Effect of Auxin and Anti-auxin (4-Phenyl-3-carbostyriloxyacetic Acid) on the Growth of Escherichia coli

Indole-3-acetic acid (IAA) inhibited specifically the growth of a wild strain of Escherichia coli IFO 3545 in a glucose-free polypeptone medium adjusted to pH below 6.3. When 50 ppm of IAA was combined with 10 ppm of 4-phenyl-3-carbostyriloxyacetic acid (V-OCH₂COOH), an anti-auxin, inhibitory effect of IAA on the bacterial growth was markedly increased though V-OCH₂COOH alone had no effect. When 30 ppm of IAA was combined with 10 ppm of V-OCH₂COOH, inhibition increased initially, but soon decreased and disappeared. Riboflavin also increased the inhibitory effect of IAA under fluorescent light. Cysteine restored not only the effect of IAA alone but also the combination effect of IAA with riboflavin or V-OCH₂COOH. An intermediary metabolite of IAA in bacteria that appeared to be identical with a photooxidation product from IAA may actually inhibit the bacterial growth. It was suggested that V-OCH₂COOH stimulated the induction of IAA-metabolizing enzymes in bacteria, as in the case of plants.     

Gibberellin-induced ethylene production and its effect on cowpea epicotyl elongation

The effect of gibberellin A₁ (GA₁) on production of ethylene by cowpea (Vigna sinensis cv Blackeye pea no. 5) epicotyl explants and its relationship to epicotyl elongation was investigated. The explants were placed upright in water and incubated in sealed culture tubes or in large jars. GA, and IAA in ethanol solution were injected into the subapical tissues of the decapitated epicotyls. Cowpea epicotyl explants elongated after GA but not after IAA treatment, and they were very sensitive to exogenous ethylene. As little as 0.14 1/1 ethylene reduced significantly GA₁-induced epicotyl elongation.Treatment with GA₁ induced the production of ethylene which began 10 h after GA application, showed a peak at about 22 h and then declined. The yield of ethylene was proportional to the amount of GA, injected. The inhibition of epicotyl elongation in closed tubes was avoided by absorbing ethylene released with Hg(Cl0₄)₂ , or by adding AVG to the incubation solution to inhibit ethylene production. Treatment with IAA elicited a rapid production of ethylene which ceased about 10 h after application. The effects of IAA and GA₁ on ethylene production were additive.Abbreviations AVG aminoethoxyvinylglycine 2-amino-4-(2-aminoethoxy)-trans-3butenoic acid - ACC 1-aminocyclopropane-1-carboxylic acid - GA gibberellin - IAA indole-3-acetic acid     

Interaction of gibberellic and indole-3-acetic acid on root formation in pea (Pisum sativum L.) epicotyl cuttings

Indole-3-acetic acid (IAA) strongly enhanced rooting of etiolated pea epicotyl cuttings while gibberellic acid (GA₃) enhanced rooting only slightly. The promoting effects of the hormones appeared not until 14 d after the onset of treatment. When GA₃ and IAA were applied together, the initiation of rooting started already after 6 d after onset of treatment. It is suggested that gibberellin plays an important role, in combination with auxin, in the initiation of root formation in Pisum cuttings.Abbreviations IAA Indole-3-acetic acid - GA₃ Gibberellic acid     

Untersuchungen zum Wirkungsmechanismus von Indolyl-3-Essigsäure mit Hilfe von schwerem Wasser

Summary The auxin-induced cell elongation and the formation of indoleacetyl-aspartic acid (IAAsp) of pea epicotyl sections and Agrostemma hypocotyl sections are inhibited by heavy water. The formation of IAAsp requires a specific enzyme. The lack of IAAsp in D₂O-treated plant tissues may be due to an influence of D₂O on the induction or on the synthesis of that enzyme. Treatment of plant sections with synthetic IAAsp has no effect on the growth of the sections in D₂O. Indole-3-acetic acid (IAA) increases the incorporation of ³²P-orthophosphate into ribosomal and soluble RNA of pea epicotyl sections in H₂O but not in D₂O. The synthesis of ribosomal RNA is decreased by heavy water.The effects of IAA and D₂O on the soluble proteins of pea sections have been studied by PAA-gel electrophoresis. D₂O does not change the pattern of protein bands in comparison with the H₂O-control, but prevents the probably IAA-induced alteration of the Rf-value of one protein band on the pherogram. It is assumed that the inhibition of auxin-induced reactions in the D₂O-medium is due to the stabilizing effect of heavy water on allosteric proteins. The results of this work support the hypothesis that IAA acts as allosteric effector.     

Possible explanation of IAA-stimulated transport of14C-ABA in long pea (Pisum sativum L.) epicotyl segments

Effects of 2,3,5-triiodobenzoic acid (TIBA) and age of etiolated pea epicotyl segments on the indol-3-ylacetic acid (IAA) stimulated transport of¹⁴C-abscisic acid (ABA) was studied. In spite of a slight decrease of IAA transport after the application of TIBA, the IAA stimulation of¹⁴C-ABA transport did not change. In segments excised from epicotyls of different age,³H-IAA transport was identical and the induction of prolongation growth by IAA in segments from the upper part of the epicotyl was observed. The IAA ap{ie226-01}ation to the growing segments was connected with intensive attraction of¹⁴C-ABA to the site {ie226-02}AA application, while the application of IAA to the older segments was growth ineffective ana no stimulation of¹⁴C-ABA transport by IAA was observed.     

Inhibitory effects of monoamine oxidase inhibitors on the growth of pea and rice seedlings

Two monoamine oxidase inhibitors of the hydrazine-type, safrazineand nialamide, inhibited growth in seedlings of rice and pea.We demonstrated histochemically that monoamine oxidase is locatedchiefly in sieve tubes and in the epidermis of pea seedling.Activity of this enzyme was high in the apical part of the epicotyl,decreasing toward the base. Inhibition of pea monoamine oxidaseby safrazine and nialamide was observed histochemically andwith an extract from the epicotyl. This supports the hypothesisthat indole-3-acetic acid (IAA) is formed from tryptamine byamine oxidase and that inhibition of this enzyme causes loweringof the auxin level, resulting in growth inhibition. Inhibitionof growth in rice seedlings by safrazine was reversed by theaddition of IAA to the culture medium. (Received May 6, 1970; )     

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg²⁺ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K _m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k _cat/K _m = 682,608.7 and 5080 s⁻¹ M⁻¹, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.     

An alternative explanation for plant growth promotion by bacteria of the genus Azospirillum

Experiments were performed to identify the substances that are excreted by the soil bacterium Azospirillum brasilense Sp7 and that were reported to stimulate the formation of lateral roots and of root hairs of grasses. Azospirillum forms indole-3 acetic acid (IAA) but only in the late stationary growth phase or when tryptophan is present in the medium, but not in continuous cultures or in the logarithmic growth phase of batch cultures. Formation of IAA by Azospirillum requires aerobic conditions. Nitrite can replace IAA in several phytohormone assay, and is even more active than IAA in a test with wheat root segments in which the increase of wet weight is determined. Higher amounts of nitrite are necessary for activity in other classical auxin assays. Nitrite shows 40–60% of the activity of IAA in the straight-growth test of Avena coleoptiles and in the formation of C₂H₄ by pea epicotyl segments. Like IAA, nitrite is inactive in promoting C₂H₄ formation by ripe apple tissues. Since nitrite alone can hardly exert phytohormonal effects, it is postulated that nitrite reacts with a substance in the cells and that a product formed by this reaction functions as auxin. Such a substance could be ascorbate. Exogenously added ascorbate enhances the rate of nitrite-dependent C₂H₄ formation by pea epicotyl sections and the nitrite-dependent increase in the wet weight of wheat root segments. Nitrite is formed by nitrate respiration of Azospirillum. The findings that nitrite can have phytohormonal effects offers an alternative explanation of the promotion of the growth of roots and the enhancement of mineral uptake of grasses by Azospirillum. Indole-acetic acid completely and nitrite partly substitute for an inoculation with Azospirillum in an assay where the increase of the dry weight of intact wheat roots is determined after an incubation for 10 d. Nitrite and IAA are, therefore, possibly the only factors causing an enhancement of the growth of roots of grasses.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid     

Occurrence of diamine oxidase in the apoplast of pea epicotyls

Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of H₂O₂ to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.Abbreviations DAO diamine oxidase - Glc6P glucose-6-phosphate     

Indole-3-acetic Acid Oxidase from Peas: I. Occurrence and Distribution of Peroxidative and Nonperoxidative Forms

Tissues of etiolated pea seedlings variety Alaska were examined for the presence of peroxidative and nonperoxidative forms of indoleacetic acid (IAA) oxidase. Enzymes were extracted in a sequence involving acetone powder preparation from pea tissues, buffer extraction of the powder, ammonium sulfate precipitation, dialysis, lyophilization, and acrylamide gel electrophoresis. Electrophoretically separable proteins were assayed for IAA oxidase activity with the Salkowski test, and peroxidase activity was based on the color reaction with benzidine and H₂O₂. Each tissue examined contained several nonperoxidative IAA oxidases. No tissue contained more than three peroxidative IAA oxidases, whereas the plumule hooks (a tissue with a high IAA oxidase activity) contained no detectable peroxidases. The results indicate that nonperoxidative IAA oxidases might play a major role in the regulation of IAA content in pea seedlings.     

Physiological activity of a viridicatin derivative, 3-(4-phenylcarbostyriloxy) acetic acid

A carboxymethylene derivative (V-OCH₂COOH) of viridicatin (V-OH)promoted the root growth of rice and sesame seedlings. V-OCH₂COOHhad no known hormonal activities, per se, but did have an inhibitoryeffect on IAA and 2,4-D-induced growth of Avena coleoptile sectionsand of carrot root callus. However, inhibition by VOCH2COOHof 2,4-D-induced growth in carrot root callus was to some extentreversed by increasing the concentration of 2,4-D. V-OCH₂C0OHseemed to competitively inhibit IAA-induced elongation of Avenacoleoptile sections. (Received September 14, 1970; )     

On the role of calcium in indole-3-acetic acid movement and graviresponse in etiolated pea epicotyls

To determine whether Ca²⁺ plays a special role in the early graviresponse of shoots, as has been reported for roots, we treated etiolated pea epicotyls with substances known to antagonize Ca²⁺ (La³⁺), to remove Ca²⁺ from the wall (spermidine, EGTA), to inhibit calmodulin mediated reactions (chlorpromazine), or to inhibit IAA transport (TIBA). We studied the effect of these substances on IAA and Ca²⁺ uptake into 7 mm long subapical 3rd internode etiolated pea epicotyl sections and pea leaf protoplasts, on pea epicotyl growth, and graviresponse and on lateral IAA redistribution during gravistimulation.Our results support the view that adequate Ca²⁺ in the apoplast is required for normal IAA uptake, transport and graviresponse. Experiments with protoplasts indicate that Ca²⁺ may be controlling a labile membrane porter, possibly located on the external surface of cell membrane, while inhibitor experiments suggest that calmodulin is also implicated in both the movement of IAA and graviresponse. Since a major transfer of Ca²⁺ through free space during graviresponse has not yet been demonstrated, and since inhibition of calcium channels does not affect IAA redistribution (Migliaccio and Galston, 1987, Plant Physiology 85:542), we conclude that no clear evidence links prior Ca²⁺ movement with IAA redistribution during graviresponse in stems.Abbreviations IAA indole-3-acetic acid - CPZ chlorpromazine - EGTA ethylene glycol bis-(aminoethyl ether) N, N, N¹, N¹-tetracetic acid - G C gravicurvature The research was supported by NASA grant NSG-7290 to AWG.     

Gas chromatography-mass spectrometry evidence for several endogenous auxins in pea seedling organs

Qualitative analysis by gas chromatography-mass spectrometry (GC-MS) of the auxins present in the root, cotyledons and epicotyl of 3-dold etiolated pea (Pisum sativum L., cv. Alaska) seedlings has shown that all three organs contain phenylacetic acid (PAA), 3-indoleacetic acid (IAA) and 4-chloro-3-indoleacetic acid (4Cl-IAA). In addition, 3-indolepropionic acid (IPA) was present in the root and 3-indolebutyric acid (IBA) was detected in both root and epicotyl. Phenylacetic acid, IAA and IPA were measured quantitatively in the three organs by GC-MS-single ion monitoring, using deuterated internal standards. Levels of IAA were found to range from 13 to 115 pmol g^-1 FW, while amounts of PAA were considerably higher (347–451 pmol g^-1 FW) and the level of IPA was quite low (5 pmol g^-1 FW). On a molar basis the PAA:IAA ratio in the whole seedling was approx. 15:1.Abbreviations IAA 3-indoleacetic acid - 4Cl-IAA 4-chloro-3-indoleacetic acid - IBA 3-indolebutyric acid - IPA 3-indolepropionic acid - PAA phenylacetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFB pentafluorobenzyl ester - PFBBr pentafluorobenzyl bromide - SIM single-ion monitoring - TMSI trimethylsilyl ester     

Physiology of gibberellin-induced elongation of epicotyl explants from Vigna sinensis

The physiological characteristics of the response of excised cowpea (Vigna sinensis cv Blackeye pea No. 5) epicotyls to gibberellins (GAs) were studied. Epicotyl explants, retaining the petioles and a 2-cm portion of hypocotyl, were placed upright in small vials containing water. Plant growth substances were injected into the subapical tissues as ethanol solutions.Epicotyl elongation resulting from treatment with 0.5 g of GA ranged between 5 and 13 times that of the control, depending on the GA applied. With GA₁, no differences were obtained with explants prepared from 5 to 9-day-old seedlings. The increase in elongation could be detected within 6 h of treatment, and the stimulus of a single application lasted at least 4 days. Final elongation was proportional to the logarithm of the amount of GA, applied, 0.01 to lug. The response to GA treatment was limited to the upper part, the most sensitive zone being located between 2 to 4 mm below the apex of the epicotyl; this effect was entirely due to cell elongation.The induction of epicotyl elongation by GAs seems to be specific and independent of the effect of auxin. IAA had no effect on elongation and 4-chloro-phenoxyisobutyric acid (PCIB) did not affect the response to GA₁ Abbreviations ABA abscisic acid - GA gibberellin - IAA Indole-3-acetic acid - TIBA 2,3,5-triiodobenzoic acid - PCIB 4-chloro-phenoxyisobutyric acid     

Particulate glucan synthetase activity: generation and inactivation after treatments with indoleacetic acid and cycloheximide

Growing regions from epicotyls of Pisum sativum L. var Alaska contain a particulate enzyme which transfers glucose from guanosine diphosphate glucose to alkali-soluble and -insoluble products (glucan synthetase activity). When the epicotyl is decapitated to remove the source of natural hormone, the tissue below ceases growth and loses synthetase activity as well as the capacity to continue forming cellulose in vivo. If indoleacetic acid (IAA) is added to the cut apex, massive amounts of cellulose are deposited in the next few days. Particulate glucan synthetase activity is either maintained or greatly increased depending on whether endogenous activity levels are relatively high or low at the time of hormone addition. These effects appear to be due in part to IAA-dependent generation of a protein essential for synthetase activity since they are severely inhibited by concentrations of cycloheximide which are effective at preventing protein synthesis. Nevertheless, the addition of cycloheximide alone to the epicotyl reduces the rate of disappearance of synthetase activity, i.e., a protective effect. Also, a soluble thermolabile component is present in the aging epicotyl which promotes loss of synthetase activity when added to the particulate enzyme in vitro. Accordingly, turnover of pea glucan synthetase activity may be controlled in part by an inactivating protein which is itself subject to turnover.     

Proposed Enzymes of Auxin Biosynthesis and Their Regulation III. Some Properties of Pea Indolylacetaldehyde Oxidase

Indole-3-acetaldehyde oxidase (IAAld-oxidase) occurs in pea in two forms, of which the first, more active enzyme, has its pH optimum at 4.5, while the second, barely half as active, has a pH optimum at 7.0. Only the pH 4.5 oxidase can be resolved from the acetone powder. Besides IAA1d the more stable IA1d was used as substrate in testing the enzymatic activity. The pea enzyme seems not to be a dismutase since indolylmethanol or indolylethanol were not formed as products. Pyridine nucleotide coenzymes did not activate the partially purified enzyme. The pH 4.5 oxidase was inhibited by more than 50 % by IAA > L-asp > tryptophol > indoleacetylaspartic acid > 2,4-D (at 1 mM concentration). The pH 7.0 oxidase was inhibited relatively more weakly, a stronger than 50 % inhibition was caused only by NAA > L-asp. The oxidases were clearly distinguished by the response to L-asparagine (1 mM): the activity of the pH 4.5 oxidase was increased (+ 12 %), while the activity of the pH 7.0 oxidase was decreased (-71 %). In preliminaryin vitro experiments the phytohormones (1 mM) kinetin and GA₃ increased the conversion of IAAld to IAA, while ABA decreased it.     

The Auxins IAA and 4-Cl-IAA Differentially Modify Gibberellin Action via Ethylene Response in Developing Pea Fruit

This study explores the unique growth-regulatory roles of two naturally occurring auxins, indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA), and their interactions with gibberellin (GA) during early pea (Pisum sativum L.) fruit development. We have previously shown that 4-Cl-IAA can replace the seed requirement in pea pericarp growth (length and fresh weight), whereas IAA had no effect or was inhibitory. When applied simultaneously, gibberellin (GA₃ or GA₁) and 4-Cl-IAA had a synergistic effect on pericarp growth. In the present study, we found that simultaneous application of IAA and GA₃ to deseeded pericarps inhibited GA₃-stimulated growth. The inhibitory effect of IAA on GA-stimulated growth was mimicked by treatment with ethephon (ethylene releasing agent), and the inhibitory effects of IAA and ethylene on GA-mediated growth were reversed by silver thiosulfate (STS), an ethylene action inhibitor. Although pretreatment with STS could retard senescence of IAA-treated pericarps, STS pretreatment did not lead to IAA-induced pericarp growth. Although 4-Cl-IAA stimulated growth whereas IAA was ineffective, both auxins induced similar levels of ethylene evolution. However, only 4-Cl-IAA-stimulated growth was insensitive to the effects of ethylene. Gibberellin treatment did not influence the amount of ethylene released from pericarps in the presence or absence of either auxin. We propose a growth regulatory role for 4-Cl-IAA through induction of GA biosynthesis and inhibition of ethylene action. Additionally, ethylene (IAA-induced or IAA-independent) may inhibit GA responses under physiological conditions that limit fruit growth.     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

RECOVERY OF NATIVE AND APPLIED AUXIN FROM THE LIGHT-GROWN ‘ALASKA’ PEA SEEDLING

Scott , Tom K., and Winslow R. Briggs . (Stanford U., Stanford, Calif.) Recovery of native and applied auxin from the light-grown ‘Alaska’ pea seedling. Amer. Jour. Bot. 49(10): 1056–1063. Illus. 1962.—The physiological status of both endogenous and exogenously applied auxin was compared in the epicotyl of the 9-day-old light-grown ‘Alaska’ pea (Pisum sativum L.) by means of agar-diffusion and short-term ether extraction. A detailed analysis of endogenous auxin revealed a linear basipetal decrease in diffusible auxin within the growing region. A decrease in extractable auxin occurred only within the most mature region. The capacity for uptake of indole-3-acetic acid (IAA), applied in lanolin paste, was compared in different regions of the epicotyl. The fifth and most apical internode had the greatest capacity for uptake as measured by extraction. A reduced capacity was found in more basal internodes. The transport rate of applied IAA, under conditions of optimal uptake, was 10–12 mm/hr. An application of IAA for 24 hr resulted in a dramatic increase in auxin content throughout the length of the epicotyl compared to that found in the normal control. There was no apparent gradation in content from apex to base. An increase of diffusible auxin was also found, but only in the fourth and third internodes. That no such increase was detected in the basal 3 internodes suggested that the auxin transport system within this region had special properties related to a transition between shoot and root vascular patterns.