Isolation and Characterization of Golgi Membranes from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

A simple and rapid technique was developed for the isolationof the vesicular Golgi membranes from suspension-cultured cellsof sycamore (Acer pseudoplatanus L.). The procedure involvespreparation of protoplasts and differential centrifugation ofdisrupted protoplasts followed by the sucrose density gradientcentrifugation. Starting from broken protoplasts, sedimentableat two different centrifugal forces (10,000g and 100,000 g),two Golgi-enriched fractions of lower density, GF₁ and GF'₁,and higher density, GF₂ and GF'₂, were separated. Purity ofthe fraction was assessed by determining the marker enzyme activitiesas well as the electron microscopy of the specimens obtained. Inosine diphosphatase was enriched about 15- and 6-fold, respectively,in the GF₂ fraction from 10,000g and the GF'₂ one from 100,000gpellets, whereas the enrichment in GF₁ and GF'₁ was approximately6–7 fold. Galactosyl-transferase in GF₂ was enriched about25-fold. GF₁ and GF₂ account for 3–4% of the total proteinof 10,000g pellets, and GF'₁ and GF'₂ for about 6–7%of the total protein of 100,000g pellets. Electron microscopicobservations show that GF₂ and GF'₂ consisted principally ofvesicular Golgi membranes without an internal matrix althoughGF₁ and GF'₁ were contaminated with ER membranes and ribosomes. (Received March 11, 1985; Accepted June 17, 1985)     

Enzyme Sets of Glycolysis, Gluconeogenesis, and Oxidative Pentose Phosphate Pathway Are Not Complete in Nongreen Highly Purified Amyloplasts of Sycamore (Acer pseudoplatanus L.) Cell Suspension Cultures

Differential centrifugation and Percoll-gradient centrifugation of protoplast lysates of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) yielded pure amyloplasts. Contamination of the final amyloplast preparation by foreign compartments was assessed by measuring marker enzyme activities. The activity of alkaline pyrophosphatase was taken as a 100% plastid marker; relative to this marker, mitochondria (cytochrome c oxidase) averaged 0.34%, microbodies (catalase) 0.61%, and cytosol (alcohol dehydrogenase) 0.09%. Enzymatic activities of the glycolytic, gluconeogenic, pentose phosphate and the starch degradation pathways were found to be present in these amyloplast extracts in appreciable amounts. But the pyrophosphate-dependent phosphofructokinase and phosphoglyceromutase were judged to be essentially absent from amyloplasts because the activities of these enzymes were not enriched above the level of contaminating enzymatic activities in the amyloplast fractions. Additionally, the in vitro activities of starch phosphorylase, ATP dependent phosphofructokinase, NAD dependent glyceraldehyde-3 phosphate dehydrogenase, and glucose-6 phosphate dehydrogenase did not seem to support carbon fluxes from starch to triose phosphates as calculated from the rate of starch disappearance during carbon starvation of the cells. These results provide additional, indirect evidence for the recently emerged view that, in addition to the well known phosphate-triosephosphate translocator, another hexose phosphate and possibly also an ATP/ADP translocating system play major roles in nongreen plastids.     

Isolation and Characterization of the Amyloplast Envelope-Membrane from Cultured White-Wild Cells of Sycamore (Acer pseudoplatanus L.)

To study the characteristic features of the amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates reserve starch, in comparison with those of the chloroplast, these two types of plastids were isolated from white-wild and green-mutant protoplasts of cultured sycamore (Acer pseudoplatanus L.) cells, respectively. The intactness of the isolated amyloplast preparations was 70%. Electron microscopic ultrastructural analysis of both plastid types revealed unique structural features of the green-mutant chloroplasts, including well developed grana membranes and abundant ribosomal particles and plastoglobuli. After osmotic rupture of the isolated amyloplasts and chloroplasts, a clear separation of the envelope-membranes was achieved by discontinuous sucrose density gradient centrifugation. Although the visible absorption spectra of the envelope lipid components were indistinguishable between the amyloplasts and chloroplasts, the envelope-membrane polypeptide patterns were clearly distinct as judged by denaturing electrophoresis. By immunoblotting analysis using the specific antiserum raised against the pea chloroplast 29-kilodalton Pi-translocator, the amount of this carrier-protein (31-kilodalton) in the white-wild amyloplast envelope-membranes was estimated to be at least 10-fold less than in the green-mutant envelopes.     

Occurrence,Parasitism, and Pathogenicity of Nematodes Associated with Sycamore (Platanus occidentalis L.)

Ten species of stylet-bearing nematodes were recovered in a survey of sycamore (Platanus occidentalis L. ) stands in Georgia. Helicotylenchus, Xiphinema, and Criconemoides were the genera found most frequently. Populations of Hoplolaimus galeatus, Scutellonema brachyurum, Helicotylenchus dihystera and H. pseudorobustus increased on greenhouse-grown sycamore, but Trichodorus christiei, Xiphinema americanum, Meloidogyne hapla, M. arenaria and M. incognita did not. Hoplolaimus galeatus and S. brachyurum are semi-endoparasites; H. dihystera and H. pseudorobustus are migratory endoparasites. Hoplolaimus galeatus caused extensive root necrosis and marked decrease of fresh weights of seedling roots and tops. Helicotylenchus dihystera and S. brachyurum produced only qualitatively different sparse and unhealthy root growth. Helicotylenchus pseudorobustus caused only a reduction in root surface area.     

Stress Responses in Alfalfa (Medicago sativa L.): II. Purification, Characterization, and Induction of Phenylalanine Ammonia-Lyase Isoforms from Elicitor-Treated Cell Suspension Cultures

l-Phenylalanine ammonia-lyase has been purified from elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures using two protocols based on different sequences of chromatofocusing and hydrophobic interaction chromatography. Three distinct forms of the intact enzyme were separated on the basis of affinity for Octyl-Sepharose, with isoelectric points in the range pH 5.1 to 5.4. The native enzyme was a tetramer of M_r 311,000; the intact subunit M_r was about 79,000, although polypeptides of M_r 71,000, 67,000 and 56,000, probably arising from degradation of the intact subunit, were observed in all preparations. Two-dimensional gel analysis revealed the presence of several subunit isoforms of differing isoelectric points. The purified isoforms of the native enzyme had different K_m values for l-phenylalanine in the range 40 to 110 micromolar, although mixtures of the forms in crude preparations exhibited apparent negative rate cooperativity. The enzyme activity was induced approximately 16-fold within 6 hours of exposure of alfalfa cells to a fungal elicitor or yeast extract. Analysis by hydrophobic interaction chromatography revealed different proportions of the different active enzyme isoforms, depending upon either time after elicitation or the elicitor used. The elicitor-induced increase in enzyme activity was associated with increased translatable phenylalanine ammonia-lyase mRNA activity in the polysomal fraction.     

Stress Responses in Alfalfa (Medicago sativa L.) : V. Constitutive and Elicitor-Induced Accumulation of Isoflavonoid Conjugates in Cell Suspension Cultures

The isoflavonoid conjugates medicarpin-3-O-glucoside-6″-O-malonate (MGM), afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6″-O-malonate (AGM) were isolated and characterized from cell suspension cultures of alfalfa (Medicago sativa L.), where they were the major constitutive secondary metabolites. They were also found in alfalfa roots but not in other parts of the plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured cells treated with elicitor from Colletotrichum lindemuthianum, and this was subsequently accompanied by an increase in the levels of MGM. In contrast, net accumulation of afrormosin conjugates was not affected by elicitor treatment. Labeling studies with [¹⁴C]phenylalanine indicated that afrormosin conjugates were the major de novo synthesized isoflavonoid products in unelicited cells. During elicitation, [¹⁴C]phenylalanine was incorporated predominantly into medicarpin, although a significant proportion of the newly synthesized medicarpin was also conjugated. Treatment of ¹⁴C-labeled, elicited cells with l-α-aminooxy-β-phenylpropionic acid, a potent inhibitor of PAL activity in vivo, resulted in the initial appearance of labeled medicarpin of very low specific activity, suggesting that the phytoalexin could be released from a preformed conjugate under these conditions. Our data draw attention to the involvement of isoflavone hydroxylases during the constitutive and elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa cell cultures.     

2,4-D Resistance in a Tobacco Cell Culture Variant: Cross-Resistance to Auxins and Uptake, Efflux and Metabolism of 2,4-D

A tobacco (Nicotiana tabacum L.) variant selected as a cellline resistant to 2,4-D was found to possess cross-resistanceto auxins including IAA, naphthalene-1-acetic acid (NAA), and4-amino-3,5,6-trichloropicolinic acid (picloram). The uptakeof 2,4-D by the variant and two wild-type cell lines was essentiallylinear in relation to 2,4-D concentration, and the variant tookup 2,4-D more rapidly than the wild types. Analysis of the 2,4-Dmetabolism revealed some diversity in the metabolic patternamong the cell lines but no significant differences which couldexplain the resistance of the variant. Although the variantpossesses a much higher capacity to metabolize 2,4-D than thewild types, this is most likely a result rather than a causeof the resistance. We conclude that neither the uptake nor themetabolism is responsible for the resistance. The variant, onthe other hand, exhibited a significantly lower rate of effluxout of the cells, particularly that of free 2,4-D, than thewild types upon washing with and transfer to 2,4-D-free medium.We suggest that immobilization of 2,4-D or auxins within cellsby compart mentation may be related to but not solely responsiblefor the resistance of this tobacco cell culture variant. (Received June 18, 1984; Accepted November 21, 1984)     

Micromorpho-Anatomical Examination of 2,4-D Phytotoxicity in Grapevine (Vitis vinifera L.) Leaves

The anatomical and micro-morphological alterations as induced by the auxinic herbicide, 2,4-D (2,4-dichlorophenoxy acetic acid) have not yet been elucidated for a commercially important fruit crop such as grapevine despite its super sensitivity to 2,4-D. Light and scanning electron microscopy techniques were employed to examine 2,4-D induced internal and external structural abnormalities in Merlot grapevines (Vitis vinifera L.). Healthy leaves were dorsiventrally flattened with well developed patterns of cellular structure and composition involving adaxial palisade parenchyma and abaxial spongy mesophyll. Dorsiventral variations in epidermal features involved large epidermal cells on the adaxial surface, and trichomes and stomata with turgid elliptical guard cells on the abaxial surface. The 2,4-D injured leaves were small and enated; the veins were fasciated with rugose bands of lamina existing between fasciated veins. The epidermal cells aggregated instead of being positioned coplanar to the epidermal plane. The adaxial elongated palisade parenchyma cells were transformed into an ovoid shape with intercellular spaces. An extensive development of replacement tissues took place on the abaxial surface wherein the stomata became roundish and were either raised or sunken with collapsed and cracked guard cells that developed abnormal outer stomatal ledges. These abnormalities are expected to severely perturb the vital functions of photosynthesis and transpiration ultimately leading to vine death attributable, at least in part, to the injured leaves.     

Kinetin Transport to Axillary Buds of Dwarf Pea (Pisum sativum L.)

Decapitation resulted in the transport of significant amountsof ¹⁴C to the axillary buds from either point of application,but pretreatment of the cut internode surface of decapitatedplants with IAA (alone or in combination with unlabelled kinetin)inhibited the transport of label to the axillary buds and resultedin its accumulation in the IAA-treated region of the stem. Inintact plants to which labelled kinetin was applied to the apicalbud there was little movement of ¹⁴C beyond the internode subtendingthis bud; when labelled kinetin was applied to the roots ofintact plants, ¹⁴C accumulated in the stem and apical bud butwas not transported to the axillary buds. A considerable proportionof the applied radioactivity became incorporated into ethanol-insoluble/NaOH-solublecompounds in the apical bud of intact plants, in internodestreated with IAA, and in axillary buds released from dominanceby removal of the apical bud. The results are discussed in relation to the possible role ofhormone-directed transport of cytokinins m the regulation ofaxillary bud growth.     

Marker enzyme assessment in the liver of cyprinus carpio (L.) exposed to 2,4-D and azinphosmethyl

The potential utility of antioxidant enzymes and lipid peroxidation as indicators of exposure to 2,4-D and azinphosmethyl together with the toxic effects of these compounds in freshwater fish Cyprinus carpio were evaluated. Biochemical parameters were recorded spectrophotometrically in fish liver, which were exposed to a single dose of 2,4-D and azinphosmehtyl (1/3 LC(50)), and their mixture at 1:1 ratio for 24, 48, 72, and 96 h. The most sensitive parameter was glutathione S-transferase (GST) activity, which significantly increased with experimental exposures. Glucose 6-phosphate dehydrogenase activity did not change after 24 and 48 h while there was an elevation after 72 h in all exposure groups. The activity decreased only when these were applied in combination at 96 h. Superoxide dismutase activity increased after azinphosmethyl exposure for 48 and 96 h. 2,4-D decreased the activity after 24 h while the activity remained at the same level with control after 48 h. An elevation was found between 72 and 96 h. Mixture treatment did not changed the activity. Glutathione reductase and catalase enzyme activities, and malondialdehyde levels remained constant in all the treatment groups compared with controls. These results suggest that induction of GST activity may be used as biomarker for the assessment of water pollution in C. carpio.     

Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-β-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.     

Multiple Metal Resistance and Co-resistance in Acer pseudoplatanus L. (Sycamore) Callus Cultures

In vitro growth of Acer pseudoplatanus L. (sycamore) callustissue derived from shoot-tip explants and screened on metal-enrichedmedia was studied in an attempt to identify resistance traitswhich may explain the survival of trees at metal-contaminatedsites. Copper and Cd-resistance traits were identified in celllines originating from trees at a site with a relatively recenthistory of severe contamination by these metals, and Cd- andZn-resistance were identified in cell lines originating frommature trees at a mining spoil site with a much longer historyof exposure to elevated concentrations of these metals. In Zn-resistantcell lines, co-resistance to Ni was also found, even thoughthis metal was not elevated at the study site. This is the firstreport of multiple resistance and co-resistance to metals occurringat the cellular level in trees. The mechanisms of the measuredresistance traits remain unclear, although there was evidenceof reduced Cu and Ni uptake by resistant cell lines. It is concludedthat facultative adaptations allowing acclimation to metal stressmay be particularly significant for survival of mature trees;induction of metal resistance probably occurs in vivo in treesat metal-contaminated sites.Copyright 1995, 1999 Academic Press Acer pseudoplatanus, sycamore, callus, heavy metals, metal resistance, trees     

Physiological and Cellular Responses of the 2,4-D Degrading Bacterium, Burkholderia cepacia YK-2, to the Phenoxyherbicides 2,4-D and 2,4,5-T

Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp. YK-2 in response to sublethal concentrations of 2,4-D stress [Cho et al. (2000) Curr Microbiol 41:33–38]. In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T. Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T. SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide. These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells. Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T. Strain YK-2 could degrade 2.25 mM 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested. BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B. cepacia YK-2. Received: 7 February 2002 / Accepted: 7 March 2002     

Uptake, transport and metabolism of 14C-2,4-dichlorophenoxyacetic acid (14C-2,4-D) in cucumber (Cucumis sativus L.) explants

The uptake, distribution and metabolism of 2,4-D using 14C-labelled 2,4-dichlorophenoxyacetic acid (14C-2,4-D) was studied in isolated hypocotyl and cotyledon explants of cucumber (Cucumis sativus L.) in vitro. Cotyledons had a higher uptake capacity than hypocotyls; the uptake in cotyledons increased linearly up to 20 h, while in hypocotyls only up to 8 h. The distribution of 14C-activity in both organs was basipetal, but more pronounced in cotyledons. The 2,4-D taken up by cotyledons was metabolized very rapidly: only 7% of 14C-activity was associated with free 2,4-D after 20 h exposure. Hypocotyls metabolized 2,4-D more slowly: after 20 h 50% of the 14C-activity was still associated with free 2,4-D. After short incubation periods (2–5 h) 2,4-D-aspartate was a predominant metabolite, after longer incubation (8–20 h) 2,4-D-glucosyl ester prevailed. Roots or callus were formed on the base of cotyledons depending on the length of exposure to 2,4-D.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-(3)H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.Apparently, processes involved in biosynthesis of primary cell wall continued to produce pectic substance during cell enlargement while processes leading to biosynthesis of typically secondary cell wall polysaccharide such as 4-0-methyl glucuronoxylan were not activated.     

Use of N-Bromoacetyl-l-ornithine to Study l-Ornithine and l-Arginine Biosynthesis in Soybean (Glycine max L.) Cell Cultures

The effect of the inhibitor N²-bromoacetyl-l-ornithine (NBAO) on the biosynthesis of ornithine in higher plants, was investigated using soybean cells (Glycine max L. var Mandarin), grown in suspension culture. The NBAO was found to reduce the specific activity of the enzyme N²-acetyl-l-ornithine: l-glutamate N-acetyltransferase (EC 2.3.1.35). In contrast, the specific activity of the enzyme acetyl coenzyme A:L-glutamate N-acetyltransferase (EC 2.3.1.1), which is also involved in N-acetylglutamate biosynthesis, was not significantly changed. Estimation of the concentrations of free amino acids in the soluble fraction of the cells showed that while ornithine levels were decreased, glutamic acid levels were increased in the presence of NBAO. While arginine levels initially increased in the presence of NBAO, they finally decreased near the end of the growth period. Evidence was obtained that the initial increase in arginine levels was due to the inhibition of arginase (EC 3.5.3.1) by N²-bromoacetyl l-ornithine. We conclude that the reaction catalyzed by N²-acetyl-l-ornithine:l-glutamate N-acetyl transferase is a rate limiting reaction in vivo.     

高产花色苷玫瑰茄细胞系的筛选

花色苷在植物中呈现粉红、红、紫红、紫等颜色,可以用作食品、药品及化妆品的着色剂,亦有药用价值。作为食品添加剂,颜色较合成色素自然,且安全无毒性。早在１９８７年,Ｍｉｚukami^[1]就建议用植物细胞培养物生产花色苷类代替合成色素。所有的植物培养细胞都是异源性的。各细胞之间产花色苷的能力相差很大^[2].因为产花色苷的细胞系带有颜色标记,所以容易识别并通过肉眼选择即可获得高产花色苷的细胞系。筛选的方法很多,如平板饲喂法^[3]、小细胞团法^[4]、细胞块法^[5]、肉眼观察直接挑选法及细胞分栋器法^[6]等。高产系花色苷的含量可增加几倍到几十倍,而且产量稳定。本文采用平板法及小细胞团法筛选高产花色苷的玫瑰茄(Hibiscus sabdariffa L.)细胞系。     

Isolation and Partial Purification of Prophenoloxidase from Daucus carota L. Cell Cultures

The enzyme, phenoloxidase, was isolated and partially purified as an inactive enzyme, a proenzyme, from plant cell cultures of Daucus carota, Nicotiana tabacum, and Haplopappus gracilis. The prophenoloxidase was found to be specifically activated by Ca²⁺ or Mn²⁺ ions in concentrations above 1 millimolar. Calmodulin was not involved in this activation. Concentrations of Ca²⁺ or Mn²⁺ below 1 millimolar could not induce activation of the prophenoloxidase, but if trypsin was added simultaneously with Ca²⁺ or Mn²⁺ at a concentration of 1 millimolar or below, the proenzyme was converted to its active form. The inactive form of phenoloxidase was found to be a soluble enzyme, whereas after activation the enzyme aggregated, and a significant amount of the enzyme activity could become pelleted.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-3H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-³H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.     

Effect of intracellular 2,4-D concentration on plantlet regeneration of rice (Oryza sauva L.) callus

Intracellular 2,4-dichlorophenoxyacetic acid (2,4-D) concentration profiles in rice (Oryza sativa L.) callus were measured for various subculture periods. 2,4-D was taken up into the callus by uphill transportation from the medium. The intracellular 2,4-D taken up was metabolized gradually during cultivation. The intracellular 2,4-D concentration markedly affected the redifferentiation process after subculture in such a way that the higher its concentration was, the more strongly the redifferentiation process was suppressed, and the lesser was the browning of the callus.