Maize root growth and localized indol-3yl-acetic Acid treatment: a new methodological approach

Resin beads loaded with indol-3yl-acetic acid (IAA) were used as asymmetrical donors along the elongation zone of intact primary Zea mays L. roots. A strong curvature, towards and above the bead, occurred when IAA was applied at a mean distance of 2.20 mm from the tip. No curvature was detected after applications at 3.89 and 5.71 mm from the tip. Correspondence analysis, a new methodological approach in plant hormone studies, permitted the evaluation of the relative influence of several factors on the curvature observed for each root. The parameters considered were the initial growth rate, the exact location of the bead (1.64-2.73 millimeters from tip) and the quantity of IAA absorbed. Roots which grew rapidly bent earlier than slowly growing ones and the more basal the treatment was, the less curvature occurred. Surprisingly, the amount of IAA taken up (between 1.2 and 2.2 times the endogenous IAA content) was found to have no influence on either the time-course or the magnitude of this growth inhibition (curvature). The usefulness of this multivariate analysis is also discussed.     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays

A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth.     

Response to Gravity by Zea mays Seedlings : I. Time Course of the Response

Gravistimulation induces an asymmetric distribution of free indole-3-acetic acid (IAA) in the cortex-epidermis of the Zea mays L. cv `Stowells Evergreen' mesocotyl within 15 minutes, the shortest time tested. IAA was measured by an isotope dilution method as the pentaflurobenzyl ester. The per cent IAA in the lower half of the mesocotyl cortex was 56 to 57% at 15, 30, and 90 minutes after stimulus initiation. Curvature is detectable in the mesocotyl within 3 minutes after beginning gravitropic stimulation. The rate of curvature of the mesocotyl increases during the first 60 minutes to a maximum of about 30° per hour. Thus, the growth asymmetry continues to increase for 45 minutes after hormone asymmetry is established.

Free IAA occurs predominantly in the stele of the mesocotyl whereas esterified IAA is mainly in the mesocotyl cortex-epidermis. This compartmentation may permit determining in which tissue the hormone asymmetry arises. Current data suggest the asymmetry originated in the stele.

     

Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles

We have investigated the transport of tritiated indole-3-acetic acid (IAA) in intact, red light-grown maize (Zea mays) coleoptiles during gravitropic induction and the subsequent development of curvature. This auxin is transported down the length of gravistimulated coleoptiles at a rate comparable to that in normal, upright plants. Transport is initially symmetrical across the coleoptile, but between 30 and 40 minutes after plants are turned horizontal a lateral redistribution of the IAA already present in the transport stream occurs. By 60 minutes after the beginning of the gravitropic stimulus, the ratio of tritiated tracer auxin in the lower half with respect to the upper half is approximately 2:1. The redistribution of growth that causes gravitropic curvature follows the IAA redistribution by 5 or 10 minutes at the minimum in most regions of the coleoptile. Immobilization of tracer auxin from the transport stream during gravitropism was not detectable in the most apical 10 millimeters. Previous reports have shown that in intact, red light-grown maize coleoptiles, endogenous auxin is limiting for growth, the tissue is linearly responsive to linearly increasing concentrations of small amounts of added auxin, and the lag time for the stimulation of straight growth by added IAA is approximately 8 or 9 minutes (TI Baskin, M Iino, PB Green, WR Briggs [1985] Plant Cell Environ 8: 595-603; TI Baskin, WR Briggs, M Iino [1986] Plant Physiol 81: 306-309). We conclude that redistribution of IAA in the transport stream occurs in maize coleoptiles during gravitropism, and is sufficient in degree and timing to be the immediate cause of gravitropic curvature.     

The effect of light on metabolism of IAA in maize seedlings

Carbon 14-labelled indole-3-acetic acid (IAA) was fed to segments of shoots of Zea mays seedlings grown in light or dark to find the effect of light on IAA metabolism. The seedling parts coleoptile, with enclosed leaf, and mesocotyl were also used to examine differences in IAA metabolism between tissue types. The rate of metabolite formation as a function of time ranging from 1 to 12 hours was determined. Light did not significantly influence the amount of IAA taken up, but significantly increased its rate of metabolism and greatly increased the content of amide conjugates formed. There were also differences in metabolism depending on tissue type. In all tissues, IAA was metabolized mainly into six compounds. Four were tentatively identified as IAA-glucose (IAGlc), IAA-myo-inositol} (IAInos), indole acetamide (IAAm) and IAA-aspartic acid (IAAsp). 1-O-IAA-D-glucose (1-O-IAGlc) was the first conjugate formed and, except for mesocotyls in the light, it was the most abundant conjugate in maize tissue. In mesocotyl tissue the conversion of IAA into IAAsp was greatly stimulated by light, and the biosynthesis of IAAsp exceeded that of IAGlc. Since light strongly inhibited the growth of the mesocotyl, it is possible that the stimulation of IAAsp synthesis by light causes depletion of free IAA with resultant inhibition of mesocotyl growth.     

Role of IAA conjugates in inducing ethylene production by tobacco leaf discs

Indole-3-acetic acid (IAA) labeled in its carboxyl group was metabolized by tobacco leaf discs (Nicotiana tabacum L. cv. Xanthi) into three metabolites, two of which were preliminarily characterized as a peptide and an ester-conjugated IAA. Reapplication of each of the three metabolites (at 10 μM) resulted in a marked stimulation of ethylene production and decarboxylation by the leaf discs. Similarly, these three IAA metab olites could induce elongation of wheat coleoptile segments, which was accompanied by decarboxylation. Both the exogenously supplied esteric and peptidic IAA conjugates were converted by the leaf discs into the same metabolites as free IAA. (1-¹⁴C)IAA, applied to an isolated epidermis tissue, was completely metabolized to the esteric and peptidic IAA conjugates. This epidermis tissue showed much higher ethylene production rates and lower decarboxylation rates than did the whole leaf disc. The results suggest that the participation of IAA conjugates in the regulation of various physiological processes depends on the release of free IAA, which is obtained by enzymatic hydrolysis of the conjugates in the tissue. The present study demonstrates biological activity of endogenous IAA conjugates that were synthesized by tobacco leaf discs in response to exogenously supplied IAA.     

The role of the conversion of IBA into IAA on root regeneration in apple: introduction of a test system

A test system for root regeneration was developed that consists of stem slices of apple shoots (ca. 0.5 mm thick; fresh weight ca. 1 mg). Roots regenerated synchronously without intervening callus formation and without interference with compounds originating from other parts of the plant. Supply of indolebutyric acid (IBA) or indoleacetic acid (IAA) induced maximally an average of 8 or 4.5 roots per slice, respectively. After uptake of IBA, a high degree of conjugation resulted in a recovery of 2.5% as internal free IBA (ca. 2 times the medium concentration). Due to conversion of absorbed IBA into IAA a fraction of 0.4% was recovered as (physiologically active) free IAA. After incubation on medium with IAA, 0.5% of the absorbed hormone was recovered in the free acid form. No conversion of IAA into IBA was observed. Equimolar contents of internal free IAA after incubation on IBA or IAA resulted for IBA in a higher number of roots than for IAA. This means that IBA may also act via internal free IBA or may synergistically modify the action or endogenous synthesis of IAA.Abbreviations BHT butyl hydroxy toluene - EtOH ethanol - FW fresh weight - HAc acetic acid - HPLC high performance liquid chromatography - IAA indoleacetic acid - IAA^int free (internal) IAA in the tissue derived from IBA or IAA uptake - IBA indolebutyric acid - IBA^int free (internal) IBA in the tissue derived from IBA or IAA uptake - MeOH methanol - PAR photosynthetic active radiation - SEM standard error of the mean - TLC thin layer chromatography - UV ultra violet     

Kinetic properties of IAA oxidase from mung bean cotyledons

A crude enzyme preparation from mung bean cotyledons was separated into peroxidative and non-peroxidative IAA oxidase on a DEAE-cellulose column. Both fractions differed in their pH optima, K_m and V_max. The K_m and V_max of non-peroxidative IAA oxidase were higher than those of peroxidative IAA oxidase. Peroxidative IAA oxidase showed a linear increase in absorption at 247 and 254 nm after a short lag of 2–3 min. The addition of catalytic amounts of hydrogen peroxide eliminated the lag period and also enhanced the rate of IAA degradation. The non-peroxidative IAA oxidase fraction, however, did not exhibit any significant increase in absorption at 247 and 254 nm and showed a lag period of 5 min which was not affected by hydrogen peroxide. Instead, addition of the same catalytic amount of hydrogen peroxide inhibited the rate of IAA degradation. The peroxidative IAA oxidase fraction exhibited the reaction kinetics characteristic of peroxidase-catalysed IAA degradation. The rate of IAA oxidation by purified non-peroxidative IAA oxidase was very low. The slow rate of catalysis shown by non-peroxidative IAA oxidase appears to be due to the presence of inhibitor(s).     

Distribution and transport of IAA in tomato plants

We determined as to indole-3-acetic acid (IAA), endogenous levels byliquid chromatography-mass spectrometry using ¹³C₆-IAA, diffusible levels byfluorometric detection using indole-propionic acid, and polar transportactivityby radioactive IAA in 3-month-old tomato plants (stems, leaves or roots). TheIAA concentration in the apoplast (AP) solution was higher than those in thesymplast (SP) solution in both the upper and lower parts of stems, showing thatIAA analysis of AP solution is important. Younger leaves exported much morediffusible IAA than older leaves. The IAA concentration in the main roots wasalmost at the same level as in the stems. The results suggested that thetransport capacity of IAA is probably the limiting factor for the amount of IAAtransported in stems and the amount of polar IAA transport might be only 19% ofthe endogenous IAA amount in stems.     

Biochemical Bases for the Loss of Basipetal IAA Transport with Advancing Physiological Age in Etiolated Helianthus Hypocotyls: Changes in IAA Movement, Net IAA Uptake, and Phytotropin Binding

Basipetal transport of [¹⁴C]IAA in hypocotyl segments isolated from various regions of etiolated Helianthus annuus L. cv NK 265 seedlings declines with increasing physiological age. This decline was the result of a reduction in both transport capacity and apparent velocity. Net IAA uptake was greater and the abilities of auxin transport inhibitors to stimulate net IAA uptake were reduced in older tissues. Net IAA accumulation by microsomal vesicles exhibited a similar behavior with respect to age. Specific binding of [³H]N-1-naphthylphthalamic acid (NPA) to microsomes prepared from young and older hypocotyl regions was saturable and consistent with a single class of binding sites. The apparent affinity constants for NPA binding in microsomes prepared from young versus older tissues were 6.4 and 10.8 nanomolar, respectively, and the binding site densities for young versus old tissues were 7.44 and 3.29 picomoles/milligram protein, respectively. Specific binding of [³H]NPA in microsomes prepared from both tissues displayed similar sensitivities toward unlabeled flurenol and exhibited only slight differences in sensitivity toward 2,3,5-triiodobenzoic acid. These results demonstrate that the progressive loss of basipetal IAA transport capacity in etiolated Helianthus hypocotyls with advancing age is associated with substantial alterations in the phytotropin-sensitive, IAA efflux system and they suggest that these changes are, at least partially, responsible for the observed reduction of polar IAA transport with advancing tissue age.     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Evidence That IAA Conjugates Are Slow-Release Sources of Free IAA in Plant Tissues

Evidence that indoleacetic acid (IAA) conjugates are metabolized via enzyme-catalyzed hydrolysis to free IAA and that their biological activities are related to the rates at which they are hydrolyzed by the tissue is presented. These conclusions are based on the following observations. Slow but continuous decarboxylation of the IAA moiety of IAA-l-alanine and IAA-glycine occurs when these conjugates are applied to pea (Pisum sativum L. cv. Alaska) stem segments. Inasmuch as IAA conjugates are protected from peroxidase-catalyzed oxidative decarboxylation, the conjugates are probably hydrolyzed and the freed IAA then further metabolized. Free IAA and IAA-l-alanine are converted, by pea stem tissue, into the same metabolites. The metabolism is enzymic, since conjugates of IAA with the d-isomers of the amino acids are inactive. Ethylene production induced by IAA-l-alanine and by IAA-glycine is correlated with their hydrolysis, as indicated by their decarboxylation and with the appearance or nonappearance of IAA metabolites in the tissues.     

Effect of indoleacetic acid,abscisic acid,root tips and coleoptile tips on growth and curvature of maize roots

The effect of externally applied indoleacetic acid (IAA) and abscisic acid (ABA) on the growth of roots of Zea mays L. was measured. Donor blocks of agar with IAA or ABA were placed laterally on the roots and root curvature was measured. When IAA was applied to vertical roots, a curvature directed toward the donor block was observed. This curvature corresponded to a growth inhibition at the side of the root where the donor was applied. When IAA was applied to horizontal roots from the upper side, normal geotropic downward bending was delayed or totally inhibited. The extent of retardation and the inhibition of curvature were found to depend on the concentration of IAA in the donor block. ABA neither induced curvature in vertical roots nor inhibited geotropic curvature in horizontal roots; thus the growth of roots was not inhibited by ABA. However, when, instead of donor blocks, root tips or coleoptile tips were placed onto vertical roots, a curvature of the roots was observed.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

Subcellular localization of endogenous IAA during poplar leaf rhizogenesis revealed by in situ immunocytochemistry

Poplar 741 [Populus alba × (P. davidiana + P. simonii) × P. tomentosa] leaves were rooted within 8 days when cultured on 1/2 MS medium. The subcellular localization of endogenous indole-3-acetic acid (IAA) in the rhizogenesis was investigated, using an immunocytochemical approach. The results of IAA subcellular localization revealed organelle-specific distribution. Three days after root induction, IAA in vascular cambium cells of the basal region of the petiole was distributed mainly in the plasma membrane, endoplasmic reticulum (ER), and nucleus, with a lesser amount in the cytoplasm. In phloem of the basal region of the petiole, IAA was detected in the plasma membrane and ER of the companion cell and in the plasma membrane of the sieve element. In xylem of the basal region of the petiole, no IAA gold particles were labeled. In mesophyll cells IAA was distributed in the chloroplast starch grains before root induction, and the amount in the chloroplast starch grains increased after 3 days after root induction. This suggests that the plasma membrane and nucleus of cambium cells may be the target sites where IAA performs its physiological activities during poplar leaf rhizogenesis. IAA polar transport from lamina mesophyll to the basal region of the petiole during rhizogenesis is mediated by phloem. The starch grains of mesophyll chloroplasts appeared to accumulate IAA and may be a source of IAA during poplar leaf rhizogenesis. Novel and direct evidence regarding the function of IAA during rhizogenesis is provided in this study.     

Interaction of indoleacetic Acid with its inositol and glucoside conjugates in Avena coleoptile curvature

Avena coleoptile curvature is promoted by indoleacetic acid (IAA) IAA-glucoside, and IAA-inositol when these substances are applied in agar to the decapitated apical end of deseeded plantlets. Absorption of [³H]IAA-inositol over a wide range of concentrations during the 20 hour period of incubation is only 20 to 50% of the applied amount, compared with 85 to 92% of uptake of the applied [³H]IAA at equimolar concentrations. The absorption of IAA-glucoside could not be readily measured. The stimulation by both IAA-conjugates is very similar to that of free IAA at low concentrations (0.2 and 0.4 micromolar), but much less at higher concentrations. The interaction of free IAA with IAA-glucoside is additive or synergistic (depending on concentration). The interaction of free IAA with IAA-inositol is an inhibition (i.e. less than additive). The simultaneous application of equimolar concentrations of free IAA does not change the chromatographic pattern of the metabolic products of [³H] IAA-inositol. One of the more polar metabolites of [³H]IAA-inositol has chromatographic characteristics similar to the major polar metabolite of free [³H]IAA on an isocratically eluted reversed phase C₁₈ high performance liquid chromatography system that separates a number of IAA sugar and amino acid conjugates from each other, and from free IAA.     

Polarity of IAA Effect on Sieve-Tube and Xylem Regeneration in Coleus and Tomato Stems

A technique is described for the processing of regenerated xylem and sieve tubes from the same wound area for microscopic and quantitative comparison.

Regeneration was examined in internodes of 2 developmental stages in Coleus: internode 2, elongating, characteristic of primary growth; and internode 5, non-elongating, characteristic of secondary growth.

Transport of indoleacetic acid (IAA) in excised number 5 internodes of Coleus is strictly polar, in a basipetal direction, judging by a regeneration bioassay involving both sieve tube strands and xylem cells. Similar results were obtained with tomato.

If isolated number 5 Coleus internodes are not treated with hormone, they regenerate no xylem cells and a small number of sieve tube strands. With increasing concentrations of IAA added apically, the number of regenerated sieve tube strands (and, with higher concentrations, of xylem cells) increases progressively up to 1% IAA, the highest concentration applied.

Internode 2 of Coleus regenerates fewer xylem cells or sieve tube strands than internode 5, whether on the otherwise intact plant or with a given concentration of IAA added apically. The amount of regenerated xylem increases with added apical IAA, except that the highest concentration gives no further increase. The number of xylem cells regenerated in intact plants occurs at the same interpolated IAA concentration as in number 5 internodes. No concentration of IAA tried provided replacement of intact number of sieve tube strands in internode 2.

IAA can exert a regenerative stimulus on both xylem and sieve tubes in the area immediately adjacent to the site of its application.

     

Free and Conjugated Indoleacetic Acid (IAA) Contents in Transgenic Tobacco Plants Expressing the iaaM and iaaH IAA Biosynthesis Genes from Agrobacterium tumefaciens

The Agrobacterium tumefaciens T-DNA gene iaaM was introduced by leaf-disc transformation into transgenic tobacco (Nicotiana tabacum) plants expressing the iaaH gene. Regenerated calli were screened for the presence of indole-3-acetamide (IAM), by gas chromatography-multiple ion monitoring-mass spectrometry, and IAM-containing calli were further analyzed for free and conjugated indoleacetic acid (IAA). It was found that transgenic calli on average contained twice as much free IAA and three times more conjugated IAA than calli from wild-type plants. About 40% of the transformed calli could be regenerated to plants. The distribution of free and conjugated IAA was measured in transformed plants with a normal phenotype and compared with equivalent wild-type plants. The IAA content of transgenic plants was only slightly increased, whereas IAA-conjugate levels were enhanced significantly. These data suggest that conjugation of IAA may serve as a regulatory mechanism, contributing to maintenance of steady-state IAA pool sizes during tobacco growth and development.