Indole-3-acetic acid biosynthesis in Lemna gibba studied using stable isotope labeled anthranilate and tryptophan

IAA biosynthesis in many plants, including Lemna gibba, has been shown to involve at least two different pathways, one from tryptophan and a tryptophan-independent route. To study the kinetics of IAA biosynthesis in Lemna, we simultaneously measured the incorporation of label from [15N]-anthranilate and [2H5]-tryptophan into IAA by Lemna plants in short term feeding studies. The data show that label from anthranilate rapidly goes into IAA and tryptophan. Labeling of the IAA pool by [15N]-anthranilate slightly precedes labeling of the tryptophan pool, confirming that more than one route to IAA exists in these plants. Longer term feeding studies (5–25 h) suggest that exogenous tryptophan is used preferentially to label IAA as compared to tryptophan made by the plant. This is indicated by the fact that the IAA pool was more enriched than the tryptophan pool in [2H5]-label, but less enriched than the tryptophan pool in [15N] (which comes about by de novo synthesis of tryptophan from [15N]-anthranilate by the plant).     

Biosynthesis of indole-3-acetic acid in protoplasts,chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-³H]tryptophan to [³H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-³H]tryptophan, contained [³H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-³H]tryptophan, also synthesized [³H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [³H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - RC radiocounting     

The biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

Transport of Benzyladenine and Gibberellin A(1) from Roots in Relation to the Dominance between the Axillary Buds of Pea (Pisum sativum L.) Cotyledons

In etiolated, 5-day-old pea (Pisum sativum L.) seedlings a significantly more intensive growth of buds situated in the axil of the excised cotyledons was observed as early as 4 hours after decapitation and excision of one cotyledon of each pair. If [8-¹⁴C]benzyladenine ([¹⁴C]BA) was applied to roots of intact plants 10 hours prior to such decapitation and excision, significantly higher both total and specific ¹⁴C activities were observed in buds situated on the side of the excised cotyledons as early as 4 hours after decapitation and excision. Although the removal of a substantial part of the root system carried out simultaneously with decapitation and excision of one cotyledon resulted in a decrease in total ¹⁴C activity of buds, nevertheless a higher accumulation of ¹⁴C activity was maintained in buds situated on the side of excised cotyledon. If [¹⁴C]BA was applied to roots of seedlings after they were decapitated and deprived of one cotyledon, both total and specific ¹⁴C activities of buds situated on the side of excised cotyledons were significantly higher as early as the end of uptake of [¹⁴C]BA by roots, i.e. after 10 hours. On the other hand, [1,2-³H]gibberellin A₁ applied to roots of intact and/or decapitated and one-cotyledon-deprived seedlings in the same way as [¹⁴C]BA did not appear in the buds until very much later and only in negligible amounts (i.e.³H activity). This indicates that the release of buds from apical dominance represents an active and selective process which can result from the ability of buds to utilize and/or synthesize only certain growth substances within a certain time interval.     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact plants and stem segments

The transport of [¹⁴C]phenylacetic acid (PAA) in intact plants and stem segments of light-grown pea (Pisum sativum L. cv. Alderman) plants was investigated and compared with the transport of [¹⁴C]indiol-3yl-acetic acid (IAA). Although PAA was readily taken up by apical tissues, unlike IAA it did not undergo long-distance transport in the stem. The absence of PAA export from the apex was shown not to be the consequence of its failure to be taken up or of its metabolism. Only a weak diffusive movement of PAA was observed in isolated stem segments which readily transported IAA. When [1-¹⁴C]PAA was applied to a mature foliage leaf in light, only 5.4% of the ¹⁴C recovered in ethanol extracts (89.6% of applied ¹⁴C) had been exported from the leaf after 6.0 h. When applied to the corresponding leaf, [¹⁴C]sucrose was readily exported (46.4% of the total recovered ethanol-soluble ¹⁴C after 6.0 h). [1-¹⁴C]phenylacetic acid applied to the root system was readily taken up but, after 5.0 h, 99.3% of the recovered ¹⁴C was still in the root system.When applied to the stem of intact plants (either in lanolin at 10 mg·g^-1, or as a 10^-4 M solution), unlabelled PAA blocked the transport through the stem of [1-¹⁴C]IAA applied to the apical bud, and caused IAA to accumulate in the PAA-treated region of the stem. Applications of PAA to the stem also inhibited the basipetal polar transport of [1-¹⁴C]IAA in isolated stem segments. These results are consistent with recent observations (C.F. Johnson and D.A. Morris, 1987, Planta 172, 400–407) that no carriers for PAA occur in the plasma membrane of the light-grown pea stem, but that PAA can inhibit the carrier-mediated efflux of IAA from cells. The possible functions of endogenous PAA are discussed and its is suggested that an important role of the compound may be to modulate the polar transport and-or accumulation by cells of IAA.Abbreviations IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - IIBA 2,3,5-triiodobenzoic acid     

Biosynthesis of Camptotheca acuminata alkaloids

Tracer feeding experiments with Camptotheca acuminata plants show that [1′-¹⁴C]L-tryptophan, [Ar-³H₄]L-tryptophan, [Ar-³H₄,1′-¹⁴C]tryptophan, [1′-¹⁴C]-tryptamine, [2-¹⁴C]DL-mevalonate, and [2-¹⁴C]geraniol-[2-¹⁴C]nerol are incorporated into camptothecin. Direct stem injection of the labeled precursors into C. acuminata plants resulted in a substantial increase in the activity of isolated Camptotheca alkaloids as compared to root feeding of the same tracer.     

The effect of cycloheximide on IAA-stimulated transport of 14C-ABA and 14C-sucrose in long pea epicotyl segments

The effect of cycloheximide (CH) on the indol-3yl-acetic acid (IAA)-stimulated transport of ¹⁴C-labelled abscisic acid (ABA) and ¹⁴C-labelled sucrose was studied in 110 mm long pea epicotyl segments. IAA application resulted in elongation growth of the segments. This effect was decreased by CH treatment which also reduced [¹⁴C] ABA and [¹⁴C] sucrose accumulation in the growing apical part of the segments. A reduction in [¹⁴C] IAA uptake and in protein synthesis in this part of the segments was also observed. The simultaneous inhibition of protein synthesis and reduction of [¹⁴C] ABA and [¹⁴C] sucrose transport suggests that IAA can stimulate the transport of ABA and sucrose through a protein synthesis-based elongation growth.     

Effect of gibberellic acid on sterol production in Corylus avellana seeds

Gibberellic acid-induced germination of hazel seeds was accompanied by little change in the sterol content of the cotyledons. Dormant and germinating cotyledons rapidly incorporated [2-¹⁴C]MVA into squalene which was slowly converted to sterols. Gibberellin treatment induced an increase in the incorporation of [2-¹⁴C]MVA into cotyledon esterified sterols. An increase in free sterols occurred in the germinating embryonic axes, with increased relative amounts of stigmasterol and campesterol in the free 4-desmethylsterols. Germination was accompanied by increased incorporation of [2-¹⁴C]MVA into free and esterified sterols in the embryonic axes.     

Shikimate Pathway Activity during Shoot Initiation in Tobacco Callus Cultures

The activity of the shikimic acid pathway during shoot initiation in tobacco (Nicotiana tabacum L. Wisconsin 38) callus was examined. Enhancement of the activities of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase, shikimate kinase, chorismate mutase, and anthranilate synthase was observed during culture of tobacco callus under shootforming conditions in comparison to tissue cultured under non-organforming conditions. Confirmation of these findings was obtained by examining the incorporation of d-[¹⁴C]glucose into quinic and shikimic acids and of [¹⁴C]shikimic acid into tyrosine, phenylalanine, and tryptophan.     

On the question of β-indolyl-acetic acid synthesis from indole without a tryptophan intermediate

Summary Experiments with sterile grown maize coleoptiles were carried out to decide whether or not a biosynthetic path for -indolyl-acetic acid (IAA) from indole exists without tryptophan occurring as an intermediate. -Indolyl-acrylic acid as a tryptophan synthetase inhibitor significantly reduces the yield of [³H]tryptophan obtained from [³H]indole while the reduction in the [³H]IAA yield is considerably less pronounced. This, however, indicates only a non-linear relationship between the tryptophan concentration and the IAA yield and not the sought path. Moreover, double labelling combined with isotope competition methods in experiments with [³H]indole and L-[¹⁴C]serin show that all IAA synthesized from [³H]indole is produced on a path involving the synthesis of tryptophan as an intermediate.Abbreviation IAA -indolyl-acetc acid     

Biosynthesis and metabolism of indole-3-ethanol and indole-3-acetic acid by Pinus sylvestris L. needles

Combined gas chromatography-mass spectrometry has been used to identify indole-3-ethanol (IEt) in a purified extract from needles of Pinus sylvestris L. Quantitative estimates obtained by high-performance liquid chromatography with fluorescence detection, corrected for samples losses occurring during purification, indicate that Pinus needles contain 46±4 ng g^-1 IEt. This compares with 24.5±6.5 ng g^-1 indole-3-acetic acid (IAA) and 2.3±0.4 ng g^-1 indole-3-carboxylic acid (ICA) (Sandberg et al. 1984, Phytochemistry, 23, 99–102). Metabolism studies with needles incubated in a culture medium in darkness revealed that both [3-¹⁴C]-tryptophan and [2-¹⁴C]tryptamine mine are converted to [¹⁴C]IEt. It was also shown that [3-¹⁴C]IEt acted as a precursor of [¹⁴C]IAA. The observed metabolism appears to be enzymic in nature. The [2-¹⁴C]IAA was not catabolised to [¹⁴C]ICA in detectable quantities implying that, at best, only a minor portion of the endogenous ICA pool in the Pinus needles originates from IAA.Abbreviations DEAE diethylaminoethyl - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - ICA indole-3-carboxylic acid - IEt indole-3-ethanol - PVP polyvinylpyrrolidone     

Auxin transport and the regulation of petiole abscission in cotton (Gossypium hirsutum L.): A comparison of indol-3yl-acetic acid and phenylacetic acid

Distal applications of indol-3yl-acetic acid (IAA) to debladed cotyledonary petioles of cotton (Gossypium hirsutum L.) seedlings greatly delayed petiole abscission, but similar applications of phenylacetic acid (PAA) slightly accelerated abscission compared with untreated controls. Both compounds prevented abscission for at least 91 h when applied directly to the abscission zone at the base of the petiole. The contrasting effects of distal IAA and PAA on abscission were correlated with their polar transport behaviour-[1-¹⁴C]IAA underwent typical polar (basipetal) transport through isolated 30 mm petiole segments, but only a weak diffusive movement of [1-¹⁴C]PAA occurred.Removal of the shoot tip substantially delayed abscission of subtending debladed cotyledonary petioles. The promotive effect of the shoot tip on petiole abscission could be replaced in decapitated shoots by applications of either IAA or PAA to the cut surface of the stem. Following the application of [1-¹⁴C]IAA or [1-¹⁴C]PAA to the cut surface of decapitated shoots, only IAA was transported basipetally through the stem. Proximal applications of either compound stimulated the acropetal transport of [¹⁴C]sucrose applied to a subtending intact cotyledonary leaf and caused label to accumulate at the shoot tip. However, PAA was considerably less active than IAA in this response.It is concluded that whilst the inhibition of petiole abscission by distal auxin is mediated by effects of auxin in cells of the abscission zone itself, the promotion of abscission by the shoot tip (or by proximal exogenous auxin) is a remote effect which does not require basipetal auxin transport to the abscission zone. Possible mechanisms to explain this indirect effect of proximal auxin on abscission are discussed.     

Presence of indole-3-acetic acid in chloroplasts ofNicotiana tabacum andPinus sylvestris

The compartmentation and metabolism of indole-3-acetic acid (IAA) was examined in protoplasts derived from needles ofPinus sylvestris L., leaves of normal plants ofNicotiana tabacum L., leaves ofN. tabacum plants carrying the T-DNA gene 1 (rG1 plants) and leaves ofN. tabacum plants carrying the T-DNA gene 2 (rG2 plants) by using a rapid cell-fractionation method. In all tissues, 30%–40% of the IAA pool was located in the chloroplast, while the remainder was found in the cytosol. Quantitative analysis of indole-3-ethanol (IEt) showed that in bothPinus andNicotiana the IEt pool was located exclusively in the cytosol. The only plant that contained endogenous indoleacetamide (IAAm) was therG1-mutant ofN. tabacum, expressing theAgrobacterium tumefaciens T-DNA gene 1. Cellular fractionation of protoplasts from this transgenic plant showed that the entire IAAm pool was located in the cytosol. Feeding experiments utilizing [5-³H]tryptophan, [5-³H]IEt, [1′-¹⁴C] and [2′-¹⁴C]IAA demonstrated that the biosynthesis and catabolism of IAA occurred in the cytosol in bothPinus and in the wild type and the different mutants ofNicotiana. Furthermore, the biosynthesis of IAAm in therG1 plants was also shown to be localized in the cytosol.     

Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.)

The transport of exogenous indol-3yl-acetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-¹⁴C]-or [5-³H]IAA occurred at velocities (approx. 8–15 mm·h^-1) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [1-¹⁴C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled IAA or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-¹⁴C]IAA was also completely prevented through regions of the intact stem treated with N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid.Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol·m^-3). Transport velocity increased with concentration up to 83 mmol·m^-3 IAA but fell again with further increase in concentration.Stem segments (2 mm) cut from intact plants transporting apically applied [1-¹⁴C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol·m^-3 NPA was included in the efflux medium. Long (30 mm) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-¹⁴C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol·m^-3 NPA to the external medium completely prevented this basal efflux.These results support the view that the slow long-distance transport of IAA from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of IAA transmembrane transport predicted by the chemiosmotic polar diffusion theory.Abbreviations IAA indol-3yl-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Metabolism of carbohydrate and lipid reserves in germinated cotton seeds

Utilization of reserve lipid and carbohydrates during germination (0–12 h) and postgerminative growth (12–48 h) was studied in cotton (Gossypium hirsutum L.) seedlings. Raffinose and stachyose were utilized during the germination period and early growth; mobilization was associated with -galactosidase (EC 3.2.1.22) activity. Results from pulse-chase experiments with [³H]raffinose supplied exogenously to 4-h soaked seeds indicated that raffinose-derived catabolites contributed to the coincident increase in cotyledon sucrose and starch, and to the small increase in axis dry weight. Starch appears to be an alternative sink for end products of hydrolysis of reserve carbohydrates prior to the onset of rapid axis growth and cotyledon expansion. Mobilization of neutral lipid commenced at about 16 h after soaking, concomitant with development of key glyoxylate-cycle and other gluconeogenesis-related enzyme activities. Axis dry weight increased three-fold between 24 and 48 h. Results from pulse-chase (3 h, 16 h) experiments in which [2-¹⁴C]acetate was supplied to cotyledons of intact 22-h-old seedlings showed that acetate-derived metabolites were not transported exclusively to the axes, but were partitioned between axes and cotyledons. Only 27% of total incorporated radioactivity was recovered in axes following the chase, 18% was evolved as CO₂, and the rest was recovered in water-soluble substances (20%) and polymers (31%) within the cotyledons. Of the polymers, 55% of the activity was in polysaccharides (Starch, pectic substances, hemicellulose, cellulose), 25% in protein, and 20% in unidentified neutral and acidic compounds. Considering these data, the amount of lipid mobilized, and various routes by which supplied [2-¹⁴C]acetate could be metabolized, it appears that lipidderived compounds contribute only 25–40% of axis dry-weight gain. Lipid-derived substances retained in the cotyledons likely are utilized for expansion and differentiation of the cotyledons into photosynthetic organs.     

Indoleacetic Acid synthesis in soybean cotyledon callus tissue

Growth of an auxin-requiring soybean cotyledon callus tissue (Glycine max L., Merr. var. Acme) was promoted by tryptophan, tryptamine, indole, indoleacetamide and, to a very slight degree, anthranilic acid. When tryptophan-3-¹⁴C was supplied in the growth medium, labeled indoleacetic acid (IAA) was found in both the tissue and the medium. Medium, from which the cells had been removed, was also found to convert labeled tryptophan to IAA. Soybean callus contained 0.044 μmole/g free tryptophan, but this is apparently not available for conversion to IAA. These results suggest that while exogenously supplied trytophan could elevate a specific internal pool where IAA synthesis occurs some of the growth on a tryptophan medium can be accounted for by external conversion.     

Transport of cyanogenic glucosides: linustatin uptake by Hevea cotyledons

The ¹⁴C-labelled cyanogenic glucosides linustatin (diglucoside of acetone cyanohydrin) and linamarin (monoglucoside of acetone cyanohydrin), prepared by feeding [¹⁴C]valine to plants of Linum usitatissimum L., were applied to cotyledons of Hevea brasiliensis Muell.-Arg. in order to study their transport. Both [¹⁴C]-linustatin and [¹⁴C]linamarin were efficiently taken up by the cotyledons. Whereas ¹⁴C was recovered completely when [¹⁴C]linustatin was applied to the seedling, only about one-half of the radioactivity fed as [¹⁴C]linamarin could be accounted for after incubation. This observation is in agreement with the finding that apoplasmic linamarase hydrolyzes linamarin but not the related diglucoside linustatin. These data prove that, in vivo, linamarin does not occur apoplasmically and that linustatin, which is exuded from the endosperm, is taken up by the cotyledons very efficiently. Thus, these findings confirm the linustatin pathway (Selmar et al. 1988, Plant Physiol. 86, 711–716), which describes mobilization and transport of the cyanogenic glucoside linamarin, initiated by the glucosylation of linamarin to yield linustatin. When linustatin is metabolized to non-cyanogenic compounds, in Hevea this cyanogenic diglucoside is hydrolyzed by a diglucosidase which splits off both glucose molecules simultaneously as one gentiobiose moiety (Selmar et al. 1988). In contrast, [¹⁴C]linustatin, which is taken up by the cotyledon, is not metabolized but is reconverted in high amounts to the monoglucosidic [¹⁴C]linamarin, which then is temporarily stored in the cotyledons. These data demonstrate that in Hevea, besides the simultaneous diglucosidase, there must be present a further diglucosidase which is able to hydrolyze cyanogenic diglucosides sequentially by splitting off only the terminal glucose moiety from linustatin to yield linamarin. From this, it is deduced that the metabolic fate of linustatin, which is transported into the source tissues, depends on the activities of the different diglucosidases. Whereas sequential cleavage — producing linamarin — is purely a part of the process of linamarin translocation (using linustatin as the transport vehicle), simultaneous cleavage, producing acetone cyanohydrin, is part of the process of linamarin metabolization in which the nitrogen from cyanogenic glucosides is used to synthesize non-cyanogenic compounds.     

Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [¹⁵N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[¹⁵N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[¹⁵N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants.