Metabolism of Indole-3-acetaldehyde

Infiltration of indolcacelaldehyde (IAAId) into living tissues of sonic lower and higher plants gives rise simultaneously to both indoleacetic acid (IAA) and Iryptophol (T-ol). But on a molar basis, there is no correlation between the products indicating a disimitation. Expressed juice of Avena coleoptiles by itself, exhibits only IAA forming activity. Approximately two moles of IAAld are consumed for each mole of IAA formed at pH 4.5, but only if necessary corrections are made for losses of substrate and products. Addition of reduced NAD or NADP readily induces tryptophol formation. But even at pH 4.5, adding reduced NADP causes greater tryptophol formation, leading to a marked divergence in the acid-alcohol ratio. Varying the pH in the presence of reduced coenzymes also alters the ratio, with alcohol formation predominating. NAD and NADP have no influence on the formation of IAA from IAAld by the whole cytoplasm of Avena coleoptiles. Whole cytoplasm of Asparagus shoots forms both IAA and tryptophol from IAAld, but in widely varying amounts, devoid of any suggestive stoichiometry between the products. With the acetone powder of Avena coleoptiles including the first leaf, data indicating an apparent disimitation of IAAld are obtainable only at pH 4.5. On altering the pH however, unequal amounts of the two products, namely IAA and tryptophol, are formed and hence a different ratio results. Acetone powders of wheat coleoptiles and Asparagus shoots do not yield data supporting disimitation either at pH 4.5 or 7.2. IAA formation in Avena is aerobic while tryptophol formation is seemingly independent of oxygen supply. The former activity is selectively abolished by 10^?3M dithionite while the latter activity suffers a similar suppression in the presence of 10^?3M manganese sulphate. Varying the IAAld concentration results in unequal amounts of the two products, revealing the dissimilar affinity of the two activities for the common substrate Saturation to a level of 30 percent with ammonium sulphate throws out most of the acid-forming activity whereas the alcohol-forming system appears mostly in the protein fraction precipitated between 30 and 40 percent saturation. The enzyme system of Avena coleoptiles oxidizing IAAld to IAA can also be easily separated by Sephadex gel filtration and its independent activity demonstrated in the total absence of tryptophol formation. Based on the heterogeneous properties of the two activities, metabolism of IAAld in Avena coleoptiles is believed to be mediated by two independent enzyme systems without the intervention of a mutase or a dismutation mechanism.     

The formation of tryptophol glucoside in the tryptamine metabolism of pea seedlings

Summary Tryptamine was converted by etiolated pea seedlings into IAA, tryptophol, and an appreciable amount of an unknown metabolite. This latter compound was characterised by TLC and electrophoresis and identified, by mass spectrometry and enzymatic cleavage, as tryptophol glycoside: indole-3-ethyl--d-glycopyranoside.Abbreviations IAA indole-3-acetic acid - IAAld indole-3-acetaldehyde - TOH tryptophol - TO-glc tryptophol glucoside     

Metabolism of indole-3-acetaldoxime in plants

Summary Living tissues of diverse plants representing 17 families were infiltrated with indole-3-acetaldoxime (IAAld oxime) in phosphate buffer, pH 6, and incubated for 3 hours at 25°C. Indole compounds were then extracted, separated and identified by paper or thin-layer chromatography (TLC). Indole-3-acetic acid (IAA) was quantitatively determined. Every tissue tested converted the oxime to IAA and tryptophol (T-ol). While accumulation of indole-3-acetonitrile (IAN) was observed in the non-acidic fractions of extracts of tissues of 8 species, indole-3-acetaldehyde (IAAld) accumulated in only a single tissue viz. Amaranthus shoot.IAAld oxime undergoes spontaneous hydrolysis at pH values below 4.7 leading to the formation of IAAld. Ce l-free preparations of etiolated Avena coleoptiles appear to contain an enzyme system capable of hydrolysing the oxime to IAAld. In the presence of such preparations, more IAAld and IAA are formed at all tested durations than the spontaneously formed IAAld. In the presence of bisulfite or semicarbazide, no IAA is formed, suggesting the intermediary formation of IAAld. The compound trapped with sodium bisulfite resembles very closely synthetic IAAld in its IR spectrum.In intact tissues, therefore, IAAld oxime appears to be first hydrolysed to IAAld which is then partly oxidized to IAA and mostly reduced to T-ol. Besides other evidence, formation of T-ol in every instance is believed to indicate the intermediary formation of IAAld. The nitrile pathway is considered to be only of minor importance in normal IAA biogenesis in the majority of higher plants.     

Pathways of IAA Production from Tryptophan by Plants and by Their Epiphytic Bacteria: A Comparison

Metabolites of tryptophan were investigated using 2 systems: a bacterial (Peastem homogenates containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). The plant system produces: indolepyruvic acid (IPyA), indoleacetaldehyde (IAAld) indoleacetic acid (IAA), indoleethanol (tryptophol, IAAol), indolecarboxylie acid (ICA), indolecarboxaldehyde (ICAld). Bacteria produce additionally: indoleactic acid (ILA), tryptamine (TNH₂) and the unknown Xb and Yb, but IAAld was not detected. A nonacidic inhibitor extract from pea stems decreases the gain of IAA, IPyA, ILA, Yb. It increases the gain of IAAld, IAAol, TNH₂, Xb, and (only in the bacterial system) ICA and ICAld. Three sites of inhibitor action are suggested, namely the steps Try → IPyA, TNH₂→ IAAld, IAAld → IAA.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Occurrence of Indoleacetaldehyde and Tryptophol in the Extracts of Etiolated Shoots of Pisum and Helianthus Seedlings

Presence of a non-acidic, growth-promoting indole compound, identical in R_f and colour reaction and closely similar in UV spectrum to synthetic indoleacetaldehyde (IAAId) has been demonstrated in extracts of etiolated shoots of Pisum and Helianthus. On chemical as well as enzymic oxidation of this neutral extract, activity passes over into the acidic fraction. This neutral substance is believed to be IAAId and occurs in the free state as short-term, low temperature extractions indicate. On the basis of R_f, colour reaction and UV spectrum, tryptophol has also been identified in Helianthus extracts. Its identification in pea extracts, however, rests only on R_f, colour reaction and inactivity in Avena bioassays.     

Quantitative Determination of Indole-3-Acetaldehyde. II.

The method by Larsen and Klungsöyr (1964) for the quantitative determination of indole-3-acetaldehyde (IAAld) was modified for the purpose of eliminating the need for filtration after oxidation of the IAAld to indole-3-acetic acid (IAA). The essentials of the modified method are as follows: Samples of IAAld or IAA containing 0.015 to 0.15 μmol (ca. 2.5 to 25 μg) dissolved in peroxide-free ether are evaporated to dryness and redissolved in 1.5 ml 0.02 M Ag2SO4. The oxidation is carried out in dim light by adding 0.5 ml 0.12 N NaOH. After 1.5 min, 2 ml of a modified Salkowski reagent are added. The optical density at 525 nm is read on a spectrophotometer after 75 min. The modified Salkowski reagent consists of 100 ml 0.05 M Fe2(SO4)3 in 1.5 N H2SO4; 240 ml H2O; and 160 ml cone. H2SO4 (sp. gr. 1.84). O.D. readings are identical for equal samples of IAAld and IAA (the latter used as a standard) up to 0.08 μmol (O.D. = 0.32). Larger quantities of IAAld may be determined when using pure IAAld as a standard, but at 0.20 μmol the O.D. for IAAld is lower than for IAA (0.69 as against 0.72). Indole-3-acetonitrile, tryptophol, indole-3-carboxylic acid, and indole-3-aldehyde all give O.D. values lower than 0.1 when tested at 0.20 μmol under the same conditions as described for IAAld and IAA.     

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.     

La biosynthèse de l'acide indolyl-3-acétique en liaison avec le métabolisme du tryptophol et de l'indolyl-3-acétaldéhyde chez Rhizobium

Several indolic compounds are formed when tryptophan or tryptamine is metabolized by Rhizobium. Among these are indole-3-acetaldehyde (IAAld), tryptophol (Tr-ol), and indole-3-acetic acid (IAA). The metabolic relationship among the three compounds was investigated. The experiments were carried out either in the culture medium of growing Rhizobium cultures or in suspensions of washed bacterial cells. In neither case Tr-ol would function as a precursor of IAA, but tryptophan-2-¹⁴G gave rise to the formation of both IAA and Tr-ol. The ratio of IAA to Tr-ol depended on the experimental conditions, shaking favoring the formation of IAA. Also IAAld gave rise to the formation of IAA and Tr-ol when incubated with suspensions of washed cells. The ratio of the two compounds depended on experimental conditions such as pH value and shaking, the latter reducing the formation of Tr-ol. These results cannot be explained by the assumption of a dismutation mechanism catalyzed by a single enzymatic unit. The operation of two enzyme systems, responsible for the reduction and the oxidation, respectively, of IAAld is suggested and discussed.     

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30–40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.     

Metabolism of Indole-3-acetaldehyde.

Aerobic infiltration of synthetic indoleacetaldehyde (IAAld) in buffered medium of pH 4.55, into living tissues of lower and higher plants, leads in the majority of cases to the formation of both indoleacetic acid and tryptophol. This activity is evinced by etiolated as well as green tissues. Besides, all parts of higher plants tested — roots, cotyledons, hypocotyls and leaves possess this activity. Abolishment of this activity by boiling indicates its enzymic nature. Coupled with the established occurrence of indoleacetaldehyde, the widespread distribution of such activity, strengthens the probability that indoleacetaldehyde may be the normal and natural precursor in the biosynthesis of indoleacetic acid.     

Isolement d'une alcool déshydrogénase chez Rhizobium et rôle dans le métabolisme indolique

Rhizabium meliloti contains an alcohol dehydrogenase (E.C.1.1.1.1.) which can be isolated by breaking the cells. This soluble enzyme was purified 16.1-fold by fractional precipitations with ammonium sulfate followed by gel filtration on Sephadex. The activity of the enzyme was tested with various aldehydes as substrates in the presence of NADH. Indole-3-acetaldehyde (IAAld) can be reduced to tryptophol (Tr-ol), and the optimal pH for this reaction is ca. 6.5. The reaction can be reversed, and Tr-ol is oxidised in the presence of NAD, but is was found that the yield was very poor; the optimal pH was ca. 8.6. This alcohol dehydrogenase is responsible for Tr-ol formation in Rhizobium, but under our experimental conditions tryptophol cannot really be considered as a precursor of IAAld and indole-3-acetic acid.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Homogenates of epicotyls or roots of nonsterile pea plants incubated with tryptophan produce IAA within 1 to 4 hours, which was detected by means of the Avena curvature test and thin layer chromatography. Three results prove this short-term IAA production to be mainly caused by epiphytic bacteria: 1) Homogenates of sterile plant parts catalyze a conversion of tryptophan to IAA, a hundredfold lower. 2) Chloramphenicol or streptomycin very actively reduce the IAA gain obtained with nonsterile homogenates. 3) Washing solutions of nonsterile plant parts which do not contain plant enzymes but only epiphytic bacteria, produce IAA from tryptophan, too. IAA synthesis from tryptophan in vitro by enzymes of the pea plant occurs with lower intensity than hitherto known; possibly it is physiologically unimportant. It is discussed to what extent the hitherto existing research work about the IAA biogenesis in higher plants might be incriminated by disregarding tbe rôle of epiphytic bacteria.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-¹⁴C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a ¹⁴C-product as IAA was by (a) co-chromatography of authentic IAA and ¹⁴C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Using hydrocultured pea plants, 109 bacterial strains (42 from shoots) were isolated from shoots, roots, and from the hydroculture medium. 58 different strains (26 from shoots) were able to produce IAA from tryptophan, 15 different strains (7 from shoots) were able lo destroy IAA. (Included are 13 strains possessing both properties.) As far as they could be identified, the IAA-producing and -destroying strains belong to Pseudomonas (by far dominating), Achromobacter, Alcaligenses, Bacillus, and Flavobacterium. The IAA-destroying activity strongly depends on the physiological state of the bacteria and the milieu conditions. Bacterial IAA production (but not IAA-degradation) is supposed to be important for the plant.     

Auxin Enhancement of mRNAs in Epidermis and Internal Tissues of the Pea Stem and Its Significance for Control of Elongation

The epidermis has been considered the site of auxin action on elongation of stems and coleoptiles. To try to identify mRNAs that might mediate auxin stimulation of cell enlargement, we compared, using in vitro translation assays, mRNA enhancement by indoleacetic acid (IAA) in the epidermis, with that in the internal tissues, of pea (Pisum sativum L., cv Alaska) third internode segments. We used seedlings that had been grown under red light, which enables the epidermis to be peeled efficiently from the internode. Most of the `early' IAA enhancements previously reported using etiolated peas, plus several hitherto undescribed enhancements, occur in both the epidermis and the internal tissue of the light-grown plants after 4 hours of IAA treatment. These enhancements, therefore, do not fulfill the expectation of elongation-specific mRNAs localized to the epidermis. One epidermis-specific IAA enhancement does occur, but begins only subsequent to 1 hour (but before 4 hours) of auxin treatment. Similarly, the previously mentioned IAA enhancements common to epidermis and internal tissue do not begin, in the light-grown plants, within 1 hour of IAA treatment. Since IAA stimulates elongation in light-grown internodes within 15 minutes, it appears that none of these mRNAs can be responsible for auxin induction of elongation. We confirmed, with our methods, the previous reports that some of these mRNAs are enhanced by IAA within 0.5 hour in etiolated internodes. This indicates that we could have detected an early enhancement in light-grown tissue had it occurred.     

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.     

Regulation of Phospholipase D Activity by Light and Phytohormones in Oat Seedlings

The effect of synthetic analogs of phytohormones and red light absorbed by phytochrome on the phospholipase D activity (PLD) was studied in oat (Avena sativa L.) seedlings. ABA manifested a short-term stimulating effect on PLD activity in the green seedlings and inhibited phospholipase activity in the etiolated plants. Kinetin inhibited enzyme activity in the etiolated seedlings and did not affect its activity in light. GA did not markedly affect PLD activity in the etiolated plants and activated this enzyme in the green seedlings. Finally, IAA did not affect the enzyme activity. The relationship of the regulatory effects of phytohormones and light on PLD activity is discussed.     

Cell length,light and14C-labelled indol-3yl-acetic acid transport inPisum satisum L. andPhaseolus vulgaris L

The putative auxin-transporting cells of the intact herbaceous dicotyledon are the young, differentiating vascular elements. The length of these cells was found to be considerably greater in dwarf (Meteor) than in tall (Alderman) varieties ofPisum sativum L., and to be greater in etiolated than in light-grown plants ofP. sativum cv Meteor andPhaseolus vulgaris L. cv Mexican Black. Under given light conditions during transport these large differences in cell length did not influence the shapes of the transport profiles or the velocity of transport of¹⁴C-labelled indol-3yl-acetic acid (IAA) applied to the apical bud. However, in both etiolated and light-grown bean and dwarf pea plants the velocity of transport in darkness was ca. 25% lower than that in light. Under the same conditions of transport velocities in bean were about twice those observed in the dwarf pea. Exposure to light during transport increased the rate of export of¹⁴C from the labelled shoot apex in green dwarf pea plants but not in etiolated plants. The light conditions to which the plants were exposed during growth and transport had little effect on the rates of uptake of IAA from the applied solutions. The results indicate that the velocity of auxin transport is independent of the frequency of cell-to-cell interfaces along the transport pathway and it is suggested that in intact plants auxin transport is entirely symplastic.