Overproduced ethylene causes programmed cell death leading to temperature-sensitive lethality in hybrid seedlings from the cross<Emphasis Type="Italic"> Nicotiana suaveolens</Emphasis> ×<Emphasis Type="Italic"> N. tabacum</Emphasis>

Reproductive isolation mechanisms (RIMs) often become obstacles in crossbreeding. Hybrid lethality is a subtype of RIM but its physiological mechanism remains poorly elucidated. Interspecific hybrids of Nicotiana suaveolens Lehm. x N. tabacum L. cv. Hicks-2 expressed temperature-sensitive lethality. This lethality was induced by programmed cell death (PCD) that was accompanied by the characteristic changes of animal apoptosis in hybrid seedlings at 28 degrees C but not at 36 degrees C. When hybrid seedlings were cultured at 28 degrees C, DNA fragmentation started in the cotyledon, and nuclear fragmentation subsequently progressed with lethal symptoms spreading throughout the seedlings. At 28 degrees C, ethylene production in hybrid seedlings was detectable at a high level compared with the level in parental seedlings. In contrast, the ethylene production rate in hybrid seedlings cultured at 36 degrees C was equal to that in parental seedlings. Treatment with ethylene biosynthetic inhibitors, amino-oxyacetic acid and amino-ethoxyvinyl glycine, suppressed lethal symptoms and apoptotic changes, and also prolonged survival of hybrid seedlings. Thus, the increase in the ethylene production rate correlated closely with expression of lethal symptoms and apoptotic changes in hybrid seedlings. From these observations, we conclude that overproduced ethylene acts as an essential factor mediating PCD and subsequent lethality in hybrid seedlings. Furthermore, the present study has provided the first evidence that ethylene is involved in the phenomenon of hybrid lethality.     

5,6-二氯吲哚乙酸对水稻幼苗芽鞘向地性的影响

5,6-Cl_2-IAA可以诱导水稻芽鞘产生斜向地性生长。其倾斜角度与外加5,6-Cl_2-IAA的剂量呈线性关系,其反应具有特异性(IAA,6-BA,ABA和BR均不能诱导这种反应)。反应可在2～3h后产生,它可作为5,6-Cl_2-IAA的生物检定依据。切除芽鞘顶尖或外加TIBA对这一反应无明显影响,另外乙烯生物合成抑制剂AVG也不能消除5,6-Cl_2-IAA诱导的斜向地性生长。     

Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens×N. tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. ×N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproductive isolation and it is temperature-sensitive. Apoptotic changes were detected in the cells of hybrid seedlings and calli expressing lethality at 28 °C but not under high-temperature conditions (36 °C), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apoptosis leading to hybrid lethality. Fragmentation of nuclei was correlated with the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal symptoms revealed a specific ladder pattern suggesting nucleosomal fragmentation which is one of the biochemical changes of apoptosis. In-situ detection using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct stages on each organ of hybrid seedlings and centripetally in hybrid calli. From these results, we confirmed that cell death inducing hybrid lethality was indeed apoptosis. Received: 23 December 1999 / Accepted: 5 April 2000     

PIS1, a negative regulator of the action of auxin transport inhibitors in Arabidopsis thaliana

In order to clarify the mechanism underlying the polar auxin transport system, the pis1 mutant in Arabidopsis thaliana that is hypersensitive to N -1-naphthylphthalamic acid (NPA), an auxin transport inhibitor was isolated and characterized. Whereas the pis1 mutant is normally sensitive to phytohormones, auxins, cytokinin and ethylene precursor, this mutant is hypersensitive to NPA over the broad spectrum of its effects such as growth of seedlings, root elongation, root gravitropism, root phototropism and root curling. This result indicates that the pis1 mutant is specifically affected in the polar auxin transport system. This result also defines a genetic factor controlling both gravitropism and phototropism, and strongly indicates the involvement of auxin transport during both tropic responses. NPA, 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) represent different classes of auxin transport inhibitors. The pis1 mutation conferred hypersensitivity to both NPA and TIBA but not to HFCA. These results show the genetic separation of the actions of NPA/TIBA and of HFCA. The PIS1 gene product might be specifically involved in the response pathway of NPA/TIBA, leading to interference with auxin-efflux carriers, and might act as a negative regulator of the action of NPA/TIBA.     

Sites of auxin action: regulation of geotropism, growth, and ethylene production by inhibitors of auxin transport

The inhibitors of auxin transport-NPA (N-1-naphthylphthalamic acid), DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-a] isoindol-8-one), and TIBA (2,3,5-triiodobenzoic acid)-inhibited geotropism in roots of intact Pisum sativum L. seedlings. NPA and DPX1840 also caused cellular swelling in the roots. The swelling was due to a greater inhibition of elongation than increase in weight and looked identical to the one caused by ethylene. However, ethylene did not act as an intermediate in the action of auxin transport inhibitors because all three failed to stimulate ethylene production and some of their growth-inhibiting effect was retained in the presence of saturating levels of ethylene. In the presence of 10 mum indoleacetic acid the growth-inhibiting effect of auxin transport inhibitors was lost after 18 hours. On the other hand, auxin transport inhibitors did not interfere with the ability of auxin to promote ethylene production. Growth inhibition caused by auxin transport inhibitors was reversible. Pea root sections resumed normal growth following flushing of treated sections with inhibitor-free solutions. Experiments with (14)C-2, 4-dichlorophenoxyacetic acid revealed that the herbicide and auxin transport inhibitors may have the same binding site. It was concluded that a class of structurally dissimilar compounds may share a similar physiological role since they all appear to compete with endogenous auxin for certain binding sites and they all have similar growth-regulating activities.     

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

Facile induction of apoptosis into plant cells associated with temperature-sensitive lethality shown on interspecific hybrid from the cross Nicotiana suaveolens x N. tabacum

Two lines of suspension culture cells were obtained from a hybrid seedling of Nicotiana suaveolens Lehm. x N. tabacum L. cv. Hicks-2 expressing temperature-sensitive lethality. One of them (LH line) was inducible cell death in accordance with the lethality at 28 degrees C but not under high-temperature conditions (36 degrees C). Another one (SH line) lost the lethality and survived at 28 degrees C. The cells of LH line showed apoptotic changes when they were cultured at 28 degrees C. Fragmentation of nuclei was correlated with the lethality in the cells, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from the cells expressing the lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation that is one of the biochemical characteristics of apoptosis. From these facts, we confirmed that the process of cell death leading to hybrid lethality in the cells is certainly apoptosis. Hybrid cells were used in the experiments to estimate the point of no return in temperature-sensitive lethality and to examine the influence of cation in DNA fragmentation during apoptosis. The utility of hybrid cells as an experimental system for studies of hybrid lethality and apoptosis in plants was confirmed.     

Involvement of Ethylene in Gravity-Regulated Peg Development in Cucumber Seedling

Peg development on the lower side of the transition (TR) zoneof the hypocotyl and the root in cucumber seedlings was inhibitedby two inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine and aminooxyacetic acid, and by an inhibitor of ethyleneaction, Ag-thiosulfate. These ethylene inhibitors also inhibitedplumular hook formation of the cucumber hypocotyl. When cucumberseeds were germinated in a vertical position or on a horizontalclinostat, the seedlings exhibited straight growth without formationof a plumular hook and failed to develop a protuberant peg.In the seedlings germinated in a vertical position, exogenousIAA induced a distinct peg-like protuberance, whereas ACC andethylene stimulated overall swelling around the TR zone, whichobviously differed from the normal peg. In horizontally placedseedlings, however, peg development was more pronounced dueto treatment with 5µl/liter of ethylene. These resultsindicate that a high ethylene level in the hook region playssome role in peg development. TIBA, an inhibitor of auxin transport,at 10^–4 M inhibited peg development, as reported previouslyby Witztum and Gersani (1975), but a somewhat lower concentrationof TIBA induced two distinct pegs, on both the lower and uppersides of horizontally grown seedlings. (Received June 12, 1987; Accepted December 11, 1987)     

Inhibition of IAA-induced ethylene production in etiolated mung bean hypocotyl segments by 2,3,5-triiodobenzoic acid and 2-(p-chlorophenoxy)-2-methyl propionic acid

The effect of two auxin antagonists, 2,3,5-triiodobenzoic acid (TIBA) and 2-( p -chlorophenoxy)-2-methyl propionic acid (CMPA) on IAA-induced ethylene production in etiolated mung bean hypocotyl ( Vigna radiata L. Rwilcz cv. Berken) segments was studied. Both TIBA and CMPA inhibited IAA-induced ethylene production and CO₂ production at concentrations from 0.001 m M to 0.1 m M and 0.01 m M to 1.0 m M , respectively. The optimum concentration for inhibition of ethylene production by TIBA was 0.05 m M and CMPA was 0.5 m M . At the optimum concentration of TIBA and CMPA, there was a significant decrease in IAA-induced ethylene production without a decrease in respiration rates below control levels. After 18 h, mung bean hypocotyl segments treated with 0.05 m M TIBA for 6 h or 0.5 m M CMPA for 8 h showed a maximum inhibition of IAA-induced ethylene production. Treatments longer than 8 h caused no further inhibition. The uptake of [¹⁴C]-naphthaleneacetic acid by mung bean segments was greatly reduced by the addition of either TIBA (0.05m M ) or CMPA (0.5 m M ) to the incubation media. The results of treatment sequences showed that TIBA needed to be applied prior to IAA in order to inhibit IAA-induced ethylene production, but CMPA caused the same inhibitory effect whether applied before or after IAA treatment. These findings provide evidence that TIBA inhibits auxin-induced ethylene production in etiolated mung bean hypocotyl segments by blocking auxin movement into the tissue whereas CMPA may work on both auxin transport and action.     

Triiodobenzoic acid, an auxin polar transport inhibitor, suppresses somatic embryo formation and postembryonic shoot/root development in Eleutherococcus senticosus

The effect of auxin polar transport inhibitor on somatic embryo development and postembryonic growth in Siberian ginseng (Eleutherococcus senticosus) was examined. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin polar transport inhibitor, embryo formation from embryogenic cells was suppressed, while cell division was not affected. When globular embryos at different stages were transferred onto medium containing TIBA, development of axial and bilateral polarity was suppressed in a stagespecific manner. In abnormal embryos induced by TIBA, further development of shoot and root apical meristems and vascular differentiation was also suppressed. Thus, abnormal development of embryos induced by inhibition of auxin polar transport resulted in plantlets without shoots and roots.     

The Effects of Ascorbate on Root Regeneration in Seedling Cuttings of Tomato

The changes in ascorbate (ASC) and dehydroascorbate (DHA) levels and the activities of ascorbate metabolising enzymes were examined during adventitious root formation in cuttings of tomato (Lycopersicon esculentum Mill. cv. Paw) seedlings. The effects of ASC, DHA and the immediate ascorbate precursor – galactono-γ-lactone (GalL) supplemented to the culture medium on the rooting response, ascorbate content and the activities of the ASC-metabolising enzymes were also investigated. The cuttings treated with abovementioned compounds formed more roots then control plants. However, in contrast to the number of regenerated organs, the elongation of newly formed roots was markedly inhibited. Treatment with auxin (IAA) resulted in a similar phenotype. The inhibitor of auxin polar transport-TIBA (2,3,5-triiodobenzoic acid) effectively blocked rooting. The inhibitory effect of TIBA was reversed by auxin and ASC treatments, while DHA and GalL were ineffective. Both auxin and ASC stimulated cell divisions in an area of pericycle layer of TIBA-treated rooting zones, that enabled cuttings to form roots in the presence of the inhibitor of auxin polar transport. It has been found that the first stages of rooting, preceding the emergence of roots, are accompanied by an increase in endogenous content of ASC with a peak in the 3rd day of rooting. Subsequent stages, when elongation of newly formed roots occurs, are characterised by low level of ASC. The activities of the ascorbate peroxidase (APX), ascorbate oxidase (AOX), ascorbate free radical reductase (AFRR) and dehydroascorbate reductase (DHR) increased in the first 3 days of root formation. The initial period of rooting was also accompanied by the increase of the hydrogen peroxide content and the activities of catalase (CAT) and guaiacol peroxidase (GPX) in the rooting zones. IAA, ASC, DHA as well as Gal stimulated the APX activity, however the rise of the enzyme's activity induced by ASC, DHA and Gal was reversed by TIBA, which was found to inhibit APX. Only exogenous IAA was able to maintain the high level of APX activity in the TIBA-treated cuttings. AOX was strongly affected by ASC and GalL – treatments, its activity increased in the cuttings grown on the media containing ASC in the absence as well as in the presence of TIBA. On the other hand, GalL-dependent stimulation of its activity was suppressed if TIBA was present in a rooting medium.     

Hormonal control of root development on epiphyllous plantlets of Bryophyllum (Kalanchoe) marnierianum: role of auxin and ethylene

Epiphyllous plantlets develop on leaves of Bryophyllum marnierianum when they are excised from the plant. Shortly after leaf excision, plantlet shoots develop from primordia located near the leaf margin. After the shoots have enlarged for several days, roots appear at their base. In this investigation, factors regulating plantlet root development were studied. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) abolished root formation without markedly affecting shoot growth. This suggested that auxin transport from the plantlet shoot induces root development. Excision of plantlet apical buds inhibits root development. Application of indole-3-acetic acid (IAA) in lanolin at the site of the apical buds restores root outgrowth. Naphthalene acetic acid (NAA), a synthetic auxin, reverses TIBA inhibition of plantlet root emergence on leaf explants. Both of these observations support the hypothesis that auxin, produced by the plantlet, induces root development. Exogenous ethylene causes precocious root development several days before that of a control without hormone. Ethylene treatment cannot bypass the TIBA block of root formation. Therefore, ethylene does not act downstream of auxin in root induction. However, ethylene amplifies the effects of low concentrations of NAA, which in the absence of ethylene do not induce roots. Ag(2)S(2)O(3), an ethylene blocker, and CoCl(2), an ethylene synthesis inhibitor, do not abolish plantlet root development. It is therefore unlikely that ethylene is essential for root formation. Taken together, the experiments suggest that roots develop when auxin transport from the shoot reaches a certain threshold. Ethylene may augment this effect by lowering the threshold and may come into play when the parent leaf senesces.     

Hormonal Studies of fass,an Arabidopsis Mutant That Is Altered in Organ Elongation

We have isolated an allele of fass, an Arabidopsis thaliana mutation that separates plant development and organ differentiation from plant elongation, and studied its hormonal regulation. Micro-surgically isolated fass roots elongate 2.5 times as much as the roots on intact mutant plants. Wild-type heart embryos, when cultured with a strong auxin, naphthaleneacetic acid, phenocopy fass embryos. fass seedlings contain variable levels of free auxin, which average 2.5 times higher than wild-type seedling levels, and fass seedlings evolve 3 times as much ethylene as wild-type seedlings on a per-plant basis over a 24-h period. The length-to-width ratios of fass seedlings can be changed by several compounds that affect their endogenous ethylene levels, but fass is epistatic to etr1, an ethylene-insensitive mutant. fass's high levels of free auxin may be inducing its high levels of ethylene, which may, in turn, result in the fass phenotype. We postulate that FASS may be acting as a negative regulator to maintain wild-type auxin levels and that the mutation may be in an auxin-conjugating enzyme.     

Cell death processes during expression of hybrid lethality in interspecific F1 hybrid between Nicotiana gossei Domin and Nicotiana tabacum

Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F(1) hybrid seedlings from Nicotiana gossei Domin and Nicotiana tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26 degrees C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37 degrees C. Seedlings grown at 26 degrees C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37 degrees C. A number of stress response marker genes were expressed at 26 degrees C but not at 37 degrees C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant.     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

The Effect of 2,3.5 Triiodobenzoic Acid on Caulogenesis in Callus Cultures of Tomato and Pelargonium

Stem explants from winter grown tomatoes cultured on a cytokinin, auxin-free medium, developed one or two adventitious shoots at the top end of the explant. Addition of the auxin transport inhibitor. 2,3,5-triiodobenzoic acid (TIBA) to the medium stimulated caulogenesis with loss of polarity. Callus, initiation in pelargonium and ‘geranium’ petiole explants requires both auxin and cytokinin. On transfer, after callus induction to an auxin-free medium, rhizogenesis occurs in pelargonium cultures followed by caulogenesis. Few shoots develop and unless these are removed, further caulogenesis is suppressed. Bud-like structures were formed in the callus. Subculture on auxin-free medium containing cytokinin and TIBA resulted in shoot formation from these bud-like organs. An analogy with apical dominance is suggested. In ‘geranium’ callus, shoots developed with a low frequency (c. in 2% of the cultures): caulogenesis was increased to 80% when calli were subcultured from auxin-free, cytokinin medium after green nodule formation to cytokinin-TIBA medium. Histological studies of green nodules in ‘geranium’ callus indicated a variation in morphological development within and between nodules. It is suggested that auxin synthesis may occur at some microscopic stage in morphogenesis in ‘geranium’ cultures which suppresses further caulogenesis. This may be overcome by the addition of TIBA to the medium at the appropriate stage in morphogenesis. The possible interaction of endogenous auxin in morphogenesis is discussed.     

Developmental changes in the secondary xylem ofAcer rubrum induced by gibberellic acid,various auxins and 2,3,5-tri-iodobenzoic acid

Summary The effect of GA on the development of the secondary xylem ofAcer rubrum was studied. GA was applied to seedlings (1) separately, (2) simultaneously with TIBA, and (3) simultaneously with TIBA and an auxin. GA alone is without effect on the development of the secondary xylem. In seedlings treated with TIBA-GA or TIBA-GA-auxin, the differentiation of the xylem elements is in general similar to that of seedlings treated with TIBA or TIBA-auxin respectively. GA however, does stimulate cambial activity in a localized region of the stem if the endogenous rate of cambial division is low.The following abbreviations will be used TIBA (2,3,5-tri-iodobenzoic acid) - IAA (indole-3-acetic acid) - GA (gibberellic acid) - NAA (naphthaleneacetic acid) - 2,4-D (2,4-dichlorophenoxyacetic acid) This material was included in a doctoral thesis submitted by P. R.Morey to the graduate school of Yale University, New Haven.     

A Mutation Altering Auxin Homeostasis and Plant Morphology in Arabidopsis

Many aspects of plant development are associated with changing concentrations of the phytohormone auxin. Several stages of root formation exhibit extreme sensitivities to exogenous auxin and are correlated with shifts in endogenous auxin concentration. In an effort to elucidate mechanisms regulating development of adventitious roots, an ethyl methanesulfonate-mutagenized M2 population of Arabidopsis was screened for mutants altered in this process. A recessive nuclear mutant, rooty (rty), displayed extreme proliferation of roots, inhibition of shoot growth, and other alterations suggesting elevated responses to auxin or ethylene. Wild-type Arabidopsis seedlings grown on auxin-containing media phenocopied rty, whereas rty seedlings were partially rescued on cytokinin-containing media. Analysis by gas chromatography-selected ion monitoring-mass spectrometry showed endogenous indole-3-acetic acid concentrations to be two to 17 times higher in rty than in the wild type. Dose-response assays with exogenous indole-3-acetic acid indicated equal sensitivities to auxin in tissues of the wild type and rty. Combining rty with mutations conferring resistance to auxin (axr1-3) or ethylene (etr1-1) suggested that root proliferation and restricted shoot growth are auxin effects, whereas other phenotypic alterations are due to ethylene. Four mutant alleles from independently mutagenized populations were identified, and the locus was mapped using morphological and restriction fragment length polymorphism markers to 3.9 centimorgans distal to marker m605 on chromosome 2. The wild-type RTY gene product may serve a critical role in regulating auxin concentrations and thereby facilitating normal plant growth and development.