Elisa determination of IAA using antibodies against ring-linked IAA

An enzyme-linked immunosorbent assay (ELISA) for indole-3-acetic acid (IAA) is described which uses antibodies raised against IAA conjugated to carrier protein on the indolic ring of IAA. As little as 0.5 pmol of IAA is detectable with the ELISA. There is no significant cross-reactivity with amide conjugates of IAA and samples do not need methylation, in contrast to an ELISA using antibodies raised against carboxyl-linked IAA. Affinity chromatography on IAA-agarose was used to purify antibody preparations. Measurements of IAA levels in crown gall tumour tissue lines were made using the assay.     

Evidence That IAA Conjugates Are Slow-Release Sources of Free IAA in Plant Tissues

Evidence that indoleacetic acid (IAA) conjugates are metabolized via enzyme-catalyzed hydrolysis to free IAA and that their biological activities are related to the rates at which they are hydrolyzed by the tissue is presented. These conclusions are based on the following observations. Slow but continuous decarboxylation of the IAA moiety of IAA-l-alanine and IAA-glycine occurs when these conjugates are applied to pea (Pisum sativum L. cv. Alaska) stem segments. Inasmuch as IAA conjugates are protected from peroxidase-catalyzed oxidative decarboxylation, the conjugates are probably hydrolyzed and the freed IAA then further metabolized. Free IAA and IAA-l-alanine are converted, by pea stem tissue, into the same metabolites. The metabolism is enzymic, since conjugates of IAA with the d-isomers of the amino acids are inactive. Ethylene production induced by IAA-l-alanine and by IAA-glycine is correlated with their hydrolysis, as indicated by their decarboxylation and with the appearance or nonappearance of IAA metabolites in the tissues.     

Indole-3-acetic Acid (IAA) and IAA Conjugates Applied to Bean Stem Sections: IAA Content and the Growth Response

High resolution growth recording techniques and reverse isotope dilution analysis were used to study the relationship between indole-3-acetic acid (IAA) concentration and curvature of excised bean (Phaseolus vulgaris L. cv Bush Burpee Stringless) first internode sections unilaterally treated with hormone. The maximum rate of curvature occurred rapidly (within 25 minutes) and was proportional to the log of the amount of applied IAA recovered in the tissue. The rate of curvature decreased after 30 minutes although little or no lateral migration of applied IAA occurred and tissue levels of IAA increased. The biologic activity of IAA-amino acid conjugates was found to be directly related to the amount of free IAA, resulting from their hydrolysis, which could be recovered from the tissue.     

Epiphytic microorganisms and IAA synthesis

Summary Epiphytic microorganisms present on cotton plants synthesized 3-indoleacetic acid (IAA) from tryptophan. Microorganisms from the root zone synthesized 3 times the amount of IAA when compared with the shoot zone and the root zone contained a much higher number of microorganisms. IAA-synthesizing activity was eliminated when the tissues were treated with a weak solution of mercuric chloride. Various tests on the possible accumulation of IAA from external sources showed that IAA synthesized outside the plant does not accumulate in the plant. Although epiphytic microorganisms synthesize IAA in large amounts, they do not influence the IAA content of the plant due to (1) lack of available tryptophan, (2) destruction of the auxin by the microflora, and (3) the polar movement of the auxin.     

Effects of IAA and four IAA conjugates on morphogenesis and callus growth from tomato leaf discs

The effects of indole-3-acetic acid (IAA) and four IAA conjugates, indoleacetylalanine (IAAla), indoleacetylaspartic acid (IAAsp), indoleacetylglycine (IAGly), and indoleacetylphenylalanine (IAPhe), on growth and morphogenesis in tomato leaf discs in vitro were examined. Free IAA stimulated root initiation in the absence of cytokinin and stimulated callus growth in the presence of 0.89 M benzylaminopurine (BAP). Free IAA also inhibited shoot initiation obtained with 8.9 M BAP. The activities of the IAA conjugates depended on the conjugating amino acid, the concentration of the conjugate, and the response being measured. IAAsp had little or no activity in promoting root initiation or callus growth or in inhibiting shoots, while IAPhe was similarly inactive except at the highest concentration tested (100 M). IAAla and IAGly were both very active in inhibiting shoots and promoting callus growth, but were much less active in stimulating rooting, except at 100 M, at which concentration they were as effective as free IAA. Thin-layer chromatography of the IAA conjugates revealed that IAAla, IAGly and IAPhe were largely stable to autoclaving, but that IAAsp underwent some hydrolysis to products identical with free IAA and aspartic acid. Pretreatment of seedlings with IAA, IAAla or IAGly altered the subsequent shoot initiation response from leaf discs on media with and without IAA.     

Ethylene and IAA transport in potato

Although elongation growth is reduced by ethylene, swelling responses do not occur. Ethylene reduces neither transport nor metabolism of applied IAA in either mesocotyl or coleoptile. We propose that maintenance of high auxin levels within these tissues sustains polar transport and contributes to the relative insensitivity of maize to applied ethylene.     

IAA对盐胁迫下大豆幼苗膜伤害及抗盐力的影响

采用溶液培养法研究了ＩＡＡ（吲哚乙酸）对盐胁迫下豆幼苗膜伤害及抗盐力的影响。结果表明：一定浓度的ＩＡＡ处理可促进盐胁迫下大豆幼苗的生长,使其干物质产量增加,叶面积增大,提高叶片光合速率,增强了保护酶系统活性,降低膜脂过氧化产物ＭＤＡ含量及膜相对透性（相对导一）,增强了幼苗对盐渍环境的抵抗能力,缓解了盐害。     

Specificity and similarity of functions of the Aux/IAA genes in auxin signaling of Arabidopsis revealed by promoter-exchange experiments among MSG2/IAA19, AXR2/IAA7, and SLR/IAA14

As indicated by various and some overlapped phenotypes of the dominant mutants, the Aux/IAA genes of Arabidopsis (Arabidopsis thaliana) concomitantly exhibit a functional similarity and differentiation. To evaluate the contributions of their expression patterns determined by promoter activity and molecular properties of their gene products to Aux/IAA function, we examined phenotypes of transgenic plants expressing the green fluorescent protein (GFP)-tagged msg2-1/iaa19, axr2-1/iaa7, or slr-1/iaa14 cDNA by the MSG2 or AXR2 promoter. When driven by the MSG2 promoter (pMSG2), each GFP-tagged cDNA caused the msg2-1 phenotype, that is, the wild-type stature in the mature-plant stage, long and straight hypocotyls in the dark, reduced lateral root formation, relatively mild agravitropic traits in hypocotyls, and a normal gravitropic response in roots. However, development of one or two cotyledonary primordia was often arrested in embryogenesis of the pMSG2::axr2-1::GFP and pMSG2::slr-1::GFP plants, resulting in monocotyledonary or no cotyledonary seedlings. Such defects in embryogenesis were never seen in pMSG2::msg2-1::GFP or the msg2-1, axr2-1, or slr-1 mutant. The MSG2 promoter-GUS staining showed that expression of MSG2 started specifically in cotyledonary primordia of the triangular-stage embryos. When driven by the AXR2 promoter (pAXR2), each GFP-tagged mutant cDNA caused, in principle, aberrant aboveground phenotypes of the corresponding dominant mutant. However, either the axr2-1::GFP or slr-1::GFP cDNA brought about dwarf, agravitropic stems almost identical to those of axr2-1, and the pAXR2::msg2-1::GFP and pAXR2::slr-1::GFP hypocotyls exhibited complete loss of gravitropism as did axr2-1. These results showed functional differences among the msg2-1, axr2-1, and slr-1 proteins, though some phenotypes were determined by the promoter activity.     

Biochemical Bases for the Loss of Basipetal IAA Transport with Advancing Physiological Age in Etiolated Helianthus Hypocotyls: Changes in IAA Movement, Net IAA Uptake, and Phytotropin Binding

Basipetal transport of [¹⁴C]IAA in hypocotyl segments isolated from various regions of etiolated Helianthus annuus L. cv NK 265 seedlings declines with increasing physiological age. This decline was the result of a reduction in both transport capacity and apparent velocity. Net IAA uptake was greater and the abilities of auxin transport inhibitors to stimulate net IAA uptake were reduced in older tissues. Net IAA accumulation by microsomal vesicles exhibited a similar behavior with respect to age. Specific binding of [³H]N-1-naphthylphthalamic acid (NPA) to microsomes prepared from young and older hypocotyl regions was saturable and consistent with a single class of binding sites. The apparent affinity constants for NPA binding in microsomes prepared from young versus older tissues were 6.4 and 10.8 nanomolar, respectively, and the binding site densities for young versus old tissues were 7.44 and 3.29 picomoles/milligram protein, respectively. Specific binding of [³H]NPA in microsomes prepared from both tissues displayed similar sensitivities toward unlabeled flurenol and exhibited only slight differences in sensitivity toward 2,3,5-triiodobenzoic acid. These results demonstrate that the progressive loss of basipetal IAA transport capacity in etiolated Helianthus hypocotyls with advancing age is associated with substantial alterations in the phytotropin-sensitive, IAA efflux system and they suggest that these changes are, at least partially, responsible for the observed reduction of polar IAA transport with advancing tissue age.     

Subcellular Localization of IAA Oxidase in Peas

Indoleacetic acid (IAA) oxidase has been reported to be involved in plant growth because of its alleged role in the control of endogenous IAA levels. This purported role was reevaluated in terms of the properties and subcellular location of the enzyme in etiolated pea (Pisum sativum L. var. Alaska) epicotyls.     

Endogenous IAA and morphogenesis in tobacco petiole cultures

Short duration (48 h) dark or high light treatment of the donor plants has been shown to influence the pattern of auxin metabolism in tobacco petioles in culture. In explants from dark treated donor plants there was a peak of IAA detectable at day 3 in culture. This was associated with reduced morphogenetic potential of the explant. Inclusion of 2,3,5-triiodobenzoic acid in the medium prevented this IAA accumulation and eliminated the inhibitory effect of the dark pretreatment. Inclusion of IAA in the culture medium reduced the morphogenetic potential of explants from high light treated donor plants but had no effect on the morphogenetie potential of explants from dark treated donor plants. Explants cultured for one day on auxin-free medium and then transferred to IAA-containing medium were sensitive to auxin; those transferred after five days were insensitive. Studies on the interaction between media sucrose concentrations, endogenous IAA and peroxidase, and morphogenesis are reported. The results are discussed in relation to the influence of endogenous auxin on the receptivity of explants to exogenous (media) morphogenetic stimuli.     

Distribution and transport of IAA in tomato plants

We determined as to indole-3-acetic acid (IAA), endogenous levels byliquid chromatography-mass spectrometry using ¹³C₆-IAA, diffusible levels byfluorometric detection using indole-propionic acid, and polar transportactivityby radioactive IAA in 3-month-old tomato plants (stems, leaves or roots). TheIAA concentration in the apoplast (AP) solution was higher than those in thesymplast (SP) solution in both the upper and lower parts of stems, showing thatIAA analysis of AP solution is important. Younger leaves exported much morediffusible IAA than older leaves. The IAA concentration in the main roots wasalmost at the same level as in the stems. The results suggested that thetransport capacity of IAA is probably the limiting factor for the amount of IAAtransported in stems and the amount of polar IAA transport might be only 19% ofthe endogenous IAA amount in stems.     

水分胁迫对棉花叶片中IAA从含量、IAA氧化酶和过氧化物酶活性的影响

整株干旱降低盐棉46号叶片中的IAA总量,叶龄愈小下降愈多。幼叶中IAA总量的下降主要是结合态IAA减少的结果。气干和-1.7MPa PEG溶液渗透胁迫处理也降低离体成熟叶片的IAA总量,其变化与叶片含水量呈直线相关(r=0.905)。整株干旱处理提高各叶片的过氧化物酶活性,叶龄愈小提高愈多,但IAA氧化酶活性无显著变化。离体和整株干旱时IAA总量的下降,可能是过氧化物酶活性增加所致。     

A suitable method for localization of IAA oxidase

The method for histochemical detection of IAA oxidase proposed by Sagi was tested, based on colour reaction of natural products of IAA oxidation with Ehrlich-like reagent.     

A hypothetical route for the biogenisis of IAA

Summary A method is described for using apical sections of Avena coleoptiles grown in the dark for 70 hours as an assay for IAA and for potential precursors of IAA. The experiments were carried out under as sterile conditions as possible.Tryptophan appeared not to act as a precursor of IAA. Tryptamine however showed a marked stimulation of growth indicating its possible conversion to IAA. Other compounds that stimulated growth and hence may be regarded as potential precursors of IAA were anthranilic acid and indole. Kynurenine and shikimic acid were among those without an effect. A hypothetical route for the biogenisis of IAA is suggested based on the findings of this work.     

IAA Metabolism in Embryogenic and Non-Embryogenic Carrot Cells

Carrot somatic embryos can readily be induced from embryogeniccells transferred from auxin-containing medium to auxin-freemedium, but not from transferred non-embryogenic cells. It iswell-known that IAA, a natural auxin, plays important rolesin many physiological responses including somatic embryogenesis,but, there is no report of the IAA metabolism in embryogenicand non-embryogenic cells. Therefore, we examined IAA metabolismin embryogenic and nonembryogenic carrot cells. In this paper the IAA metabolism in embryogenic cells and non-embryogeniccells is described. The induction of IAAsp formation was clarifiedin both cells. On the other hand, in non-embryogenic cells,an unknown metabolite was detected and identified as oxindole-3-acetylasparticacid (oxIAAsp). OxIAAsp formation may be induced to eliminateexcess auxin. Furthermore, endogenous IAA contents in both cellswere quantified and the relationship between somatic embryogenesisand IAA metabolism is discussed. (Received May 2, 1994; Accepted August 30, 1994)     

Metabolism and Synthesis of Indole-3-Acetic Acid (IAA) in Zea mays (Levels of IAA during Kernel Development and the Use of in Vitro Endosperm Systems for Studying IAA Biosynthesis)

Kernels of Zea mays on an intact plant accumulate indole-3-acetic acid (IAA) at the rate of 190 ng g-1 fresh weight h-1. Of the IAA synthesized, 97% is in the esterified form and less than 3% remains as the free acid. The site of biosynthesis of the IAA, whether synthesized in the leaf and transported to the kernel, or in the kernel and remaining in the kernel, has not been established. In an attempt to determine the locus of synthesis, we grew isolated kernels on agar media not containing tryptophan or other possible aromatic precursors of IAA and observed IAA synthesis of 99 ng g-1 fresh weight h-1, approximately 52% of the in situ rate. Thus, the kernel contains all of the enzymes required for de novo aromatic biosynthesis of IAA and its ester conjugates. Furthermore, endosperm cells in suspension culture, grown on hormone-free media and in the absence of aromatic precursors, are able to synthesize IAA at a rate of 9.2 ng g-1 fresh weight h-1, or 4.8% of the in situ rate. This finding establishes that all of the enzymes of IAA biosynthesis occur in the endosperm and that the endosperm is a site of IAA biosynthesis. Isolated endosperm, prepared from developing kernels, synthesized IAA from labeled anthranilate at a rate of 8.6 ng g-1 fresh weight h-1, or 4.5% of the in situ rate. Frozen endosperm preparations maintained the ability to synthesize labeled IAA from labeled anthranilate. The identity of the synthesized IAA was established by mass spectral analysis. We suggest that endosperm preparations of Z. mays are suitable for study of the mechanism(s) of IAA biosynthesis because they (a) have high rates of synthesis; (b) show stability to freezing, enabling enzyme storage; (c) provide a system with a known rate of in situ synthesis; and (d) are available in large amounts for use as an enzyme source.