Metabolism of Pyrimidines in Protoplasts from Cultured Catharanthus roseus Cells

The metabolism of [2-¹⁴C]thymine, [2-¹⁴C]thymidine, [2-¹⁴C]uraciland [¹⁴C]uridine was investigated in protoplasts obtained fromsuspension cultures of Catharanthus roseus. Most of the exogenouslysupplied thymine, thymidine and uracil was degraded, and salvageof these pyrimidines accounted for 5–36 per cent of thetotal amount of ¹⁴C-labelled precursors which was metabolized.However, more than 80 per cent of the labelled uridine was utilizedfor the biosynthesis of nucleotides and nucleic acids, and therest was degraded. In contrast to the results from protoplastsof sugar cane cells in suspension culture, the data indicatethat protoplasts possess a pathway for the degradation of pyrimidines,and that the overall metabolism of these pyrimidines in protoplastsis very similar to the metabolism in the intact cells. Catharanthus roseus, madagascar periwinkle, protoplasts, pyrimidine metabolism     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Calcium-Sensitivity of Cortical Microtubules in the Green Alga Mougeotia

Membrane ghosts were prepared from protoplasts of the greenalga Mougeotia, and the Ca²⁺-sensitivity of microtubules onthe ghosts was examined. Microtubules on the protoplast ghosts were not depolymerizedby 3 min treatment with 1 mM Ca²⁺. As the treatment was prolonged,some depolymerization of microtubules became evident, but evenafter 10 min about 50% of the ghosts showed no depolymerization.Ca²⁺ introduced into intact protoplasts seemed to be ineffectivein depolymerizing microtubules; abundant microtubules were presenton membrane ghosts prepared from protoplasts which had beentreated with 2x10^–5M Ca²⁺-ionophore A23187 [GenBank] plus 1 mM Ca²⁺for 20 or 30 min. Neither 3 min treatment with 0.2% Triton X-100 nor with 1 mMCa²⁺ solution containing 5 min MgSO₄ and 100 mM KCl caused depolymerisationof microtubules on protoplast ghosts. However, when given successively,these treatments caused complete depolymerization of microtubules. These results suggest that Mougeotia microtubules are stableto Ca²⁺ and that the stability is conferred by a microtubule-associatedfactor which can easily be removed by Triton X-100 treatment. (Received July 19, 1985; Accepted October 25, 1985)     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Carbon-specific phytoplankton growth rates: a comparison of methods

Measurements of biomass and growth rate of two axenic algalcultures were carried out using three different methodologicalapproaches: the specific ¹⁴C-labelling of chlorophyll a, [³H]adenineincorporation into DNA and net organic carbon assimilation.Time-course experiments revealed that the specific activitiesof chlorophyll a were significantly higher than the specificactivity of total algal carbon in six of seven experiments.When the specific activity of chlorophyll a is used to calculatethe carbon biomass and growth rate, the carbon biomass of thealgae will thus be underestimated and the specific growth ratewill be too high. Determination of growth rates from incorporationof [³H]adenine gave lower values than those obtained from netorganic carbon assimilation and from ¹⁴C incorporation intochlorophyll a. Problems with adenine saturation are suggested.When [³H]adenine is used to measure growth rates in dense algalcultures, additions of >1 µM [³H]adenine are oftenrequired to maximally label the extracellular and intracellularadenine pools and hence DNA.     

Formation of L-(+)-Tartaric Acid in Leaves of the Bean, Phaseolus vulgaris L.: Radioisotopic Studies with Putative Precursors

The biosynthetic pathway from D-glucose to L-(+)-tartaric acid(TA) in detached leaves of the bean, Phaseolus vulgaris L.,was studied in three cultivars, two of which were known to containTA and one of which lacked TA, with the aid of several putativeradiolabeled intermediates, namely D-[l-¹⁴C]glucose, D-[6-¹⁴C]glucose,D-[U-¹⁴C]glucose, D-[U-¹⁴C]gluconate, L-[U-¹⁴C]-ascorbic acid,L-[l-^l4C]idonate, D-xylo-5-[U-¹⁴C]hexulosonate, D-xylo-5-[l-¹⁴C]hexulosonate,D-xylo-5-[6-^l4C]hexulosonate and L-[U-^l4C]threonate. D-[U-¹⁴C]Glucoseand D-[U-^l4C]gluconate were converted to TA with low isotopicyield but this yield was further reduced when leaf tissues weresupplied with unlabeled D-gluconate or D-xylo-5-hexulosonate.D-xylo-5-[U-¹⁴C]Hexulosonate and D-xylo-5-[l-¹⁴C]hexulosonatewere good precursors of TA. D-xylo-5-[6-¹⁴C]Hexulosonate didnot furnish ¹⁴C to TA. Addition of a metabolic product of D-xylo-5-hexulosonate(which was labeled by D-xylo-5-[l-¹⁴C]hexulosonate but not byD-xylo-5-[6-¹⁴C]hexulosonate) to leaves labeled with D-xylo-5-[l-¹⁴C]hexulosonatedoubled the incorporation of ¹⁴C into TA. L-[U-¹⁴C]Ascorbicacid, L-[l-¹⁴C]idonate and L-[U-¹⁴C]threonate failed to producelabeled TA. A metabolic scheme to accommodate these observationsis presented. (Received October 21, 1988; Accepted March 29, 1989)     

Cultivation of Rice Protoplasts and Their Transformation Mediated by Agrobacterium Spheroplasts

Improvement of the cultivation of rice (Oryza saliva L.) protoplastsisolated from suspension cultures led to their division at afrequency of 5 to 10%. Rapidly growing colonies were obtainedon a hormone-free medium when Agrobacterium tumefaciens spheroplastswere introduced into the protoplasts by polyethylene glycoltreatment. Opines corresponding to the strains of A. tumefaciensused for the spheroplast treatments were detected in some ofthese colonies at a frequency of about 10^–4. Using radioactiveprecursors, [¹⁴C]--ketoglutaric acid and [³H]-arginine, activitiesof nopaline synthase, a marker enzyme of nopaline-type crowngall, were also detected in some of these clones. These resultsshow that the rice cells were transformed by Ti plasmid introducedby the spheroplast method. (Received September 6, 1985; Accepted January 24, 1986)     

Cytological studies on mitochondria-induced cytoplasmic transformation in yeasts

In Saccharomyces cerevisiae, protoplasts from respiratory-deficient(rho^–) cytoplasmic mutant cells were transformed intorespiratory-sufficient (rho⁺) cells by incubation with mitochondriaprepared from rho⁺ cells in the presence of polyethylene glycoland CaCl₂. Mitochondria prepared from different species, Hansenulawingei and Schizosaccharomyces pombe, also caused the transformationof S. cerevisiae rho^– protoplasts into the rho⁺ cellsas previously reported (14) The obtained transformants wereconfirmed to contain one nucleus and several mitochondrial DNAsby fluorescent staining of DNA. The transformants clearly restoredcytochromes a and b while untransformed recipient cells lackedthe cytochromes. In order to know the mechanism of the transformation,physiological measurement of endocytotic activity of protoplastsand cytological examination of mitochondria-protoplast aggregatesunder the transforming condition were performed. Protoplastshad significant endocytotic activity under this condition. Onthe other hand, fluorescence and electron microscopic observationsindicated that mitochondria forming aggregates with protoplastswere subsequently integrated into recipient protoplasts throughfusion rather than endocytosis. However, the possibility ofendocytosis could not be completely excluded when the low frequencyof the transformation (about 10^–6 to 10^–7) was takeninto account. This is discussed in this paper. A new convenientmethod for measuring endocytosis is also presented. (Received September 27, 1979; )     

Ti Plasmid Homologous Sequences Present in Tissues from Agrobacterium Plasmid-transformed Petunia Protoplasts

Suspension cell protoplasts of albino Petunia hybrida have beentransformed by isolated Agrobacterium tumefaciens Ti plasmid.Uptake of octopine Ti plasmid (pTiACH5) into protoplasts wasstimulated by poly-L-ornithine and polyethylene glycol (PEG).The frequency and efficiency of transformation of protoplaststo phytohormone autotrophy was compared using the two uptakeagents with various concentrations of plasmid. Transformationwas most efficient with PEG-mediated uptake, 5 µg of Tiplasmid per 10⁶ protoplasts giving a frequency of 6?10^–5.Octopine was not synthesised in any of the transformants afterthe second subculture on hormone-free medium. DNA-DNA hybridisationshowed the presence of DNA homologous to the T-DNA region ofpTiACH5 in all clones analysed. (Received November 9, 1981; Accepted January 29, 1982)     

The Development of Fast-Start Performance in Fishes: Escape Kinematics of the Chinook Salmon (Oncorhynchus tshawytscha)

C-starts are high acceleration swimming movements critical forpredator avoidance by fishes. Since larval fishes are particularlyvulnerable to predation, C-start behavior is likely to be especiallyimportant during early life history stages. This paper examinesthe developmental changes in C-start performance with kinematicdata on immature chinook salmon (Oncorhynchus tshawytscha) (eleuthroembryostage, sensu Balon, 1975). The scaling of C-start kinematicsof immature fishes differs from that of adults. Adult C-startdurations increase with increasing body length while C-startdurations of immature fishes decrease (e.g., adult stage 1 duration[sec] = 0.0019.length [L] [cm] $ 0.026 [R² = 0.77] [Webb, 1978];eleuthroembryos stage 1 duration [sec] = –0.026L [cm]$ 0.100 [R² = 0.81]). Distance traveled during stage 2 alsodiffers between adult and immature fishes. Adult distance traveledscales directly with length (distance [cm] = 0.38L^1.01 [cm],R² = 0.96 [Webb, 1978]) while chinook eleuthroembryo distancetraveled is positively allometric with length (distance [cm]=0.37L¹³¹ [cm], R² = 0.83). There are similarities in the developmentof C-starts and burst swimming. For example, mean velocity scalessimilarly between the two locomotor modes (For burst swimming:U_mean [cm/sec] = 8.1 ± 1.1L [cm] $ 4.89 [R² = 0.86] [Webband Corolla, 1981]. For C-start stage 2: U_mean [cm/sec] = 10.96L[cm] - 14.09 [R² = 0.70]). This study demonstrates that C-startescape performance improves during early post-hatching development.Comparisons of immature chinook salmon fast-starts with dataon larval burst swimming and on adult C-starts suggest thatchanges specific to developing fish affect the scaling of kinematicparameters.     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata     

Lactoperoxidase-Catalyzed Iodination of Plasmamembrane Lipids and Proteins in Arabidopsis Protoplasts

Lactoperoxidase-catalyzed iodination was evaluated as a techniqueto identify plasma membrane components in protoplasts isolatedfrom leaf tissue of Arabidopsis thaliana (L.) Heynh. Restrictionof the probe to the plasma membrane was assessed by determiningradioactivity in lipids characteristic of intracellular organellesafter labeling intact versus broken protoplasts. Intact protoplastswere purified on a sucrose-sorbitol gradient and iodinated usingNa[¹²⁵I] with lactoperoxidase and H₂O₂. Lipids as well as proteinswere shown to be iodinated in a lactoperoxidase-catalyzed reaction.Labeling of intracellular lipids was low in the intact protoplastswhich indicated that the iodination reaction was restrictedto the plasma membrane. (Received March 18, 1988; Accepted May 25, 1988)     

Direct Gene Transfer in Psophocarpus tetragonolobus Resistance to Kanamycin

Epicotyl-derived protoplasts of Psophocarpus tetragonolobuswere isolated and regenerated to plants. These protoplasts weretransformed to kanamycin resistance following uptake of plasmid(pABDl or pHP23) DNA in combination with PEG treatment. Protoplast-derivedtransformed colonies were selected on kanamycin (75 mg l^–1).The transformed calli expressed NPT II activity and also exhibitedthe presence of the plasmid gene integrated into the plant genome.However, none of the transformed clones showed regenerationof shoot buds. Psophocarpus tetragonolobus, winged bean, naked DNA transformation, protoplast culture, regenerated plants     

Compartmentation of Aldolase Isoforms in Maize Leaves

The leaves of maize seedlings contain two principal isozymesof fructose 1,6-bisphosphate aldolase (E.C. 4.1.2.13 [EC] ), one chloroplasticand one cytosolic (Gasperini and Pupillo, 1982). Mesophyll protoplastswere separated from bundle sheath (BS) strands of both light-grownand dark-grown maize leaves. Aldolase isozymes were separatedfrom extracts of chloroplasts, etioplasts, protoplasts and BSstrands by column isoelectric focusing. The major isozyme ofgreen leaves (pI 4.2) was exclusively in BS chloroplasts, andthere was no evidence of other isozymes occurring in BS tissue.The cytosolic isozyme (pI 6.7) was present in protoplasts ofmesophyll cells, where it may limit the synthesis of hexose-phosphates(estimated activity of 9.4 µmol h^–1 g^–1 fr.wt.) together with lower activities of an acidic form (pI 4.6).Etiolated leaves contained significant amounts of the pI 6.7isozyme in both mesophyll and BS cells, but also minor activitiesof one or more acidic forms with pI values of 4.4–4.7(average pI 4.6) which appear to be located partly in BS etioplasts.The main developmental events for maize leaf aldolase afterillumination were a moderate decrease of cytosolic isozyme (pI6.7) which disappears from the BS within hours and a large,gradual increase of the BS plastid isozyme (pI 4.2). The isoformwith a pI 4.6 also increased rapidly to a low, steady activityin greening mesophyll protoplasts. Key words: C₄, fructose 1,6-bisphosphate, aldolase, Zea mays     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Isoforms of NADP-dependent Malic Enzyme in Tissues of the Greening Maize Leaf

Scagliarini, S., Pupillo, P. and Valenti, V. 1988. Isoformsof NADP-dependent malic enzyme in tissues of the greening maizeleaf.—J. exp. Bot. 39: 1109–1119. The compartmentation of the isoforms of NADP-dependent malicenzyme (E.C. 1.1.1.40 [EC] ) has been studied in cell-free extractsand in enzymatically-isolated protoplasts of mesophyll tissue(MT) and bundle sheath (BS) strands of greening maize leaves.The etiolated leaf of 10-d-old seedlings contains a cytosolicisozyme with a pl of 5.4 ?0.1 at low specific activity (s.a,45 ? 3 nmol min^–1 mg^–1 protein), found both in MTand BS. The green leaf on the other hand contains the dominantBS chloroplast isozyme with pl 4.6 ? 0.2 at a s.a, of 370 ?40 nmol min^–1 mg^–1 protein (3.2 ? 0.5 µmolmin^–1 mg^–1 chl) and a minor, previously undescribedisoform with pl 6.5 ? 0.1 also localized in the BS at a s.a.of 38 ? 6 nmol min^–1 mg^–1 protein. Green MT protoplastshave only traces of pl 4.6 isozyme. After illumination of dark-grown seedlings, the total leaf activityshows a rapid increase (1.5-fold within 2 h), attributed mainlyto the pl 5.4 isozyme of MT protoplasts and BS strands. Thisis followed by a large increase of enzyme activity due to thecontinued rise of pl 5.4 isozyme for about 24 h and, after aninitial lag of a few hours, to the accumulation of pl 4.6 isozyme.After 18 h illumination, pl 4.6 and 5.4 isozyme activities tendto decline in the MT whereas they are still increasing in theBS, particularly the former. This pl 4.6 species has becomethe major one by 48 h illumination. The final pattern of greenleaves is established around 96 h light, when the chloroplastisozyme has attained its maximum level, the pl 5.4 isozyme ofBS strands has been superceded by the pl 6.5 species (also supposedto be cytosolic) and MT protoplasts retain little residual activity.Some metabolic implications of the changing pattern of NADP-dependentmalic isozymes during maize leaf greening are discussed. Key words: C₄, isozymes, malic enzyme, photodifferentiation, Zea mays     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets

Human and rabbit platelets fully aggregated byplatelet-activating factor (PAF) underwent slow disaggregation but wererapidly disaggregated by the PAF receptor antagonists WEB-2086,Y-24180, SM-12502, and CV-3988. Whereas the1-alkyl-2-[³H]acetyl-sn-glycero-3-phosphocholine([³H]acetyl-PAF)specifically bound to platelet receptors underwent slow and spontaneousdissociation, it dissociated promptly from its receptor when WEB-2086was added, in parallel with platelet disaggregation and disappearanceof P-selectin on the cell surface. Extracellular[³H]acetyl-PAF wasrapidly deacetylated by normal rabbit platelets; some of the[³H]acetyl-PAF wasbound to the cells and a very small amount of [³H]acetate wasdetected in the cells. In contrast, when1-[³H]alkyl-2-acetyl-sn-glycero-3-phosphocholinewas added to the platelets, the radioactivity was rapidly incorporatedinto the 1-alkyl-2-acyl-sn-glycero-3-phosphocholinefraction. These results indicate that1) continuous binding of PAF to itsreceptor is necessary for prolonged platelet aggregation, which may bemediated through an unknown signaling system for a long-term cellresponse rather than a transient signaling system, and2) most of the[³H]acetyl-PAF boundto platelets is metabolized extracellularly by ecto-type PAFacetylhydrolase, with the lyso-PAF generated being incorporated rapidlyinto the cells and converted to1-alkyl-2-acyl-sn-glycero-3-phosphocholine.

     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)