Conjugation of aspartic acid with 4-bromophenylacetic acid,an auxin analogue of aspartic acid

Tobacco mesophyll protoplasts conjugate the auxins indoleacetic acid and naphthaleneacetic acid with aspartic acid very efficiently. This conjugation was found to be correlated with the toxicity of these molecules to protoplast-derived cells grown at low densities. Among a series of halogenated phenylacetic acids, 4-bromophenylacetic was toxic to cells grown at low densities although not able to stimulate proliferation at high cell densities, as opposed to indoleacetic acid and naphthaleneacetic acid. [¹⁴C-car☐yl]-4-bromophenylacetic acid was conjugated with aspartic acid by tobacco protoplasts. Although 4-bromophenylacetic acid is not an auxin, this molecule shares with auxins some of their properties.     

Auxin Conjugation by Tobacco Mesophyll Protoplasts : Correlations between Auxin Cytotoxicity under Low Density Growth Conditions and Induction of Conjugation Processes at High Density

Auxin induction of the proliferation of Nicotiana tabacum (cv Xanthi) mesophyll protoplasts and of protoplast-derived cells was studied. The growth-promoting properties and cytotoxicities at high concentrations of IAA and naphthaleneacetic acid were strongly affected by cell density. The induction of growth by 2,4-dichlorophenoxyacetic acid and picloram was not affected by cell density. The comparison of catabolism of these [¹⁴C]-labeled auxins by protoplasts showed that IAA and naphthalene-acetic acid were rapidly accumulated and conjugated unlike 2,4-dichlorophenoxyacetic acid and picloram. The major catabolite derived from naphthaleneacetic acid was identified as naphthaleneacetyl-l-aspartate. The biosynthesis of this conjugate in protoplasts was inducible by naphthaleneacetic acid concentrations found to be cytotoxic under low density growth conditions. However, although it was taken up by cells, the conjugate was not cytotoxic at concentrations as high as 0.2 mm under low density growth conditions. The relationship between conjugation processes and auxin cytotoxicity is discussed.     

Modified hormonal balance in rooting-recalcitrant rac mutant tobacco shoots

ABSTRACT

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the D8 wild-type. The mutant shoots grew at a lower rate and did not root whatever the treatments, whereas the wild-type shoots rooted spontaneously during the culture cycle without auxin treatment. The mutant and wild-type shoots showed similar peroxidase variations along the culture cycle (21 days) but with higher levels of activity for the rac mutant: minimum peroxidase activity occurred at day 14 in whole shoots of both tobacco genotypes, but already at day 7 in the basal parts of the stems (where roots appear) of the wild-type tobacco, while it was delayed in the mutant. Free and conjugated auxin and polyamine levels were also determined in whole shoots and basal parts of the stems. The rac mutant was characterised by higher auxin and polyamine contents. A peak of auxins and polyamines appeared at day 14 in the whole shoots whatever the tobacco genotype. This peak was delayed in the basal parts of the rac stems compared to the wild-type ones. The mutant shoots contained higher levels of benzyladenine and isopentenyladenosine at the end of the culture cycle, whereas zeatin riboside was more abundant in wild-type shoots. In response to increasing concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), only the wild-type shoots responded by an increase in growth rate followed by inhibition at high concentrations. The rac shoot responses were very low or nonexistent. Peroxidase activity was also measured in the basal parts of tobacco stems grown in the presence of IBA. Results suggest growth inhibition related to auxin accumulation, possibly combined with elevated putrescine content. Second, rooting induction seems to take place in both tobacco genotypes; however, the process of root formation is blocked in the mutant. The lack of initiation and expression phases of rooting in relation to auxin content in the mutant is discussed.     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division     

Host and T-DNA determinants of cytokinin autonomy in tobacco cells transformed byAgrobacterium tumefaciens

The hormone autonomy of tobacco (Nicotiana tabacum L.) cells transformed byAgrobacterium tumefaciens containing mutations attmr (the “rooty” locus) of the pTiT37 plasmid has been examined. These cells require cytokinin, but not auxin for continuous growth in culture, indicating that the function of thetmr locus is to specify or induce cytokinin autonomy. Examination of tissues from plants regenerated from cells transformed by the mutant bacteria showed that the auxin independent phenotype is suppressed, but can be reinitiated in culture by exposure to an exogenous supply of auxin. In addition the developmental state of the cells from such regenerated plants can exert a profound influence on their cytokinin autonomy phenotype.     

UGT74D1 Is a Novel Auxin Glycosyltransferase from Arabidopsis thaliana

Auxin is one type of phytohormones that plays important roles in nearly all aspects of plant growth and developmental processes. The glycosylation of auxins is considered to be an essential mechanism to control the level of active auxins. Thus, the identification of auxin glycosyltransferases is of great significance for further understanding the auxin regulation. In this study, we biochemically screened the group L of Arabidopsis thaliana glycosyltransferase superfamily for enzymatic activity toward auxins. UGT74D1 was identified to be a novel auxin glycosyltransferase. Through HPLC and LC-MS analysis of reaction products in vitro by testing eight substrates including auxins and other compounds, we found that UGT74D1 had a strong glucosylating activity toward indole-3-butyric acid [IBA], indole-3-propionic acid [IPA], indole-3-acetic acid [IAA] and naphthaleneacetic acid [NAA], catalyzing them to form corresponding glucose esters. Biochemical characterization showed that this enzyme had a maximum activity in HEPES buffer at pH 6.0 and 37°C. In addition, the enzymatic activity analysis of crude protein and the IBA metabolite analysis from transgenic Arabidopsis plants overexpressing UGT74D1 gene were also carried out. Experimental results indicated that over-production of the UGT74D1 in plants indeed led to increased level of the glucose conjugate of IBA. Moreover, UGT74D1 overexpression lines displayed curling leaf phenotype, suggesting a physiological role of UGT74D1 in affecting the activity of auxins. Our current data provide a new target gene for further genetic studies to understand the auxin regulation by glycosylation in plants.     

Relationship between Auxin and Amino Acid Metabolism of Tobacco Protoplast-Derived Cells

Single amino acids were found to be highly toxic to protoplast-derived cells of tobacco (Nicotiana tabacum cv Xanthi) cultured at low density in a culture medium containing a low naphthaleneacetic acid concentration (0.05 micromolar). The cytotoxicities of alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, proline, and valine were reduced when the naphthaleneacetic acid concentration of the culture medium was increased to 1 micromolar. This selective modification of amino acid toxicity by naphthaleneacetic acid could not be correlated with modifications of uptake rates or incorporation of these amino acids into protein or amino acid-auxin conjugates. A mutant clone resistant to high naphthaleneacetic acid concentrations and affected in root morphogenesis did not display, at the cellular level, the naphthaleneacetic acidmediated modification of amino acid cytotoxicity.     

LIGHT AND HORMONAL CONTROL OF ROOT FORMATION IN ZEA MAYS CALLUS CULTURES

Callus cultures of Zea mays were used to study the interaction of light with exogenous cytokinin/auxin levels in the initiation, growth and development of roots. Three auxins, indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4 dichlorophenoxyacetic acid (2,4 D) were remarkably different in their effects on callus growth and root initiation. NAA at concentrations of 5 and 25 μM produced the highest combined yields of callus and roots under low light conditions. No significant morphological effects on roots were observed with the three auxins tested nor did low and intermediate light intensities alter root development.
At intermediate light levels the addition of the cytokinin, zeatin, was also able to influence the differentiation of the callus tissue. Increasing the cytokinin/auxin ratio from low to high shifted the development from callus growth to abundant root formation. High light caused the formation of short, thick roots. This effect could be counteracted in part by zeatin which promoted elongation. These observations suggest that both, the cytokinin/auxin ratio and light play an important role in the development of monocotyledonous roots.     

Auxin-dependent cell division and cell elongation. 1-Naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid activate different pathways

During exponential phase, the tobacco (Nicotiana tabacum) cell line cv Virginia Bright Italia-0 divides axially to produce linear cell files of distinct polarity. This axial division is controlled by exogenous auxin. We used exponential tobacco cv Virginia Bright Italia-0 cells to dissect early auxin signaling, with cell division and cell elongation as physiological markers. Experiments with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated that these 2 auxin species affect cell division and cell elongation differentially; NAA stimulates cell elongation at concentrations that are much lower than those required to stimulate cell division. In contrast, 2,4-D promotes cell division but not cell elongation. Pertussis toxin, a blocker of heterotrimeric G-proteins, inhibits the stimulation of cell division by 2,4-D but does not affect cell elongation. Aluminum tetrafluoride, an activator of the G-proteins, can induce cell division at NAA concentrations that are not permissive for division and even in the absence of any exogenous auxin. The data are discussed in a model where the two different auxins activate two different pathways for the control of cell division and cell elongation.     

Auxin effect on the transmembrane potential difference of wild-type and mutant tobacco protoplasts exhibiting a differential sensitiity to auxin

The effects of 1-naphthaleneacetic acid (NAA) and other auxin analogs on the transmembrane potential difference (Em) were compared on tobacco protoplasts isolated from two genotypes differing in their sensitivity to auxins. For both types, NAA modifies Em by inducing at low doses a hyperpolarization, the amplitude of which increased with auxin concentration. Above an optimal concentration this hyperpolarization was reduced and even nullified. However, for the mutant type, this electrical response was shifted toward higher NAA concentrations, as its growth response. In the presence of structural analogs of auxin which have been showed to modify the dose-response curve for growth, the Em was altered: the growth-stimulatory molecule (picloram) initiated hyperpolarization, whereas the growth-inhibitory substance (4-bromophenylacetic acid) caused depolarization. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity.     

A morphological and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryogenic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid

Summary Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron microscopy. Both auxins promoted enhanced cell division, particularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus growth, and embryo morphology. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal morphology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heterogeneous regions of cell division. Proliferating cell regions were composed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medium with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meristems were often lacking or partially obliterated due to callus proliferation. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - SEM scanning electron microscopy     

Tissue culture and plant regeneration of okra (Abelmoshus esculentus)

Explants (hypocotyl, cotyledon, cotyledonary node and leaf segment) were excised from aseptically grown okra (Abelmoschus esculentus) seedlings. The explants were cultured on a Murashige and Skoog basal nutrient medium supplemented with auxins, cytokinins and auxin-cytokinin combinations. Callus formation and root differentiation occurred in a medium containing naphthaleneacetic acid (NAA) or indoleacetic acid. There was a greater proliferation of roots on medium supplemented with NAA. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the growth medium suppressed root formation. No shoot bud or shoot development was observed at any of the auxin levels tested. Both kinetin (KN) and zeatin (Z) also proved ineffective in inducing shoot buds or shoots. Shoots were produced on cotyledon and cotyledonary node explants cultured in a medium supplemented with benzyladenine and NAA. These shoots developed roots on the same medium. The plantlets, on transfer to soil, grew normally.     

The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance

Mutagenized populations of Arabidopsis thaliana seedlings were screened for plants capable of root growth on inhibitory concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Four of the mutant lines recovered from this screen display a defect in root gravitropism as well as hormone resistance. The aerial portions of these plants are similar to wild-type in appearance. Genetic analysis of these four mutants demonstrated that hormone resistance segregated as a recessive trait and that all four mutations were alleles of the auxin-resistant mutation aux1 [Maher HP, Martindale SJB (1980) Biochem Genet 18: 1041-1053]. These new mutants have been designated aux1-7, 1-12, 1-15, and 1-19. The sensitivity of wild-type and aux1-7 roots to indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and ethylene was determined. The results of these assays show that aux1-7 plants require a 12-fold (indole-3-acetic acid) or 18-fold (2,4-dichlorophenoxyacetic acid) higher concentration of auxin than wild-type for a 50% inhibition of root growth. In addition, ethylene inhibition of root growth in aux1-7 plants is approximately 30% that of wild-type at saturating ethylene concentrations. These results indicate that aux1 plants are resistant to both auxin and ethylene. We have also determined the effect of ethylene treatment on chlorophyll loss and peroxidase activity in the leaves of aux1 and wild-type plants. No difference between mutant and wild-type plants was observed in these experiments, indicating that hormone resistance in aux1 plants may be limited to root growth. Our studies suggest that the AUX1 gene may have a specific function in the hormonal regulation of gravitropism.     

Effects of natural and synthetic auxins on the gravitropic growth habit of roots in two auxin-resistant mutants of Arabidopsis, axr1 and axr4: evidence for defects in the auxin influx mechanism of axr4.

The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium. Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect. Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA. The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells. The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins. These results suggest that the lesion of axrl mutants is different from that of axr4.     

Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.     

THE EFFECT OF AUXINS UPON PROTOPLASMIC STREAMING

1. Evidence has accumulated that the action of auxins in promoting growth is exerted not upon the cell wall but upon the cell contents; i.e., the protoplasm. Following indications previously obtained, therefore, the effect of auxins on the rate of protoplasm streaming in the Avena coleoptile was studied. 2. Indole-3-acetic acid, the most active auxin available in pure form, was found to increase the rate of streaming in the epidermal cells of the Avena coleoptile at concentrations between 0.5 and 0.002 mg. per liter, the maximum increase being brought about at 0.01 mg. per liter. This concentration is approximately that which, applied in agar to one side of the decapitated coleoptile, would give a curvature of 1°; i.e., it is well within the range of concentrations active in growth promotion. It is, however, much less than that which produces maximum elongation in immersed sections of Avena coleoptiles. 3. This accelerating effect is readily determined quantitatively by comparison with the streaming in control coleoptiles in pure water, which, if thoroughly aerated, maintain a constant rate for over an hour. The accelerating effect takes place immediately and is over within about 30 minutes. 4. Concentrations of indole-3-acetic acid greater than 0.5 mg.per liter inhibit the streaming, the effect being also over in about 30 minutes, and its extent increasing with increasing auxin concentration. This parallels the effect of high auxin concentrations in inhibiting elongation, although the inhibition of streaming is obtained at much lower concentrations than inhibit elongation. 5. The effects of indole-3-acetic acid on streaming are not specific for that substance, but appear to be common to auxins in general. Thus coumaryl-3-acetic acid and allocinnamic acid, both of which bring about cell enlargement, root formation, and bud inhibition, i.e. are typical auxins, also cause an immediate acceleration of the rate of streaming, and as with indole-acetic add the effect is over in about 30 minutes. The concentrations of these two substances which produce the maximum effect are about ten times that of indole-acetic acid, which approximately corresponds with their relative auxin activities. The curves relating concentrations of these substances to their effects on streaming are very similar to that for indole-acetic acid. 6. On the other hand, certain substances which are known to affect streaming in other materials do not produce any effect comparable to that of auxin. Ethylene chlorhydrin, histidine, and urea in all concentrations were without effect on streaming in the Avena coleoptile within the first 30 minutes of treatment. 7. The effects produced by the auxins were not due to pH. 8. The action on streaming here studied is evidently quite different from the re-starting of streaming after its cessation, studied by Fitting in Vallisneria. Correspondingly histidine, which in Fitting''s experiments showed activity down to 10^–7 M, is inactive here. 9. Per contra, the effect of auxin here studied is on normal streaming. It takes place immediately and at concentrations in the same range as those which produce growth. The curve of effect against concentration parallels that for growth although the actual concentration values differ. It is therefore reasonable to suppose that the effect of auxin on streaming is closely connected with one of the first stages of its effect on the growth process.     

Control and Kinetics of Branch Root Formation in Cultured Root Segments of Haplopappus ravenii

Branch root formation required only the presence of minerals, sucrose as a carbon source, and an auxin. The number of primordia formed was a function of auxin concentration. With naphthaleneacetic acid at 0.1 mg/l, up to 60 or more branches were formed per centimeter of Haplopappus ravenii root segment. Under our conditions, pea root segments formed only five or six branches per centimeter, but tomato and radish, like H. ravenii, formed large numbers of branches. Cytokinin inhibited branch formation, while gibberellic acid was without effect. Vitamins were not required for branch formation, although they enhanced elongation. Up to 5 days were required for the maximum number of stable branch primordia to form under the influence of naphthaleneacetic acid. If naphthaleneacetic acid was withdrawn earlier, fewer branch primordia developed. The requirement for a lengthy exposure to naphthaleneacetic acid, the kinetics of the response, and the ease with which naphthaleneacetic acid could be rinsed out of the tissue with consequent cessation of branch root formation, were similar to other hormone-regulated developmental systems. Anatomical and cytological studies were made of segments exposed for various times to auxin. The segments were mostly diarch, and branches formed obliquely to protoxylem poles. While primarily only pericycle-endodermis cells divided, both these and cortex cells responded in the first 24 hours exposure to naphthaleneacetic acid with enlarged nuclei and nucleoli, and a few cortical cells divided. Maximum nucleus and nucleolus size was reached approximately 9 hours after exposure to naphthaleneacetic acid. Branches rarely elongated more than 5 cm before their meristems died. The H. ravenii culture is maintained only by the frequent formation of new naphthaleneacetic acid-induced branches.     

The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis)

The effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H4]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled internal standards. [13C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [2H4]IAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. The presence of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.