Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.     

Cadmium causes the oxidative modification of proteins in pea plants

In pea (Pisum sativum L.) leaves from plants grown in the presence of 50 µm CdCl₂ the oxidative production of carbonyl groups in proteins, the rate of protein degradation and the proteolytic activity were investigated. In leaf extracts the content of carbonyl groups measured by derivatization with 2,4‐dinitrophenylhydrazine (DNPH), was two‐fold higher in plants treated with Cd than in control plants. The identification of oxidized proteins was carried out by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of proteins derivatized with DNPH and immunochemical detection with an antibody against DNPH. The intensity of the reactive bands was higher in plants exposed to Cd than in controls. By using different antibodies some of the oxidized proteins were identified as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase. The incubation of leaf crude extracts with increasing H₂O₂ concentrations showed a progressive enhancement in carbonyl content and the pattern of oxidized proteins was similar to that found in Cd‐treated plants. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment. In peroxisomes purified from pea leaves a rise in the carbonyl content similar to that obtained in crude extracts from Cd‐treated plants was observed, but the functionality of the peroxisomal membrane was not apparently affected by Cd. Results obtained demonstrate the participation of both oxidative stress, probably mediated by H₂O₂, and proteolytic degradation in the mechanism of Cd toxicity in leaves of pea plants, and they appear to be involved in the Cd‐induced senescence previously reported in these plants.     

Translocation and Complex Formation of Picloram and 2,4-D in Rape and Sunflower

Uptake, translocation and complex formation of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of rape (Brassica napus L. cv. Nilla) and sunflower (Helianthus annuus L. var. uniflorus) were studied. Sunflower is susceptible both to 2,4-D and picloram, while rape is susceptible to 2,4-D but more tolerant to picloram. The uptake of the herbicides through the leaves was almost complete in both species. Translocation of 2,4-D into the roots took place more readily than that of picloram. In sunflower about 50 per cent of the applied 2,4-D was extruded through the roots into the nutrient solution after 9 days. In the picloram-treated sunflower most of the activity was found in the aerial parts, while in picloram-treated rape most of the activity still occurred in the treated leaf after 9 days. No activity at all was found in the roots or in the nutrient solution of the picloram-treated rape seedlings. While the major part of 2,4-D always was found in the state of free herbicide, a large fraction of picloram was rapidly bound into water-soluble complexes. This binding was especially pronounced in rape. Separation by paper chromatography showed that different radioactive compounds were formed. Most of these could be hydrolyzed, thereby releasing free herbicide. The results support the hypotheses that complex formation could counteract herbicide translocation and toxicity of auxin herbicides.     

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.     

Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exogenous auxin

The activation of cell division and oxidative stress responses has been investigated in the case of leaf protoplast-derived cells. Initiation of protoplast culture was found to be associated with oxidative stress as indicated by the rate of H₂O₂ release into the medium and/or by catalase and ascorbate peroxidase activities. Both cell division frequency and the above stress-related parameters were dependent on the exogenous auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) concentrations used. In addition, the well known oxidative stress-inducing agent paraquat (1 μM) could promote cell division at suboptimal auxin concentration but not in the absence of exogenous auxin. The H₂O₂ scavenger dimethylthiourea and the NADPH oxidase inhibitor diphenyleneiodonium inhibited not only the activation of cellular defense reactions but cell division as well. Based on the above experimental observations, it is suggested that exogenous auxin (2,4-D) enhances cellular defense reactions in parallel with cell division activation.     

三种除草剂对五爪金龙的防除作用及2,4-D丁酯对环境的影响

用3种除草剂(2,4-D丁酯、麦草畏和塔隆)对五爪金龙(Ipomoea cairica L.Sweet)进行化学防除试验。结果表明:1.00 mL L-1的2,4-D丁酯可以彻底杀灭五爪金龙。喷施1.00 mL L-12,4-D丁酯20 d后,五爪金龙茎叶枯死率接近100%;60 d后五爪金龙的总生物量显著低于其它处理及对照;90 d后未出现生长恢复,最终盖度防效为99.8%。而喷施1.00 mL L-1的麦草畏40 d后,五爪金龙的茎叶枯死率为99.0%,但仍有少量存活的根,90 d后再次萌生率为10.0%;喷施1.00 mL L-1塔隆40 d后,五爪金龙的茎叶枯死率为100%,90 d后再次萌生率为100%。土壤残留分析表明:在有机质含量较高[(10.14±1.01)g kg-1]的土壤中2,4-D丁酯降解速率较快,半衰期为14 d,施药后80 d的土壤中已检测不到2,4-D丁酯。此外,在野外喷洒1.0 mL L-1的2,4-D丁酯对其它植物是安全的,施药1年后,样地内的植物均能恢复生长。因此,实践中可用1.00 mL L-1的2,4-D丁酯来防除五爪金龙。     

The transport of auxin and regeneration of xylem in okra and pea stems

Internodes were isolated from okra and pea plants grown from seed under controlled conditions. Isolated internodes were treated apically or basally with IAA-C¹⁴ or 2,4-D-C¹⁴ and several vascular bundles were severed in the middle of the internodes. Transport of auxins was basipetally polar, IAA moving with greater facility than 2,4-D. Apically applied auxin stimulated vascular regeneration in the wound area; basally applied auxin did not, but radioactivity from basally applied IAA reached the wound. This suggests that the path of transport or mode of presentation to cells is important unless basally and apically applied auxin are metabolized differently.     

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506–508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4–17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways. Experiment station: Plant Biotechnology Centre, Primary Industries Research Victoria, Department of Primary Industries, La Trobe University, Bundoora, Victoria 3086, and the Victorian Microarray Technology Consortium (VMTC).     

Influence of certain herbicides and a forest fertilizer on the nitrogen fixation by the lichen Peltigera praetextata

Summary Effects of herbicides (Garlone 3A, MCPA, 2,4-D and Krenite) and nitrogen fertilizer (NH₄NO₃), commonly used in Swedish forestry, on nitrogen fixation (C₂H₂-reduction) by Peltigera praetextata (Sommerf.) Zopf. (field and laboratory) and its phycobiont Nostoc sp. (laboratory) were studied. The alga was affected by the herbicides 2,4-D and Krenite and the fertilizer, with a decrease in nitrogenase activity. Nitrogen fixation by the lichen was not affected by herbicides but treatment with NH₄NO₃ led to depression of nitrogenase activity and serious disturbance of the symbiosis, the latter effect due to the fertilizer's lethal effects on the mycobiont (electron microscopy).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - Garlone 3A triclopyre - Krenite Ammoniumethylcarbamylphosphonate     

Translocation and Metabolism of Picloram and 2,4-D in Populus tremula

Translocation and metabolism of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in small plants of aspen (Populus tremula L.) were studied. In most experiments ¹⁴C-carboxyl-labelled herbicides were used. Considerable quantities of both herbicides were retained in the treated leaf. Translocation was mainly upwards into the growing shoot tip. Only minute quantities were found in the roots. Injection of the herbicides through a cut stem surface increased the translocation into the roots very little. One of the reasons for the limited downward translocation is considered to be a ready transfer of the herbicides between the phloem and the xylem. Both herbicides were incorporated into complexes from which the active herbicides could be released. However, this complex formation can only partly account for the retention of the herbicides in the treated leaves. The differences in metabolism found between 2,4-D and picloram cannot explain the considerable difference in toxicity between the compounds.     

Herbicidal activity of 4-chloroindoleacetic acid and other auxins on pea, barley and mustard

The natural auxins, 4-chloroindoleacetic acid and its methyl ester have strong herbicidal effects on pea, Pisum sativum , a plant in which they occur naturally. The standard herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) is only 5 times more effective than 4-chloroindoleacetic acid. The I₅₀, the dose inhibiting yield by 50%, for 4-chloroindoleacetic acid and its methyl ester is 0.5 kg ha⁻¹ or 15 mg kg⁻¹ fresh weight, close to the concentration of 4-chloroindoleacetic acid methyl ester in maturing pea seeds. Naphthaleneacetic acid and indoleacetic acid are also inhibitory, but at much higher concentrations. In its inhibiting effect on white mustard, Sinapis alba , 4-chloroindoleacetic acid approximates that of 2,4-D, whereas in barley, Hordeum vulgare , it is a stronger herbicide than 2,4-D. All auxins tested killed white mustard at low doses, but none killed barley. Both 4-chloroindoleacetic acid and 2,4-D killed pea. The chloroindole auxins of pea may be the hypothetic death hormones or senescence factors that are secreted from the developing seeds into the parent plant which is strongly inhibited or killed and from which the nutrients are mobilized and translocated to the seeds. The action mechanism of auxin type herbicides may be to simulate the action of endogenous herbicides.     

Genetic transformation of wheat using mature seed tissues

A protocol for biolistic transformation of bread wheat based on using mature seed tissues as explants has been developed. Embryogenic callus obtained from mature seed tissues was transformed with a psGFP-BAR plasmid containing gfp reporter gene and bar selectable marker gene. The influence of hormone composition of the medium on the efficiency of transformation of mature wheat seed tissues has been demonstrated. The use of auxin 2,4-D resulted in the formation of transgenic plants with a frequency of 0.75%, while the use of Dicamba auxin for the regeneration of plants did not result in transformant development. The transgenic status of the plants obtained in the experiments has been confirmed by PCR and RT-PCR. Stable inheritance of transgenic features in the following generations of wheat (T₁, T₂) has been demonstrated and transgenic plants exhibiting high resistance to herbicides have been obtained. The protocol developed allows for a simplified transformation of wheat in order to obtain transgenic plants with novel features.     

The specificity of auxin transport in intact pea seedlings (Pisum sativum L.)

Summary When eight ¹⁴C-labelled auxin and non-auxin compounds were applied to the apical buds of intact dwarf pea seedlings (Pisum sativum L.), only [1-¹⁴C]indoleacetic acid ([¹⁴C]IAA) and -[1-¹⁴C] naphthaleneacetic acid ([¹⁴C]NAA) underwent appreciable basipetal transport during the first 24 h; over a longer period (72 h) considerable basipetal transport of the auxin [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([¹⁴C]2,4-D) also occurred, but at a very much lower velocity (ca. 1.4–2.2 mm·h^-1). The movement of 2,4-D possessed many of the characteristics of a typical auxin transport. During uptake and transport IAA and NAA were extensively metabolised to the corresponding aspartates, and to ethanol-insoluble/NaOH-soluble compounds; little metabolism of 2,4-D was observed. None of the non-auxin compounds applied (sorbose, sucrose, leucine, adenine and kinetin) underwent appreciable basipetal transport from the apical bud. All but sorbose were extensively metabolised by the apical tissues. Little metabolism of sorbose itself was detected.The results suggest that the long-distance basipetal auxin transport system from the apical bud of intact plants is specific for auxins; the specificity may result from the affinity of auxins for specific transport sites.     

Effect of galactoglucomannan-derived oligosaccharides on elongation growth of pea and spruce stem segments stimulated by auxin

Galactoglucomannan-derived oligosaccharides (GGMOs) (degree of polymerization 4–8) isolated from the wood of poplar (Populus monilifera Ait.) were shown to be inhibitors of the 2,4-dichlorophenoxyacetic acid-stimulated elongation growth of pea (Pisum sativum L. cv. Tyrkys) and spruce [Picea abies (L.) Karst] stem segments. A dependence on the concentration of GGMOs (between 10^-5-10^-10M) as well as plant species was ascertained. Pea stem segments were much more sensitive (10^-10M) than spruce (10^-8M). The GGMOs did not exhibit toxicity even at high concentrations and during long-term bioassays. The timing of the action of GGMOs and auxin in the growth process was also studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - d.p degree of polymerization - GGMOs galactoglucomannan-derived oligosaccharides This research was supported by the Slovak Grant Agency for Science.     

GH3 expression and IAA-amide synthetase activity in pea (Pisum sativum L.) seedlings are regulated by light,plant hormones and auxinic herbicides

The formation of auxin conjugates is one of the important regulatory mechanisms for modulating IAA action. Several auxin-responsive GH3 genes encode IAA-amide synthetases that are involved in the maintenance of hormonal homeostasis by conjugating excess IAA to amino acids. Recently, the data have revealed novel regulatory functions of several GH3 proteins in plant growth, organ development, fruit ripening, light signaling, abiotic stress tolerance and plant defense responses. Indole-3-acetyl-aspartate (IAA-Asp) synthetase catalyzing IAA conjugation to aspartic acid in immature seeds of pea (Pisum sativum L.) was purified and characterized during our previous investigations. In this study, we examined the effect of auxin and other plant hormones (ABA, GA, kinetin, JA, MeJA, SA), different light conditions (red, far-red, blue, white light), and auxinic herbicides (2,4-D, Dicamba, Picloram) on the expression of a putative GH3 gene and IAA-amide synthesizing activity in 10-d-old pea seedlings. Quantitative RT-PCR analysis indicated that the PsGH3-5 gene, weakly expressed in control sample, was visibly induced in response to all plant hormones, different light wavelengths and the auxinic herbicides tested. Protein A immunoprecipitation/gel blot analysis using anti-AtGH3.5 antibodies revealed a similar pattern of changes on the protein levels in response to all treatments. IAA-amide synthetase activity determined with aspartate as a substrate, not detectable in control seedlings, was positively affected by a majority of treatments. Based on these results, we suggest that PsGH3-5 may control the growth and development of pea plants in a way similar to the known GH3 genes from other plant species.     

Translocation and Complex Formation of Root-applied 2,4-D and Picloram in Susceptible and Tolerant Species

Translocation and complex formation of ¹⁴C-labelled 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram) in seedlings of sunflower (Helianthus annuus L. var. uniflorus), rape (Brassica napus L. cv. Nilla), wheat (Triticum aestivum L. cv. Starke), and Norway spruce (Picea abies (L.) H. Karst.) were studied. The herbicides were absorbed through the roots from the nutrient solution, Picloram was well translocated to the shoots of the four species; while the acropetal translocation of 2,4-D was small except in rape. In 2,4-D-susceptible sunflower and rape and in picloramsusceptible sunflower and spruce the herbicides were recovered mainly in the uncomplexed form. In 2,4-D tolerant wheat and spruce most of the absorbed 2,4-D was converted into water-soluble or TCA-insoluble complexes. In picloram-tolerant wheat and in relatively picloram-tolerant rape, the absorbed picloram was also converted into complexes recovered predominantly in the water-soluble fraction. Most of the complexes released free herbicides by hydrolyzing in NaOH or HCI. The results further support the hypothesis that complex formation counteracts herbicide toxicity.     

Effects of pH Changes on Ion and 2,4-D Uptake of Wheat Roots

The quantitative relationships between pH-dependent ion and 2,4-D uptake in winter wheat seedlings (Triticum aestivum L. cv. Yubileynaya 50) have been investigated. The movement of various ions (potassium, phosphate, nitrate and ammonium) and 2,4-D across the root membranes was monitored with radioactive and stable isotope tracer methods. It was found that the H₊ ion concentration of the absorption solution strongly influences the 2,4-D uptake of the roots. Simultaneously, the 2,4-D uptake stimulates secretion of H⁺ into the absorption solution, that is, a H⁺ efflux can accompany the uptake of 2,4-D. This finding is consistent with the acid secretion theory of auxin and fusicoccin action. At pH 4 the 2,4-D uptake was much higher than at pH 6, thereby inhibiting the ion uptake and increasing the phytotoxicity in the plant. The results indicate that 2,4-D enters the root cells rapidly at the lower pH, mostly as undissociated molecules. With reference to the 2,4-D concentration in the roots at pH 4, a possible transport mechanism of the auxin herbicide is briefly discussed.     

An auxin-auxotrophic mutant of Nicotiana plumbaginifolia

Summary Auxin (indole-3-acetic acid) is considered to be an important signalling molecule in the regulation of plant growth and development but neither auxin synthesis nor its mode of action is clearly understood. To identify genes involved in these processes, mutations were sought that altered the auxin requirement of plant tissues for growth. For the first time mutant plants were obtained that carry a recessive mutation at a single nuclear locus (auxl) which results in an absolute requirement for exogenous auxin for normal growth. In the absence of auxin treatment, mutant plants undergo premature senescence and die.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FUdR 5-fluorodeoxyuridine - IAA-EE indole-3-acetic acid ethyl ester - IMS indole-3-methanesulfonic acid