An auxin-induced polypeptide in dicotyledonous plants

Antisera were raised to a 70-kD (kilodalton) soybean (Glycine max) protein encoded by a 2,4-dichlorophenoxyacetic acid (2,4-D) inducible mRNA, GH3. These antisera have been used to probe protein blots to study the kinetics and specificity of the GH3 induction response as well as the species specificity and intracellular location of the protein. Detectable levels of the GH3 protein are induced by 2,4-D within 2 h in elongating hypocotyl sections, root sections, and etiolated plumules, and within 30–60 min in soybean suspension cells. Synthesis of the GH3 protein is induced by a variety of auxins. Other plant hormones such as gibberellic acid, cytokinin and ethylene added with or whithout 2,4-D do not alter the level of GH3 protein induction. The GH3 protein is found only in the S100 fraction and is not associated with the nucleus or cell wall. This antiserum also reacts with a 2,4-D-inducible 70-kD protein in other dicots.     

Characterization of a class of small auxin-inducible soybean polyadenylated RNAs

Four new auxin-responsive RNAs from soybean (Glycine max (L.) Merr., var. Wayne) are described. The RNAs were identified by hybridization to three cDNA probes obtained from a library enriched for sequences which increase in abundance within 60 min after 2,4-D (2,4-dichlorophenoxyacetic acid) treatment. These RNAs appear to define a new class of small (i.e. approximately 550 nucleotides) RNAs that respond extremely rapidly to application of exogenous auxin. In excised elongating hypocotyl sections, an increase in the abundance of these RNAs can be detected 2 to 5 min after treatment with 50 M 2,4-D. This response is half maximal after 10 min and reaches steady state in 60 min. RNA blot analysis shows that these RNAs are expressed differentially in various parts of the seedling. The degree of inducibility by auxin is also organ-specific, with the elongating hypocotyl being the most responsive of the organs tested. The RNAs display identical response specificities with one exception. Accumulation of one RNA, designated 10A, is completely abolished by simultaneous addition of cycloheximide and 2,4-D. This RNA also displays a different 2,4-D dose response than other RNAs examined. These results suggest that more than one mechanism is involved in rapid modulation of gene expression by auxin.     

Selective Inhibition of Auxin-Stimulated NADH Oxidase Activity and Elongation Growth of Soybean Hypocotyls by Thiol Reagents

The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the thiol reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of thiol reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of reagent addition. If the thiol reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the thiol reagents were delayed or higher concentrations of thiol reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site thiols in the auxin-stimulated but not in the constitutive activity.     

Timing of the response of coleoptiles to the application and withdrawal of various auxins

Summary A study was made of the time courses of growth promotion and the reversal of growth promotion upon the addition and withdrawal of various auxins. Growth promotion by 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) occurs more slowly and is less vigorous than growth promotion by the same concentration of indoleacetic acid (IAA).The time required for the reversal of the stimulation of elongation by auxin is many times greater for 2,4-D-stimulated growth than for IAA- or NAA-stimulated growth (80 min vs. about 10 min). This difference appears to be due to the sluggish exit of 2,4-D since (1) experiments with labeled auxins show that 2,4-D moves out of the tissue more slowly than IAA, and (2) it is possible to shorten the time required for a decline in elongation rate after the removal of 2,4-D to 13 min by adding an auxin antagonist (p-chlorophenoxyisobutyric acid).The rapid reversal of the hormonal stimulation of growth is discussed in relation to possible mechanisms of action of auxin.     

Induction of somatic embryos in cotyledonary tissue of soybean,Glycine max L. Merr

A method for the induction of somatic embryos in soybean tissue cultures is described. Cotyledons from immature embryos were utilized as explant source. Supplementing the culture medium with auxins (2,4-D, MCPA, 2,4,5-T, NAA, IAA, IBA) caused formation of meristematic tissue on cotyledon explants. The extent of meristematic tissue formed depended on the kind and concentration of auxin in the culture medium. With 2,4-D and MCPA, embryoids originated from meristematic tissue. Embryoid formation rates were influenced by the developmental stage of the embryos serving as explant source and auxin concentration. Addition of cytokinins to the medium containing 2,4-D or supplementing it with high sugar concentrations inhibited the formation of meristematic tissue and of embryoids on cotyledon explants.Abbreviations BA 6-benzyladenin - 2,4-D 2,4-dichlorphenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - L2 Phillips and Collins (1979) medium Present and correspondence address: Akademie der Landwirtschaftswissenschaften der DDR, Institut für Pflanzenernährung, DDR-6909, Jena     

Enhancement of ethylene biosynthesis and germination with thidiazuron and some selected auxins in Striga asiatica seeds

Witchweed [ Striga asiatica (L.) Kuntze], an economically important parasitic weed on several poaceous crops, is difficult to control. In nature, germination and subsequent morphogenesis of Striga are cued to specific host-derived chemical signals. Seeds (approximately 2.4 mg) treated with thidiazuron (TDZ) or the auxins 2,4-dichlorophenoxy-acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), or 2-(4-chloro- o -tolyloxy) propionic acid (MCPP) produced little ethylene (66-138 nl l⁻¹). Combinations of TDZ with the auxins increased ethylene production by 4- to 18-fold. Ethylene production was strongly inhibited (86–92%) by aminoethoxyvinylglycine (AVG), inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. Ethylene evolved from seeds treated with TDZ in combination with 2,4-D increased after a lag period and was promoted by a pretreatment in 2,4-D. TDZ or any of the auxins, at the rates tested, effected negligible to low levels of germination (0 to 16%), whereas mixtures of TDZ with the above auxins stimulated 38 to 84% germination. Test solutions containing TDZ and indole-3-acetic acid (IAA) were, however, less effective. TDZ/auxin-induced germination was inhibited by AVG and the ethylene action inhibitor silver thiosulfate (STS). The inhibitory effect of the former was reversed by treatment with ACC. In vitro studies revealed negligible germination (< 1%) on control medium. Seeds germinating on media containing TDZ alone developed into seedlings with distinct shoots and rudimentary roots. Seeds germinating on media containing 2,4-D, irrespective of TDZ concentration, were induced to form calli. The results are consistent with a model in which both germination and subsequent morphogenesis in Striga are associated with exogenous and endogenous phytohormones.     

Auxin-Stimulated NADH Oxidase Purified from Plasma Membrane of Soybean

NADH oxidation by plasma membrane vesicles purified from hypocotyls of etiolated soybean seedlings by two-phase partition was stimulated 2- to 3-fold by auxins, indole-3-acetic acid, 2,4-dichlorophenoxy acetic acid (2,4-D), and α-naphthaleneacetic acid. The stimulation was concentration dependent in the presence or absence of detergent with a maximum for 2,4-D at 1 micromolar. The NADH oxidation activity was solubilized with the zwitterionic detergent CHAPS and purified by ion exchange chromatography and gel filtration approximately 2000-fold over the total homogenate. Both the partially purified fraction and an active band from nondenaturing gel electrophoresis revealed the same three bands when analyzed by denaturing gel electrophoresis. When obtained from plasma membrane vesicles from the region of rapid cell elongation, the NADH oxidase complex retained auxin responsiveness throughout purification (3- to 5-fold stimulation by 1 micromolar 2,4-D).     

Ethylene production by auxin-deprived, suspension-cultured pear fruit cells in response to auxins, stress, or precursor

Auxin-deprived, mannitol-supplemented, suspension-cultured pear (Pyrus communis L. Passe Crassane) fruit cells produce large quantities (20-40 nanoliters ethylene per 10⁶ cells per hour) of ethylene in response to auxins, CuCl₂ or 1-amino-cyclopropane-1-carboxylic acid (ACC). Maximum rates of production are achieved about 12 hours after the addition of optimal amounts of indoleacetic acid (IAA), naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4 to 5 hours after the addition of CuCl₂ and 1 to 2 hours after the addition of ACC. Supraoptimal concentrations of IAA result in a lag phase followed by a normal response. High concentrations of NAA and 2,4-D result in an early (4-5 hours) stress response and injury.

Continuous protein and RNA synthesis are essential for elaboration of the full IAA response; only protein synthesis is necessary for the response to CuCl₂ and ACC. Based on polysomal states and rates of amino acid incorporation, CuCl₂ partially inhibits protein synthesis while nonetheless stimulating ethylene production. In general, ethylene production by the pear cells resembles that of other plant systems. Some differences may reflect the sensitivity of the cells and are discussed. The relatively high levels of ethylene produced and the experimental convenience of the cultured cells should make them especially suitable for further investigations of ethylene production and physiology.

     

Differential regulation of Ku gene expression in etiolated mung bean hypocotyls by auxins

Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca(2+) chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and alpha-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.     

Regulation of organogenesis and somatic embryogenesis by auxin in melon,Cucumis melo L.

Various tissues of seeds and seedlings of melon were cultured in vitro to study the effects of auxin concentration on organogenesis and embryogenesis. Adventitious shoots and somatic embryos were formed from explants of cotyledons of mature seeds, hypocotyls of seedlings, and leaves and petioles of young plantlets. Expanded cotyledons of seedlings formed only adventitious shoots. All tissues responded similarly to the 2,4-D concentration in the media, that is, adventitious shoots were formed at low concentration, callus proliferated without differentiation at intermediate concentration and somatic embryos were induced at high concentration. Cotyledons of mature seeds formed both adventitious shoots and somatic embryos more efficiently than any other tissues cultured.Effects of three auxins, 2,4-D, NAA and IAA, on organogenesis and embryogenesis were compared using cotyledons of mature seeds. Adventitious shoots were formed at low level of auxins (0 to 0.01 mg/l 2,4-D; 0 to 0.1 mg/l NAA; 0 to 1.0 mg/l IAA), and embryos were formed at high level of auxins (1.0 to 2.0 mg/l 2,4-D; 3.0 to 10.0 mg/l NAA; 20.0 to 100.0 mg/l IAA). IAA gave more efficient shoot formation and embryogenesis than the other auxins.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA 3indoleacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog     

Somatic embryogenesis from immature embryos in meadowfoam (Limnanthes alba)

Developing embryos from immature seeds were excised and cultured. Optimal proliferation of differentiated secondary embryos occurred on Murashige-Skoog media containing 7% sucrose, 0.1 M 2,4-D, and 0.1–1.0 M zeatin. Higher levels of auxins inhibited embryo proliferation. Secondary embryos were subcultured to produce more embryos. The results indicate the feasibility of clonal propagation of meadowfoam.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-Benzyladenine     

Physiological characteristics of two auxin-resistant mutants of Arabidopsis thaliana,aux-2 and Dwf

Two auxin-resistant mutants of Arabidopsis thaliana L. have been characterized physiologically: aux-2 is a recessive mutation and is unlinked to a dominant mutation, Dwf, which is apparently lethal when homozygous. The progeny of selfed Dwf plants segregate into Dwf (agravitropic) and dwf ⁺ (normal) phenotypes. aux-2 phenotype was indistinguishable from the wild-type on criteria other than resistance to exogenous auxins: 3-fold to 2,4-D and 2-fold to IAA. On the other hand, Dwf plants had a typical dwarf phenotype with single unbranched roots which lacked hairs. Compared to the wild-type, Dwf seedling roots were highly resistant to exogenous auxins: 2000-fold to 2,4-D and 360-fold to IAA. Both aux-2 and Dwf were normal in their response to exogenous ABA. The dwarf phenotype was insensitive to gibberellins but root hair formation was restored by application of auxins.The results indicate that altered auxin phsysiology can lead to agravitropism and dwarfism.Abbrevations ABA Abscisic acid - GA₃ Gibberellic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

A rapid response to a plant hormone: auxin stimulates phospholipase A2 in vivo and in vitro

Addition of the active auxins indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid or alpha-naphthylacetic acid to cultured soybean (Glycine max L.) cells prelabeled with ethanolamine or choline increased the radioactivity in the lysophosphatidylethanolamine (LPE) or lysophosphatidylcholine (LPC) pool within 5 min. The inactive auxin analogue, beta-naphthylacetic acid, was inactive in this response. In membranes prelabeled in vivo, either with ethanolamine or choline, and subsequently isolated from zucchini (Cucurbita pepo L.) hypocotyls, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid stimulated the conversion of phosphatidylethanolamine (PE) to LPE and of phosphatidylcholine (PC) to LPC in vitro whereas the inactive auxin analogue 2,3-dichlorophenoxyacetic acid did not.     

Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.)

This study developed a plant regeneration protocol for Trifolium nigrescens (Viv.) via somatic embryogenesis (SE). Immature zygotic embryos at torpedo (TsE) and cotyledonary (CsE) stage were cultured on media with different auxins and cytokinins at different concentrations. The cultural requirements for SE differed between the explants used: the addition of 6-furfurylaminopurine (kinetin) or N⁶-[2-isopentenyl]-adenine (2iP) along with 2,4-dihydrophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) was needed to elicit the embryogenic response of CsE, but an exogenous cytokinin totally inhibited 2,4-D-induced SE from TsE. When applied alone, neither the cytokinin nor NAA induced SE in TsE or CsE. In all effective cultures the first somatic embryos appeared directly from the upper part of the hypocotyl (TsE and CsE) and from the margin of cotyledons (TsE) on day 7. Embryogenic callus occurred on CsE after 10 days. At comparable concentrations 2,4-D was a more potent SE inducer than NAA, but most of the embryoids induced on media with 2,4-D displayed morphological abnormalities, whereas those produced in the presence of NAA generally resembled zygotic embryos. Plant regeneration was achieved after transfer of somatic embryos or embryo-derived first shoots to medium without plant growth regulators (PGRs). The frequency of plant recovery was about 30% for embryoids obtained on media containing 2,4-D, and for material from media with NAA the recovery rates were 44–68% (somatic embryos) and 72–100% (embryoid-derived shoots). Regenerants appeared identical to each other and to wild plants; they produced flowers and had the chromosome complement typical for the species, 2n = 16, in root tip cells.