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PubMed1981

Subcellular localization of proteases in wheat and corn mesophyll protoplasts.

Mesophyll protoplasts were isolated from the leaves of wheat and corn seedlings. After purification the protoplasts were judged to be free of contaminating proteases in the isolation enzymes based on specific activity of the proteases in comparison to leaf tissue and their response to inhibitors that "differentiated" between leaf and isolation enzyme proteases. Wheat protoplasts showed rates of photosynthesis of 95 to 100 micromoles O(2) per milligram chlorophyll per hour, while corn exhibited rates of 35 to 85 micromoles O(2) per milligram chlorophyll per hour, indicating the intactness of the chloroplasts within the protoplasts. These chloroplasts were isolated from the protoplasts using the procedure of Robinson and Walker (1979 Arch Biochem Biophys 196: 319-323). Yields of 91 and 82% intact chloroplasts were obtained from wheat and corn, respectively, based on the distribution of ribulose bisphosphate carboxylase in wheat and NADP-malate dehydrogenase in corn. Vacuoles were obtained from the protoplasts using a modification of the techniques of Wagner and Siegelman (1975 Science 190: 1298-1299) and Saunders (1979 Plant Physiol 64: 74-78). The vacuoles were at least 98% free of protoplast contamination as determined by assaying for "marker" enzymes of chloroplasts, mitochondria, and endoplasmic reticulum. Assuming one vacuole per protoplast, the vacuoles contained 4% of the soluble protein of the protoplasts in wheat and 8% in corn. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuoles. Although the isolated chloroplasts always retained about 13% of the proteolytic activity of the protoplasts, this could be accounted for by that which became associated with the chloroplasts during their isolation.

Journal Article↗
PubMed1981

The synthesis of polyamines from methionine in intact and disrupted leaf protoplasts of virus-infected chinese cabbage.

In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-(14)C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.Protein synthesis is similar for 4 hours in both healthy and infected protoplasts. Accumulation of labeled spermidine stops after 2 hours in healthy protoplasts but continues in the infected protoplasts. Much of the newly synthesized protein and spermidine is present in the easily sedimentable fraction of the readily disrupted protoplasts.Disrupted and diluted protoplasts have a decreased ability to metabolize methionine to protein and spermidine. The residual synthetic activity is essentially entirely in the easily sedimentable fraction. However, this fraction is unable to synthesize spermine, an activity found in protoplasts and disrupted protoplasts. Disrupted protoplasts contain spermidine synthase (EC 2.5.1.16) and about a quarter of this activity is present in a low-speed sedimentable fraction containing the chloroplasts. The protoplast system is suitable for an analysis of polyamine synthesis in turnip yellow mosaic virus infection and appears particularly suitable for study of the distribution of the enzymes involved.

Journal Article↗
PubMed1990

Cryopreservation of rye protoplasts by vitrification.

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20 degrees C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0 degrees C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN(2)); and (d) recovery of the protoplasts from LN(2) and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN(2) if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3+/-2%. In contrast, survival of ACC protoplasts following recovery from LN(2) was 34 +/- 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 +/- 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.

Journal Article↗
PubMed2006

A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.

BACKGROUND: Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking. RESULTS: We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7-14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60-70% transformation efficiency. In contrast, using 50 microg of a 12 kb plasmid we obtained a maximum of 25-30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes. CONCLUSION: We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.

Journal Article↗
PubMed2026

Characterization of oxidative status in maize protoplasts under temperature and saline-alkali stresses.

BACKGROUND: Protoplasts have emerged as a powerful model system in plant functional genomics, offering significant utility in functional gene analysis, protein interaction studies, and transient expression platforms for gene editing. Despite their versatility, inherent limitations restrict their broader application, highlighting the need for systematic investigations into their responses to abiotic stressors, such as temperature fluctuations and saline-alkali conditions (200 mM saline mixture: 170mM NaCl and 30mM Na2CO3, pH = 9.1). RESULTS: In this study, we comprehensively examined the effects of varying temperatures and saline-alkali stress on the integrity, viability, and reactive oxygen species (ROS) metabolism of maize protoplasts. Key markers of oxidative stress-including ROS accumulation, lipid peroxidation (measured as malondialdehyde, MDA), antioxidant enzyme activity (superoxide dismutase, SOD), and hydrogen peroxide (H2O2) levels-were quantified to assess the oxidative stress response. Protoplasts maintained at 4 °C demonstrated enhanced stability and antioxidant capacity, preserving cell viability and endogenous protein integrity for up to 16 h. Conversely, exposure to 37 °C significantly compromised protoplast viability, while incubation at 28 °C exerted minimal effects within 16 h. CONCLUSIONS: Our study investigated the effects of various temperature stresses and salt-alkali stress on maize protoplasts. The results demonstrated that both temperature and salt-alkali stress significantly impacted protoplast production, viability, and the expression of endogenous proteins. These findings not only characterize the redox response of maize protoplasts, but also provide guidance for protoplast isolation and other procedures: 4 °C is suitable for short-term maintenance, 25-28 °C for routine functional assays, and 37 °C should be avoided. These findings provide valuable insights into the stress responses of protoplasts and establish a foundation for future research aimed at improving plant stress tolerance through protoplast-based techniques.

Zea mays↗
PubMed2000

Mycelial protoplast isolation and regeneration of Lentinus lepideus.

Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%.

Cell Fusion↗
PubMed1995

Inhibition of brome mosaic virus (BMV) amplification in protoplasts from transgenic tobacco plants expressing replicable BMV RNAs.

Transgenic tobacco plants (V123 plants) expressing a set of full-length brome mosaic virus (BMV) genomic RNAs from the cauliflower mosaic virus 35S promoter were produced. The accumulation level of BMV RNAs in V123 plant cells was approximately 1% of that in nontransgenic tobacco protoplasts inoculated with BMV RNAs. The level of BMV RNA in V123 protoplasts did not increase after inoculating the protoplasts with BMV RNAs, whereas V123 protoplasts supported the accumulation of cucumber mosaic virus (CMV) RNAs to a level similar to that in non-transgenic tobacco protoplasts after inoculation with CMV RNA. Such BMV-specific resistance was also observed in protoplasts from V12 plants expressing full-length BMV RNA1 and RNA2, both of which are required and sufficient for BMV RNA replication. On the other hand, protoplasts from M12 plants, expressing truncated BMV RNA1 and RNA2 in which the 3' 200 nucleotides required for BMV RNA replication were deleted, exhibited weaker resistance to infection with BMV RNA than V12 protoplasts, although the accumulation level of truncated BMV RNA1 and RNA2 in M12 protoplasts was higher than that of BMV RNA1 and RNA2 in V12 protoplasts. These results suggest that expression of BMV RNA replicons is involved in the induction of resistance, rather than high-level accumulation of BMV RNAs and/or their encoded proteins.

Bromovirus↗
PubMed1981

Protoplast fusion in Streptomyces: conditions for efficient genetic recombination and cell regeneration.

Protoplasts from four different species of Streptomyces regenerated cells efficiently in hypertonic soft agar medium overlaid on partially dehydrated regeneration medium. The efficiencies of regeneration were strongly dependent upon the incubation temperatures for cell growth and for protoplast regeneration. Cell growth temperatures (before protoplast formation) required for efficient protoplast regeneration varied from species to species, and did not necessarily correlate with the optimum temperatures for protoplast regeneration. Under the best conditions, protoplasts from all four species were able to regenerate viable cells at nearly 100% efficiency and also formed confluent lawns of mycelia when plated in high concentrations. The temperatures for cell growth and protoplast regeneration also affected the frequencies of genetic recombinants obtained by protoplast fusion in S. fradiae, and highest recombinant frequencies were obtained under conditions which favoured efficient protoplast regeneration. With the modified procedure described, maximum frequencies of genetic recombinants were obtained by treating parental protoplasts with 40 to 60% polyethylene glycol 1000.

Agar↗
PubMed1988

Fluorescent vital stains for complementary labelling of protoplasts from Trichoderma spp.

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.

Fluorescent Dyes↗
PubMed1993

Evaluation of DAPI as a fluorescent probe for DNA in viable Petunia protoplasts.

4',6-Diamidino-2-phenyl-indole (DAPI), is a fluorescent probe that specifically and quantitatively stains DNA. Electroporation of viable Petunia protoplasts in the presence of DAPI revealed integral fluorescence that was similar for both the electroporated and fixed protoplasts, indicating quantitative staining of DNA. DAPI fluorescence was localized in the nuclei of viable protoplasts of Petunia. Protoplasts had a short term viability of 56-65% of the control (non-electroporated, unstained) protoplasts as determined by fluorescein diacetate staining 24 hr following electroporation in the presence of DAPI. The majority (84% of the number originally cultured) of these protoplasts subjected to electroporation were able to form a cell wall, but most did not form microcalli because they were blocked in cell division. The three week plating efficiency for protoplasts exposed to DAPI was 4% of the original number of protoplasts initially cultured compared to 30% for the control. DAPI should not be used as a fluorescent probe for plant protoplasts when the protoplasts are cultured for sustained growth because the levels of DAPI required to obtain quantitative staining of the DNA resulted in inhibition of the cell cycle. DAPI may, however, be used as a fluorescent DNA probe for short term (24 hr) studies.

Cell Division↗
PubMed2004

Analysis of gene expression in yeast protoplasts using DNA microarrays and their application for efficient production of invertase and alpha-glucosidase.

The global gene expression of cultured Saccharomyces cerevisiae protoplasts was compared with that of cells using DNA microarray. Quantitative and qualitative analyses revealed that after 6 h of cultivation, 416 gene transcript levels (about 7.1% in all) in the cultured protoplasts were different from those in the cells. Various characteristics and functions of the protoplasts were predicted from the analysis of the gene functions. The cultured protoplasts were more sensitive to oxidative stress than the cultured cells. Their cell cycles were arrested at the G1 phase and cell wall synthesis was promoted. Carbohydrate metabolism was activated in cultured protoplasts, while amino acid biosynthesis was inhibited. Furthermore, some genes associated with the secretory pathway of metabolites were activated, leading to active secretion of these metabolites into the broth. As an example of the application of DNA microarray analysis, we developed two novel methods for the production of useful enzymes based on the characteristics of protoplasts. One was the production of invertase based on the activated secretory pathway, while the other was the production of alpha-glucosidase based on the activated carbohydrate metabolism. The secretion of invertase and alpha-glucosidase was promoted in cultured protoplasts. The invertase and alpha-glucosidase productivities in the cultured protoplasts were 657 U and 218 U, respectively. On the other hand, only 227 U of invertase was produced, while alpha-glucosidase was not detected, in the cultured cells. The fragile protoplasts were immobilized in agarose gel to protect them from hydrodynamic stress. Four repeated-batch cultures with the immobilized protoplasts were performed, leading to the production of 1574 U of invertase and 739 U of alpha-glucosidase. The same productivities were obtained when this system was scaled up by 10-fold (invertase: 13304 U; alpha-glucosidase: 7688 U).

Journal Article↗
PubMed1994

The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration.

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.

Journal Article↗
PubMed1978

Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures.

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to -3.9 C resulted in 80% lysis of the protoplasts. At -3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl >/= Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.

Journal Article↗
PubMed1983

Effect of cold acclimation on intracellular ice formation in isolated protoplasts.

When cooled at rapid rates to temperatures between -10 and -30 degrees C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation-cooling to -15 or -20 degrees C at rates of 10 or 16 degrees C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at -5 degrees C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of -15 degrees C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of -42 degrees C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution.

Journal Article↗
PubMed1989

Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves.

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT(50) (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from -5 degrees C to -25 degrees C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT(50). However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10 degrees C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.

Journal Article↗
PubMed1989

Transient gene expression in electroporated Solanum protoplasts.

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts--250 V/cm; Désirée mesophyll protoplasts--225 V/cm; Désirée suspension culture protoplasts--225 V/cm; and Désirée tuber protoplasts--150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36-48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.

Chloramphenicol O-Acetyltransferase↗
PubMed1993

Iron uptake by Bifidobacterium thermophilum protoplasts.

Protoplasts of Bifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out. Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface. This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process. It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium. Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies. The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only. Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact. The amount of iron so bound was dose-dependent on the amount of iron entering the cell. The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO4. It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.

Bifidobacterium↗
PubMed1995

Response of yeast protoplasts to their mating partners.

The mating process between two protoplasts or between a protoplast and a cell in the yeast Saccharomyces cerevisiae was manifested by a specific morphological response of only the cell partner. The cells produced projections, up to 5 micrometers long, to meet their protoplast partners. The protoplasts responded, after a period of nonspecific hernia-like growth, by ceasing to grow and assuming oval or spherical shapes. They never formed mating projections, apparently due to the absence of complete cell walls. Similarly to the cells, nuclear division in protoplasts was arrested and the nucleus migrated towards the plasma membrane at the site of protoplast-cell contact. Cytoplasmic microtubules were directed to this site, indicating the position of the spindle pole body (SPB) on the nucleus adjacent to the plasma membrane. Actin patches accumulated also in this region. These cytological features of the protoplasts were reminiscent of the reorganization of the cytoskeleton and nucleus characteristic of mating cells. This implies that the ability of protoplasts to produce and receive mating signals was unaffected by protoplasting. Fusion, however, was not initiated due to the absence of the complete cell wall in one of the partners. Thus, the cell wall appeared to be necessary for the expression of polarized growth during mating and for cell fusion.

Mating Factor↗