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PubMed2000

Fumonisin B1-induced cell death in arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways.

We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.

Arabidopsis↗
PubMed2004

Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.

Base Sequence↗
PubMed1990

Platelet-interactive products of Streptococcus sanguis protoplasts.

To isolate a more native, platelet-interactive macromolecule (class II antigen) of Streptococcus sanguis, cultured protoplasts were used as a source. Protoplasts were optimally prepared from fresh washed cells by digestion with 80 U of mutanolysin per ml for 75 min at 37 degrees C while osmotically stabilized in 26% (wt/vol) raffinose. Osmotically stabilized forms were surrounded by a 9-nm bilaminar membrane, as shown by transmission electron microscopy. Protoplasts were cultured in chemically defined synthetic medium and osmotically stabilized by ammonium chloride. Spent culture media were harvested daily for 7 days. Each day, soluble proteins were isolated from media, preincubated with platelet-rich plasma, and tested for inhibition of platelet aggregation induced by S. sanguis cells. Products released from S. sanguis protoplasts and reactive with an anti-class II antigen immunoaffinity matrix were able to inhibit S. sanguis-induced platelet aggregation. As resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, anti-class II-reactive protoplast products included silver-stained bands of 67, 79, 115, 216, and 248 kDa. The 115-kDa protein fraction was isolated by gel filtration and ion-exchange chromatography. This form of the class II antigen contained N-formylmethionine at its amino terminus. Rhamnose constituted 18.2% of the total residual dry weight and nearly half of its carbohydrate content. Diester phosphorus constituted 1% of this fraction. After trypsinization of the protoplast products from either preparation, a 65-kDa protein fragment was recovered. This protoplast protein fragment and the S. sanguis cell-derived 65-kDa class II antigen, previously implicated in the induction of platelet aggregation, were shown to be functionally and immunologically identical.

Amino Acids↗
PubMed1977

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state.

When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.

Bacillus subtilis↗
PubMed1986

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.

Adenosine Triphosphate↗
PubMed1962

Inhibition by antibiotics of the growth of bacterial and yeast protoplasts.

Shockman, Gerald D. (Temple University School of Medicine, Philadelphia, Pa.) and J. Oliver Lampen. Inhibition by antibiotics of the growth of bacterial and yeast protoplasts. J. Bacteriol. 84:508-512. 1962.-The characteristics and requirements for growth of bacterial (Streptococcus faecalis) and yeast (Saccharomyces cerevisiae) protoplasts were established and the effect of a variety of antibacterial and antifungal antibiotics determined. A clear differentiation was obtained between such inhibitors of bacterial cell wall synthesis as penicillin and cycloserine, which did not prevent protoplast growth, and all others, antibacterial and antifungal, which inhibited protoplasts and intact organisms at the same range of concentration. Novobiocin, previously reported to inhibit bacterial wall synthesis, was also effective against a reaction(s) essential to the growth of S. faecalis protoplasts. The antibacterial action of streptomycin, neomycin, and kanamycin was essentially eliminated by the high salt concentration needed to maintain the protoplasts. Removal of the cell wall did not significantly increase antibiotic susceptibility of a resistant species. Protoplasts of Bacillus megaterium were insensitive to the antifungal agent, nystatin, and did not bind it to any detectable degree. Thus, the yeast or bacterial cell wall does not appear to play a major role in determining relative antibiotic susceptibility by masking internal sensitive target sites. A variety of antifungal antibiotics tested on the growth of log-phase yeast cells failed to produce osmotically fragile forms.

Anti-Bacterial Agents↗
PubMed1969

Gelatin-induced reversion of protoplasts of Bacillus subtilis to the bacillary form: biosynthesis of macromolecules and wall during successive steps.

Protoplasts of Bacillus subtilis plated on SDG medium formed L colonies in quantative yield and propagated in the L-form indefinitely. Protoplasts or L bodies placed in 25% gelatin medium formed bacillary colonies. Details of the reversion of these naked bodies to the walled form are reported here. Protoplasts prepared in minimal medium reverted fairly synchronously 3 to 4 hr after inoculation into gelatin, but protoplasts preincubated in casein hydrolysate (CH)-enriched minimal medium were primed to revert within 1 hr in the gelatin. Preincubation for 1.5 hr in 0.44% CH was required for good priming. Cells must be subjected to this preincubation (step 1) in the naked state; it is effective for L bodies as well as protoplasts. Priming was blocked by chloramphenicol, puromycin, and actinomycin D but was not affected by penicillin, lysozyme, or inhibition of deoxyribonucleic acid (DNA) synthesis. It is concluded that protein and ribonucleic acid (RNA) synthesis are required during step 1, that DNA synthesis is not required, and that wall mucopeptide is not made. The reversion of well-primed protoplasts in the gelatin (step 2) proceeded undisturbed in thymine-starved cells with chromosomes arrested at the terminus. It was scarcely slowed by chloramphenicol in the gelatin but was delayed about 3 hr by both puromycin and actinomycin D. Escape from inhibition occurred while the inhibitors were still actively blocking growth. Penicillin and cycloserine inhibited and lysozyme reversed reversion. Momentary melting of the gelatin delayed reversion. It is concluded that mucopeptide synthesis occurs in step 2, that concomitant RNA, DNA, or protein synthesis is not essential, but that physical immobilization of excreted cell products at the protoplast surface is necessary early in step 2. Newly reverted cells were misshapen and osmotically sensitive. Processes which confer osmotic stability after reversion (step 3) did not occur in the presence of chloramphenicol or actinomycin D.

Bacillus subtilis↗
PubMed1984

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part I. The synthesis of echinomycin.

Washed suspensions of Streptomyces echinatus, and protoplasts derived from them, have been shown to synthesise echinomycin in the absence of growth. Protoplast suspensions free from significant contamination with unlysed mycelia are obtained by incubation with lysozyme followed by filtration through layers of tightly packed glass wool. Although physiologically young cells produce a better yield of protoplasts, optimal antibiotic biosynthesis is achieved with protoplasts prepared from mycelia about to enter the stationary phase of growth i.e., approximately 24 h after inoculation into a nutrient broth--salts seed medium. As judged by the incorporation of label from L-[methyl-14C]methionine, echinomycin synthesis proceeds for about 1 h after preparation of washed suspensions, but the kinetics of incorporation by intact cells and protoplasts are different. Uptake of labelled methionine by protoplasts is critically dependent upon the presence of sucrose as osmotic stabiliser and is drastically reduced if galactose, calcium, or magnesium is omitted from the suspending buffer. Uptake by intact, washed cells is essentially independent of nutrients in the medium. Small quantities of 11 materials other than echinomycin are detectable in chloroform extracts after labelling with L-[methyl-14C]methionine; some of these may represent precursors in the biosynthesis of the antibiotic. All amino acid constituents of echinomycin as well as tryptophan, a putative precursor of the quinoxaline chromophores, are actively incorporated into echinomycin by protoplasts and resting cells, but not with equal efficiency.

Amino Acids↗
PubMed2004

A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304.

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and beta-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and beta-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the alpha-1,3-glucanase. The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-1,3-glucanase and the chitinase preparation. The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with alpha-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.

Amino Acid Sequence↗
PubMed2006

Isolation, culture, and plant regeneration from leaf protoplasts of Passiflora.

The family Passifloraceae contains many species exploited in the food, pharmaceutical, and ornamental plant industries. The routine culture of isolated protoplasts (naked cells) followed by reproducible plant regeneration, is crucial to the genetic improvement of Passiflora spp. by somatic cell technologies. Such procedures include somatic hybridization by protoplast fusion to generate novel hybrid plants, and gene introduction by transformation. Seedling leaves are a convenient source of totipotent protoplasts. The protoplast-to-plant system developed for Passiflora edulis fv. flavicarpa is summarized in this chapter. The procedure involves enzymatic degradation of leaf tissue using commercially-available Macerozyme R10, Cellulase R10, and Driselase. Isolated protoplasts are cultured in Kao and Michayluk medium, semi-solidified with agarose. The medium containing the suspended protoplasts is dispensed as droplets or thin layers and bathed in liquid medium of the same composition. Shoot regeneration involves transfer of protoplast-derived tissues to Murashige and Skoog-based medium. The protocols developed for P. edulis are applicable to other Passiflora spp. and will underpin the future biotechnological exploitation of a range of species in this important plant family.

Cell Culture Techniques↗
PubMed2001

Formation and regeneration of protoplasts for protease production in Streptomyces rimosus.

To improve the formation and regeneration frequency of protoplasts for protease production, experiments were performed using a cultivation of Streptomyces rimosus TM-55 (CCRC 940061) in a Tryptic-soy broth (TSB) containing 2% of glycine for 2 days. It was found that the protoplast formation decreased with increased incubation temperature and increased ratio of culture broth to vessel volume. The optimal incubation temperature was 28 degreesC and the ratio of culture broth to vessel volume was 2:5. The hypertonic medium containing 10 mM MgCl2, 25 mM CaCl2 and 500 mM sucrose provided high stability for protoplasts. Supplementation with MgSO4, KCl and NaNO3 improved the regeneration frequency of protoplasts. The smear method had a higher protoplast regeneration frequency than the pour plate method. Protoplasts had protease productivity which was similar to that obtained with fresh mycelia, with each milliliter of culture broth yielding 141 units of protease with 3.5 x 10(8) protoplasts and 148 units of protease with 14.25 mg fresh mycelia respectively in a shaking culture, while the values were 15 and eight units of protease in a static culture.

Agar↗
PubMedDate not supplied

[Production of Candida lipolytica protoplasts].

Optimal conditions were found for the production and isolation of the protoplasts of Candida lipolytica. The maximum amount of the protoplasts was produced after 90 minutes of the incubation with a crude preparation of the enzyme from Helix pomatia (100 mg/g wet biomass). Longer incubation results in lysis of the protoplasts and structural damages of the intracellular components. The yield of the protoplasts does not depend on the nature of stabilizing agent. A decrease in the stabilizer concentration increases the yield of the protoplasts four times. Preliminary treatment of the yeast cells with a 0.1 M solution of SH compounds (cysteine, beta-mercaptoethanol) does not increase the yield of the protoplasts; and 0.2 M solution of these compounds decreased the yield of the protoplasts.

Candida↗
PubMed2004

[Protoplast culture and plant regeneration of the methionine resistant variant of Astragalus melilotoides Pall].

An efficient protocol for plant regeneration from protoplasts of the methionine resistant variant of Astragalus melilotoides was established. The friable calli induced from internode segments of variant plants were used for protoplast preparation. The protoplasts were isolated through enzyme digestion. Calli were formed after sustained divisions of protoplasts. High frequency of shoot differentiation was obtained from the protocalli on differentiated medium. The effects of different media, culturing methods and plating densities on protoplast divisions and plant regeneration were studied. The results show that agarose-beads culture method, KM8p medium supplemented with 1.0 mg/L 2,4-D, 0.5mg/L 6BA, 0.3 mol/L mannitol, 2% (W/V) sucrose and 500 mg/L casein hydrolysate at a plating density of 3 x 10(5)/mL are the appropriate conditions for protoplast division of the methionine resistant cell line. The division frequency is over 38%. The protoplast-regenerated plants still preserve resistance to methionine and ethionine.This research builds up the foundation for the resistant cell line as a parent of somatic hybridization.

Astragalus Plant↗
PubMed1985

Cell fusion between L-forms and protoplasts of Staphylococcus aureus.

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.

Adhesiveness↗
PubMed1989

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells.

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Expression of resistance to potato virus X in isolated potato protoplasts.

Resistance to Potato Virus X (PVX) in potato cultivars is conferred by the genes Nx and/or Nb. The Nx gene is activated by group 3 strains of PVX. When protoplasts from the cultivars King Edward containing the Nx gene and Pentland Crown containing neither resistance gene were inoculated with a group 3 strain of PVX resistance was expressed in the King Edward protoplasts. Replication of PVX occurred in King Edward protoplasts but only at a very low level as compared with the level observed in the susceptible Pentland Crown protoplasts. Similar results were obtained when protoplasts obtained from leaf discs mechanically inoculated with PVX were used. Virus levels were assayed by immunofluorescence and ELISA. Maintenance of resistance in protoplasts of King Edward following inoculation with PVX RNA was confirmed suggesting that resistance probably operates at the transcription stage in replication. These results clearly demonstrate single gene resistance in potato protoplasts inoculated with PVX.

Immunity, Innate↗
PubMed1989

Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein.

Tobacco mosaic virus (TMV)-like pseudovirus particles containing mRNA for Escherichia coli beta-glucuronidase (GUS) were electroporated into mesophyll protoplasts from control or TMV coat protein (CP)-transgenic tobacco (Nicotiana tabacum cv. Xanthi). GUS-particles were expressed 100-fold less efficiently in CP-transformed than in control protoplasts whereas unencapsidated GUS mRNA was expressed only 2.8-fold less efficiently. Lower transient expression of packaged GUS mRNA is probably due to inhibited disassembly of nucleocapsids in CP-transgenic protoplasts. Control and U1 CP-transformed protoplasts are equally susceptible to infection by cowpea strain TMV (Cc), as well as unencapsidated Cc or U1 RNA. In contrast, native or in vitro reconstituted U1 TMV particles result in 5- to 6-fold fewer infected CP-transgenic than control protoplasts. When Cc RNA was transcapsidated in U1 CP in vitro, the hybrid virions were equally infectious in both classes of protoplasts. We conclude that although compatible U1 protein-protein interactions significantly inhibit (GUS) nucleocapsid disassembly in CP-transgenic protoplasts, the endogenous CP must also interfere with a later stage of infection involving the homologous viral RNA.

Capsid↗
PubMed1995

Comparison of flash-induced light-scattering transients and proton efflux from auxinic-herbicide resistant and susceptible wild mustard protoplasts: a possible role for calcium in mediating auxinic herbicide resistance.

Comparison of flash-induced light-scattering signals from protoplasts of auxinic herbicide-resistant ('R') and -susceptible ('S') wild mustard biotypes suggest that ATP-dependent activity manifested as a conformational change differs between the biotypes. The signal obtained from R protoplasts is unaffected by 50 microM picloram, an auxin analogue. In comparison, the signal amplitude from the S protoplasts decreases 40% in 20 microM picloram and is abolished in 50 microM picloram. Signal amplitude from the R protoplasts can be decreased by simultaneous incubation with 50 microM picloram and 50 microM verapamil, a calcium channel blocker. Incubation of the S protoplasts with the calcium ionophore A23187 in the presence of 0.5 mM calcium reduced the inhibitory effect of picloram. Absorbance spectrophotometry using acridine orange as a pH indicator suggests that picloram affects the proton flux in the S biotype only. Based on these results, we hypothesize that picloram affects the calcium and hydrogen dynamics in the S biotype. Because the scattering characteristics of S protoplasts incubated with picloram can be nearly reproduced in the R protoplasts by simultaneous incubation with picloram and verapamil, calcium may have a significant role in modulating auxinic herbicide resistance. To our knowledge, this is the first report to implicate such a role for calcium.

Acridine Orange↗