Amino-acid transport in suspension-cultured plant cells

The carrier system which transports L-leucine (L-leu) into suspension-culturedNicotiana tabacum L. cv. Wisconsin 38 cells appeared to be constitutive since it was always present and was not induced by L-leu even in nitrogen-starved cells. However, L-leu uptake rates for cells grown in medium containing L-leu were transiently reduced as a result of either transinhibition or repression. Growth-phase cells appeared to have more L-leu carriers per unit area of membrane than stationary-phase cells, and for this reason growing-phase cells exhibit higher L-leu uptake rates. These higher rates reflect a physiological or developmental condition since growth-phase cells did not dramatically change their L-leu uptake rates when subcultured, while stationary-phase cells doubled their rates within 6 h after being subcultured. Cells grown in a medium lacking a useable carbon souce had uptake rates higher than control rates for several days. These higher rates peaked after about 1 d and then decreased over the next several days. Cells grown in a medium lacking a nitrogen souce responded similarly except that the increased rates peaked after about 3 d and persisted longer. Kinetic analysis of uptake rates in cells grown without a carbon souce for 1 d or without a nitrogen souce for 3 d indicated that the L-leu carrier had K_ms similar to those of untreated cells. These results indicate that cultured tobacco cells respond to their environment by increasing or decreasing the number or activity of kinetically similar L-leu carriers.Abbreviations L and S medium Linsmaier and Skoog (1965) medium with additions - L-leu L-leucineIV=McDaniel et al. 1981     

Catabolism of adenine nucleotides in suspension-cultured plant cells

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspension-cultured Catharantus roseus cells with [8-14C]adenosine, were catabolized rapidly and most of the radioactivity appeared in 14CO2. Allantoin and allantoic acid, intermediates of the oxidative catabolic pathway of purines, were temporarily labelled. When the cells, prelabelled with [8-14C]adenosine, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nucleotides increased. The results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent on the concentration of adenine nucleotides in the cells. Studies using allopurinol, coformycin and tiazofurin, inhibitors of enzymes involved in purine metabolism, suggest that participation of AMP deaminase and xanthine oxidoreductase in the catabolism of adenine nucleotides in plant cells. AMP deaminase was found in extracts from C. roseus cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells from different species of plants.     

Inositol-containing lipids in suspension-cultured plant cells: an isotopic study

Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [³²P]orthosphosphate, [2-³H]glycerol, and myo-[2-³H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [³²P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported.     

Novel O-D-galacturonoyl esters in the pectic polysaccharides of suspension-cultured plant cells.

Driselase digestion of uronate-6-14C-labeled primary walls of cultured spinach (Spinacia oleracea L.) cells yielded about 18 novel uronate-containing compounds, most of which could be hydrolyzed by cold dilute alkali to yield oligo-[14C]galacturonides. One typical Driselase digestion product (compound 17) yielded alpha-(1-->4)-D-[14C]galacturonotriose(GalA3) upon very mild treatment with alkali (50% yield of GalA3 in 7.2 min at pH 11 and 25 degrees C). One of the three galacturonate residues in compound 17 was reducible to a galactose residue with sodium borohydride, indicating that that GalA residue was esterified, via its--COOH group, to a putative alcohol. Compound 17 had a higher mobility than GalA3 on paper chromatography, indicating that the putative alcohol was relatively nonpolar. The putative alcohol could not have been methanol because Driselase readily hydrolyzed mono-, di-, and trimethyl esters of GalA3 to yield free galacturonic acid. Another Driselase digestion product (compound 12) was a derivative of GalA3 that apparently possessed two nonpolar esterified substituents: one about as labile as in compound 17, and the other approximately 10 times more stable. Compounds 12 and 17 could not labeled by in vivo feeding of [U-14C]cinnamate, suggesting that they were not phenolic conjugates. Similar but chromatographically distinguishable uronate-14C-labeled esters were obtained by Driselase digestion of walls of cultured carrot (Daucus carota L.), Paul's Scarlet rose (Rosa sp.), and tall fescue (Festuca arundinacea Schreber) cells. In spinach, the novel compounds constituted about 5% of the total galacturonate residues of the cell wall. The observations suggest that pectic polysaccharides are linked, via O-D-galacturonoyl ester bonds, to relatively hydrophobic constituents of the primary cell wall. Their possible role in wall architecture is discussed.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Stable-isotope labeling using an inducible viral infection system in suspension-cultured plant cells

We established a novel strategy for preparing uniformly stable isotope-labeled proteins by using suspension-cultured plant cells and an inducible virus vector encoding the research target. By using this new method, we demonstrated the expression of three proteins, namely, Escherichia coli dihydrofolate reductase (DHFR), chicken calmodulin (CaM), and porcine protein kinase C-dependent protein phosphatase-1 inhibitor with a molecular mass of 17-kDa (CPI-17). In addition, we successfully expressed bovine pancreatic trypsin inhibitor (BPTI), which contains three pairs of disulfide bonds, as the soluble form. In the most efficient case, as little as 50 ml culture yielded 3–4 mg ¹⁵N-labeled protein suitable for NMR experiments. The ¹H-¹⁵N HSQC spectra of all of these proteins clearly indicated that their structures were identical to those of their counterparts reported previously. Thus, the present results suggest that our novel protocol is a potential method for NMR sample preparation.     

Structural features and biological activity of xyloglucans from suspension-cultured plant cells.

Different xyloglucan (XG) fractions were isolated from Rubus fruticosus cells cultured in suspension. Sequential extraction showed that two distinct xyloglucans existed in the primary walls. The first could be easily extracted in alkali and the second was tightly associated to cellulose. A third fraction was isolated from the extracellular polysaccharides of the culture medium. The alkali-soluble XG and the extracellular XG showed many structural features in common. By use of an anti-XG polyclonal antibody, electron microscopy examination suggests that the extracellular hemicellulose is progressively released from the wall by a sloughing mechanism. Oligosaccharides prepared from the extracellular XG were purified and their structure examined by FAB-ms technique. When the nonasaccharide was added at low concentrations (10(-5) mg/ml) to the culture medium it was able to elicit several different glycanohydrolase activities associated to the cell wall.     

Induced net Ca2+ uptake and callose biosynthesis in suspension-cultured plant cells

In suspension-cultured cells of Glycine max and Catharanthus roseus, marked callose synthesis can be induced by digitonin and chitosan. Leakage of a limited pool of electrolytes precedes callose formation, K⁺ representing the major cation lost. Poly-L-ornithine, as well as the ionophores A 23187 and ionomycin, also induces some callose synthesis but to a lesser extent. Digitonin increases the net uptake of Ca²⁺ from the external buffer with a time course parallel to callose synthesis but lagging behind the leakage of K⁺. Nifedipine partly blocks callose synthesis as well as the digitonin-induced increase in net Ca²⁺ uptake. Taken together, the data support the hypothesis that addition of the various substances might indirectly lead to membrane perturbation causing the common event of an increase in net Ca²⁺ uptake which results in callose deposition by a direct activition of the Ca²⁺-dependent and plasma-membane-located 1,3--glucan synthase.     

Identification and characterization of an 18-kilodalton, VAMP-like protein in suspension-cultured carrot cells

Polyclonal antibodies raised against rat vesicle associated membrane protein-2 (VAMP-2) recognized, in carrot (Daucus carota) microsomes, two major polypeptides of 18 and 30 kD, respectively. A biochemical separation of intracellular membranes by a sucrose density gradient co-localized the two polypeptides as resident in light, dense microsomes, corresponding to the endoplasmic reticulum-enriched fractions. Purification of coated vesicles allowed us to distinguish the subcellular location of the 18-kD polypeptide from that of 30 kD. The 18-kD polypeptide is present in the non-clathrin-coated vesicle peak. Like other VAMPs, the carrot 18-kD polypeptide is proteolyzed by tetanus toxin after separation of coatomers. Amino acid sequence analysis of peptides obtained by digestion of the 18-kD carrot polypeptide with the endoproteinase Asp-N confirms it to be a member of the VAMP family, as is suggested by its molecular weight, vesicular localization, and toxin-induced cleavage.     

Phosphocreatine production coupled to the glycolytic reactions in the cytosol of cardiac cells

Phosphocreatine production catalyzed by a cytosolic fraction from cardiac muscle containing all glycolytic enzymes and creatine kinase in a soluble form has been studied in the presence of creatine, adenine nucleotides and different glycolytic intermediates as substrates. Glycolytic depletion of glucose, fructose 1,6-bis(phosphate) and phosphoenolpyruvate to lactate was coupled to efficient phosphocreatine production. The molar ratio of phosphocreatine to lactate produced was close to 2.0 when fructose 1,6-bis(phosphate) was used as substrate and 1.0 with phosphoenolpyruvate. In these processes the creatine kinase reaction was not the rate-limiting step: the mass action ratio of the creatine kinase reaction was very close to its equilibrium value and the maximal rate of the forward creatine kinase reaction exceeded that of glycolytic flux by about 6-fold when fructose 1,6-bis(phosphate) was used as a substrate. Therefore, the creatine kinase raction was continuously in the state of quasiequilibrium and the efficient synthesis of phosphocreatine observed is a result of constant removal of ADP by the glycolytic system at an almost unchanged level of ATP ([ATP] ? [ADP]), this leading to a continuous shift of the creatine kinase equilibrium position.When phosphocreatine was added initially at concentrations of 5–15 mM the rate of the coupled creatine kinase and glycolytic reactions was very significantly inhibited due to a sharp decrease in the steady-state concentration of ADP. Therefore, under conditions of effective phosphocreatine production in heart mitochondria, which maintain a high phosphocreatine: creatine ratio in the myoplasm in vivo, the glycolytic flux may be suppressed due to limited availability of ADP restricted by the creatine kinase system. The possible physiological role of the control of the glycolytic flux by the creatine kinase system is discussed.     

Protein isoprenylation in suspension-cultured tobacco cells.

Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid. Isoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity. In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins. Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins. Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro. These studies indicate the presence of at least two distinct prenyl:protein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAIM carboxy terminus (farnesyl:protein transferase) and one that utilizes geranylgeranyl pyrophosphate and preferentially modifies a substrate protein with a CAIL carboxy terminus (geranylgeranyl:protein transferase type I). This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis.     

Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

Nucleotide pools in suspension-cultured cells of Datura innoxia

A general picture of the metabolic events which govern to growth behaviour of a batch culture of suspended dedifferentiated cells of Datura innoxia is obtained by following both the uptake and accumulation of the medium phosphate and sucrose by the cells, and the synthesis of RNA, protein and starch. The results are compared with the changes in the nucleotide pools described in the preceding paper. The sequence of formation and the regulatory dependencies of cellular pools of phosphate, sucrose, nucleotides and RNA in the production of proteins, starch and cell mass, and in the control of proliferation and cell growth are discussed. Furthermore, the importance of the maintenance pools for metabolic survival during starvation is emphasized.     

Cell-cycle-related variation in proteins in suspension-cultured rice cells

To understand the cell cycle process in plants, we searched for proteins that quantitatively change during the cell cycle in suspension-cultured rice (Oryza sativa L.) cells. The proteins were analyzed by a two-dimensional polyacrylamide gel electrophoresis image-analysis system. We detected 11 proteins that quantitatively changed during the cell cycle, among which -tubulins and a calreticulin-like protein were identified. The amounts of -tubulin proteins were low in the M phase and high in the G1 phase. In contrast, mRNAs for two of the three types of -tubulin were high in the M phase of the cell cycle. The addition of protease inhibitors MG132 or E64d to the cells decreased the -tubulin proteins during 24 h, suggesting that -tubulin proteins are degraded in vivo by proteases other than those whose activities are inhibited by MG132 or E64d.     

Extracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Hydroxyproline-rich glycoprotein was isolated from the used media of tobacco XD-6 cells cultured in suspension and purified by repeated ion exchange- and gel filtration-chromatography. The preparation was judged to be homogeneous from ultracentrifugation and isoelectric focusing. The dry wt of the glycoprotein was composed of 94% polysaccharide and 6% protein. Hydroxyproline accounted for 23% of the amino acids in the protein moiety. The polysaccharide moiety consisted of 44% galactose, 30% arabinose, 5% rhamnose, and 21% uronic acid. About 10% of the uronic acid residues were present as methyl esters.     

Intracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Two soluble glycoproteins containing hydroxyproline were extractedfrom cultured tobacco cells (cell line XD-6S) and purified byion-exchange and gel-filtration chromatography. On DEAR-cellulosecolumn chromatography in the final step of the purification,one was eluted at 90 mM NaCl and the other at 120 mM as singlepeak. Both purified glycoproteins were also sedimented as singlepeak with an ultracentrifugation. The S_20,w values were 6.1for the former and 7.0 for the latter. These glycoproteins were composed of 94% polysaccharide and6% protein in the former, and 87% polysaccharide and 13% proteinin the latter. The sugar moiety consisted of galactose, arabinose,rhamnose, and uronic acid in both. Hydroxyproline accountedfor 12% in the former and 20% in the latter amino acid composition.A high content of alanine in both (14 and 15%) was one of thedistinctive characteristics of these soluble glycoproteins. These intracellular soluble hydroxyproline-containing glycoproteinswere not labelled within 30 min of incubation with ₃H-proline,although the radioactivity was rapidly incorporated (within15 min) into the intracellular macromolecules. (Received February 21, 1978; )     

Abscisic Acid-induced chilling tolerance in maize suspension-cultured cells

The induction of chilling tolerance by abscisic acid (ABA) in maize (Zea mays L. cv Black Mexican Sweet) suspension cultured cells was examined. Cell viability during exposure to chilling was estimated by triphenyl tetrazolium chloride reduction immediately after chilling and a filter paper growth assay. Both methods yielded comparable results. Chilling tolerance was induced by transferring 5-day-old cultures (late log phase) to a fresh medium containing ABA (10 to 100 micromolar). The greatest chilling tolerance was achieved with ABA at 100 micromolar. Growth of cells was inhibited at this concentration. After a 7-day exposure to 4°C in the dark, the survival of ABA-treated cells (100 micromolar ABA, 28°C for 24 h in the dark) was sevenfold greater than untreated cells. Effective induction of chilling tolerance was first observed when cells were held at 28°C for 6 hours after adding ABA. No tolerance was induced if the culture was chilled at the inception of ABA treatment. Induction of chilling tolerance was inhibited by cycloheximide. These results indicate that ABA is capable of inducing chilling tolerance when ABA-treated cells are incubated at a warm temperature before exposure to chilling, and this induction requires de novo synthesis of proteins.     

Suramin inhibits oxidant signalling in tobacco suspension-cultured cells

Plant cells respond to ultraviolet radiation and other oxidant‐generating agents by mobilizing cellular defences, but the signal network linking perception of redox perturbation with defence remains unknown. Irradiation of tobacco suspension‐cultured cells with UVC was found to induce the activation of a specific MAPK⁴⁶ (salicylic acid‐induced protein kinase) within 1 min. To explore where UVC and other oxidants might initially act to trigger this signal response, we employed suramin, a non‐membrane‐permeable reagent that interferes with membrane receptor‐mediated signalling in mammalian cells. Pre‐treatment of tobacco cells with suramin strongly attenuated the UVC‐induced activation of MAPK⁴⁶ in a concentration‐dependent manner. Suramin was also able to interdict both ozone‐ and hydrogen peroxide‐induced activation of MAPK⁴⁶, indicating that reactive oxygen species (ROS) signalling to the MAPK cascade in general may be initiated at the cell membrane, perhaps through oxidative activation of membrane receptors.