Pea Xyloglucan and Cellulose : III. Metabolism during Lateral Expansion of Pea Epicotyl Cells

Lateral expansion of the third internodes of pea epicotyls was evoked by treatment with either 2,4-dichlorophenoxyacetic acid (2,4-D) or ethylene gas. During growth, 2,4-D enhanced and ethylene inhibited the deposition of xyloglucan and cellulose in the cell wall, with the result that the wall framework (ghost) from ethylene-treated swollen tissue was much thinner than that from 2,4-D-treated. The level of activity of xyloglucan synthase, alkali-insoluble β-glucan synthases, and endo-1,4-β-glucanases were all enhanced by 2,4-D treatment but not by ethylene. Both 2,4-D and ethylene treatments led to increased osmotic potential in the swelling tissues. Accordingly, swelling after 2,4-D treatment was accompanied by xyloglucan degradation, concomitant with substantial net synthesis, but swollen tissue as a result of ethylene treatment was characterized by walls whose integrity was weakened by relatively low levels of newly deposited polysaccharides rather than by the degradation.     

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.     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

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)     

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.     

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.     

Activated sludge treatment of a xenobiotic with or without a biogenic substrate during start-up and shocks

Lab-scale continuous flow activated sludge systems that were acclimated to 2,4-dichlorphenoxyacetic acid (2,4-D) under sole 2,4-D influent and without sludge wastage, were able to maintain successful 2,4-D treatment when both 2,4-D and a biogenic substrate were fed and the systems operated with finite mean cell residence times (theta(c)). When the systems were fed dual 2,4-D and biogenic substrates and operated with finite theta(c) from the start, treatment of 2,4-D fluctuated noticeably long after acclimation. At the reintroduction of 2,4-D after its absence from the influent for a period of time (2,4-D shock), the systems under both the sole and dual substrate conditions suffered similar treatment losses; the extent of treatment losses was related to the length of 2,4-D absence time. When shocked, systems with sole 2,4-D influent had a slight advantage over dual substrates by showing a faster recovery from shocks with the help of re-acclimation.     

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.     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline     

Formation of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) embryogenic callus involves peroxide-generating germin-like oxalate oxidase

In wheat ( Triticum aestivum L.), embryogenic callus formation comprises suppression of precocious germination by the zygotic embryo and the initiation of dedifferentiated cellular proliferation within it. Embryogenic calli are induced by treating immature embryos with 2,4-dichlorophenoxyacetic acid (2,4-D). Upon withdrawal from 2,4-D, somatic embryos develop from the periphery of the callus. Prior to visible callus formation, there is a striking induction of "germin-like" oxalate oxidase ("gl-OXO": EC 1.2.3.4) gene expression. Accumulation of gl-OXO mRNA is rapidly stimulated upon auxin treatment, with a consequent development of apoplastic enzyme activity producing H(2)O(2) within the cell wall. Within the dedifferentiated calli, gl-OXO enzyme activity becomes widespread over the surface of embryogenic calli. Differentiation of somatic embryos is initiated in regions of densely cytoplasmic, meristematic cells that are marked by highly localised expression of gl-OXO activity within these embryogenic cell masses. We suggest that this localised generation of H(2)O(2) by gl-OXO promotes peroxidative cross-linking of cell wall components, thereby preventing cellular expansion and maintaining these cell masses in an embryogenically competent condition.     

2,4-D Resistance in a Tobacco Cell Culture Variant II. Effects of 2,4-D on Nucleic Acid and Protein Synthesis and Cell Respiration

The effects of 2,4-D on nucleic acid and protein synthesis andcell respiration were compared between a 2,4-D-resistant variantand its wild-type cell lines of tobacco (Nicotiana tabacum L.).The variant continued cell division and growth in the presenceof 100 µM 2,4-D which was strongly inhibitory to the wild-typecell lines. Among the macromolecular syntheses studied, DNAsynthesis was the most sensitive and protein synthesis was theleast sensitive to inhibitory concentrations of 2,4-D. The variantdisplayed threefold higher resistance to 2,4-D than the wild-typecell line based on the 50% inhibitory concentrations of 2,4-Don DNA synthesis. No significant differences which could explainthe 2,4-D resistance were found between the variant and thewild-type cell lines in 2,4-D concentrations required to inhibitRNA and protein synthesis. The effect of 2,4-D on cell respirationwas detectable without a noticeable lag. The resistance of thevariant based on the effect on cell respiration also was apparentimmediately after 2,4-D addition. According to the 50% inhibitoryconcentrations of 2,4-D on cell respiration, the variant showeda level of resistance similar to that estimated by DNA synthesis.These results indicate that the resistance of the variant isdue to a modification which reduces the cellular sensitivityto phyto-toxic concentrations of 2,4-D with respect to, at least,DNA synthesis and respiration. (Received August 6, 1985; Accepted November 27, 1985)     

Identification of a Phosphoprotein Expressed During Somatic Embryogenesis in Wheat Leaf Base Cultures

2,4-D mediated induction of somatic embryogenesis in wheat is enhanced in the presence of Ca⁺⁺ and its removal by EGTA reduces the response significantly. Changes that occur at the polypeptide level following 2,4-D treatment were analysed. Intense cell division activity was discernable in the leaf base explants within an hour of treatment. Changes in protein profiles were prominent in the membrane fraction as compared to the soluble fraction. The protein profile of the leaf base culture with somatic embryos was distinct from the calli induced from mature embryos on a 2,4-D containing medium. The role of Ca²⁺ in the induction of somatic embryogenesis was demonstrated by the use of EGTA (a calcium chelator), verapamil, nifedipine (calcium channel blockers), W7 (calmodulin antagonist) and Li (PI inhibitor). ^{In vitro} protein phosphorylation studies showed that 2,4-D, calcium and related treatments inhibit phosphorylation of proteins. In the membrane fraction proteins, accumulation of polypeptides at the low molecular weight range was seen in samples treated with verapamil and W7, and a 30 kO polypeptide in the samples treated with calmodulin antagonist, W7. Autoradiography of membrane fraction proteins displayed the presence of a 16 kO protein phosphorylated in samples treated with verapamil, nifedipine and W7. It thus appears that 2,4-D and Ca⁺⁺ prevent the phosphorylation of this phosphoprotein. These results thus indicate the action of 2,4-D via the Ca²⁺-CaM signaling pathway in triggering the induction of somatic embryogenesis.     

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.     

The influence of phytohormones on zeta potential and electrokinetic charges of winter wheat cells

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2 x 10(-5) MCa2+. Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.     

Role of the epidermis in auxin-induced elongation of light-grown pea stem segments

1. Effects of auxin on elongation and cell wall properties werestudied using 5th internode segments of light-grown pea epicotyl.The results were:
2. The optimum concentration of 2,4-D for elongationinductionwas about 1 µg/ml, both for unpeeled and peeledsegments.
3. Using stress-relaxation analysis, mechanical propertiesof thecell wall were expressed by the parameters 1/₁, To andT_m. Unpeeledsegments were first treated with 2,4-D, then theepidermis waspeeled off. Parameters of the epidermal cell wallwere conspicuouslychanged by 2,4-D but those of the inner tissuewere not.
4. Actinomycin D and cycloheximide inhibited 2,4-D-inducedchangesin cell wall parameters, as well as in elongation, ofunpeeledsegments apd of the epidermis.
5. 2,4-D did not induceelongation of the isolated epidermis butpromoted that of peeledsegments. This promotion was smalleras compared with unpeeledsegments. 2,4-D did not significantlyinfluence the diffusionpressure deficit of peeled segmentsbut did increase their elasticand plastic extensibilities.
6. We conclude that auxin primarilyinduces cell wall looseningof the epidermis, most likely throughnucleic acid and proteinsynthesis.

¹ Present address: Biological Institute, Department of GeneralEducation, Nagoya City University, Mizuho-ku, Mizuho-cho, Nagoya467, Japan. (Received April 22, 1971; )     

2,4-D和NAA在拟南芥细胞分裂和伸长中的作用分析

以拟南芥悬浮细胞体系为实验材料,研究了人工合成生长素NAA和2,4-D外源处理对细胞形态、细胞鲜重、细胞分裂指数等指标的影响.结果表明:2μmol/L 2,4-D可有效促进细胞分裂但不影响细胞伸长;同样浓度的NAA主要诱导细胞的伸长;细胞伸长和细胞分裂是2个不相偶联的过程;在2,4-D所诱导的细胞分裂过程中异三聚体G-蛋白α-亚基GPA1强表达.     

枸杞体细胞胚发生中Ca^2＋和ATPase的超微结构定位研究

研究2,4-D诱导枸杞体细胞胚发生中的作用及其与Ca^2 含量和ATPase活性时空分布动态之间的关系,以探讨2,4-D诱导植物体细胞胚发生的作用机理。采用超微细胞化学定位的方法,跟踪分析了体细胞胚发生与发育的不同时期,Ca^2 和ATPase活性的时空分布动态。结果表明：2,4-D是诱导离体培养的枸杞体细胞进入胚胎状态的关键激素。在含有2,4-D和不含2,4-D的培养条件下,分别诱导枸杞体细胞脱分化后,再转入除去2,4-D的MS培养基上,进行分化培养,结果前者可分化形成体细胞胚,因而称为胚性愈伤组织。后者在相同条件却不能分化形成胚,故称为非胚性愈伤组织。在2,4-D诱导枸杞的胚性愈伤组织中,胚性细胞分化早期的细胞间隙和细胞壁上均有Ca^2 沉淀。随着胚性细胞的分化、分裂和多细胞原胚形成,这时Ca^2 在细胞内的分布主要集中在细胞膜和液泡膜上;球形胚期在细胞核中Ca^2 呈弥散性分布。在此过程中,ATPase活性时空分布与Ca^2 的定位变化具有高度一致性,仅仅稍滞后于Ca^2 出现的时间。而在胚性细胞分化早期,ATPase活性同样位于质膜上,随后在液泡和细胞核都可见ATPase活性分布。而在非胚性愈伤组织中,则未见Ca^2 和ATPase活性呈时空动态分布,而且随着非胚性细胞的液泡化,无论是Ca^2 含量,还是ATPase活性都呈逐渐降低的趋势。表明Ca^2 和ATPase活性变化与2,4-D诱导的胚性细胞分化和发育密切相关。并由此推测,Ca^2 和ATPase的时空分布对胚性细胞分化中的信息传递和调控相关基因表达起着关键性作用。     

枸杞体细胞胚发生中Ca2+和ATPase的超微结构定位研究

研究2,4-D诱导枸杞体细胞胚发生中的作用及其与Ca~(2+)含量和ATPase活性时空分布动态之间的关系,以探讨2,4-D诱导植物体细胞胚发生的作用机理。采用超微细胞化学定位的方法,跟踪分析了体细胞胚发生与发育的不同时期,Ca~(2+)和ATPase活性的时空分布动态。结果表明:2,4-D是诱导离体培养的枸杞体细胞进入胚胎状态的关键激素。在含有2,4-D和不含2,4-D的培养条件下,分别诱导枸杞体细胞脱分化后,再转入除去2,4-D的MS培养基上,进行分化培养,结果前者可分化形成体细胞胚,因而称为胚性愈伤组织。后者在相同条件却不能分化形成胚,故称为非胚性愈伤组织。在2,4-D诱导枸杞的胚性愈伤组织中,胚性细胞分化早期的细胞间隙和细胞壁上均有Ca~(2+)沉淀。随着胚性细胞的分化、分裂和多细胞原胚形成,这时Ca~(2+)在细胞内的分布主要集中在细胞膜和液泡膜上;球形胚期在细胞核中Ca~(2+)呈弥散性分布。在此过程中,ATPase活性时空分布与Ca~(2+)的定位变化具有高度一致性,仅仅稍滞后于Ca~(2+)出现的时间。而在胚性细胞分化早期,ATPase活性同样位于质膜上,随后在液泡和细胞核都可见ATPase活性分布。而在非胚性愈伤组织中,则未见Ca~(2+)和ATPase活性呈时空动态分布,而且随着非胚性细胞的液泡化,无论是Ca~(2+)含量,还是ATPase活性都呈逐渐降低的趋势。表明Ca~(2+)和ATPase活性变化与2,4-D诱导的胚性细胞分化和发育密切相关。并由此推测,Ca~(2+)和ATPase的时空分布对胚性细胞分化中的信息传递和调控相关基因表达起着关键性作用。     

Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.

Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.