Plant Physiol. (1982) 69, 853-8580032-0889/82/69/0853/06/$00.50/0

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

Received for publication May 7, 1981 and in revised form October 23, 1981

VOLKER MAGNUS, SUMSKI SIMAGA, SONJA ISKRIC, AND SERGIJE KVEDERRudter Boskovic Institute, 41001 Zagreb, Yugoslavia

ABSTRACT

Metabolic reactions involving the aliphatic side chain of tryptophanwere studied in the holoparasitic dicotyledonous plants Orobanche graciisSm., 0. haea Baumg., and 0. 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-carboxylicacid. Independently, these metabolites were also formed from D-tryptophan,tryptamine, indole-3-lactic acid, and indole-3-acetic acid. As in autotrophicplants, tryptophan and tryptamine were also converted, via indole-3-acet-aldehyde, to indole-3-acetic acid, indole-3-ethanol, and its glucoside. Thebranch of tryptophan metabolism relevant to auxin biogenesis and catab-olism is, therefore, not rudimentary in Orobanche but even more complexthan in autotrophic higher plants.

The genus Orobanche, family Orobanchaceae, related to theScrophulariaceae, is constituted of Chl-free root parasites. Someof its representatives grow on crop plants and then may affectyields (3, 27). This adds economic significance to the question: Isthe heterotrophy of Orobanche only nutritional, or does it extendto other vital activities?

Studies of the enzymes present in Orobanche (16, 17, 26) haveso far shown little difference from autotrophic plants, but thereare no such data extending to the capability of the parasite tomake and to metabolize its own growth hormone, IAA.' Com-pounds known to have a key function in IAA biogenesis andmetabolism in autotrophic plants (22) were, therefore, applied toOrobanche, and the metabolites were isolated and identified.Inasmuch as this is the first examination of indole metabolism ina Chl-free root parasite, we wished to work at high substrateconcentrations to establish the metabolic potential of the plant.The data presented indicate that IAA biogenesis and metabolismare not rudimentary in Orobanche but are apparently even morecomplex than in autotrophic plants, particularly with respect tothe generation of indolic compounds with a C1 side chain.

MATERIALS AND METHODS

Chemicals. The glycosides (15) (G. Lacan, V. Magnus, unpub-lished) of tryptophol, IAA fB-D-glucopyranoside (9), 3-methoxy-methylindole (5), and indole-3-acetaldehyde (8) were synthesizedin this laboratory. The other chemicals were purchased, in analyt-ical purity from commercial sources. Column chromatographywas performed on silica gel, particle size 0.05 to 0.2 mm (Kemika,Zagreb, Yugoslavia) and Sephadex LH-20 (Pharmacia AB, Upps-ala, Sweden). Silica gel G and GF for analytical and silica gel PF

' Abbreviations: IAA, indole-3-acetic acid; tryptophol, indole-3-ethanol.

for preparative TLC were purchased from E. Merck (Darmstadt,West Germany) or Kemika (Zagreb, Yugoslavia) and coated onglass plates, according to the specifications of the supplier.

Plant Matenal. Orobanche ramosa L. growing on tobacco wascollected near Vukovar, Yugoslavia. 0. gracilis Sm., growingmainly on Lotus corniculatus L., and 0. lutea Baumg., growing onMedicagofalcata L. and M. sativa L., were collected near Zagreb.The collected plant material was protected from wilting andtemperature stress. It was comprised, for 0. gracilis and 0. lutea,of inflorescences, prior to anthesis, which were worked up withinan average of 3 to 4 h. In the case of 0. ramosa, whole floweringplants, including subterranean parts, were used. They were washedfree from adhering soil and stored overnight.The plant material was cut into sections about 1 cm in length.

Boiled plant material was prepared in flasks immersed in a boilingwater bath for 30 min.

Incubation Procedure. Substrate solutions (1 ml/g of plantmaterial) were prepared in 0.067 M KH2PO4 (pH 4.5) at approxi-mate concentrations of 1 mg/ml for D- and L-tryptophan, trypt-amine hydrochloride, indole-3-lactic acid, and tryptophol, and 0.1mg/ml for IAA, indole-3-acetaldehyde, indole-3-methanol, in-dole-3-carboxaldehyde, and indole-3-carboxylic acid. Inhibitorswere added to the substrate solutions at 1 to 2 mg/ml for cyclo-serine and semicarbazide hydrochloride and 0.1 mg/ml for KCN.These solutions were vacuum-infiltrated (at 20 mm Hg) into theplant sections. The sections took up about one-quarter of thesolutions, a great deal of which was retained in dead space, suchas that in between bracts and in flower buds. Intemal substratelevels per g of metabolically active plant tissue are, thus, smallerthan are those per ml of the external solutions by at least oneorder of magnitude. The plant material and the remaining sub-strate solutions were incubated separately at 22°C for 5 h, thelatter with aeration.

Prepurification of Plant Extracts. After incubation, the plantmaterial was homogenized in a 2-fold (w/v) amount of methanol.The homogenate was filtered, and the residue was reextracted.The combined filtrates were stored at -10°C until used. Theywere then concentrated in vacuo to a few ml; methanol (50 ml)was added, and the mixture was left at -10°C overnight. Theprecipitate was discarded, and the filtrate was concentrated invacuo, adjusted to 10 ml with methanol, and extracted, once with30 ml and a second time with 15 ml of benzene. The combinedextracts were evaporated and the residue extracted with 5 x 0.5ml of benzene:methanol (20:1, v/v).Column Chromatography. The prepurified samples were con-

centrated to 0.5 to 1 ml and applied to a column (i.d., 1 cm)containing 3.8 g of silica gel packed in benzene:methanol (20:1),which was also used as the first eluent (30 ml) to yield fraction L1. Two further fractions were collected: L 2 with 30 ml of benzene:methanol (15:1); and H with 35 ml of ether:methanol:H20 (30:10:1). By this method, the indolic compounds present in the plantextracts were eluted in three groups, as summarized in Table I.

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Plant Physiol. Vol. 69, 1982

Table I. Chromatographic Separation of Indolic Metabolites Formed by Orobanche SectionsGroups of metabolites eluted together in each of the three fractions (L l, L 2, H) obtained from the silica gel

column were separated and preliminarily identified by TLC in the solvents indicated. Detection was done byspraying with dinitrophenylhydrazine for indole-3-carboxaldehyde and with Ehrlich reagent for the othercompounds. The RF values given were determined with pure compounds; reproducibility was about ±0.03 unitsfor TLC plates from the same lot. Interference by native plant constituents with the detection of a particularcompound in an extract is indicated by values in parentheses.

Fraction RF Values on Thin-Layer Chromatogramsafrom the CompoundColumn 1 2 3 4 5 6 7

L I Indole-3-acetic acid methyl ester (0.47) (0.62) (0.79) (0.87) (0.76) (0.89) (0.86)Tryptophol 0.14 0.22 0.47 0.76 (0.73) (0.87) 0.68Indole-3-carboxaldehyde 0.14 0.22 0.44 (0.76) (0.72) (0.87) 0.68Indole-3-methanol 0.08 0.15 0.39 (0.71) (0.72) (0.86) _b3-Methoxymethylindole 0.34 (0.49) (0.67) (0.86) (0.78) (0.91) (0.80)

L 2 Indole-3-carboxylic acid (bulk) 0.00 0.02 0.02 0.43 0.64 0.35 0.56

H Indole-3-acetic acid 0.00 0.00 0.00 0.19 0.35 0.33 0.64Indole-3-acetic acid f8-D-gluco-

pyranoside 0.00 0.00 0.00 0.24 0.54 0.82C 0.07Tryptophol ,8-D-glucopyranoside 0.00 0.00 0.00 0.25 0.54 0.47 0.08Indole-3-carboxylic acid (tail) 0.00 0.02 0.02 0.43 0.64 0.35 0.56

a Solvents used: 1, Chloroform (stabilized with about 1% ethanol); 2, chloroform:ethanol, 95:5; 3, chloroform:carbon tetrachloride:methanol, 50:40:10; 4, chloroform:ethanol, 80:20; 5, 2-propanol:n-heptane:H20, 55:30:11; 6,2-propanol:ethyl acetate:25% aqueous ammonia, 35:45:20; 7, chloroform:methanol:glacial acetic acid, 90:10:1.

bDecomposition to several products.c Indole-3-acetamide formed by ammonolysis.

The reliability of this method has been shown previously (13).Thin-Layer Chromatography. The compounds eluted together

from the column were separated and preliminarily identified byTLC. The solvent systems used and the RF values of the com-pounds found are summarized in Table I. Chromatograms weresprayed with the Ehrlich reagent (1% p-dimethylaminobenzalde-hyde in ethanol:concentrated HCI [1:1, v/v]) for indoles in general,dinitrophenylhydrazine (saturated solution in 2 N HCI) for indole-3-carboxaldehyde, and 10%o ethanolic sulfuric acid with heatingfor indole-3-carboxylic acid methyl ester.

Quantities of indole-3-carboxaldehyde were estimated by co-chromatography with a series of known standards, and visualcomparison was made of the spot areas and color intensities afterspraying with dinitrophenylhydrazine, taking the general precau-tions described in Shellard (23). Amounts of this aldehyde pre-sented are arithmetic means with SE around 10%1. The amounts ofthe other metabolites formed were estimated from chromatogramsby order of magnitude.

In preparative TLC, zones were localized under a UV lamp.Exposure times were kept as short as possible, so that photochem-ical decomposition of indolic compounds was not observed.

Identification of Metabolites. The metabolites preliminarilyidentified by their elution characteristics on the silica gel columnand by the RF values and color reactions on thin-layer chromat-ograms (Table I) were purified by preparative TLC, and theiridentity was confirmed as follows.

Tryptophol. Since tryptophol and indole-3-carboxaldehydecould not be separated by silica gel chromatography, the isolateobtained by the above general procedure was applied to a columnof Sephadex LH-20 (28 x 1.5 cm) eluted with 95% ethanol.Tryptophol eluted after contaminating plant pigments but beforeindole-3-carboxaldehyde. The purified sample was acetylated withacetanhydride:pyridine (1:1, v/v [0.5 ml, room temperature, over-night]), yielding a compound with the chromatographic propertiesof O-acetyl tryptophol.

Tryptophol Glucoside. The metabolite was cleaved to tryptopholby fi-glucosidase from sweet almond emulsin under standard

conditions (0.1 M Na-citrate [pH 5.0], 1.5 h, 30°C). The corre-sponding l-D-fucopyranoside, -galactopyranoside, and -xylopy-ranoside, and the a-L-arabinoside, which would also be hydro-lyzed by the enzyme, were chromatographically distinct from themetabolite.

Indole-3-Acetic Acid. On the addition of diazomethane, themetabolite was completely transformed to a compound with thechromatographic properties of IAA methyl ester.

Indole-3-Acetic Acid Glycoside. The isolated metabolite with thechromatographic mobility of authentic IAA /i-D-glucopyranosidewas hydrolyzed by 25% aqueous ammonia, to yield IAA plusindole-3-acetamide. The metabolite also gave IAA on treatmentwith fl-glucosidase, but the small quantity of the metabolite avail-able precluded an exact identification of the sugar moiety.

Indole-3-Acetic Acid Methyl Ester. Chromatographic identifi-cation of this compound required multiple development (5-10times) with benzene to separate the ester from plant pigments,which also yielded blue colors with the Ehrlich reagent. Ammon-olysis of the compound (using 30Yo NH3 in absolute methanol)gave a mixture of indole-3-acetamide and IAA. The ester was also

_~YG M -rpoM ...... ,........_L00000I..doI-3-a^.t.debvde.8 |-- - - - M iTrypt ophoI,. l

rK.-_We3-carboxaeldh e,d.I.-._eUcaMee3 ae

ri W-_d37- I § hdoe-3-|-mn d|no | ryllc aeS |

FIG. 1. Comparison of indole metabolism in Orobanche and in auto-trophic higher plants..-, Reactions occurring in autotrophic plants butnot confirmed for Orobanche; ->, reactions occurring only in Orobanche;--, reactions occurring in autotrophic higher plants which were confirmedfor Orobanche; 1, possibly involving indole-3-pyruvic acid as the inter-mediate.

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TRYPTOPHAN METABOLISM IN OROBANCHE

Table II. Indolic Compounds Formed as Metabolites by Three Species of OrobanchePlant sections were infiltrated with solutions of the compounds given and incubated for 5 h. Metabolites were identified, and relative amounts were

estimated by TLC. Detection limits are approximately 0.1 ,ug of compound per 10 g of plant material. Identifications were confirmed by chemical andspectroscopic methods.

Metabolites Foundb

Precursor Supplied Species Ex-Trypto- IAA IAA Indole-3- Indole-3-

amined' Trypto- phol glu- IAA methyl glucA Indole-3- carboxal- carboxylicphol coside ester side dehyde acid

L-Tryptophan G ++ ++ - ++ ++L ++ ++ - + - +++ +++ +++R +++

L-Tryptophan + semicarbazide L (+) (+) - ++ ++ ++

D-Tryptophan L ++ ++ - + - +++ +++ +++G ++ +++ +++ +++

D-Tryptophan + semicarbazide L (+) (+) - - - ++ ++ ++

Indole-3-lactic acid L - - - - - ++ +++ +

Tryptamine G +++ +++ - - - +++ +++ +++L +++ +++ - - +++ +++ +++

Tryptamine + semicarbazide or cycloserine G (+) (+) - - ++ ++ ++L (+) (+) - ++ ++ ++

Indole-3-acetaldehyde L +++ +++ ++ - +++ +++ +++R +++ +++ ++ + - +++ +++ +++

Tryptophol G +++(+)

Indole-3-acetic acid L - - + - + +++R - - + + +++ +

Indole-3-methanol L - - - - - ++ +

Indole-3-carboxaldehyde L - - - - - ++ +

Indole-3-carboxylic acid L -a G, Orobanche gracilis Sm.; L, 0. lutea Baumg.; R, 0. ramosa L.b -, Below detection limit; (+), trace; +, up to about 10 jLg; ++, intermediate quantity; +++, close to 100 jig or above; void, not examined. Estimates

are for 10 g of plant material and substrate concentrations of I mg/ml.c Including 3-methoxymethylindole and 3,3'-diindolylmethane formed as artifacts.

reduced to tryptophol with lithium aluminum hydride (anhydrousether, reflux, 3 h).

Indole-3-Carboxaldehyde. The isolation of this metabolite par-allels that of tryptophol up to the step of Sephadex chromatogra-phy, where elution of the aldehyde is more retarded. Fractionscontaining the purest compound gave UV and [1H]NMR spectraidentical with those of authentic indole-3-carboxaldehyde.

Indole-3-Methanol, 3-Methoxymethylindole, and 3,3'-Diindolyl-methane. It is known that indole-3-methanol spontaneously di-merizes to give 3,3'-diindolylmethane (29) and also condenseswith the methanol used for the extraction of plant metabolites toyield 3-methoxymethylindole (13). In this work, the latter com-pound was preferentially detected and taken as evidence for thepresence of indole-3-methanol in the incubated plant material.For further identification, 3-methoxymethylindole was reduced toskatole with lithium aluminum hydride (anhydrous ether, reflux,3 h) (11). When dissolved in H20, boiled for 4 h, and left overnightat room temperature, the indolic compound from the plant extractand synthetic 3-methoxymethylindole gave an almost identicalchromatographic pattern of decomposition products, with the

most prominent spots at RF = 0.37 and 0.47 in chloroform.Indole-3-Carboxylic Acid. This metabolite showed the same UV

absorption maxima of authentic indole-3-carboxylic acid. It wasmethylated with diazomethane, and the ester formed was furtherpurified by TLC in chloroform and on a column of Sephadex LH-20 (28 x 1.5 cm) eluting with 95% ethanol. The sample obtainedgave the UV and NMR spectra of authentic indole-3-carboxylicacid methyl ester.

RESULTS AND DISCUSSION

Autotrophic higher plants synthesize IAA from indole-3-acet-aldehyde, which arises from tryptophan, as outlined in Figure 1(22). To test whether this metabolic pathway has been preservedin Orobanche, several key compounds were incubated with theplant material. The metabolites detected are presented in TableII. Although most of the substrates were incubated with only oneor two representatives of the investigated three-species group ofOrobanche, the data add up to a consistent pattern, indicating thatthere are no significant differences concerning the biogenesis andmetabolism of IAA in the genus Orobanche.

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Plant Physiol. Vol. 69, 1982

Table III. Effect of Inhibitors and Boiling the Plant Material on the Formation of Indole-3-carboxaldehyde inOrobanche

Living or boiled plant sections were infitrated with substrate-inhibitor solutions. After incubation and usualwork-up, the amount of indole-3-carboxaldehyde was estimated by TLC, by visual comparison with authenticstandards. Values given have SE of approximately 10o.

Amount

Pretreatment of Plant Material and Inhibitors of Indole-Species Substrate Added to Substrate Solutions 3-carbox-aldehydeFormeda

tg0. gracilis Tryptamine 60

Semicarbazide, I mg/ml 25Cycloserine, I mg/ml 20

0. ramosa L-Tryptophan 125KCN, 0.1 mg/ml 50Boiled plant material 44

Indole-3-acetaldehyde 265Boiled plant material 199

Indole-3-acetic acid 116KCN, 0.1 mg/ml 48Boiled plant material 27

0. luteasample 1 L-Tryptophan 158b

Boiled plant material 2lbSemicarbazide, 1.7 mg/ml 58bBoiled plant material and semicarbazide, 1.7 7bmg/ml

Tryptamine 169Boiled plant material 26Semicarbazide, 1.6 mg/ml 100Boiled plant material and semicarbazide, 1.6 6mg/ml

sample 2 L-Tryptophan 58

D-Tryptophan 34Semicarbazide, 2 mg/ml 30Boiled plant material 6

Indole-3-lactic acid 12Boiled plant material 0

a Reduced to 10 g of plant material and a substrate concentration (pure base in the case of tryptamine) of 1mg/ml. Linear dependence on substrate concentration and the amount of plant material present was assumed.

b Average of three parallel experiments.

Indolic Compounds Containing a C2 Side Chain. The formationof IAA from indole-3-acetaldehyde could be demonstrated withcertainty. The majority of the aldehyde was, however, reduced totryptophol, which was further conjugated with glucose. The pref-erential reduction ofindole-3-acetaldehyde has also been observedin nonparasitic higher plants (20). Tryptophol glucoside has beendescribed for pea seedlings (14).When tryptophan was incubated with Orobanche, the formation

of indole-3-acetaldehyde could not be shown directly, but itsinvolvement as an intermediate could be deduced from the pres-ence of its metabolites, tryptophol, and its glucoside, which werepreponderant. Free IAA was not observed, but small quantities ofits methyl ester were found. This may be due to reaction oforiginally present free or conjugate IAA with the methanol usedfor extraction.The intermediates in the conversion of tryptophan to indole-3-

acetaldehyde are still a matter of dispute; they may be tryptamine,

indole-3-pyruvic acid, or both. In the present work, this questionwas pursued only to the extent of finding possible metabolicbranching points for the formation of indolyl-C, compounds.Tryptamine, though not yielding detectable amounts of IAA, isan excellent precursor of tryptophol and its glucoside (for similarrelative efficiencies of tryptophan and tryptamine as IAA andtryptophol precursors, see Refs. 6 and 25). This transformationwas inhibited, by at least 90%o, by the carbonyl reagents semicar-bazide and cycloserine, but no trapping of the intermediate alde-hyde was observed. The action of the inhibitors should, therefore,be due to their reaction with pyridoxal phosphate present as acofactor in a tryptamine metabolizing amine oxidase which hasbeen identified in plant species unrelated to Orobanche (2, 19, 30).Indole-3-pyruvic acid could not be incubated with the plantmaterial. The acid is unstable at pH 4.5, so substrate solutionscontaining this compound would decompose. Indole-3-pyruvicacid was not detected as a tryptophan metabolite in Orobanche.

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TRYPTOPHAN METABOLISM IN OROBANCHE

Ofits possible precursors, indole-3-lactic acid (4) did not yield anyindolyl-C2 metabolite. D-Tryptophan was metabolized to thesecompounds in qualitatively and quantitatively the same way aswas its L-isomer, which was inhibited by semicarbazide. Thisindicates the involvement of pyridoxal phosphate dependent en-zymes, i.e. either indole-3-pyruvic acid-forming tryptophan trans-aminases (7, 10, 18) or tryptamine-forming decarboxylases (7, 24),but it does not permit a distinction between these two alternatives.

Metabolites Containing a C1 Side Chain. As evident from TableII, nearly every substrate tested was converted to indolic com-pounds containing a C1 side chain: indole-3-methanol; indole-3-carboxaldehyde; and indole-3-carboxylic acid. The amounts ofindole-3-carboxaldehyde, which was most easily estimated, variedfrom sample to sample of plant material. However, when materialof the same provenance was compared (Table III), both isomersof tryptophan, tryptamine, and IAA consistently gave about thesame amounts of indole-3-carboxaldehyde. With enough semicar-bazide or cycloserine to suppress the generation of indolyl-C2metabolites from tryptophan and tryptamine by more than 90%o,the formation of indole-3-carboxaldehyde was inhibited by only30 to 60%1o. Evidently, this aldehyde, which does not react with theabove carbonyl reagents under the present conditions, also arisesfrom tryptophan and tryptamine by pathways not involving IAA.The responsible enzyme, or enzyme complex, is inhibited byKCN.When indole-3-carboxaldehyde was incubated with Orobanche,

it was reversibly reduced to indole-3-methanol and irreversiblyoxidized to indole-3-carboxylic acid (Table II), as established forpea seedlings (13). The ready interconvertibility of indole-3-car-boxaldehyde and indole-3-methanol, however, makes it difficultto decide which compound is the primary indolyl-C, metabolite.While an aldehyde would be a common product of an oxidative

Table IV. Influence of Streptomycin on Bacterial Contamination andIndole-3-carboxaldehyde Formation in Orobanche

L-Tryptophan and tryptamine were incubated with Orobanche luteaBaumg., D-tryptophan with 0. gracilis Sm. Bacterial counts were obtainedby the conventional plating method using oxo-agar. Dilutions were pre-pared with 0.067 M K-phosphate buffer (pH 7.0), and colonies werecounted after 2 d at 37°C. SE were about 10%o for aldehyde amounts andabout 5 units of the least significant digit given for bacterial counts.

Number of Bacteria Micro-per ml of Substrate grams' of

Concentra- Solution Indole-3-

Substrate tion of carboxal-Streptomy- Before dehydecin Sulfate After in- Detected inincuba- cubation the Planttion Material

mg/mlL-Tryptophan 0.0 7.3 x 105 7.9 x 105 15.2

0.5 4.1 x 103 <10b 12.65.0 7.3 x 103 <102b 18.4

D-Tryptophan 0.0 1.4 x 105 5.5 x 105 26.55.0 4.2 x 102 9.5 x 101 43.65.0 2.4 x 103 8.9 x 102 47.4

Tryptamine 0.0 NDC 1.4 x 105 181.25.0 ND 1.0 X 102 175.2

aReduced to 10 g of plant material and a substrate concentration (purebase in the case of tryptamine) of 1.0 mg/ml. Linear dependence onsubstrate concentration, and the amount of plant material present wasassumed.

b Lowest dilution tested was 1:102.cNot determined.

chain-cleaving reaction, parallel formation of both alcohol andaldehyde via 3-methyleneindolenine has also been proposed (1).

Possible Artifact Formation during Metabolism or Extraction.To ascertain that the compounds claimed to be metabolites didnot arise by chemical decomposition of the respective substrates,experiments with boiled plant material were performed. The onlycase of significant artifact production established was the conver-sion of indole-3-acetaldehyde to indole-3-carboxaldehyde andindole-3-carboxylic acid (but not to indole-3-methanol), whichwas about the same in both living and boiled plant material. Itmight, therefore, be suspected that indolyl-C1 compounds arise byartifactual decomposition of indole-3-acetaldehyde and, possibly,indole-3-pyruvic acid, which are themselves formed as the truemetabolites. However, when the enzymes producing these unstablemetabolites were inhibited, the formation ofc&mpounds with a C1side chain was not substantially diminished. This possibility must,therefore, be insignificant.

Bacterial Contamination. Speculating upon the mechanism ofindole-3-carboxaldehyde formation from tryptophan and trypt-amine in Orobanche, a reasonable first step, preceding the cleavageof their side chains, would be hydroxylation at the positionadjacent to the indole ring. This hydroxylation has been describedfor the microorganism Pseudomonasfluorescens (21, 28). It may,thus, be suspected that the indolyl-C, compounds observed inexperiments with Orobanche are actually formed by contaminantbacteria (12). This suspicion is favored by the fact that, theepiphytic bacteria isolated from the plant material were flagellateand resistant to chloroamphenicol and, thus, may belong to thegenus Pseudomonas.

In our experiments, substrate solutions saved after the infiltra-tion of Orobanche sections contained about 105 to 106 bacteria perml, this number increasing maximally four times on incubation.Comparable amounts of bacteria must be present in the infiltratedplant material. When streptomycin was added to the substratesolutions, the number of viable bacteria decreased by three ormore orders of magnitude, whereas the amount of indole-3-car-boxaldehyde formed from D- and L-tryptophan and from trypt-amine was not significantly affected (Table IV). This was alsoobserved, by qualitative comparison of chromatograms, for theother metabolites of these compounds.

CONCLUDING REMARKS

Metabolic pathways involving the tryptophan side chain, whichare common to autotrophic higher plants, and the reactions dem-onstrated here for Orobanche are compared in Figure 1. Not alltransformations could be examined, but, in general, the parasiteappears to be potentially independent from its host with regard toIAA biogenesis and metabolism. Under field conditions, however,the two plants may well exchange any common intermediate, sothe real auxin metabolism in Orobanche may depend on thephysiological state of the host plant. Indole-3-carboxaldehyde andthe corresponding alcohol and acid have so far been consideredmetabolites of IAA, except for some rare cases, where they can beformed from indole-3-acetonitrile (31). Orobanche seems to beunique in accumulating appreciable quantities of these indolyl-C,compounds, on incubation with D- and L-tryptophan, tryptamine,and indole-3-lactic acid.

Acknowledgments-We thank Mr. I. Z. Plaviic for providing material of Oro-banche ramosa and Dr. Lj. ljanic for an authoritative determination of species. Weare also grateful to Dr. M. Wrischer for electron micrographs of epiphytic bacteriaand to Mr. D. Duraiin for NMR spectra.

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858 MAGNUS ET AL.

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