Changes in Endogenous Gibberellin Contents during Growth of Cultured Tobacco Cells

Changes in the contents of endogenous gibberellins (GAs) wereexamined in three kinds of cultured tobacco cells; a crown gallcell and two cultured cells derived from normal tissue of Nicotianatabacum. The relative amounts of the GAs were analyzed by systematicchromatographic purifications followed by GC-SIM. In all thecell lines examined, the content of GAj was the highest duringthe logarithmic phase of growth, indicating that GA₁ has a physiologicalrole in the growth of dedifferentiated cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received April 11, 1984; Accepted July 10, 1984)     

Regulation of Turgor Pressure by Suspension-Cultured Tobacco Cells

Turgor regulation and effects of high NaCl and water deficiton growth and internal solutes were studied after transferringtobacco cells from control culture medium (osmotic pressure= 0.13–0.15 MPa at time of transfer) to culture mediumcontaining either 82 mol m^–3 NaCl or 150 mol m^–3melibiose (osmotic pressure of media = 0.62 MPa). Followingtransfer to media with higher osmotic pressure, expansion rateand turgor pressure were reduced. Within 24 h of imposing thewater deficit, expansion rate had returned to that of cellsin control culture medium. However, by 24 h, turgor pressurehad only risen from 0.2 MPa to 0.65 MPa in the NaCl treatmentand to 0.53 MPa in the melibiose treatment, while it was 0.73MPa in the control treatment. Furthermore, turgor pressure remainedwithin 0.05 MPa of these respective values for the rest of the(75 h) experiment. These results suggest differences in bothcell wall properties (extensibility and/or threshold turgor)and the level at which turgor is maintained for cells in thevarious treatments. Solutes contributing nearly all (82–97%) of the osmoticpressure in cells were identified. The initial (up to 24 h)increases in turgor pressure were mainly due to increases insolute concentrations caused by relatively slow expansion rates.However, increased Na⁺ and Cl ^– uptake contributed toincreased turgor pressure in the NaCl treatment and caused turgorpressure of cells in this treatment to increase faster thanin the melibiose treatment. Likewise, expansion rate rose morequickly in the NaCl than in the melibiose treatment. After 24h, maximum expansion rate was reached and concentrations ofmost internal solutes began to decrease. Nevertheless, turgorpressure remained relatively constant. The constancy of turgorpressure was due to increased glucose uptake rates relativeto controls, with consequent increases in concentrations ofsucrose, glucose and fructose and, in cells in the melibiosetreatment, of organic acids. Glucose uptake was slower in theNaCl than in the melibiose treatment but higher turgor pressurewas maintained in the NaCl treatment due to high uptake of Na⁺and Cl^–. Glucose uptake appears to respond to a systemof turgor regulation, but further experiments are required toconfirm this and to determine whether Na⁺ and Cl^– uptakealso respond to a system of turgor regulation. Key words: Salinity, water deficit, growth     

Autophagy in Tobacco Suspension-Cultured Cells in Response to Sucrose Starvation

The response of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2) to nutrient starvation was investigated. When the cells that were grown in Murashige-Skoog medium containing 3% (w/v) sucrose were transferred to the same medium without sucrose, 30 to 45% of the intracellular proteins were degraded in 2 d. An analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that proteins were degraded nonselectively. With the same treatment, protease activity in the cell, which was measured at pH 5.0 using fluorescein thiocarbamoyl-casein as a substrate, increased 3- to 7-fold after 1 d. When the cysteine protease inhibitor (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane (10 [mu]M) was present in the starvation medium, both the protein degradation and the increase in the protease activity were effectively inhibited. Light microscopy analysis showed that many small spherical bodies accumulated in the perinuclear region of the cytosol 8 h after the start of the inhibitor treatment. These bodies were shown to be membrane-bound vesicles of 1 to 6 [mu]m in diameter that contained several particles. Quinacrine stained these vesicles and the central vacuole; thus, both organelles are acidic compartments. Cytochemical enzyme analysis using 1-naphthylphosphate and [beta]-glycerophosphate as substrates showed that these vesicles contained an acid phosphatase(s). We suggest that these vesicles contribute to cellular protein degradation stimulated under sucrose starvation conditions.     

Melibiose, A Suitable, Non-Permeating Osmoticum for Suspension-Cultured Tobacco Cells

Dracup, M., Gibbs, J. and Greenway, H. 1986. Melibiose, a suitablenon-permeating osmoticum for suspension-cultured tobacco cells.—J.exp. Bot. 37: 1079–1089. A neutral, non-permeating osmoticum with low molecular weightwas required for studies involving responses to water deficitand salinity by suspension-cultured cells of tobacco. For thispurpose, raffinose, sorbitol, mannitol and melibiose were evaluated. Raffinose was hydrolysed by cells which were then able to useone of the products, namely fructose, for growth. Sorbitol andmannitol were not used for growth but were taken up by cells.After 96 h in media containing 50 mol m^–3 of sorbitolor mannitol as the carbon source, cells contained 85 mol m^–3sorbitol or 45 mol m^–3 mannitol. At least part of theuptake of sorbitol would have been active as sorbitol was transportedagainst a concentration gradient. Melibiose was one of the products of hydrolysis of raffinoseand proved to be an effective osmoticum. When supplied as thesole source of sugar for cells, melibiose was neither hydrolysednor taken up by cells. Furthermore, melibiose was not toxicsince adding 50 mol m^–3 to a culture medium containingglucose did not affect growth of cells. Key words: Sorbitol, mannitol, uptake     

Identification of Chitinase and Osmotin-Like Protein as Actin-Binding Proteins in Suspension-Cultured Potato Cells

Cytoplasmic aggregation is an early resistance-associated eventthat is observed in potato tissues either after penetrationof an incompatible race of Phytophthora infestans, the potatolate blight fungus, or after treatment with hyphal wall components(HWC) prepared from P. infestans. In potato cells in suspensionculture, the number of cells with cytoplasmic aggregation increasedupon treatment with HWC, but such an increase was suppressedby treatment with cytochalasin D prior to treatment with HWC.This result suggested that cytoplasmic aggregation in culturedpotato cells might be connected with the association of actinfilaments. To identify the molecular basis of cytoplasmic aggregation,we purified actin and actin-related proteins by affinity chromatographyon a column of immobilized DNase I from cultured potato cellsand isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysisof the amino-terminal amino acid sequences indicated that the43 kDa, 32 kDa and 22 kDa proteins were potato actin, basicchitinase and osmotin-like protein, respectively. This conclusionwas supported by the results of Western blotting analysis ofthe 43 kDa and 32 kDa proteins with antibodies against actinand basic chitinase. Binding analysis with actin coupled toactin-specific antibodies and biotinylated actin suggested thatthe 32 kDa and 22 kDa proteins had actin-binding activity. Inaddition, examination of biomolecular interactions using anoptical biosensor confirmed the binding of chitinase to actin.These results imply the possibility that basic chitinase andosmotin-like protein might be involved in cytoplasmic aggregation,hereby participating in the potato cell's defense against attackby pathogen. (Received June 11, 1996; Accepted January 27, 1997)     

Role of the Plasmalemma H-ATPase in Pseudomonas syringae-Induced K/H Exchange in Suspension-Cultured Tobacco Cells

Activation of a host plasma membrane K⁺ efflux/net H⁺ uptake exchange by pathogenic pseudomonads plays an important role in the development of hypersensitivity in tobacco (Nicotiana tabacum). Involvement of the plasmalemma H⁺-pumping ATPase in this response was investigated. The exchange response of suspension-cultured tobacco cells to Pseudomonas syringae pv syringae was reduced 90% or more by ATPase inhibitors including vanadate, N-ethylmaleimide, and N,N′-dicyclohexylcarbodiimide. The exchange was also strongly inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone and by slightly alkaline external pH. Respiratory inhibitors such as oligomycin and sodium azide reduced the exchange by 50% to 75%, while glycolysis inhibitors such as sodium arsenite and sodium iodoacetate decreased exchange by approximately 90%. These results suggest that plasmalemma H⁺-ATPase activity is required for the exchange response and that this may reflect a requirement for a plasmalemma pH and/or electrical potential gradient.     

Oxidative Damage to Membranes by a Combination of Aluminum and Iron in Suspension-Cultured Tobacco Cells

Aluminum (Al) and ferrous iron [Fe(II)] are separately non-toxicto cultured tobacco cells in nutrient solution. However, Aland Fe(II) together cause the peroxidation of membrane iipids,the accumulation of Al and Fe, and the loss of viability [Onoet al. (1995) Plant Cell Physiol. 36: 115]. We investigatedthe cause-and-effect relationships of these various responses.In cells exposed to Fe(TT) or Fe(III)-EDTA, both the peroxidationof ipids and the loss of viability were similarly enhanced byAlCl₃ in a dose-dependent manner. During exposure to AlCl₃,the accumulation of Al and the loss of viability became apparentrapidly and simultaneously at 8 h, whereas both the peroxidationof lipids and the accumulation of Fe occurred at later times.However, lipophilic antioxidants protected cells efficientlynot only from the peroxidation of Iipids but also from the lossof viability and the accumulation of Al and Fe. These resultssuggest that the peroxidation of Iipids in the plasma membranethat is caused by both Al and Fe leads to the accumulation ofAl and Fe and the loss of viability. (Received April 18, 1997; Accepted October 1, 1997)     

Changes in Triterpenoid Content during the Growth Cycle of Cultured Plant Cells

Similar changes in the pentacyclic triterpenoid contents wereobserved during the growth cycle of Datura innoxia, Luffa cylindricaand Lycopersicon esculentum seedling callus cells in batch culture.Triterpenoid contents decreased for several days after callusinoculation, then increased rapidly during the mid and lateexponential phases of growth. (Received May 28, 1984; Accepted September 13, 1984)     

Kinetics of l-Valine Uptake in Suspension-Cultured Cells and Protoplast-Derived Cells of Tobacco: Comparison of Wild-Type and the Val-2 Mutant

A kinetic analysis was made of l-valine uptake in protoplast-derived cells (mesophyll protoplasts cultured for 6 days) and in suspension-cultured cells of tobacco (Nicotiana tabacum L., cv Xanthi). Cells from wild-type and Val^r-2 mutant plants were compared. A low-K_m component was found in protoplast-derived cells (K_m = 45 ± 5 micromolar) as well as in suspension-cultured cells (K_m = 84 ± 21 micromolar). In the mutant cells the V_max of this component was 12- to 14-fold less than in wild-type cells. A second component (K_m = 2.4 ± 0.7 millimolar) was found in suspension-cultured cells but not in protoplast-derived cells; its V_max was the same in wild-type and mutant cells. A third component was apparently unsaturable (linear component). It was present in protoplast-derived cells but not in suspension-cultured cells, and had the same magnitude in wild-type and mutant cells. The results are discussed with reference to the uptake of l-valine in leaf tissue, in which the three kinetic components have been found simultaneously. The reduced V_max of the low-K_m component in the Val^r-2 mutant, and the differential expression of the other two components in suspension-cultured cells and protoplast-derived cells indicate that the kinetically distinguishable components represent physically distinct transport systems.     

Interactions between Cadmium and Nickel in Phytochelatin Biosynthesis and the Detoxification of the Two Metals in Suspension-Cultured Tobacco Cells

We examined the effects of simultaneous treatments with Cd and Ni on the phytochelatin (PC) biosynthesis in suspension-cultured tobacco (Nicotiana tobacum cv. Bright Yellow-2) cells. The induction of PC biosynthesis in response to Cd was not affected by the coexistence of Ni. Cd and Ni formed complexes with different compounds in cells.     

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C₁, xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO₃, an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O₂-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A₂, which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.     

Putrescine Accumulation is Associated with Growth Inhibition in Suspension-Cultured Rice Cells under Potassium Deficiency

The effects of potassium deficiency on the growth and the polyaminelevels of suspension-cultured rice cells were investigated.Our results indicate that putrescine accumulation is a factor,among others, causing growth inhibition of suspension-culturedrice cells under potassium deficiency. (Received September 27, 1993; Accepted November 20, 1993)     

Variations in the Chilling Sensitivity of Suspension-Cultured Cells of Mung Bean (Vigna radiata [L.] Wilczek) during the Growth Cycle

Variations in the chilling sensitivity of mung bean (Vigna radiata[L.] Wilczek) cells in suspension culture were studied withreference to the growth cycle. At the initial stages after subculture,cells were relatively insensitive to chilling, but chillingsensitivity increased abruptly thereafter and reached a maximumat the early stage of exponential growth, namely 6 days aftersubculture. Upon further culturing, cells became chilling-tolerant,becoming most tolerant at the late stage of exponential growth,namely 14–15 days after subculture. The results of thereciprocal exchange of conditioned culture medium between thecells at the early and the late exponential phases of growthprior to cold incubation revealed that the change in sensitivitywas due primarily to changes in physiological features of cellsand not to changes in the chemical composition of the culturemedium. In support of this possibility, we observed that thecold sensitivity of the tonoplast H⁺-ATPase in vivo changedmarkedly as a function of growth cycle: it was very sensitiveat the early stage of exponential growth and less sensitiveat the late stage. ¹Contribution no. 3668 from The Institute of Low TemperatureScience, Hokkaido University.     

Protein Phosphorylation in Amyloplasts Isolated from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Highly purified amyloplasts were isolated from cultured cells of sycamore (Acer pseudoplatanus L.). Incubation of amyloplasts with [γ-³²P]-ATP resulted in the labeling of more than ten polypeptides. Pulsechase experiments showed the reversibility of the process with some but not all of the polypeptides. The phosphorylation reaction of one polypeptide, M_r 100, was shown to be calcium dependent. Although exogenously added pig brain calmodulin had no effect, the calmodulin antagonist W-7 strongly inhibited phosphorylation of the 100 kilodaltons polypeptide. The presence of endogenous calmodulin, about 1 to 3 micrograms per milligram protein, in the amyloplast preparation was estimated by activation of phosphodiesterase in vitro.     

Physiological Changes of Carrot Cells in Suspension Culture during Growth and Senescence

Changes in the synthetic activities of nucleic acids and protein and in the amount of adenine nucleotides in Daucus carota cells have been examined during a period of rapid cell division and during the stationary phase. Cell growth and the syntheses of those macromolecules in the logarithmic phase were significantly enhanced by increasing the concentration of phosphate in the medium. In a phosphate-rich medium, RNA and protein rapidly degraded and the number of cells with denatured cytoplasm increased when the culture entered into a stationary phase because of exhaustion of glucose. On the other hand, when the growth ceased by exhaustions of phosphate and nitrogen, the cells slowly underwent physiological changes leading to cellular senescence. The value of adenylate energy charge during the growth period was about 0.8–0.9 irrespective of the growth rate. After cessation of growth the value declined to about 0.5.     

Growth Inhibition in Suspension-Cultured Rice Cells under Phosphate Deprivation Is Mediated through Putrescine Accumulation

The effects of phosphate deprivation on the growth and polyamine levels of suspension-cultured rice (Oryza sativa) cells were investigated. When rice suspension cells were deprived of phosphate, cell growth was markedly inhibited. Phosphate deprivation resulted in a higher putrescine level and lower spermidine and spermine levels in rice suspension cells. The growth of rice cells cultured in the absence of phosphate did not recover as a result of spermidine and spermine addition. D-Arginine and [alpha]-methylornithine, inhibitors of putrescine biosynthesis, caused a reduced level of putrescine in rice suspension cells cultured under phosphate deprivation. The growth of rice cells cultured in the absence of phosphate was completely recovered after the addition of D-arginine but not [alpha]-methylornithine. Our results indicate that putrescine accumulation is a factor causing growth inhibition of suspension-cultured rice cells under phosphate deprivation.     

Changes in Some Enzyme Activities during Growth and Organogenesis in Dark-Grown Tobacco Callus Cultures

Enzymes involved in malate metabolism, viz., glutamic-oxalacetictransaminase, malate dehydrogenase, malic enzyme and phosphoenolpyruvatecarboxylase, had severalfold higher specific activities in organ-formingcallus cultures of tobacco compared to non-organ-forming cultures.These activities increased considerably during the days precedingshoot and root differentiation. While malate accumulated untilday 15 in non-organ-forming callus, it accumulated up to day6 in shoot-and root-forming callus. Total and reducing sugarsaccumulated until day 3 and declined thereafter in all the cultures.Thus, tobacco callus may utilize this pathway for deriving reducingpower which is required for organogenetic processes. (Received April 30, 1987; Accepted December 1, 1987)     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells I. Intracellular Site of Synthesis and Transport

The distribution of arabinose-containing macromolecules in suspension-culturedtobacco cells was examined using sucrose density gradients.Exogenously applied ¹⁴Carabinose was scarcely converted intoother sugars, and concentrated in the Golgi-rich fraction (1.15g/cm³) and then secreted to the cell wall. ¹⁴C-Arabinose wasalso incorporated in a lower sucrose density fraction (1.11g/cm³), which contains small vesicles presumably originatedfrom the Golgi apparatus. The arabinose-containing macromoleculesin this fraction was more easily solubilized in water than thosein the Golgi-rich fraction. Alkaline hydrolysis of the macromoleculesindicated that cell-wall glycoprotein is a major component ofthe macromolecules and that the degree of glycosylation is slightlygreater in the lower density fractions than in the Golgi-richfraction. Based on these results, a scheme is suggested in whichthe glycoproteins and polysaccharides are glycosylated in theGolgi apparatus and secreted to the cell wall via secretionvesicles in the low density fraction. The possibility of ¹⁴C-arabinose-containingmacromolecules, in the early phase of synthesis, being a markerof the plant Golgi apparatus is also proposed. (Received September 21, 1980; Accepted January 27, 1981)