Studies on the production of indole-3-acetic acid with auxin-heterotrophic mutants derived from cultured crown gall cells

Mutants in the indole-3-acetic acid metabolism derived fromcultured crown gall cells were tested to see whether they couldutilize any one of eight indolic compounds in place of indole-3-aceticacid. Two auxin-heterotrophic mutant cell lines could not utilizeindolepyruvic acid, but growth recovered when there was a supplementof indole-3-acetic acid. Indoleacetonitril and indoleacetaldoximeinhibited the growth of mutant cell lines and their parentalcrown gall cells. Cultured crown gall cells may have synthesizedindole-3-acetic acid from tryptophan via indolepyruvic acidand indole-acetaldehyde, and also may be able to produce indole-3-aceticacid from tryptophan via tryptamine (Received May 6, 1980; )     

Biosynthesis of auxin by the gram-positive phytopathogen Rhodococcus fascians is controlled by compounds specific to infected plant tissues

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.     

Phytohormones in Japanese Mugwort Gall Induction by a Gall-Inducing Gall Midge

A variety of insect species induce galls on host plants. Liquid chromatographic/tandem mass spectrometric analyses showed that a gall midge (Rhopalomyia yomogicola) that induces galls on Artemisia princeps contained high levels of indole-3-acetic acid and cytokinins. The gall midge larvae also synthesized indole-3-acetic acid from tryptophan. Close observation of gall tissue sections indicated that the larval chamber was surrounded by layers of cells having secondary cell walls with extensive lignin deposition, except for the part of the gall that constituted the feeding nutritive tissue which was composed of small cells negatively stained for lignin. The differences between these two types of tissue were confirmed by an expression analysis of the genes involved in the synthesis of the secondary cell wall. Phytohormones may have functioned in maintaining the feeding part of the gall as fresh nutritive tissue. Together with the results in our previous study, those presented here suggest the importance of phytohormones in gall induction.     

Biosynthesis of Auxin by the Gram-Positive Phytopathogen Rhodococcus fascians Is Controlled by Compounds Specific to Infected Plant Tissues

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.     

The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid

Gene 2 from the T region of Ti plasmids appears to be expressed both in eucaryotic and in procaryotic systems. In transformed plant cells it participates in auxin-controlled growth and differentiation, and in bacteria it is expressed into a defined protein of Mr 49000. We investigated the possibility that it codes for an enzyme involved in auxin biosynthesis. Only extracts from Escherichia coli cells expressing gene 2 hydrolyzed indole-3-acetamide into a substance which was unambiguously identified as indole-3-acetic acid. The same reaction was found in Agrobacteria containing gene 2, but not in strains lacking the gene. Extracts from tobacco crown gall cells, but not from non-transformed cells, showed the same enzyme activity, and the reaction product was also identified as indole-3-acetic acid. The results indicate that gene 2 of the T region, which participates in tumorous growth of plant cells, codes both in bacteria and in plants for an amidohydrolase involved in the biosynthesis of the plant hormone indole-3-acetic acid.     

l-Tryptophan metabolism in wound-activated and Agrobacterium tumefaciens-transformed potato tuber cells

The in vivo metabolism of L-tryptophan in wound-activated and Agrobacterium tumefaciens , strain C 58, transformed tissues of white potato tubers ( Solanum tuberosum L. cv. Saskia) was investigated. The following metabolites of L-tryptophan were identified in both tissues by co-chromatography with authentic standards in several thinlayer chromotography (TLC) and high pressure liquid chromatographic (HPLC) systems: indole-3-acetic acid (IAA), indole-3-acetaldehyde, indole-3-ethanol, indole-3-acetamide and tryptamine. Labelled indole-3-acetaldoxime was only found in transformed tissue. Crown gall tissue generally incorporated [¹⁴C]-L-tryptophan into precursors of IAA at a distinctly higher rate than did wound tissue. Tryptamine and indole-3-ethanol accumulated about ten-fold more label in crown gall cells than in cells from wounded tissue. The incorporation of radioactivity into indole-3-acetamide as determined by 2 consecutive TLC systems followed by HPLC analysis was rather low, though consistently observed in both tissues. An indole-3-acetamide hydrolyzing enzyme, the putative product of gene 2 on the T-DNA, could be extracted from the transformed tissue only. The indole-3-ethanol level was 4.3 nmol (g dry weight)⁻¹ and 41 nmol (g dry weight)⁻¹ for wounded tissue and primary crown gall tissue, respectively, as determined by HPLC with a [¹⁴C]-labelled internal standard. The experiments are critically discussed in relation to recent reports on a T-DNA encoded enzyme of IAA biosynthesis in crown gall tumors.     

Primary Action of Indole-3-acetic Acid in Crown Gall Tumors: Increase of Solute Uptake

Exogenously added indole-3-acetic acid at a concentration of 100 micromolars stimulates d-glucose uptake (or 3-O-methyl-d-glucose uptake) by 25% in crown gall tumors induced on potato tuber tissue by Agrobacterium tumefaciens strain C 58. The titration of the endogenous IAA with the auxin antagonist 2-naphthaleneacetic acid at 100 micromolars reduces d-glucose uptake by about 80%. The apparent inhibition constant K_i is 21 micromolars. Other auxin antagonists like 1-naphthoxyacetic acid and 2-(p-chlorophenoxy)-2-methylpropionic acid show similar effects. The uptake of the amino acids leucine, methionine, tryptophan, lysine, and aspartic acid is also inhibited by 2-naphthaleneacetic acid to similar degrees. The auxins 1-naphthaleneacetic acid and 2-naphthoxyacetic acid at concentrations between 10 and 100 micromolars inhibit solute uptake only slightly (inhibition less than 20%). The impact of the results on the postulated role of indole-3-acetic acid as a modifier of the electrochemical proton gradient across the plasmalemma in crown gall tumor tissue is discussed.     

Measurement of the Rates of Oxindole-3-Acetic Acid Turnover, and Indole-3-Acetic Acid Oxidation in Zea mays Seedlings

Nonhcbcl, H. M. 1986. Measurement of the rates of oxindole-3-aceticacid turnover and indole-3-acetic acid oxidation in Zea maysseedlings.—J. exp. Bat. 37: 1691–1697. Oxindole-3-acetic acid is the pnncipal catabolite of indole-3-aceticacid in Zea mays seedlings. In this paper measurements of theturnover of oxindole-3-acetic acid are presented and used tocalculate the rate of indole-3-acetic acid oxidation. [³H]Oxindolc-3-acetic acid was applied to the endosperm of Zeamays seedlings and allowed to equilibrate for 24 h before thestart of the experiment. The subsequent decrease in its specificactivity was used to calculate the turnover rate. The averagehalf-life of oxindole-3-acetic acid in the shoots was foundto be 30 h while that in the kernels had an average half-lifeof 35 h. Using previously published values of the pool sizesof oxindole-3-acetic acid in shoots and kernels from seedlingsof the same age and variety, and grown under the same conditions,the rate of indole-3-acetic acid oxidation was calculated tobe I-I pmol plant^–1 h^–1 in the shoots and 7·1pmol plant^–1 h^–1 in the kernels. Key words: Oxindole-3-acetic acid, indole-3-acetic acid, turnover, Zea mays     

Metabolism of Tryptophan, Indole-3-acetic Acid, and Related Compounds in Parasitic Plants from the Genus Orobanche

Metabolic reactions involving the aliphatic side chain of tryptophan were studied in the holoparasitic dicotyledonous plants Orobanche gracilis Sm., O. lutea Baumg., and O. ramosa L. Unlike known autotrophic plants, the parasite metabolized l-tryptophan directly to indole-3-carboxaldehyde, which was further converted to indole-3-methanol and indole-3-carboxylic acid. Independently, these metabolites were also formed from d-tryptophan, tryptamine, indole-3-lactic acid, and indole-3-acetic acid. As in autotrophic plants, tryptophan and tryptamine were also converted, via indole-3-acetaldehyde, to indole-3-acetic acid, indole-3-ethanol, and its glucoside. The branch of tryptophan metabolism relevant to auxin biogenesis and catabolism is, therefore, not rudimentary in Orobanche but even more complex than in autotrophic higher plants.     

Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase.

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent against soil-borne fungal plant pathogens. In this study, indole-3-acetic acid (IAA) biosynthesis in strain CHA0 was investigated. Two key enzyme activities were found to be involved: tryptophan side chain oxidase (TSO) and tryptophan transaminase. TSO was induced in the stationary growth phase. By fractionation of a cell extract of strain CHA0 on DEAE-Sepharose, two distinct peaks of constitutive tryptophan transaminase activity were detected. A pathway leading from tryptophan to IAA via indole-3-acetamide, which occurs in Pseudomonas syringae subsp. savastanoi, was not present in strain CHA0. IAA synthesis accounted for less than or equal to 1.5% of exogenous tryptophan consumed by resting cells of strain CHA0, indicating that the bulk of tryptophan was catabolized via yet another pathway involving anthranilic acid as an intermediate. Strain CHA750, a mutant lacking TSO activity, was obtained after Tn5 mutagenesis of strain CHA0. In liquid cultures (pH 6.8) supplemented with 10 mM-L-tryptophan, growing cells of strains CHA0 and CHA750 synthesized the same amount of IAA, presumably using the tryptophan transaminase pathway. In contrast, resting cells of strain CHA750 produced five times less IAA in a buffer (pH 6.0) containing 1 mM-L-tryptophan than did resting cells of the wild-type, illustrating the major contribution of TSO to IAA synthesis under these conditions. In artificial soils at pH approximately 7 or pH approximately 6, both strains had similar abilities to suppress take-all disease of wheat or black root rot of tobacco. This suggests that TSO-dependent IAA synthesis is not essential for disease suppression.     

Effect of water-soluble vitamins on the production of indole-3-acetic acid by Azospirillum brasilense

The effects of six water-soluble vitamins on tryptophan-dependent synthesis of indole-3-acetic acid in Azospirillum brasilense were investigated. A multifactorial regression analysis was employed to produce models of indole-3-acetic acid synthesis versus concentrations of tryptophan and the vitamins added to the growth medium. Very low levels of the B-group vitamins added at 10 to 100 microg l(-1) affected production of indole-3-acetic acid in A. brasilense. The largest release of this phytohormone was observed after amendment with pyridoxine and nicotinic acid. Results of the study suggest a role these vitamins may fulfil in the regulation of indole-3-acetic acid synthesis in A. brasilense.     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

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.     

Activity of 1,2-benzisoxazole-3-one and indole-2,3-dione on plant regeneration in vitro and on cell elongation

We tested the morphogenetic and cell elongating activity of 1,2-benzisoxazole-3-one, a compound similar to 1,2-benzisoxazole-3-acetic acid but lacking the lateral carbon chain. For comparison, we tested also the activity of indole 2,3-dione, having the same indolic ring as indole 3-acetic acid but no lateral carbon chain. The tests were made on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. We found that 1,2 benzisoxazole-3-one retains part of the high shoot inducing activity of 1,2-benzisoxazole-3-aceticacid, while indole-2,3-dione is inactive. Both compounds have no effect on root induction or cell elongation. It seems therefore that the activity of 1,2 benzisoxazole-3-acetic acid is partly related to the structure of its ring, and that also in this respect 1,2 benzisoxazole-3-acetic acid differs from other auxinlike compounds.Abbreviations BOA 1,2-benzisoxazole-3-acetic acid - BOO 1,2-benzisoxazole-3-one - IAA in-dole-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.     

Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.     

Tryptophan and indole-3-acetic acid accumulation in Acer cell cultures and its relationship with cell autolysis

The accumulation and decline of free indole-3-acetic acid (IAA) and tryptophan has been monitored in cells of Acer pseudoplatanus L. grown in batch suspension cultures. The period of maximal IAA accumulation per cell or per unit dry weight of tissue was found to precede the peak of tryptophan accumulation by several days. A study of cell viability throughout a growth passage indicated the presence of a basal level of non-viable cells of 5–7%, with only minor increases occurring during the first week of the three-week growth passage. The results suggest that IAA biosynthesis is not regulated by substrate availability arising from proteolysis in dead cells.Abbreviation GC-MS Gas chromatography-mass spectrometry - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - TLC thin-layer chromatography     

Plant regeneration from cotyledon protoplasts of Xinjiang muskmelon

Cotyledon protoplasts were isolated from seedlings of Xinjiang muskelon (Cucumis melo var.saccharinus) grown under sterile conditions and cultured in modified Miller medium. High frequency division of the protoplast-derived cells was observed. Agarose bead culture with B₆S₃ tobacco crown gall nurse cells was found most suitable for the cotyledon protoplasts when compared with other culture methods. Intact plants were regenerated from the protocalli by a two-step culture procedure with liquid and then solid media.Abbreviations BAP 6-benzylaminopurine - B₆S₃ crown gall tumor cells of tobacco - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MES 2(Nmorpholino) ethanesulfonic acid - MS Murashige and Skoog medium(1962) - NAA -naphthaleneacetic acid - N6 Zhu et al. medium (1975)