Interaction between Pseudomonas syringae pv. Pisi and Isolated Pea Mesophyll Protoplasts

The interaction between five races of Pseudomonas syringae pv. pisi (PSP) and isolated mesophyll protoplasts obtained from five pea cultivars was studied. There was no trend in the attachment of bacterial cells to surfaces of compatible and incompatible host protoplasts. The viability of protoplasts from compatible and incompatible host-pathogen interactions did not differ significantly; however, changes in the viability of cultivar Kelvedon Wonder protoplasts, compatible with all five races, was relatively more stable following inoculation with bacteria than those of cultivar Fortune, incompatible with all of the five races. Protoplast cell wall regeneration did not take place until 24–48 h after isolation. It is concluded that the use of pea protoplasts as a model system for studying the pea-PSP interaction appears to have considerable potential for the future, but more basic research is required.     

A Revised Medium for Growth of Pea Mesophyll Protoplasts

The nutrient requirements of mesophyll protoplasts from Pisum sativum L. cv. Timo have been investigated and a synthetic and completely defined medium has been designed. A high calcium concentration (12 mM) stimulated both protoplast survival and cell division. The content of iron and zinc was also critical. Additions of nicotinic acid, pyridoxine and thiamine were necessary. The protoplast growth was enhanced when some amino acids were included in the medium. An absolute requirement for auxin and cytokinin was shown. In the revised medium about 90% of the isolated protoplasts survived and formed a cell wall. The first divisions were observed after 5 days and after 1 week 10–20% of the cells had divided at least once.     

Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum)

The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition.     

Light-Enhanced Uptake of Metabolites in Mesophyll Protoplasts: Evidence for a Novel Pyruvate Transport System

3 ) and sorghum (C₄) leaves for the measurements of osmotic volume change and metabolite uptake. We first investigated whether the silicone oil layer filtering centrifugation method could be applied to the protoplasts. The density of the silicone oil was optimized (ρ =1.026) and 0.5M betaine was chosen as an osmoticum in the protoplast suspending medium. By using [¹⁴C] sorbitol and [¹⁴C] inulin as the marker of the medium carried over into the pellet, protoplast osmotic or internal volume was estimated to be 200–300 μl (mg Chl)⁻¹, with the medium space in the pellet of 8–15 μl (mg Chl)⁻¹. Lowering of the osmotic pressure of the medium induced protoplast swelling as expected. Light also induced swelling. Using this system, we could detect light-enhanced uptake of ascorbate, glutamate and pyruvate in both barley and sorghum protoplasts. Pyruvate uptake was far higher in barley than in sorghum and inhibited by various inhibitors, showed saturation kinetics and, therefore, seemed to be mediated by a translocator protein. Received 10 August 1999/ Accepted in revised form 6 December 1999     

Factors Influencing the Growth and Division of Pea Mesophyll Protoplasts

High yields of viable protoplasts were produced from pea leaves provided that only leaves of the same age were used in each preparation. The conditions under which the pea plants were grown and the age of the plants were also important. The protoplasts were cultured in a medium supplemented with 1 mg/1 2iP and 1 mg/1 2,4-D. They were able to regenerate cell walls within two days. After 5 days cell divisions were apparent and sustained divisions led to callus formation. Special emphasis has been given in this paper to the choice of leaf material for protoplast isolation.     

Evidence for HCO(3) Transport by the Blue-Green Alga (Cyanobacterium) Coccochloris peniocystis

The possibility of HCO₃⁻ transport in the blue-green alga (cyanobacterium) Coccochloris peniocystis has been investigated. Coccochloris photosynthesized most rapidly in the pH range 8 to 10, where most of the inorganic C exists as HCO₃⁻. If photosynthesis used only CO₂ from the external solution the rate of photosynthesis would be limited by the rate of HCO₃⁻ dehydration to CO₂. Observed rates of photosynthesis at alkaline pH were as much as 48-fold higher than could be supported by spontaneous dehydration of HCO₃⁻ in the external solution. Assays for extracellular carbonic anhydrase were negative. The evidence strongly suggests that HCO₃⁻ was a direct C source for photosynthesis.     

Mechanism of L-Lysine Transport by Pea Protoplasts

Dureja, I., Guha-Mukherjee, S. and Prasad, R. 1986. Mechanismof L-lysine transport by pea protoplasts.—J. exp. Bot.37: 549–555. L-Lysine uptake was studied in pea protoplasts to characterizethe transport process. The uptake was pH dependent with optimumat pH 5?8. A kinetic analysis of uptake showed that L-lyslneuptake was biphasic. The respiratory inhibitors, sodium arsenate,azide, iodoacetate and 2, 4, dinitrophenol, inhibited the uptakeof L-lysine at a final concentration of 0?1 mol m^–3 suggestingit to be mediated in part by an active process. Competitiveinhibition of L-lysine uptake by only L-arglnine and of L-leucineand glycine uptake by several amino acids indicated that L-lysineuptake occurs via a specific system whereas the uptake of L-leucineand glycine was mediated through a relatively non-specific permease. Key words: Pea protoplasts, L-lysine transport, active transport, specific system     

Characteristics of Transport of L-Leucine and Glycine in Pea Protoplasts

The uptake of L-leucine and glycine into pea protoplasts wasstudied under various conditions. The uptake of both L-leucineand glycine was pH dependent with the optimal pH being 4.0 and5.0 for L-leucine and glycine, respectively. A kinetic studyof L-leucine uptake showed that uptake is multiphasic; K_m valuesof different phases were 1.1 mol m^–3, 33.3 mol m^–3and 100 mol m³. A similar analysis for glycine at a concentrationrange of 0.1–10 mol m^–3 also showed a multiphasictransport system for it. The uptake of L-leucine at lower concentrations(between0.1–2.0 mol m^–3) was energy dependent, since arsenate,azide, dinitrophenol and iodoacetate inhibited the uptake. However,the uptake of L-leucine was not inhibited by ouabain at anyconcentration of L-leucine employed. The uptake of glycine wasnot inhibited by any of these inhibitors suggesting that glycineuptake was not mediated by an active process. Key words: Pea protoplast, L-Leucine, Glycine transport, Active transport, Mediated transport     

Regeneration of Mesophyll Protoplasts Isolated from Dihaploid Clones of Solanum tuberosum

Protoplasts have been isolated from leaves of shoot cultures of six dihaploid clones of Solanum tuberosum L. (2n = 2x = 24). In the KM medium (Kao and Michayluk 1975), sustained cell divisions were obtained in up to 50% of the plated protoplasts of four clones, whereas only a few divisions occurred in the other two clones. The first mitosis appeared 2–8 days after plating, dependent on the clones. In the clones showing sustained cell divisions, a protoplast titre of about 5 × 10³ per ml turned out to be optimal. The culture conditions for protoplasts of one of the poorly growing clones, clone H² 140, have been improved using modified KM media, plating at a concentration of as high as 5 × 10⁴ cells per ml, and subsequent diluting at intervals 5 days. The dilutions were carried out with media containing 0.25% agar. Up to 60% of the plated protoplasts underwent divisions within 10 days under these conditions. After about 15 days, the regenerants were transferred onto media inducing organogenesis. Shoots and roots were formed on modified media MS (Murashige and Skoog 1962) and B5 (Gamborg et al. 1968). Plants have been regenerated in four of the investigated clones. Countings of chromosomes revealed a satisfactory stability of the karyotype in shoot culture and protoplast regeneration.     

Reproducibly High Plating Efficiencies of Isolated Mesophyll Protoplasts from Shoot Cultures of Tobacco

Sterile shoot cultures of Nicotiana tabacum L. cv. Sansum proved to be a highly appropriate source for the isolation of viable protoplasts. High yields of isolated protoplasts and high plating efficiencies are guaranteed by the absence of contaminaction, by controlled culture conditions and by the rhythmic rejuvenation of the shoots at each transfer to fresh agar medium.     

Evidence for Na-Independent HCO(3) Uptake by the Cyanobacterium Synechococcus leopoliensis

At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na⁺. The rate of photosynthesis greatly exceeded the CO₂ supply rate and indicated that HCO₃⁻ was taken up by a Na⁺-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na⁺ and exceeded the CO₂ supply rate by 8 to 45 times. The apparent photosynthetic affinity (K_½) for DIC was high (6-40 micromolar) and was not markedly affected by Na⁺ concentration, whereas with air-grown cells K_½ (DIC) decreased by more than an order of magnitude in the presence of Na⁺. Lithium, which inhibited Na⁺-dependent HCO₃⁻ uptake in air-grown cells, had little effect on Na⁺-independent HCO₃⁻ uptake by standing culture cells. A component of total HCO₃⁻ uptake in standing culture cells was also Na⁺-dependent with a K_½ (Na⁺) of 4.8 millimolar and was inhibited by lithium. Analysis of ¹⁴C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na⁺-independent CO₂ transport system. The conversion from Na⁺-independent to Na⁺-dependent HCO₃⁻ uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO₃⁻ for subsequent photosynthetic fixation.     

A Large-scale Isolation Procedure for Cereal Mesophyll Protoplasts

A method suitable for the large-scale isolation of cereal protoplastsfrom up to 50 g of leaf material is described. Surface-sterilizedleaves from cultivars of wheat, barley, maize, sorghum, andTriticale were diced and vacuum infiltrated with enzyme mixturecomposed of cellulysin (1 per cent w/v), hemicellulase (1 percent w/v), and macerozyme (0.5 per cent w/v). With this procedure,yields of between 10⁶ to 10⁷ protoplasts per gram of leavescan be reproducibly obtained after only 1.5–3 h of enzymatictreatment. These protoplasts were almost 100 per cent viable(as determined by fluorescein diacetate staining) and incorporationof ³H-uridine and ¹⁴C-leucine into an acid-insoluble fractionwas demonstrated. Almost one-third of the ribosomes of theseisolated protoplasts were present as polysomes. cereals, leaf mesophyll, protoplast isolation     

Comparative Studies of Leaf Tissue and Isolated Mesophyll Protoplasts: II. ION RELATIONS

Freshly isolated tobacco mesophyll protoplasts had contentsof K^$, Cl^– and Na^$ slightly higher (on a cell basis) thanthe original leaf tissue, and had a high K^$/Na^$ ratio similarto that of the leaf tissue. Influxes of these ions into theprotoplasts were of similar magnitudes to the correspondingfluxes in leaf tissue when compared on the basis of the respectiveplasmalemma surface areas. In both systems the K^$ influx wasstrongly inhibited by CN^– and by DNP. These results suggestthat the ion relations of the freshly isolated mesophyll protoplastswere similar to those of the original leaf tissue and that isolatedmesophyll protoplasts should be a useful system for the studyof ion transport processes in leaf cells.     

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using ¹⁴C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.     

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.     

Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625

The active transport and intracellular accumulation of HCO₃⁻ by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na⁺.Na⁺-dependent HCO₃⁻ accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O₂ evolution and CO₂ fixation. However, when Synechococcus was grown in standing culture, high rates of HCO₃⁻ transport and photosynthesis were observed in the absence of added Na⁺. The internal HCO₃⁻ pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO₃⁻ transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO₃⁻ from the medium in the absence of Na⁺ was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO₂ transport involving a high-affinity transport system which was reversibly inhibited by H₂S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO₂ transport system, whereas Na⁺-dependent and Na⁺-independent HCO₃⁻ transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO₃⁻ was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO₂ fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO₃⁻ + CO₂. The pattern of fluorescence quenching observed in response to added HCO₃⁻ and Na⁺ in air-grown and standing culture cells was highly characteristic for Na⁺-dependent and Na⁺-independent HCO₃⁻ accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO₃⁻ transport and the dynamics of intracellular HCO₃⁻ accumulation and dissipation.     

l-Aspartate Transport into Pea Chloroplasts : Kinetic and Inhibitor Evidence for Multiple Transport Systems

The kinetics of l-aspartate transport into pea chloroplasts was studied in the presence and absence of transport inhibitors to determine whether multiple aspartate carriers exist. Transport was measured by the silicone oil centrifugation technique. Reciprocal plots of concentration-dependent transport rates were biphasic, indicating the presence of two transport components, distinguishable on the basis of their affinity for aspartate. These transport components, called high affinity and low affinity transport could also be distinguished on the basis of their apparent substrate saturability and their sensitivity to media pH. The apparent K_m for high affinity transport was 30 micromolar. The K_m for low affinity transport was not determined. To test whether these transport components could also be distinguished on the basis of inhibitor sensitivity and to assess the value of inhibitors for distinguishing multiple aspartate translocators, a survey of several classes of potential inhibitors was conducted. High affinity aspartate transport was inhibited by p-chloromercuribenzenesulfonate and mersalyl, both sulfhydryl-reactive reagents; diethyl pyrocarbonate, a histidine-reactive reagent; and nigericin and carbonyl cyanide m-chlorophenylhydrazone, both ionophores. Low affinity aspartate transport was not inhibited by p-chloromercuribenzenesulfonate or nigericin, but preliminary results suggest it was sensitive to diethyl pyrocarbonate. Because the high and low affinity transport components could be distinguished not only by their sensitivity to media pH and substrate saturability, but also by their sensitivity to various inhibitors, we concluded that they may represent different transport systems or carriers.     

ATP Sulphurylase and O-Acetylserine Sulphydrylase in Isolated Mesophyll Protoplasts and Bundle Sheath Strands of S-deprived Maize Leaves

In Zea mays L. (cv. XL 72 A) leaves sulphur deficiency causedreduction of soluble protein and chlorophyll contents, whereasATP sulphurylase (EC 2.7.7.4 [EC] ) and O-acetylserine sulphydrylase(EC 4.2.95.9 [EC] ) activities increased with the increasing of S-deprivationtime. The two enzymes exhibited the maximum activity after 5d (ATP sulphurylase) and 3 d (O-acetylserine sulphydrylase)from the beginning of deprivation period. The activities weredifferently distributed between mesophyll protoplasts and bundlesheath strands. The results suggest that the activity of thetwo enzymes may be induced sequentially and differently regulatedin the two types of cells. Key words: ATP sulphurylase, Bundle sheath strands, Mesophyll protoplasts, O-acetylserine sulphydrylase, Sulphur deprivation, Zea