was developed and evaluated. The aqueous phase at pH 2.7 of a methanolic plant extract constituted the 1st phase, from which IAA and ABA were transferred via diethyl ether, the 2nd phase, to a 3rd phase consisting of an alkaline buffer, enclosed in a dialysis tube. Partitioning of the free forms of the two hormones among the three phases in one container were carried out simultaneously and efficiently. The method also proved to be satisfactory when used as a combined step for both extraction and partitioning, at which the plant homogenate in buffer at pH 2.7 constituted the 1st phase. The content of IAA and ABA in kernels of Zea mays and in hypocotyls of Beta vulgaris were tested with the new method. The method presented is reliable and time-saving, and the demand for chemicals is less than for most of the conventional procedures used.     

Azospirillum brasilense SP245 mutants in production of anthranilic and indolyl-3-acetic acids]

The mutants of Azospirillum brasilense Sp245 altered in the production of anthranilic (Ant) and indolyl-3-acetic (IAA) acids were selected after the chemical or transposon facilitated mutagenesis and divided into the following three classes: Ant+IAA+, Ant+IAA- and Ant-IAA-. A hypothesis on the existence of a pattern for tryptophan conversion to anthranilate that is different from the classic pattern, and on the connection of the indolyl-3-acetic synthesis with this process is suggested.     

Study of Parameters Involved in the Determination of IAA and ABA in Plant Materials

A procedure for the combined purification of both indolyl-3-aceticacid (IAA) and abscisic acid (ABA) is presented. The procedureis designed to obtain accurate quantifications with minimallosses and within a relatively short time. The prepurification procedure makes optimal use of C18 prepackedcartridges. A purification step by high pressure liquid chromatographyis included and discussed. The quantification of IAA is performed with the 2-methylindolepyroneassay (2-MIP-assay). An alternative way of correcting for aspecificfluorescence is proposed and tested. ABA is analysed by GLC-ECD. The accuracy of the overall method is tested using differentapproaches for both ABA and IAA. Using this method, extractionby homogenization is compared to extraction by diffusion withouthomogenization. Key words: IAA, ABA, Purification     

The role of auxins in differentiation of rice tissues culturein Vitro

Effects of various auxins on callus induction (dedifferentiation) and organ redifferentiation from the callus were studied by using various tissues of rice,Oryza sativa L. cv. Kyoto Asahi. 2,4-D, NAA and IAA were used as auxins for the test of their ability to induce callus. All of these were active. This callus induction by auxin was successful in all tissues used; seed, root, shoot nodule, anther and ovary. In all of the calluses induced by various auxins such as 2,4-D, NAA and IAA and derived from various tissues such as seed, root, shoot nodule, anther and ovary, organ redifferentiation, i.e., formation of shoots and roots was achieved by removing the auxins from the medium used for the callus calture. Cytokinins were not necessary for the organ redifferentiation in these calluses. These results suggest that auxin is the only exogenous factor that determines dedifferentiation and redifferentiation in rice plant tissues culturedin vitro.     

Identification of 4-Chloroindole-3-acetic Acid and Its Methyl Ester in Immature Seeds of Vicia amurensis (the Tribe Vicieae), and Their Absence from Three Species of Phaseoleae

The natural chlorinated auxins 4-chloroindole-3-acetic acid(4-Cl-IAA) and its methyl ester (4-Cl-IAA Me ester) were found,in addition to IAA and its Me ester, by gas chromatography-massspectrometry in immature seeds of Vicia amurensis, a Vicieaespecies. In contrast, only non-chlorinated, IAA and IAA Me esterwere present in immature seeds of three Phaseoleae species.These results are further evidence of the wide distributionof 4-Cl-IAA and its Me ester in various Vicieae. (Received October 3, 1986; Accepted December 22, 1986)     

Changes in diffusible indole-3-acetic acid from various parts of tulip plant during rapid elongation of the flower stalk

In order to chemically identify the putative indole-3-acetic acid (IAA) and to confirm the native source of auxins account for rapid elongation of the floral stalk of tulip, we examined diffusible IAA from various parts of tulip plant during rapid elongation of the flower stalk. IAA was identified in the diffusates collected from the leaves, internodes, and floral organs with gas chromatography (GC)–mass spectrometry. The amount of diffusible IAA from different plant organs followed the order of that the internodes > flower organs > leaves during the period of rapid elongation of the floral stalk. The diffusible IAA from internodes reached its peak amount at different time than did diffusible IAA from the flower. The results obtained indicated that the top internode is probably the major source of auxins account for rapid elongation of the flower stalk.     

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.     

Tryptophan-dependent biosynthesis of auxins in soil

The presence of auxins in soil may have an ecological impact affecting plant growth and development. A rapid and simple colorimetric method was used to assess California soils for their potential to produce auxins upon the addition of L-tryptophan (L-TRP). The auxin content measured by colorimetry was expressed as indole-3-acetic acid (IAA)-equivalents. A substrate (L-TRP) concentration of 5.3 g kg^-1, glucose concentration of 6.7 g kg^-1, no nitrogen, pH 7.0, 40°C, shaking (aeration) and 48 h incubation time were selected as standardized conditions to assay for auxin biosynthesis in soil. IAA was confirmed as a major microbial metabolite derived from L-TRP in soil by use of high performance liquid chromatography (HPLC). Under standardized conditions, L-TRP-derived auxins in 19 soils varied greatly ranging from 18.2 to 303.2 mg IAA equivalents (auxins) kg^-1 soil. This study suggests that the phenotypic character of the soil microbiota has more of an influence on auxin production than the soil physicochemical properties (e.g., pH, organic C content, CEC, etc.).     

Effects of natural and synthetic auxins on the gravitropic growth habit of roots in two auxin-resistant mutants of Arabidopsis, axr1 and axr4: evidence for defects in the auxin influx mechanism of axr4.

The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium. Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect. Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA. The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells. The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins. These results suggest that the lesion of axrl mutants is different from that of axr4.     

The effect of 3-Indolylacetic acid on the differentiation of plastids in callus culture ofCichorium intybus L.

Summary The effect of 3-indolylacetic acid (IAA) on structure of plastids in cells of callus developing on the phloem explants of chicory roots was investigated. In the absence of IAA the proplastids in the initial expiant developed into typical chloroplasts. The presence of increasing IAA concentrations in the medium resulted in a gradual reduction of the thylakoid system accompanied by an increasing starch content of the plastids. Depending on the IAA concentration used, various types of plastids from typical chloroplasts to typical amyloplasts were found. A possible relationship between auxins and sugar metabolism is indicated.     

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.     

Auxin carriers in membranes of lupin hypocotyls

The pH-driven accumulation of [³H]indolyl-3-acetic acid (IAA) has been found to occur in membrane vesicles of lupin (Lupinus albus L.) hypocotyls. Most of this association of auxin with membranes is very sensitive to osmotic shock, high concentrations of permeable weak acids, incubation at 20° C for 20 min and to some ionophores. Long incubation times also depress the ability to accumulate radioactive IAA but this ability can be partially restored by a treatment that presumably reconstitutes the pH gradient across the membranes. Two specific inhibitors of auxin transport, N-1-naphtylphthalamic acid and 2,3,5-triiodobenzoic acid, stimulate net IAA uptake with an optimum at about 10^-6 M (pH 5.0). At least two auxin carriers appear to be present in the lupin membrane vesicles. An uptake carrier seems to be saturated at 10^-7 M IAA in the presence of N-1-naphtylphthalamic acid, but higher IAA concentrations are needed to saturate an efflux carrier. The uptake carrier also shows a high affinity for IAA and 2,4-dichlorophenoxyacetic acid and a low affinity for 1-naphthylacetic acid.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indolyl-3-acetic acid - NAA naphthalene-1-acetic acid - NIG nigeriein - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid - VAL valinomycin     

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.