A Mutation Altering Auxin Homeostasis and Plant Morphology in Arabidopsis

Many aspects of plant development are associated with changing concentrations of the phytohormone auxin. Several stages of root formation exhibit extreme sensitivities to exogenous auxin and are correlated with shifts in endogenous auxin concentration. In an effort to elucidate mechanisms regulating development of adventitious roots, an ethyl methanesulfonate-mutagenized M2 population of Arabidopsis was screened for mutants altered in this process. A recessive nuclear mutant, rooty (rty), displayed extreme proliferation of roots, inhibition of shoot growth, and other alterations suggesting elevated responses to auxin or ethylene. Wild-type Arabidopsis seedlings grown on auxin-containing media phenocopied rty, whereas rty seedlings were partially rescued on cytokinin-containing media. Analysis by gas chromatography-selected ion monitoring-mass spectrometry showed endogenous indole-3-acetic acid concentrations to be two to 17 times higher in rty than in the wild type. Dose-response assays with exogenous indole-3-acetic acid indicated equal sensitivities to auxin in tissues of the wild type and rty. Combining rty with mutations conferring resistance to auxin (axr1-3) or ethylene (etr1-1) suggested that root proliferation and restricted shoot growth are auxin effects, whereas other phenotypic alterations are due to ethylene. Four mutant alleles from independently mutagenized populations were identified, and the locus was mapped using morphological and restriction fragment length polymorphism markers to 3.9 centimorgans distal to marker m605 on chromosome 2. The wild-type RTY gene product may serve a critical role in regulating auxin concentrations and thereby facilitating normal plant growth and development.     

Mechanism of Indole-3-acetic Acid Conjugation: No Induction by Ethylene

Formation of indole-3-acetic acid-aspartate in detached primary leaves of cowpea (Vigna sinensis Endl.) floating on (14)C-indole-3-acetic acid (3 muc; 3.15 mum, phosphate-citrate buffer, pH 4.75), almost doubled when leaves were pretreated with 31.5 mum(12)C-indole-3-acetic acid for 17 hr and then transferred to (14)C-indole-3-acetic acid for 4 hours as compared with leaves preincubated in buffer only. When leaves were preincubated with ethylene (11.0 and 104 mul/l) instead of (12)C-indole-3-acetic acid, no induction of indole-3-acetylaspartic acid formation was observed, and the rate of indole-3-acetylaspartic acid formation decreased as compared with control leaves. Rhizobitoxine (1.87 mum) inhibited indole-3-acetic acid-induced ethylene production but did not prevent the formation of indole-3-acetylaspartic acid. In view of the similarity of these results and those previously obtained with alpha-naphthaleneacetic acid, it is concluded that ethylene has no role in the auxin-induced indole-3-acetylaspartic acid formation in cowpea leaves.     

Comparative indole-3-acetic Acid levels in the slender pea and other pea phenotypes

Free indole-3-acetic acid levels were measured by gas chromatography-mass spectrometry in three ultra-tall `slender' Pisum sativum L. lines differing in gibberellin content. Measurements were made for apices and stem elongation zones of light-grown plants and values were compared with wild-type, dwarf, and nana phenotypes in which internode length is genetically regulated, purportedly via the gibberellin level. Indole-3-acetic acid levels of growing stems paralleled growth rates in all lines, and were high in all three slender genotypes. Growth was inhibited by p-chlorophenoxyisobutyric acid, demonstrating the requirement of auxin activity for stem elongation, and also by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. It is concluded that the slender phenotype may arise from constant activation of a gibberellin receptor or transduction chain event leading directly or indirectly to elevated levels of indole-3-acetic acid, and that increased indole-3-acetic acid levels are a significant factor in the promotion of stem elongation.     

Cell elongation in the grass pulvinus in response to geotropic stimulation and auxin application

Summary Horizontally-placed segments of Avena sativa L. shoots show a negative geotropic response after a period of 30 min. This response is based on cell elongation on the lower side of the leaf-sheath base (pulvinus). Triticum aestivum L., Hordeum vulgare L. and Secale cereale L. also show geotropic responses that are similar to those in Avena shoots. The pulvinus is a highly specialized organ with radial symmetry and is made up of epidermal, vascular, parenchymatous and collenchymatous tissues. Statoliths, which are confined to parenchyma cells around the vascular bundles, sediment towards the gravitational field within 10–15 min of geotropic stimulation. Collenchymatous cells occur as prominent bundle caps, and in Avena, they occupy about 30% of the volume of the pulvinus. Geotropic stimulation causes a 3- to 5-fold increase in the length of the cells on the side nearest to the center of the gravitational field. Growth can also be initiated in vertically-held pulvini by the application of indole-3-acetic acid, 1-naphthaleneacetic acid or 2.4-dichlorophenoxyacetic acid. 2.3.5.-triiodobenzoic acid interferes with growth response produced by geotropic stimulation as well as with the response caused by auxin application. Gibberellic acid and kinetin have no visible effect on the growth of the pulvinus. Polarization microscopy shows a unique, non-uniform stretching of the elongating collenchymatous cells. Nonelongated collenchymatous cells appear uniformally anisotropic. After geotropic stimulation or auxin application, they appear alternately anisotropic and almost isotropic. Such a pattern of cell elongation is also observed in collenchyma cells of geotropically-stimulated shoots of Rumex acetosa L., a dicotyledon.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - TIBA 2.3.5-triiodobenzoic acid     

The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments

Summary In previous research here, 3,5-diiodo-4-hydroxybenzoic acid (DIHB) was shown to promote the elongation of roots of cress (Lepidium sativum) seedlings growing in light, and to inhibit the auxin-induced production of ethylene in this tissue. Although DIHB is a cofactor for the oxidation of indole-3-acetic acid (IAA) by horse-radish peroxidase, it inhibits the decarboxylation of [1-¹⁴C]IAA by segments excised from cress roots. The inhibition by DIHB of ethylene production by this tissue does not, therefore, arise from a reduction of IAA levels. These findings are discussed in relation to the effects of DIHB on cress root growth.Abbreviations IAA indole-3-acetic acid - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DCP 2,4-dichlorophenol - 2,4-D 2,4-dichlorophenoxyacetic acid This study forms part of a research project to be submitted by M.L.R. for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).     

Effects of Seselin and Coumarin on Growth, Indoleacetic Acid Oxidase, and Peroxidase, with Special Reference to Cucumber (Cucumis sativa L.) Radicles

Seselin, a natural coumarin derivative isolated from citrus roots, inhibited radicle growth in seedlings of cucumber (Cucumis sativa), lettuce (Lactuca sativum), radish (Raphanus sativus), and wheat (Triticum aestivum) grown in the dark. Coumarin similarly inhibited radicle growth of cucumber seedlings. Growth retardation of the cucumber radicles was accompanied by an increased activity of peroxidase and indole-3-acetic acid oxidase. Both compounds antagonized indole-3-acetic acid-induced growth of wheat coleoptiles, whereas coumarin was much less effective than seselin in antagonizing gibberellic acid-induced release of reducing sugars from barley endosperm. It is suggested that seselin plays an important role in the regulation of root growth, and that it is the indole-3-acetic acid oxidase cofactor previously detected in citrus roots.     

Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.     

Immunocytochemical localization of indole-3-acetic acid in primary roots of Zea mays

Colloidal gold-labelled antibody was used to localize indole-3-acetic acid in caps of primary roots of Z. mays cv. Kys. Gold particles accumulated on the nucleus, vacuoles, mitochondria, and some dictyosomes and dictyosome-derived vesicles. This is the first localization of indole-3-acetic acid in dictyosomes and dictyosome-derived vesicles and indicates that dictyosomes and vesicles constitute a pathway for indole-3-acetic acid movement in and secretion from root cap cells. Our findings provide cytochemical evidence to support the hypothesis that indole-3-acetic acid plays an important role in root gravitropism.     

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.     

Inactivity of Oxidation Products of Indole-3-acetic Acid on Ethylene Production in Mung Bean Hypocotyls

The suggestion that indole-3-acetic acid (IAA)-stimulated ethylene production is associated with oxidative degradation of IAA and is mediated by 3-methyleneoxindole (MOI) has been tested in mung bean (Phaseolus aureus Roxb.) hypocotyl segments. While IAA actively stimulated ethylene production, MOI and indole-3-aldehyde, the major products of IAA oxidation, were inactive. Tissues treated with a mixture of intermediates of IAA oxidation, obtained from a 1-hour incubation of IAA with peroxidase, failed to stimulate ethylene production. Furthermore, chlorogenic acid and p-coumaric acid, which are known to interfere with the enzymic oxidation of IAA to MOI, had no effect on IAA-stimulated ethylene production. Other oxidation products of IAA, including oxindole-3-acetic acid, indole-3-carboxylic acid, (2-sulfoindole)-3-acetic acid, and dioxindole-3-acetic acid, were all inactive. 1-Naphthaleneacetic acid was as active as IAA in stimulating ethylene production but was decarboxylated at a much lower rate than IAA, suggesting that oxidative decarboxylation of auxins is not linked to ethylene production. These results demonstrate that IAA-stimulated ethylene production in mung bean hypocotyl tissue is not mediated by MOI or other associated oxidative products of IAA.     

Inactivity of 3-methyleneoxindole as mediator of auxin action on cell elongation

The recently reported growth-promoting ability of 3-methyl-eneoxindole was examined in order to test the hypothesis that indole-3-acetic acid acts as a growth promoter only after oxidative conversion to 3-methyleneoxindole. Methyleneoxindole was synthesized from indole-3-acetic acid and N-bromosuccinimide, and its identity was confirmed by ultraviolet absorption, infrared absorption, mass spectrometry, and melting point. Methyleneoxindole was found to lack growth-promoting activity in coleoptile and pea (Pisum sativum) stem segments. Chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid, was found to have no inhibitory effect on growth promotion by indole-3-acetic acid. It is concluded that 3-methyleneoxindole is inactive as a growth promoter and therefore does not mediate the action of auxin on cell elongation.     

Auxin activity of 3-methyleneoxindole in wheat

A product of the enzymatic oxidation of indole-3-acetic acid, 3-methyleneoxindole, is at least 50-fold more effective than indole-3-acetic acid in stimulating the growth of wheat (Triticum vulgare, red variety) coleoptiles. Ethylenediaminetetra-acetic acid can antagonize the growth-stimulating properties of the parent compound, indole-3-acetic acid, presumably by chelating Mn²⁺, which is required for the enzymatic oxidation of indole-3-acetic acid. The growth stimulating effect of 3-methyleneoxindole, a product of the blocked reaction, on the other hand, is still evident in the presence of ethylenedia-minetetraacetic acid. In the presence of 2-mercaptoethanol, indole-3-acetic acid fails to stimulate the elongation of wheat coleoptiles. The property of binding to sulfhydryl compounds including 2-mercaptoethanol is unique to 3-methyleneoxindole among indole-3-acetic acid and its oxidation products. These findings suggest that 3-methyleneoxindole is an obligatory intermediate in indole-3-acetic acid induced elongation of wheat coleoptiles.     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

Ethylene-enhanced catabolism of [C]indole-3-acetic Acid to indole-3-carboxylic Acid in citrus leaf tissues

Exogenous [¹⁴C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole-3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [¹⁴C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGIu). Increased formation of ICGIu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGIu were identified by combined gas chromatography-mass spectrometry. Formation of ICGIu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene reduces endogenous IAA levels.     

Local application of indole-3-acetic acid,by resin beads to intact growing maize roots

Five types of anion-exchanger resin beads which had adsorbed indole-3-acetic acid (IAA) were tested as IAA donors. The rate of IAA-uptake by beads was a function of time and pH. The release was relatively steady during 6 h application on vertical maize roots. No IAA degradation occurred in the beads (Amberlite IRA 400 type) but 45.8% was metabolised in the roots during treatment. Beads loaded with IAA and placed on one side of the root (at 2.20±0.03 mm from the tip) induced a curvature towards and above the bead (23.3±1.1 degrees after 5.25 h application). In contrast, control beads (without IAA) did not change the axial growth rate. Applied IAA seemed to move differently from endogenous IAA. The use of resin beads loaded with IAA offers a technique to study the effects of local IAA application on intact growing roots.Abbreviations 3,3-DGA 3,3 dimethyl-glutaric acid - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Ox-IAA oxindole-3-acetic acid     

Promotion of peroxidase activity in the cell wall of Nicotiana

Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5-triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin.     

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     

Short-term aluminium-induced changes in barley root tips

The short-term exposure of barley roots to low Al concentration caused significant root growth inhibition and radial swelling of roots. During Al treatment, the radial expansion of root cells occurred in root tissues representing elongation zone and meristem. Both low pH and Al treatments caused significant disruption of cell membranes in swollen roots. In contrast to Evans blue uptake callose formation was observed only at higher Al concentrations and was detected in both swollen and adjacent root areas. Similarly to Al, exogenous short-term application of indole-3-acetic acid, polar transport inhibitor triiodobenzoic acid, ethylene precursor 1-aminocyclopropane-1-carboxylic acid or H₂O₂ evoked root growth inhibition and radial cell expansion in barley root tip too.     

Ethylene enhances reactivity of superoxide with peroxidase to form the oxy-ferrous complex

Ethylene and its analogues acetylene, carbon monoxide, and propylene inhibited the rate of oxidation of indole-3-acetic acid by peroxidase. Annulment of this effect by addition of superoxide dismutase showed that inhibition occurred only in the presence of the superoxide anion radical (O2-.). Kinetic and spectral data established that ethylene and its analogues enhanced markedly the rate of reaction of O2-. with peroxidase. This reaction resulted in the formation of compound III, an oxy-ferrous complex of peroxidase. In the presence of indole-3-acetic acid, the interaction between ethylene, peroxidase, and O2-. activated the reduced peroxidase in equilibrium compound III shuttle. O2-. is a major product of this shuttle, and compound III constitutes the dominant steady-state form of peroxidase. These interactions may help to explain the mechanism of action of ethylene as a plant growth regulator.     

Micropropagation of lemon balm

Traditional propagation of lemon balm (Melissa officinalis L.) is inefficient for establishing a good quality clonal population. Results of the presented experiments outline an effective method for micropropagation of this species. Following culture initiation from shoots of field-grown plants on growth regulator free Murashige–Skoog medium, rapid shoot multiplication with only rudimentary root formation could be achieved on media containing various concentrations of indole-3-acetic acid and 6-benzyladenine. The combination of 5.71 μM indole-3-acetic acid and 6.66 μM 6-benzyladenine resulted in the best multiplication. Transfer of propagules to media containing indole-3-acetic acid and kinetin did not result in shoot proliferation; however, single plantlets grown on media containing 5.71 μM indole-3-acetic acid and 13.9 μM kinetin developed more compact shoots and stronger roots than the control plants and were suitable for acclimatisation with an efficiency over 95%. This revised version was published online in June 2006 with corrections to the Cover Date.