Detection and characterization of glutathione S-transferase activity in rice EF-1betabeta'gamma and EF-1gamma expressed in Escherichia coli.

Plant elongation factor EF-1 consists of four subunits (EF-1alphabetabeta'gamma). EF-1alpha. GTP catalyses the binding of aminoacyl-tRNA to the ribosome. EF-1beta and EF-1beta' catalyze the GDP/GTP exchange on EF-1alpha. GDP. However, the function of EF-1gamma, a subunit detected in eukaryotes, but not in prokaryotes remained unknown. This report demonstrates that rice EF-1betabeta'gamma and recombinant EF-1gamma possess glutathione S-transferase (GST) activity. The EF-1betabeta'gamma- or EF-1gamma-dependent GST activity is about one-fiftieth of the rice GST activity. The Km values of EF-1betabeta'gamma, EF-1gamma, and rice GST for glutathione and 1-chloro-2,4-dinitrobenzene are of about the same order. Although recombinant EF-1gamma is heat labile, active EF-1gamma was obtained by purifying it in the presence of 20% glycerol.     

Meristem differentiation in Riella helicophylla (Bory et Mont.) Mont. under the influence of auxin or antiauxin

During the development of the unistratose gemmae of Riella helicophylla, the single intercalary meristem of the very young gemmae is subdivided into two lateral meristems. The duration of the cell reproduction cycle increases from the margin to the median part of the gemmae. This polarization within the meristem disappears after addition of the antiauxin PCIB to the culture medium. PCIB leads to a retardation or blockage of the cell cycle during the light period of the culture. Under the influence of PCIB the amount of starch in the chloroplasts is strikingly increased, probably because of a reduction of starch degradation. Addition of sugars compensates the effect of PCIB on the cell cycle. The effects of PCIB are counteracted by auxin. The results are taken as evidence that auxin plays a role in directing the transport of substances needed for the continuation of the cell reproduction cycle between adjacent cells of the meristem.Abbreviations IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCIB p-chlorophenoxyisobutyric acid - G-l-P glucose-l-phosphat - EDTA ethylenediaminetetraacetic acid     

Inherited phenotype instability of inflorescence and floral organ development in homeotic barley double mutants and its specific modification by auxin inhibitors and 2,4-D

Background and Aims Barley (Hordeum vulgare) double mutants Hv-Hd/tw₂, formed by hybridization, are characterized by inherited phenotypic instability and by several new features, such as bract/leaf-like structures, long naked gaps in the spike, and a wide spectrum of variations in the basic and ectopic flowers, which are absent in single mutants. Several of these features resemble those of mutations in auxin distribution, and thus the aim of this study was to determine whether auxin imbalances are related to phenotypic variations and instability. The effects of auxin inhibitors and 2,4-D (2,4-dichlorophenoxyacetic acid) on variation in basic and ectopic flowers were therefore examined, together with the effects of 2,4-D on spike structure.Methods The character of phenotypic instability and the effects of auxin inhibitors and 2,4-D were compared in callus cultures and intact plants of single homeotic Hv-tw₂ and Hv-Hooded/Kap (in the BKn3 gene) mutants and alternative double mutant lines: offspring from individual plants in distal hybrid generations (F₉–F₁₀) that all had the same BKn3 allele as determined by DNA sequencing. For intact plants, two auxin inhibitors, 9-hydroxyfluorene-9-carboxylic acid (HFCA) and p-chlorophenoxyisobutyric acid (PCIB), were used.Key Results Callus growth and flower/spike structures of the Hv-tw₂ mutant differed in their responses to HFCA and PCIB. An increase in normal basic flowers after exposure to auxin inhibitors and a decrease in their frequencies caused by 2,4-D were observed, and there were also modifications in the spectra of ectopic flowers, especially those with sexual organs, but the effects depended on the genotype. Exposure to 2,4-D decreased the frequency of short gaps and lodicule transformations in Hv-tw₂ and of long naked gaps in double mutants.Conclusions The effects of auxin inhibitors and 2,4-D suggest that ectopic auxin maxima or deficiencies arise in various regions of the inflorescence/flower primordia. Based on the phenotypic instability observed, definite trends in the development of ectopic flower structures may be detected, from insignificant outgrowths on awns to flowers with sterile organs. Phenotypically unstable barley double mutants provide a highly promising genetic system for the investigation of gene expression modules and trend orders.     

A chemical biology approach reveals an opposite action between thermospermine and auxin in xylem development in Arabidopsis thaliana

Thermospermine, a structural isomer of spermine, is produced through the action of ACAULIS5 (ACL5) and suppresses xylem differentiation in Arabidopsis thaliana. To elucidate the molecular basis of the function of thermospermine, we screened chemical libraries for compounds that can modulate xylem differentiation in the acl5 mutant, which is deficient in thermospermine and shows a severe dwarf phenotype associated with excessive proliferation of xylem vessels. We found that the isooctyl ester of a synthetic auxin, 2,4-D, remarkably enhanced xylem vessel differentiation in acl5 seedlings. 2,4-D, 2,4-D analogs and IAA analogs, including 4-chloro IAA (4-Cl-IAA) and IAA ethyl ester, also enhanced xylem vessel formation, while IAA alone had little or no obvious effect on xylem differentiation. These effects of auxin analogs were observed only in the acl5 mutant but not in the wild type, and were suppressed by the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB) and α-(phenyl ethyl-2-one)-IAA (PEO-IAA), and also by thermospermine. Furthermore, the suppressor of acaulis51-d (sac51-d) mutation, which causes SAC51 overexpression in the absence of thermospermine and suppresses the dwarf phenotype of acl5, also suppressed the effect of auxin analogs in acl5. These results suggest that the auxin signaling that promotes xylem differentiation is normally limited by SAC51-mediated thermospermine signaling but can be continually stimulated by exogenous auxin analogs in the absence of thermospermine. The opposite action between thermospermine and auxin may fine-tune the timing and spatial pattern of xylem differentiation.     

The role of auxins in somatic embryogenesis of <Emphasis Type="Italic">Abies alba</Emphasis>

The somatic embryogenesis of conifers is a process susceptible to exogenous phytohormonal treatments. We report the effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the auxin inhibitor p-chlorophenoxyisobutyric acid (PCIB) on the endogenous level of the auxin indole-3-acetic acid (IAA) and on the anatomical composition of early somatic embryos of Abies alba (European silver fir). The embryogenic suspensor mass (ESM) of Abies alba proliferated on a medium supplemented by 2,4-D as well as on an auxin-free medium. The endogenous level of IAA was significantly higher in the ESM cultivated on a medium supplemented by 2,4-D. The decrease in the endogenous level of IAA in the first week of maturation is one of the most important stimuli responsible for the subsequent development of embryos. However, suppression of IAA synthesis by an auxin inhibitor did not stimulate the development of embryos. The maturation of somatic embryos from the globular to the cotyledonary stage occurs when the concentration of endogenous auxin in the ESM (including the embryos) increases. Early somatic embryos proliferating on a medium supplemented by auxin had an increased probability of maturing successfully. Exogenous auxin treatment during maturation did not compensate for the auxin deficiency during proliferation.     

Ospdr9, which encodes a PDR-type ABC transporter, is induced by heavy metals, hypoxic stress and redox perturbations in rice roots

Little is known about the role of pleiotropic drug resistance (PDR)-type ATP-binding (ABC) proteins in plant responses to environmental stresses. We characterised ospdr9, which encodes a rice ABC protein with a reverse (ABC-TMS(6))(2) configuration. Polyethylene glycol and the heavy metals Cd (20 microM) and Zn (30 microM) rapidly and markedly induced ospdr9 in roots of rice seedlings. Hypoxic stress also induced ospdr9 in rice roots, salt stress induced ospdr9 at low levels but cold and heat shock had no effect. The plant growth regulator jasmonic acid, the auxin alpha-naphthalene acetic acid and the cytokinin 6-benzylaminopurine triggered ospdr9 expression. The antioxidants dithiothreitol and ascorbic acid rapidly and markedly induced ospdr9 in rice roots; the strong oxidant hydrogen peroxide also induced ospdr9 but at three times lower levels. The results suggested that redox changes may be involved in the abiotic stress response regulation of ospdr9 in rice roots.     

Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology

Summary Mutant lines of Arabidopsis thaliana resistant to the artificial auxin 2,4-dichloro phenoxyacetic acid (2,4-D) were isolated by screening for growth of seedlings in the presence of toxic levels of 2,4-D. Genetic analysis of these resistant lines indicated that 2,4-D resistance is due to a recessive mutation at a locus we have designated Axr-1. Mutant seedlings were resistant to approximately 50-fold higher concentrations of 2,4-D than wild-type and were also resistant to 8-fold higher concentrations of indole-3-acetic acid (IAA) than wild-type. Labelling studies with (¹⁴C)2,4-D suggest that resistance was not due to changes in uptake or metabolism of 2,4-D. In addition to auxin resistance the mutants have a distinct morphological phenotype including alterations of the roots, leaves, and flowers. Genetic evidence indicates that both auxin resistance and the morphological changes are due to the same mutation. Because of the pleiotropic morphological effects of these mutations the Axr-1 gene may code for a function involved in auxin action in all tissues of the plant.     

A gain-of-function mutation in the Arabidopsis pleiotropic drug resistance transporter PDR9 confers resistance to auxinic herbicides

Arabidopsis (Arabidopsis thaliana) contains 15 genes encoding members of the pleiotropic drug resistance (PDR) family of ATP-binding cassette transporters. These proteins have been speculated to be involved in the detoxification of xenobiotics, however, little experimental support of this hypothesis has been obtained to date. Here we report our characterization of the Arabidopsis PDR9 gene. We isolated a semidominant, gain-of-function mutant, designated pdr9-1, that exhibits increased tolerance to the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Reciprocally, loss-of-function mutations in PDR9 confer 2,4-D hypersensitivity. This altered auxin sensitivity defect of pdr9 mutants is specific for 2,4-D and closely related compounds as these mutants respond normally to the endogenous auxins indole-3-acetic acid and indole-butyric acid. We demonstrate that 2,4-D, but not indole-3-acetic acid transport is affected by mutations in pdr9, suggesting that the PDR9 transporter specifically effluxes 2,4-D out of plant cells without affecting endogenous auxin transport. The semidominant pdr9-1 mutation affects an extremely highly conserved domain present in all known plant PDR transporters. The single amino acid change results in increased PDR9 abundance and provides a novel approach for elucidating the function of plant PDR proteins.     

Establishment of synchrony by starvation and readdition of auxin in suspension cultures of Catharanthus roseus cells

A system of synchronous cell division was established by starvation of auxin and its readdition to suspension cultures of cells of Catharanthus roseus L. cv. Little-Pinky. When cells in the stationary phase were transferred to fresh medium free of 2,4-dichlorophenoxyacetic acid (2,4-D), cells were arrested preferentially at the G₁ phase. After cells had been cultured for 2 days in medium without 2,4-D, readdition of 2,4-D induced the synchronous division of cells. In this system, 70–80% of cells divided synchronously within 3 to 4h, and the mitotic index increased sharply in parallel with the increase in cell number. Active synthesis of DNA was demonstrated by measurements of incorporation of [³H]-thymidine into the DNA fraction. The induction of cell division by the addition of 2,4-D was inhibited by treating cells with analogues of auxin, such as 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxyisobutyric acid.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - IAA indole-3-acetic acid - MS Murashige & Skoog - NAA -naphthalenacetic acid - PCIB p-chlorophenoxyisobutyric acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid     

Auxin binding in roots: A comparison between maize roots and coleoptiles

Auxin binding onto membrane fractions of primary roots of maize seedlings has been demonstrated using naphth-1yl-acetic acid (NAA) and indol-3yl-acetic acid (IAA) as ligands. This binding is compared with the already well characterized interaction between auxins and coleoptile membranes. The results indicate that while kinetic parameters are of the same order for root and coleoptile binding, a number of differences occur with respect to location in cells and relative affinity. The possible significance of the existence of such binding sites in root cells is discussed in relation to auxin action.Abbreviations 4-Cl-PA 4-chlorophenoxyacetic acid - EDTA ethylene diamine tetracetic acid - IAA indol-3yl-acetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA naphth-1yl-acetic acid - 2-NAA naphth-2yl-acetic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3 diol - TIBA 2,3,5 triiodobenzoic acid - NPA naphthylphthalamic acid - PCIB 4-chlorophenoxyisobutyric acid - PCPP 4-chlorophenoxyisopropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid

2,4-dichlorophenoxyacetic acid (2,4-D), a chemical analogue of indole-3-acetic acid (IAA), is widely used as a growth regulator and exogenous source of auxin. Because 2,4-D evokes physiological and molecular responses similar to those evoked by IAA, it is believed that they share a common response pathway. Here, we show that a mutant, antiauxin resistant1 (aar1), identified in a screen for resistance to the anti-auxin p-chlorophenoxy-isobutyric acid (PCIB), is resistant to 2,4-D, yet nevertheless responds like the wild-type to IAA and 1-napthaleneacetic acid in root elongation and lateral root induction assays. That the aar1 mutation alters 2,4-D responsiveness specifically was confirmed by analysis of GUS expression in the DR5:GUS and HS:AXR3NT-GUS backgrounds, as well as by real-time PCR quantification of IAA11 expression. The two characterized aar1 alleles both harbor multi-gene deletions; however, 2,4-D responsiveness was restored by transformation with one of the genes missing in both alleles, and the 2,4-D-resistant phenotype was reproduced by decreasing the expression of the same gene in the wild-type using an RNAi construct. The gene encodes a small, acidic protein (SMAP1) with unknown function and present in plants, animals and invertebrates but not in fungi or prokaryotes. Taken together, these results suggest that SMAP1 is a regulatory component that mediates responses to 2,4-D, and that responses to 2,4-D and IAA are partially distinct.     

Indoleacetic acid enhancement of the inhibition of Lemna growth caused by abscisic acid

Summary Indoleacetic acid substantially increased the inhibitory influence of abscisic acid on growth measured on fresh weight basis of Lemna gibba L. A similar synergistic action was obtained with indolebutyric acid while neither naphthylacetic acid nor 2,4-dichlorophenoxyacetic acid showed any synergism. The antiauxin para-chlorophenoxy-isobutyric acid did not counteract the synergistic action of IAA and ABA. The results indicate that the enhancing effect of IAA on the ABA action is not a typical auxin effect.Abbreviations ABA abscisic acid - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCIB para-chlorophenoxy-iso-butyric acid     

Endogenous Auxin is Required but Supraoptimal for Rapid Growth of Rice (Oryza sativa L.) Seminal Roots, and Auxin Inhibition of Rice Seminal Root Growth is Not Caused by Ethylene

The dual effects of auxin and ethylene on rice seminal root growth were investigated in this study. Low concentrations of exogenous indole-3-acetic acid (IAA) had no effect on rice seminal root growth, whereas higher concentrations (≥0.003 μM) were inhibitory. In contrast, low concentrations of the auxin action inhibitor p-chlorophenoxyisobutyric acid (PCIB), ranging from 0.5 to 50 μM, promoted rice seminal root growth, whereas high concentrations of PCIB (≥500 μM) and the polar auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibited rice seminal root growth. These results suggest that endogenous auxin is required but supraoptimal for rapid growth of rice seminal roots. In addition, although rice seminal root growth was inhibited by the exogenous ethylene-releasing compound ethephon or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) as well as exogenous IAA, the 50% inhibition of growth (I₅₀) caused by ethephon or ACC was weakened by certain concentrations of the ethylene action inhibitor Ag⁺ (0.016-0.4 μM). However, the I₅₀ caused by exogenous IAA was strengthened by Ag⁺ or the ethylene biosynthetic inhibitor aminoethoxyvinylglycine (AVG) and weakened by certain concentrations of PCIB (0.5-50 μM). Together, the inhibitory mechanisms of auxin and ethylene on rice seminal root growth should be different, and auxin inhibition of rice seminal root growth should not be caused by ethylene. Furthermore, our results indicated that a certain threshold level of ethylene was required to maintain rice seminal root growth, and that ethylene within the threshold may antagonize auxin inhibition of rice seminal root growth.     

Translocation and Complex Formation of Picloram and 2,4-D in Wheat Seedlings

Uptake, translocation and metabolism of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of wheat (Triticum aestivum L.) were studied. The uptake of the herbicides through the upper surface of the first leaf was slow but was almost complete after nine days. Picloram was absorbed faster than 2,4-D. Picloram was also translocated into the stem and the untreated leaves to a greater extent than 2,4-D. Only small fractions of the activity were recovered from the roots and from the nutrient solution. Picloram and 2,4-D formed water-soluble conjugates in the tissues. These conjugates were very labile and hydrolyzed under release of the unchanged herbicides. The isotope from 2,4-D was also incorporated in an insoluble fraction, containing cell walls and proteins. Also from this fraction biologically active 2,4-D could be released by hydrolysis. The formation of the complexes was partly prevented by cycloheximide. It is suggested that herbicide detoxification through complex formation is of importance for the relatively low sensitivity of wheat to auxin herbicides.     

Effects of desiccation on the 77°K fluorescence properties of the liverwort Porella navicularis and the isolated lichen green alga Trebouxia pyriformis

The uptake of the auxin type herbicide 2,4-D into rice seedlings ( Oryza sativa L. cv. Dunghan Shali) and its effects on the K⁺, NH⁺₄ and NO₃ ion uptake and the K⁺ content were investigated at different pH values. A short incubation of the roots in 0.01 m M 2,4-D caused a marked ion uptake inhibition only at low pH. The non-auxin type herbicide benthiocarb did not produce such an inhibitory effect. Lowering of the pH in the external medium led to an increased 2,4-D uptake by the roots. These results can be explained by the increased H⁺ permeability of the membranes, allowing a more rapid entrance of 2,4-D into the root cells, thereby inhibiting the active ion uptake. Rice roots not subjected to 2,4-D treatment responded to H⁺ stress with an increased anomalous K⁺ uptake and a decreased K⁺ content. With reference to the effects of pH changes on the ion and 2,4-D uptake, possible transport mechanism of NH⁺₄ and 2,4-D are briefly discussed.     

The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf.

Unilateral application of indole-3-acetic acid (IAA) in a lanolin base to hypocotyls of partially etiolated seedlings of wild-type Arabidopsis thaliana induced growth curvature in a dose-dependent manner. The effects of IAA in concentrations from 1 to 1000 microM were studied, with maximum IAA-induced curvature at 100 microM. Three IAA-insensitive mutants were isolated and are all in the same locus, massugu1 (msg1). They did not undergo hypocotyl growth curvature at any of the IAA concentrations tested. msg1 is recessive and is located on chromosome 5. msg 1 hypocotyl growth is resistant to 2,4-dichlorophenoxyacetic acid (2,4-D), but the roots are as sensitive to 2,4-D as the wild type. Growth of the hypocotyl was inhibited to essentially the same extent as the wild type by 6-benzylaminopurine, abscisic acid, and 1-aminocyclopropane-1-carboxylate, an ethylene precursor. The msg1 leaves were also resistant to 2,4-D-induced chlorosis. The gravitropic response of the msg1 hypocotyl takes much more time to initiate and achieve the wild-type degree of curvature, whereas the msg1 roots responded normally to gravity. The mature plants and the etiolated seedlings of msg1 were generally wild type in appearance, except that their rosette leaves were either epinastic or hyponastic. msg1 is the first auxin-insensitive mutant in which it effects are mostly restricted to the hypocotyl and leaf, and msg1 also appears to be auxin specific.