Relationship between the activity of the ethylene-forming enzyme and the level of intracellular 2,4-dichlorophenoxyacetic acid in pear cell culturesin vitro

Ethylene production by auxin-dependent pear cells culturedin vitro falls rapidly when they are deprived of 2,4-D. This phenomenon is associated with a decrease in ACC production. Readdition of 2,4-D causes a resumption of ACC production and ethylene synthesis. Ethylene-forming enzyme (EFE) activity, although never limiting, decreases sharply during 2,4-D depletion and rises again upon addition of 2,4-D. This increase in the EFE activity is not a rapid response to 2,4-D, since it requires several hours. Changes in EFE activity follow the same pattern as changes in 2,4-D concentration; the decrease in EFE activity is also concomitant with a decrease in the ability of 2,4-dinitrophenol to inhibit ethylene production. The possible role of auxins in the modulation of EFE activity is discussed.     

Effects of 2,4-D and its metabolite 2,4-dichlorophenol on antioxidant enzymes and level of glutathione in human erythrocytes

The effects of in vitro exposure of human erythrocytes to different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) were studied. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the level of reduced glutathione (GSH) were determined. The activity of erythrocyte superoxide dismutase SOD decreased with increasing dose of 2,4-D and 2,4-DCP, while glutathione peroxidase activity increased. 2,4-D (500 ppm) decreased the level of reduced glutathione in erythrocytes by 18% and 2,4-DCP (250 ppm) by 32%, respectively, in comparison with the controls. These results lead to the conclusion that in vitro administration of herbicide-2,4-D and its metabolite 2,4-DCP causes a decrease in the level of reduced glutathione in erythrocytes and significant changes in antioxidant enzyme activities. Comparison of the toxicity of 2,4-D and 2,4-DCP revealed that the most prominent changes occurred in human erythrocytes incubated with 2,4-DCP.     

Relationship between the activity of the ethylene-forming enzyme and the level of intracellular 2,4-dichlorophenoxyacetic acid in pear cell culturesin vitro

Ethylene production by auxin-dependent pear cells culturedin vitro falls rapidly when they are deprived of 2,4-D. This phenomenon is associated with a decrease in ACC production. Readdition of 2,4-D causes a resumption of ACC production and ethylene synthesis. Ethylene-forming enzyme (EFE) activity, although never limiting, decreases sharply during 2,4-D depletion and rises again upon addition of 2,4-D. This increase in the EFE activity is not a rapid response to 2,4-D, since it requires several hours. Changes in EFE activity follow the same pattern as changes in 2,4-D concentration; the decrease in EFE activity is also concomitant with a decrease in the ability of 2,4-dinitrophenol to inhibit ethylene production. The possible role of auxins in the modulation of EFE activity is discussed.     

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.     

Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture

The activity of caffeic acid-O-methyltransferase (OMT) in carrot cells was greatly affected by the amount of 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented to the culture medium. The OMT fraction was purified by (NH₄)₂SO₄ followed by ultrafiltration and gel filtration or DEAE-Sephadex chromatography after cells were cultured in the medium containing [2-¹⁴C]-2,4-D. Thus, this purified fraction revealed high OMT activity and was still radioactive. The OMT activity was about eight-fold higher (or more) in cells cultured at 0.05 ppm 2,4-D than in those at 1.0 ppm 2,4-D. The ratio of radioactivity to OMT activity was about four-fold higher in cells cultured at 1.0 ppm 2,4-D than those at 0.05 ppm 2,4-D. On the other hand, the OMT fraction was separated into two radioactive protein fractions by DEAE-Sephadex chromatography. The radioactive fractions became Et₂O-soluble after HCl hydrolysis, but not after salt-urea treatment. From these results, it was concluded that 2,4-D is covalently bound to proteins in the OMT fraction. Such 2,4-D protein conjugates may play a role in the regulation of OMT activity.     

Auxin-activated nadh oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

Summary The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14–17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.     

An auxin induces the appearance of auxin-binding activity in artichoke tubers

Artichoke tuber tissue responds to treatment with the synthetic auxin, 2,4-D, by undergoing cell division. Specific IAA-binding activity is undetectable in crude membrane preparations made from either the intact tuber or from tissue cultures in the absence of 2,4-D. On the other hand specific IAA-binding activity is readily detectable if the tissue has first been cultured in 2,4-D. Studies of 2,4-D efflux-influx kinetics are also in agreement with this notion. Both parameters are modified and the net result is a higher level of retention of 2,4-D in this tissue if the tissue has been previously cultured in 2,4-D-containing media, suggesting the appearance of auxin-binding activity.     

Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber calli

The effects of the herbicides 1,1'-dimethyl-4,4'-bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1muM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.     

Effect of 2,4-dichlorophenoxyacetic Acid on glucosylation of scopoletin to scopolin in tobacco tissue culture

2,4-Dichlorophenoxyacetic acid (2,4-D) stimulated the formation of scopoletin and scopolin in tobacco (Nicotiana tabacum L. `Bright Yellow') cell culture. It especially stimulated the uptake of scopoletin from culture medium into the cells and the glucosylation of scopoletin to its monoglucoside, scopolin. This phenomenon is peculiar to 2,4-D, in contrast to other plant hormones. 2,4-D (1 μg/ml) stimulated the glucosylation of scopoletin to scopolin by enhancing UDP-glucose:scopoletin glucosyltransferase (SGTase) activity. The enhancement of SGTase activity caused by treatment with 2,4-D was observed when the syntheses of RNA and protein were inhibited by either actinomycin-D and/or cycloheximide. However, the stimulatory effect of 2,4-D was inhibited by treatment with dinitrophenol. Furthermore, SGTase with or without treatment by 2,4-D in vivo for 24 hours, was isolated from cultured tobacco cells. The enzymes were purified about 200-fold by precipitation with (NH₄)₂SO₄ and chromatography with Sephadex G-100, DEAE-cellulose, and hydroxyapatite. The specific activity of 2,4-D-treated SGTase was 10 times higher than that of untreated SGTase even in the purified fraction, which showed one protein band under electrophoresis. These results suggest that the enhancement of SGTase activity by 2,4-D is due to the energy-dependent activation of the enzyme already present, but not due to the de novo synthesis of the enzyme.     

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

2,4-D Hyper Accumulation Induced Cellular Responses of <i>Azolla pinnata</i> R. Br. to Sustain Herbicidal Stress

In the present experiment with ongoing concentration (0 µM, 100 µM, 250 µM, 500 µM and 1000 µM) of 2,4-D, the responses of Azolla pinnata R.Br. was evaluated based on cellular functions. Initially, plants were significantly tolerated up to 1000 µM of 2,4-D with its survival. This was accompanied by a steady decline of indole acetic acid (IAA) concentration in tissues with 78.8% over the control. Membrane bound H⁺ -ATPase activity was over expressed within a range of 1.14 to 1.25 folds with activator (KCl) and decreased within a range of 57.3 to 74.6% in response to inhibitor (Vanadate) application. With regards to IAA metabolism, plants recorded a linear increase with wall bound oxidase activity up to maximum concentration of 2,4-D. The variations were more moderated when wall bound IAA-oxidase recorded a linear increase proportionate to the 2,4-D concentrations. This was more extended with the presence of different isoforms of IAA-oxidase which was much more pronounced with distinct polymorphisms of expressed proteins, however, not independent to the 2,4-D concentrations. Polyamines like spermine, spermidine and putrescine (spm, spd and put) were not consistent in concentration with the dosages of 2,4-D. Besides these, plants were induced to apoplastic NAD(P)H oxidase activity maximally by 1.6 folds under 500 µM 2,4-D over control. Still, putrescine responded more or less consistently and recorded maximally 11.9 folds at 500 µM 2,4-D as compared to the control. NAD(P)H oxidase activity recorded maximally 1.6 folds against control and remain consistent throughout the concentrations of 2,4-D. GPX along with APX were more linear in responses through the concentration of 2,4-D except CAT as compared to control. On enzymatic antioxidative activity, peroxidases (GPX and APX) were overexpresed in a similar manner except for catalase with a non-significant rise. In stabilization of cellular redox, glutathione reductase attended maximum value by 2.45 folds at 1000 µM evidenced with significant variations in protein polymorphism. The sensitivity of 2,4- D also appeared in Azolla with a maximum loss of nucleic acids as documented by the comet assay. Moreover, the Azolla might have some DNA damage protective activity as evident using frond extract with plasmid nick assay. Therefore, Azolla plants with its cellular responses is evident to sustain against the 2,4-D herbicidal stress and may be granted in bio remediation process for the contaminated soil.     

The influence of 2,4-dichlorophenoxyacetic acid on cell cycle Kinetics and Sister-Chromatid Exchange Frequency in Garlic (Allium sativum L.) Meristem Cells

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) at low concentrations on cell cycle duration and sister-chromatid exchange (SCE) frequency was studied using meristem root-tip cells ofAllium sativum L. 2,4-D induced a marked prolongation of the cell cycle. At the same time, small but statistically significant increases in SCE frequencies were observed at 5 μM and 15 μM 2,4-D concentrations. The significance of these findings in the evaluation of mutagenic activity of 2,4-D is discussed.     

2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134

2,4-Dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2,4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an Mr of approximately 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha-KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degrees C, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+, Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha-KG were 109 and 8.9 microM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha-KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.     

2,4-D诱导固氮螺菌和慢生型大豆根瘤菌在小麦上的结瘤作用及其效果

分别在水培和砂培条件下进行了2,4-D诱导固氮螺菌和慢生型大豆根瘤菌在小麦根上的结瘤试验,结果表明2,4-D能诱发它们在小麦根系上形成“类根瘤”,扫描电镜结果证明只有个别细菌进入小麦根瘤细胞内,在细胞间隙有较多的细菌。用乙炔还原法仅检测到接种大豆根瘤菌的小麦根瘤有微量的固氮酶活性,但在盆栽植株的生长方面,看不到2,4-D,2,4-D+固氮螺菌或2,4-D+根瘤菌对小麦生长的促进作用。     

2,4-D和激动素对烟草愈伤组织IAA氧化酶和细胞分裂素氧化酶活性的影响

2,4-D和激动素(KT)均显著降低烟草愈伤组织中IAA氧化酶和细胞分裂素氧化酶的活性,KT的影响更显著.在MS中的愈伤组织IAA氧化酶活性最高,MS 2,4-D中的次之,MS KT和MS 2,4-D KT中的最低.愈伤组织在MS中继代6 d时,细胞分裂素氧化酶活性出现明显的高峰,在其它3种培养基中则没有.     

Inhibition of the indole-3-acetic acid-induced epinastic curvature in tobacco leaf strips by 2,4-dichlorophenoxyacetic acid

It has been reported that auxin induces an epinastic growth response in plant leaf tissues. Leaf strips of tobacco (Nicotiana tabacum L. 'Bright Yellow 2') were used to study the effects of indole-3-acetic acid (IAA), the principal form of auxin in higher plants, and a synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), on epinastic leaf curvature. Incubation of leaf strips with 10 micro M IAA resulted in a marked epinastic curvature response. Unexpectedly, 2,4-D showed only a weak IAA-like activity in inducing epinasty. Interestingly, the presence of 2,4-D resulted in inhibition of the IAA-dependent epinastic curvature. In vivo Lineweaver-Burk kinetic analysis clearly indicated that the interaction between IAA and 2,4-D reported here is not a result of competitive inhibition. Using kinetic analysis, it was not possible to determine whether the mode of interaction between IAA and 2,4-D was non-competitive or uncompetitive. 2,4-D inhibits the IAA-dependent epinasty via complex and as yet unidentified mechanisms.     

Correlation between Plant Growth Regulator Release Rate and Bioactivity for the Series of Newly Synthesized Phytoactive Polymers

Phytoactive polymers are high molecular weight systems in which a plant growth regulator (PGR) unit is attached to the polymeric chain by a hydrolyzable chemical bond. The release rate of the PGR is linked to the biological activity of the phytoactive polymer and can be controlled by properties inherent in the whole macromolecular system. In this study the correlation of biological activity and plant growth regulator hydrolytic release rate was investigated for the series of newly synthesized 2,4-dichlorophenoxyacetic acid (2,4-D) polymeric esters. The polymers synthesized differ in their molecular weight, side group structure, and 2,4-D residue content. The influence of these polymer characteristics on the 2,4-D hydrolytic release was investigated, and it was demonstrated that hydrolysis rate substantially depends on the polymer molecular weight, side group structure, and 2,4-D residue content. It was also demonstrated that phytoactive polymer bioactivity depends on the hydrolysis rate of the polymers, and in dependence of this parameter can provide stimulating or inhibiting activity. Biological activity was illustrated by the elongation of wheat and barley coleoptiles.     

Dissociation of Cortical Cell Walls and Enhancement of Cellulase Activity during the Emergence of Callus from Rice Roots in the Presence of 2,4-D

The separation of cortical cells from root explants of rice(Oryza saliva L. cv. Sasanishiki) was stimulated by 2,4-D andboth the plastic and the elastic properties of cell walls increasedduring the formation of callus. These events, in particular,the separation of cortical cells, may be important for the generationof clumps of callus cells that are initiated at the interiorof root tissues, specifically around the vessels. Buffer-solublecellulase activity was significantly enhanced by treatment with2,4-D (6- to 10-fold) at the early stage of formation of rootcallus (1–2 days after the start of treatment with 2,4-D).The optimum concentration of 2,4-D and time course studies indicatedthat this enhacement was correlated with and preceded both theseparation of cortical cells and loosening of the cell wall.The enhancement of cellulase activity by 2,4-D in rice is thesuch first finding in a monocot. (Received September 24, 1992; Accepted February 18, 1993)