Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : II. Partial Characterization

l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO₂ produced per minute per milligram protein at pH 8.4 and 30°C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 ± 300 daltons.     

Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures

Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 ± 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K⁺. When the enzyme is dialyzed in the presence of K⁺ and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K⁺. The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K⁺ concentrations increase. The change in threonine and K⁺ concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.     

Plant Regeneration of Protoplasts of Trifolium lupinaster L. from Cell Suspension Cultures

paper deals with regeneration of protoplasts in cell suspension cultures of hypocothl from Trifolium lupinaster L. on the SL2 basal medium with BA 0.1 mg/L and picloram 0.06 mg/L for 3--4 month,s. The protopiasts were isolated from suspensions cells subcultured for 3 days and were recuhured in modified liguid medium 8p. The first division of the regenerated cell occurred 3 days after being cultured in medium Bp. Small calli could be seen with naked eyes by one month. The calli when grew up to 2 mm long, were transferred in succession differentiation medium A and B for organ differentiation. The differentiated shoots formed their roots on 1/2 MS supplamented with NAA 1.0mg/L and then grew into plantlets.     

生姜离体叶片愈伤组织悬浮细胞系建立与植株再生

以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。     

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.     

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.Microtubules (MTs) are important cytoskeletal polymers that are conserved in eukaryotic cells and are assembled from α- and β-tubulin heterodimers (Desai and Mitchison, 1997). In plants, MTs have important functions in essential cellular processes, such as cell division, and in cell morphogenesis. MTs in plant cells adopt several distinct higher order arrays and are remodeled in response to the cell cycle, developmental programs, and environmental cues (Hashimoto, 2015). Genetic, molecular, and cell biological approaches have been used to identify cellular factors that regulate the organization and dynamics of plant MTs. Considerable effort has been devoted to simulating the organization of cortical MT arrays by computational modeling.Cell-free in vitro studies are essential for the biochemical characterization of various MT regulators and for elucidating the mechanistic principles underlying the versatility of this dynamic polymer in cellular functions. The purification of sufficient amounts of assembly-competent tubulin is a prerequisite for these in vitro studies. Tubulin is traditionally purified from mammalian brains, since these tissues contain sufficiently high concentrations of tubulin to allow MT assembly in crude cell extracts. Polymerized MTs and their associated MT-binding proteins are separated from other cellular proteins by sedimentation. Pelleted MTs are then depolymerized upon drug washout under MT-destabilizing conditions, such as high concentrations of salt and calcium and low temperature. A few rounds of assembly-disassembly cycles highly enrich for tubulin and copurify MT-associated proteins, which can subsequently be removed by column chromatography (Borisy et al., 1975). Tubulin has also been purified from several plant sources (Morejohn and Fosket, 1982; Mizuno, 1985; Jiang et al., 1992; Bokros et al., 1993; Moore et al., 1997). However, since tubulin concentrations are low in plant cells, taxol, which stabilizes MTs, is generally included in the polymerization buffer, and cytoplasm-rich miniprotoplasts, which lack vacuoles, are sometimes used as starting material (Hamada et al., 2013). Since it is technically challenging to isolate assembly-competent pure tubulin from nonneural sources (Sackett et al., 2010), general plant science laboratories may hesitate to prepare plant tubulin themselves.Although the primary amino acid sequences of eukaryotic tubulins are fairly well conserved and the biophysical mechanisms of MT assembly and disassembly are thought to be similar for all MTs, the kinetics of MT dynamic instability differ for MTs assembled from animal and plant tubulin (Moore et al., 1997). Interactions with MT-interacting proteins may differ for tubulins isolated from different biological sources, as reported for the MT-dependent activation of kinesin (Alonso et al., 2007). Posttranslational modifications of tubulin, which generate distinct tubulin signatures and may modulate the functions of MT-interacting proteins, such as kinesin (Sirajuddin et al., 2014), are extensive in brain tubulin (Janke, 2014) but may be quantitatively and qualitatively different in plant tubulin. Furthermore, MT nucleation by the γ-tubulin ring complex shows a strong preference for tubulin from the same species (Kollman et al., 2015). Thus, it is important to use plant tubulin, and not brain tubulin, for in vitro studies of plant MTs.Tubulin is folded by a series of molecular chaperones to form an αβ-tubulin heterodimer in which one structural GTP is embedded in the interdimer interface (Lundin et al., 2010). The requirement of these eukaryote-specific chaperones precludes the use of prokaryotic expression systems for synthesizing properly folded and functional tubulin. Bacterially synthesized tubulin can be folded in rabbit reticulocyte lysate to produce functional tubulin, but with moderate yields (Shah et al., 2001). A yeast (Saccharomyces cerevisiae) expression system has been developed to produce modified yeast tubulin (Uchimura et al., 2006; Johnson et al., 2011), but this system is not suitable for the synthesis of animal (Sirajuddin et al., 2014) and plant (our unpublished data) tubulin. A baculovirus-insect cell expression system was recently reported to yield functional human tubulin (Minoura et al., 2013).Tubulin-binding proteins have been used to develop affinity-purification columns. The TOG domains (named after the human MT regulator, colonic and hepatic Tumor Overexpressed Gene [ch-TOG]) are among the best-characterized tubulin-binding domains. ch-TOG and orthologs from other eukaryotes bind to the growing plus ends of MTs and accelerate MT growth (Al-Bassam and Chang, 2011). TOG domains from the yeast ortholog Stu2 were recently used to affinity purify assembly-competent tubulin from fungal and animal sources (Widlund et al., 2012). In this study, we demonstrate that a TOG-based affinity column can be used to purify functional tubulin from tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). We examined the posttranslational modifications of the isolated tubulins by mass spectrometry and immunoblot analysis and showed that a His-tagged Arabidopsis tubulin isotype could be purified using this column. These results show that wild-type and recombinant functional tubulin from plant sources can be isolated efficiently.     

Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum

A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.     

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.     

Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.     

Somatic Embryogenesis and Plant Regeneration from Papaya Suspension Cultures

Compact embryogenetic calli were obtained from explants on P3 medium after 4 weeks of culture and high-frequency somatic embryogenesis occurred after these calli were transferred into suspension culture. Experimental data showed that low level (0.2%W/V) of activated charcoal had beneficial effects on somatic embryogenesis. Abundant calli on P4 medium however, showed no embryogenesis. On the other hand, callus induction and somatic embryogenesis varied with different rarities of exptants. The efficiency of somatic embryogenesis was much higher, if roots were used as explants, whereas stems were more suitable for callus formation Mature somatic embryos with cotyledons were cultured on MS medium containing different plant hormones. The optimum medium for germination and growth of entire plantlet was Mso medium. The somatic embryos on MS2, MS and MS3 media germinated rapidly, but formed excessive callus from the surface of germinating embryos.     

Studies on the Pectic Depolymerases I. Exopolygalacturonase from Root Tissues and Cell Suspension Cultures of Carrots

Three polygalacturonases (PG) have been isolated from carrots(Daucus carota L. cv. Kintoki). Two were isolated from roottissues (PG-I and PG-II) and one from cell suspension cultures(PG-III). PG-I and PG-III were readily solubilized in a lowionic strength buffer, whereas PG-II required additional NaClto be solubilized. These seems to be a change in the propertiesof PG between the original tissue and carrot cell cultures.The three PGs were partially purified by chromatography on SephadexG-150, and characterized. Elution from a Sephadex G-150 column indicated a molecular weightof about 48,000 for all three PGs. PG-III, studied in detail,hydrolyzed the galacturonan chain in an exo-fashion, and wasnot activated by a variety of cations at concentrations of 0.5or 1.0 mM. The pH optimum, and pH and heat stability of PG-Iand PG-III were slightly different from those of PG-II. PG-Iwas also different from PG-II and PG-III in its pectin hydrolyzingactivity. These results indicate that the enzymatic properties of PG-IIIfrom cell cultures are very similar to those of PG-I or PG-IIfrom root tissues; the only significant difference seems tobe the binding properties of the PGs to the cell wall materials. (Received March 23, 1981; Accepted June 11, 1981)     

Plant Regeneration from Immature Zygotic Embryo-Derived Embryogenic Calluses and Cell Suspension Cultures of Catharanthus roseus

Culture conditions for plant regeneration in immature zygotic embryo-derived embryogenic cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) Little Bright Eye are described. Immature zygotic embryos formed off-white, friable calluses at a frequency of 20% on Murashige and Skoog (MS) medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) after 8 weeks of culture. After a second subculture using MS basal medium at 4-week intervals, off-white friable calluses formed a small quantity of yellowish, compact embryogenic calluses. Upon transfer to MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Cell suspension cultures were established with embryogenic calluses using liquid MS medium supplemented with 4.52 µM 2,4-D. Embryogenic cell clumps from cell suspension cultures developed into plantlets at a frequency of 56.7% when plated onto MS basal medium. Plantlets were transplanted to potting soil and grown to maturity in a growth chamber.     

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.     

Cell Growth and Division: I. A Mathematical Model with Applications to Cell Volume Distributions in Mammalian Suspension Cultures

A mathematical model is formulated for the development of a population of cells in which the individual members may grow and divide or die. A given cell is characterized by its age and volume, and these parameters are assumed to determine the rate of volume growth and the probability per unit time of division or death. The initial value problem is formulated, and it is shown that if cell growth rate is proportional to cell volume, then the volume distribution will not converge to a time-invariant shape without an added dispersive mechanism. Mathematical simplications which are possible for the special case of populations in the exponential phase or in the steady state are considered in some detail. Experimental volume distributions of mammalian cells in exponentially growing suspension cultures are analyzed, and growth rates and division probabilities are deduced. It is concluded that the cell volume growth rate is approximately proportional to cell volume and that the division probability increases with volume above a critical threshold. The effects on volume distribution of division into daughter cells of unequal volumes are examined in computer models.     

Somatic Embryogenesis in Sugarcane (Saccharum officinarum L.): Growth and Plant Regeneration from Embryogenic Cell Suspension Cultures

Embryogenic cell suspension cultures were established from calliderived from young leaves of sugarcane (Saccharum officinarumL.) by placing them in liquid medium containing 5 per cent coconutwater (CW), 2–3 mg 1^–1 2, 4-D and 500 mg 1^–1casein hydrolysate (CH). The cultures were maintained by transferring2.5–5.0 ml of the suspension to 35 ml of fresh mediumevery 4–5 days. Organized structures resembling the earlystages of embryogeny were formed when 2, 4-D in the medium waslowered (0.1–1.0 mg 1^–1) but these did not developbeyond the globular or early scutellar stages. High levels ofsucrose (6–10 per cent) promoted the formation of proembryoids.Plating of the suspension on MS agar medium supplemented with0.25–2.0 mg 1^–1 2, 4-D, 5 per cent CW, 500 mg 1^–1CH, with or without activated charcoal, resulted in the formationof embryogenic calli. A large number of embryoids were formedin media containing lower 2, 4-D concentrations. Transfer ofembryoids to half-strength MS medium with 6 per cent sucroseestablished plantlets which were successfully transferred tosoil. Saccharum officinarumL, sugarcane, suspension culture, embryogenesis, regeneration     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.