Studies in Plant Growth Hormones: V. CHROMATOGRAPHY OF HORMONES IN EXCISED AND INTACT ROOTS OF TOMATO SEEDLINGS

1. An examination has been made of the hormones present in extractsof excised roots and intact seedling roots of tomato. Acid andneutral ether-soluble fractions and the ether-insoluble aqueousfraction were chromatographed, and the chromatograms assayedusing oat coleoptile sections. 2. The pattern of hormone activity in excised roots differedlittle from that in seedling roots. 3. On chromatograms of the aqueous fraction developed in isopropanol/ammonia, growth promotion occurred at the position of 3-indolylaceticacid (IAA, Rf 0·5) and sometimes at the position of 3-indolylacetonitrile(IAN, Rf 0·8). When the IAA zone was eluted off the paperand rechromatographed, it formed the IAN zone and another zoneof promotion at Rf 0·1–0·2. 4. When the aqueous fraction was developed in n-butanol/ammonia,promotion occurred at the position of IAA (Rf 0·15),at Rf 0·5, and at the position of IAN (Rf 0-85). Thesezones have been called X, Y, and Z respectively. They were alsoformed when the IAA zone in wopropanol/ammonia was rechromatographedin ammoniacal n-butanol. It is shown that X and Y are interconvertible,and that each can form Z on rechromatography; also, there issome evidence that Z can form X and Y. When Z was separatedinto ether-soluble (acid and neutral) and ether-insoluble fractionswith sodium bicarbonate solution and chromatographed in iiopropanol/ammonia,growth resulted at the position of Z in the neutral and aqueousfractions, but in the acid fraction it occurred at Rf 0·24–O·35.Comparison with other chromatograms indicates that this lastzone does not occur in the aqueous fraction but has been formedas a result of extraction with sodium bicarbonate solution. 5. The zones found in the aqueous fraction also occurred insmall quantities in acid and neutral ethereal fractions in anumber of experiments. 6. The ethereal fractions gave no chromogenic reactions withferric chloride/ perchloric acid, nitrous/nitric acid, or p-dimethylaminobenzaldehyde(MeAB). In the aqueous fraction only MeAB gave a reaction (yellow)which showed any consistent correlation with biological activity.A yellow colour with this reagent is not a characteristic chromogenicreaction of indole compounds. It is suggested that a non-indolehormone system may be operating in tomato roots.     

The Auxin Activity Extractable from Excised Tomato Roots by Cold 80 per cent. Methanol

Extracts of excised tomato roots prepared with cold aqueousmethanol have been partitioned between ethyl acetate and waterand chromatograms of the two fractions bioassayed by an Avenacoleoptile straight growth test and sprayed with various reagentsincluding those giving colour reactions with indole compounds.The greater part of the auxin activity is in the aqueous fractionand chromatograms of this fraction give positive indole reactions. The aqueous fraction chromatographed with isopropanol/ammonia/watershows two zones of growth-promoting activity. The zone of lowerRf contains tryptophane and unidentified ninhydrin-positivematerial. Tryptophane accounts for the activity of the Ehrlich-positiveregion of this zone. The activity of the zone of higher Rf isassociated with ninhydrin-positive material; this activity can,by use of other solvents, be distinguished in Rf from that ofthe common amino-acids which would occur in this zone on theprimary chromatograms. Chromatograms of the aqueous fractionalso show the presence of urea and of a phenol, neither of whichare associated with the zones of growth-promoting activity. The ethyl acetate fraction, and particularly the acid fractionof this, contains growth-promoting activity which, in a rangeof solvents, always corresponds in Rf to IAA. The acid fractioncontains a growth inhibitor corresponding in Rf to the ß-inhibitorof other workers. A zone of growth-promoting activity near thesolvent front on isopropanol/ammonia/water chromatograms containsmore than one active component, but IAN appears to be absent.     

Metabolism of Some Indole Auxins in Excised Tomato Roots

Indolylacetylaspartic acid (IAAsp) and possibly indolylacetylglutamicacid (IAG) are formed by exposure of excised tomato roots toIAA. Little ‘free’ IAA accumulates in the tissue.An unidentified substance reacting pink with nitricnitrite reagentis also formed. These substances are metabolized when IAA-treatedroots are transferred to auxin-free medium. IAAsp and IAA aresimilarly inhibitory to the growth of excised tomato roots.Excised tomato roots do not interconvert IAA and IAN. IAN-feedingleads to IAN accumulation and the appearance of indolylcarboxylicacid (ICA); transference to auxin-free medium causes a declinein the IAN activity but the ICA spot persists. The inhibitoryactivity of IAN is not due to its conversion to ICA. Excisedtomato and wheat roots respond very differently to externall-tryptophane but in neither case is there evidence of the conversionof tryptophane to ethyl acetate-soluble auxins.     

The Anther Smut of Sea Campion: A Study of the Role of Growth Regulators in the Dwarfing Symptom

The sympton of dwarfing in the sea campion, Silene vulgaris(Moench) Garcke sub sp. maritima(With.) A. & D. Löveinfected with the anther smut fungus Ustilago violacea (Pers.)Fuckel, a systemic, perennial parasite, has been investigated. Extracts of both healthy and diseased plants contain IAA andGA₃, but diseased plants contain less gibberellins than healthyplants. Neither IAA nor gibberellins were detected in significantquantities in the medium when U. violacea was grown in pureculture, but IAN was present. IAN was also found m extractsof diseased plants and it is tentatively suggested that it isformed by the fungus and may accumulate in the host owing tothe inability of the plant to convert IAN to IAA The exogenousapplication of IAN to healthy plants does not produce any diseasesymptoms. The dwarfing symptom of the diseased plant may be due to thelower levels of gibberellins which it contains compared withhealthy plants since the exogenous application of GA₃, restoresthe diseased plant to normal growth     

Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene.

Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern of NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo.     

Quantification of free plus conjugated indoleacetic acid in arabidopsis requires correction for the nonenzymatic conversion of indolic nitriles.

The genetic advantages to the use of Arabidopsis thaliana mutants for the study of auxin metabolism previously have been partially offset by the complexity of indolic metabolism in this plant and by the lack of proper methods. To address some of these problems, we developed isotopic labeling methods to determine amounts and examine the metabolism of indolic compounds in Arabidopsis. Isolation and indentification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAA]) was carried out under mild conditions, thus proving its natural occurrence. We describe here the synthesis of 13C1-labeled IAN and its utility in the gas chromatography-mass spectrometry quantification of endogenous IAN levels. We also quantified the nonenzymatic conversion of IAN to IAA under conditions used to hydrolyze IAA conjugates. 13C1-Labeled IAN was used to assess the contribution of IAN to measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known to be present in Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic biochemistry, since the levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible.     

STUDIES ON THE MECHANISM OF GROWTH INHIBITING EFFECT OF LIGHT

1. A substance which inhibits indoleacetic acid (IAA)-and naphthaleneaceticacid (NAA)-induced elongation of Avena coleoptile section andIAA-induced Avena coleoptile curvature was found in an ethersoluble neutral fraction of water extract of sunflower leavesand in agar blocks containing the diffusate from young sunflowerleaves.
2. This substance also inhibits the growth of isolatedsunflowerepicotyl.
3. The R_f value (0.9) of the substance ona paper chromatogramdeveloped with ammoniacal iso-propanolindicates that it isidentical with the inhibitor reported byAUDUS et al. (1956),but not with inhibitor-ß.
4. Theinhibitor can be transported from leaf to stem, and thetransportseems to be accelerated by illuminating the leaf.
5. The auxindiffused from sunflower leaf into agar block may beidenticalwith IAA.
6. A substance, which has the same properties as theinhibitorfrom sunflower leaf, was obtained in crystalline formfrom theleaf of Jerusalem artichoke.
7. The mechanism of growthinhibition caused by this crystallinesubstance seems to involveinactivation of a sulfhydryl group.
8. The reason why the stemgrowth of sunflower seedlings is reducedby strong light isdiscussed: the amount of the inhibitor transportedfrom leafto stem is increased under strong light, and in thestem, growthinhibition is caused by a direct effect of thisinhibitor ongrowth and by its inhibiting effect on the transportof IAAfrom leaf to stem.

¹ Present address: Botanical Garden, Faculty of Science, Universityof Tokyo, Tokyo (Received February 15, 1961; )     

Growth Regulators in Picea abies

The occurrence of growth regulators active in the Avena coleoptile straight-growth test in sprouting buds and seedlings of Norway spruce (Picea abies Karst.) was investigated. The acid ether fraction contained a growth stimulator, the Rf of which in isopropanol: ammonia: water was 0.2–0.4. This substance behaved as indole-3-acetic acid (IAA) in elec-trophoresis, in chromatography in various solvent systems on paper and on a Sephadex column. It gave the colour typical of IAA when sprayed with Ehrlich reagent and its fluorescence characteristics corresponded to IAA. Acid ether-soluble inhibitors showed most activity at Rf 0.4–0.7, but due to tailing they interfered with the determination of the stimulator at the Rf of IAA in the bioassay. They also masked the activity of other stimulators. Colour reactions were obtained with Ehrlich reagent in the inhibiting chromatogram zone. When eluates from this zone were tested in high dilutions or after gel filtration growth stimulation was obtained. The acid fraction of seedling shoots also contained a stimulator with Rf 0.7–0.8. In the neutral-basic ether-soluble fraction growth stimulation was obtained at Rf 0.5–0.7. The extracts also contained stimulatory substances insoluble in ether but soluble in n-butanol and partly in ethyl acetate. When the butanol fraction was hydrolyzed in 1 M NaOH a substance behaving as IAA when chromatographed was released.     

Root-promoting Substances in Salix alba

Root-promoting substances were extracted from softwood cuttings of Salix alba L. by centrifuging them with water or by shaking the ground freeze-dried stems with water. Rooting substances were partitioned by paper chromatography or chemical fractionation and their rooting activity was tested by mung bean cuttings. Both extracts indicated three major root -promoting fractions at Rf 0-0.1, 0.7-0.8, and 0.3-0.4 in a decreasing order of their activities when paper chromatographed with isopropanol:ammonia:water 8:1:1 v/v. The strongest one indicated an apparent synergistic rooting effect with indol-3yl-acetic acid (IAA) regardless of the extraction method. These results indicate that water can extract from freeze -dried sample the similar rooting substances found in the centrifugal diffusates. The Rf 0–0.1 fraction consisted of at least four fractions and the strongest one did not move from the starting line on the chromatogram when isopropanol:ammonia:water 8:1:1 was used. This starting line fraction was extremely strong in rooting activity and its highest concentration resulted in 8.7 times as many roots as controls. More thain additive rooting effect between IAA and the fraction was found only at the highest concentration. The fraction was very soluble in water but insoluble in chloroform or ethyl ether and only stimulated rooting of mung bean cuttings when it was applied within 3 days after cuttings were made. It had no effect in lengthening roots. The starting line fraction was further found to have four root-promoting subfractions at Rf 0.05, 0.35, 0.65, and 0.85 when it was chromatographed in 60 % isopropanol. Among these four, the subfractions at Rf 0.65 and 0.35 were strongly root promotive and displayed more than additive root promotion with IAA at the highest concentrations studied.     

The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.     

Natural Occurrence of Indoleacetamide and Amidohydrolase Activity in Etiolated Aseptically-Grown Squash Seedlings

Etiolated seedling tissues of aseptically grown squash (Cucurbitamaxima Duch) contain indole-3-acetamide (IAM) as a natural endogenouscompound, conclusively identified by gas chromatography-massspectrometry (GC-MS). Roots of aseptically raised seedlingsalso contain amide hydrolysing activity, which converts IAMto IAA, indoleacetonitrile (IAN) to IAM and IAA, and 1-naphthaleneacetamideto 1-naphthaleneacetic acid. This activity was enriched 48-foldby fractional precipitation with ammonium sulphate, Sephadexgel nitration and anion exchange chromatography. Being hydrolytic,it works equally well in air and in vacuo, without added cofactors.The partially purified enzyme works optimally between pH 7 and7.5, and a K_m value of 80 µM was calculated with IAM asthe substrate. The product of this reaction was definitivelyidentified as IAA by GC-MS. The temperature optimum of thisamidohydrolase lies around 45°C, and it is stable to freezing.A comparison of its properties with the amidohydrolase of Agrobacteriumor crown gall tissue, shows it to be different. In view of thenatural occurrence of both IAM and the amidohydrolase, it issuggested that the IAM pathway of IAA biogenesis is feasiblein etiolated squash seedlings. ⁴Deceased 2/2-1993.     

Regulation of cabbage (Brassica oleracea capitata) hypocotyl elongation, and infection by Peronospora parasitica, with B-indolylacetonitrile and chloramphenicol

Untreated excised segments of the hypocotyls of dark grown cabbage seedlings are always systemically infected when inoculated with the conidia of the obligate parasite Peronospora parasitica (Downy mildew). Cabbage hypocotyl elongation is promoted by 10^–4 M indolylacetonitrile (IAN) and this elongation is inhibited by 100 g mL^–1 chloramphenicol (CAM). The fungus remains localized in 5–8 day old hypocotyl segments exposed to CAM, but this inhibition is reversed by IAN. Indol-3-acetic acid (IAA) has the same effect as IAN. Both gibberellic acid and kinetin inhibit systemic infection. Conidial spore germination is not reduced by the CAM concentration used in these experiments. The success of the pathogen in the host is not correlated with host elongation, but is probably related to a common metabolic site in either host or pathogen affected by both CAM and IAN.Abbreviations IAN indolylacetonitrile - CAM chloramphenicol - IAA indol-3-acetic acid - G gibberellic acid - K kinetin     

The Effect of Carbon Dioxide Concentratioin, light Intensity and Isonicotinyl Hydrazide on the Photosynthetic Production of Glycollic Acid by Chlorella

Glycollic acid production by Chlorella was measured by colorimetricdetermination of the acid excreted into the medium. It was foundthat glycollic acid production showed a maximum at a low concentrationof carbon dioxide but tended toward zero as the rate of photosynthesisapproached carbon dioxide saturation. Glycollic acid productionbecame measurable at light intensities approaching that requiredto saturate photosynthesis and increased steadily with furtherincrease in intensity. Treatment with isonicotinyl hydrazideresulted in an approximately threefold stimulation of glycollicacid concentration over the range of conditions used. It issuggested that the precursor of glycollic acid is ribulose diphosphate,and that isonicotinyl hydrazide acts by inhibiting the furthermetabolism of glycollic acid.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

Growth-regulator Interactions in the Growth of the Shoot System of Avena sativa Seedlings: I. THE GROWTH OF THE FIRST INTERNODE

1. Segments, 3.5 mm. long, cut from the first internode of Avenasativa seedlings grown in complete darkness respond to bothauxins and gibberellic acid by accelerated extension. 2. The optimum concentration of indole-3-acetic acid (IAA) is10 p.p.m. and of gibberellic acid (GA) is 0.1 p.p.m. 3. The degree of stimulation relative to the growth of controlsegments is affected by the inclusion in the segement of thenode between the internode and coleoptile. Thus the gibberellineffect is greatly increased while the IAA effect is decreased.The optimal concentrations are not affected by inclusion ofthe node. 4. These results can best be explained in terms of the supplyby the node tissue of an endogenous auxin which is necessaryfor the expression of GA action. 5. Numerous factorial experiments demonstrated that there isno detectable interaction between applied IAA and GA in thepromotion of first-internode extension. This implies that thepostulated endogenous auxin which synergized GAA action in (4)is either an active form of IAA produced only in the node tissueor is a completely different auxin. 6. No synergism of growth-promotive action can be detected betweenGA and the two synthetic auxins I-naphthylacetic acid and 2,4-dichlorophenoxyaceticacid. 7. p-chlorophenoxy-iso-butyric acid (PCIB) anc 2,4,6-trichlorophenoxyaceticacid (2,4,6-T) act as weak auxins and thus antagonize competitivelythe promotive action of GA. 8. The anti-auxin -(I-naphythyl-methyl-sulphide)propionic acid(NMSP) antagonizes competitively the promotive action of bothIAA and GA. 9. The facts under (5)–(8) suggest that auxins and GAare acting at the same growth-promotion centres and may competefor them. 10. Growth inhibitions are induced by high concentrations ofPCIB, 2,4,6-T and NMSP. The inhibitions produced by PCIB and2,4,6-T are both synergized by supra-optimal concentrationsof IAA while that of NMSP is synergized by supra-optimal concentrationsof both IAA and GA. This similarity of the effects of IAA andGA suggests that their inhibition actions also are of a closelysimilar nature.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

Esters of indole-3-acetic Acid from Avena seeds

The present studies showed that about 80% of the indole-3-acetic acid extractable from Avena kernels by aqueous acetone was esterified to polymers precipitable by ammonium sulfate and ethanol or acetone. The polymers were positively charged, being adsorbed to cation exchange columns at a pH of 3, or below, and eluted at a pH greater than 4. The polymers were heterogeneous with respect to size, about 5,000 to 20,000 daltons, and charge, exhibiting apparent pK_a values of 4.2 and 4.7. The polymer fractions contained esterified IAA, anthrone-reactive material that liberated glucose upon acid hydrolysis, phenolic compounds, and peptidic material with a high proportion of hydrophobic amino acids. Since the esterified IAA was unstable, establishing polymer purity was not possible, and the designation IAA-glucoprotein fraction was adopted.     

Effects of Inhibitory Concentrations of 3-Indolylacetic Acid and 3-Indolylacetonitrile on Cell Division and Tissue 2

Inhibitory concentrations of 3-indolylacetonitrile (IAN) cause,in cultured excised tomato roots, a marked decrease in the rateof cell division at the apical meristem but only a slight reductionin the lengths of mature exodermal and cortical cells. The reducedrate of cell division is associated with a decrease in the.number of meristematic cells at the root apex. By contrast,3-indolylacetic acid (IAA) causes marked reduction in the lengthsof mature cortical cells but does not markedly reduce cell-divisionrate at the apical meristem. Various lines of evidence indicate that both IAA and IAN causea relative increase in the number of longitudinal and a decreasein the number of transverse division walls in the meristematiczone of the root apex. Partial inhibition of the linear growth of excised tomato rootsby IAA and IAN is accompanied by increases in root and stelardiameters. These increases result from radial enlargement ofthe cortical cells and increase in the number of stelar cellsin the transverse section. The enlarged steles contain an increasednumber of lignified xylem elements, but only with the most inhibitoryconcentration of IAN (10^–4g./ml.) is there evidence ofthe development of secondary xylem. Both auxins increase significantlythe xylem vessel unit length.     

Changes in Levels of Naturally Occurring Gibberellin-like Substances during Germination of Seed of Lycopersicon esculentum Mill.

Naturally occurring gibberellin-like substances possessing acidic,basic, and neutral properties were detected, by paper partitionchromatography, in ethanolic extracts of tomato seed and ofetiolated seedlings after 72 and 116 hours' growth. Dwarf maizemutants of the d-1 and d-5 types, ‘Meteor’ pea seedlingsand young ‘Potentate’ tomato plants were used asbioassay material. Hydrolysis of seed and seedling proteinsby ficin in phosphate buffer, pH 6.2, after removal of ethanol-solublesubstances, liberated more and different ‘bound’gibberellin-like substances. It is suggested that protein hydrolysisduring germination is an important means of liberating thesesubstances at different stages of seedling development. Acidic substances were present in all the extracts prepared,but in general two with Rfs 0.25 and 0.55 in iso-propanol: ammonia:water : : 10:1:1 v/v were differentiated on d-2 and d-5 maizerespectively. Neutral substances in dry seed extracts chromatographedin the same solvent, had Rfs of 0.05, 0.35, and 0.95 and thesewere found only in the ethanolic (‘free’) extracts.They were active on d-1 and d-2 maize and ‘Meteor’pea. Basic gibberellin-like substances with Rfs of 0.05 and0.35 were found in ‘free’ extracts of both dry seedand etiolated seedlings after 116 hours' growth which were activeon d-2 maize only. Two others with Rfs 0.45 and 0.95 were extractedfrom seedlings after 72 hours' growth and these were activeon young ‘Potentate’ tomato plants. It is suggested that certain gibberellin-like substances, capableof reversing dwarfism in test plants, may be responsible formorphogenetic or other responses not involving stem extensionin the parent species. Changes were found in the levels of gibberellin-likesubstances but there was no evidence of changes in levels ofseed inhibitors relative to seed growth substances.     

The Timing of Growth Promotion and Conversion to Indole-3-acetic Acid for Auxin Precursors

Using a high resolution continuous recording technique, the length of the latent period preceding the growth response to two suspected precursors of indole-3-acetic acid (IAA) indole-3-acetonitrile (IAN), and indole-3-ethanol was studied in corn (Zea mays L.), wheat (Triticum aestivum L.), and peas (Pisum sativum L.). The timing of the conversion in vivo of these presumed precursors to IAA was also examined. In wheat the rate of conversion of IAN to IAA is rapid, and the latent period in the growth response to IAN is correspondingly short. In corn the rate of conversion in vivo of IAN to IAA is slow, and there is a long latent period in the growth response. In peas there is no conversion of IAN to IAA, and there is no measurable growth response to IAN. These data indicate that IAN is active in promoting growth only after conversion to IAA. This conclusion is strengthened by transport studies. Using a similar approach, we have also obtained data indicating that indole-3-ethanol is active only after conversion to IAA.