Purification, physicochemical properties, and localization of cytochrome c in energy-transducing membranes from Mycobacterium phlei.

Cytochrome c from Mycobacterium phlei has been isolated and purified to homogeneity using an isoelectric focusing technique. The purified cytochrome c has a molecular weight of 12,600 ± 400 and exhibits an isoelectric point (pI) of 4.7 ± 0.05. The amino acid composition of cytochrome c shows a higher proportion of valine and arginine residues and a greatly reduced content of lysine residues when compared to Bacillus subtilis cytochrome c. This imparts less acidic character to the cytochrome c from M. phlei. The cytochrome c from M. phlei acts as the most effective electron acceptor for M. phlei NADH-cytochrome c reductase, while yeast and horse heart cytochrome c are not as efficient electron acceptors. The absence of correlation between the oxidation-reduction potential with the observed activity of NADH-cytochrome c reductase activity indicates that the electrochemical potential is not a sufficient determinant for bacterial cytochrome c function. In order to obtain information concerning the topology of respiratory components, two membrane systems from M. phlei were used; ghost preparations in which the membrane is oriented rightside out as in whole cells and membrane vesicles in which membranes are oriented inside out. Labeling of protoplast ghosts and membrane vesicles with lactoperoxidase-catalyzed iodination reveals that cytochrome c is localized on the outer membrane of protoplast ghosts, which is similar to that observed in mammalian mitochondria. The results also show that cytochrome c from M. phlei binds preferentially to basic phospholipids and not to neutral or acidic phospholipids. Scatchard analysis of the binding of cytochrome c to phosphatidyl ethanolamine shows high affinity (K_a of 3.79 × 10⁵M^?1) and low affinity (K_a of 3.75 × 10⁴M^?1) binding.     

Development of an optical method for monitoring protoplast formation from cultured plant cells

The size distribution of cell aggregates during protoplast isolation from Catharanthus roseus and Nicotiana tabacum was measured by a Coulter counter. It was observed that a gradual reduction in the size of cell aggregates occured during protoplast formation. A previously developed specialized spectrophotometer for the photometric measurement of plant cell concentration was used for continuous monitoring of the reduction in the size distribution of cell aggregates during protoplast formation. This made it possible to use changes in optical density (O.D.) to distinguish the three stages in protoplast formation—plasmolysis, maceration and cell wall digestion. During the processes of maceration and cell wall digestion, the O.D. decreased and reached a steady value at the end of each process. Consequently, changes in the O.D. could be used to determine precisely the end of each process. The cell wall digestion process was described by a simple first order reaction model and the rate of protoplast formation (cell wall digestion) was quantitatively evaluated from the rate constant (k) of this reaction. By using the values of k, the optimal enzymatic reaction conditions for isolating protoplasts from C. roseus and N. tabacum cells were determined.     

Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

Background

Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.

Methodology/Principal Findings

We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.

Conclusions/Significance

This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.     

Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p-nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p-nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Protoplast volume:water potential relationship and bound water fraction in spinach leaves

Methods used to estimate the (nonosmotic) bound water fraction (BWF) (i.e. apoplast water) of spinach (Spinacia oleracea L.) leaves were evaluated. Studies using three different methods of pressure/volume (P/V) curve construction all resulted in a similar calculation of BWF; approximately 40%. The theoretically derived BWF, and the water potential (Ψ_w)/relative water content relationship established from P/V curves were used to establish the relationship between protoplast (i.e. symplast) volume and Ψ_w. Another method of establishing the protoplast volume/Ψ_w relationship in spinach leaves was compared with the results from P/V curve experiments. This second technique involved the vacuum infiltration of solutions at a range of osmotic potentials into discs cut from spinach leaves. These solutions contained radioactively labeled H₂O and sorbitol. This dual label infiltration technique allowed for simultaneous measurement of the total and apoplast volumes in leaf tissue; the difference yielded the protoplast volume. The dual label infiltration experiments and the P/V curve constructions both showed that below −1 megapascals, protoplast volume decreases sharply with decreasing water potential; with 50% reduction in protoplast volume occurring at −1.8 megapascals leaf water potential.     

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis>

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

Removal of browning and growth enhancement by polyvinylpolypyrrolidone in protoplast cultures ofCyamopsis tetragonoloba L.

The occurrence of browning in protoplast cultures ofCyamopsis tetragonoloba completely inhibited the growth of protoplast derived colonies. Of the various additives employed to counteract the problem of browning and subsequent necrosis, polyvinylpolypyrrolidone (PVPP) was found most effective. Simultaneous addition of polyvinylpyrrolidone (PVP) to the protoplast culture medium accentuated the effect of PVPP and also improved the frequency of protoplast division.     

Development of SCAR Markers to Determine the Mating Types of Lepista nuda Protoplast Monokaryons

Lepista nuda (Bull. ex Fr.) Cooke belongs to Tricholomataceae and is an edible fungus with both economic and medical value. Mycelia were isolated from the fruiting bodies of L. nuda and were used to prepare the protoplast monokaryons. One hundred and fifteen monokaryons were obtained and their mating types were determined using somatic incompatibility tests. Protoplast monokaryons segregated into either the A₁B₁ or the A₂B₂ mating types. Inter-simple sequence repeats and sequence-related amplified polymorphism fingerprinting were used to analyse the mating types of these protoplast monokaryons and 16 sequence-characterised amplified region primers were developed to efficiently differentiate between the monokaryon mating types. Multiplex PCR analyses were also established. The data presented here outline a method for the precise and rapid identification of protoplast monokaryon mating types, which has the promise to shorten the period required for conventional crossbreeding.     

Separation of yeast protoplasts from membrane ghosts using an aqueous two-phase system

Protoplasts, prepared from Saccharomyces cerevisiae, were separated from membrane ghosts using an aqueous two-phase system of Dextran T 500, 5% (w/w) and polyethylene glycol (PEG) 8000, 5% (w/w). The protoplast preparation was prewashed in the top phase. After resuspension in fresh top phase an equal volume of bottom phase was added. A protoplast preparation almost free from membrane ghosts was obtained as a precipitate in the bottom phase. The membrane ghosts partitioned to the interphase.     

Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture

Vacuoles were isolated from Acer pseudoplatanus cell suspension culture using a one-step procedure involving the lysis of the protoplast plasmalemma through a gradient of Ficoll containing DEAE-Dextran. The vacuole suspensions were slightly contaminated by other organelles (less than 5%) and the isolated vacuoles readily accumulated neutral red. Since α-mannosidase was located exclusively in the vacuoles it was used as a convenient marker. It was shown that the number of vacuoles per protoplast decreased as the cell aged. Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.     

Switchgrass (Panicum virgatum L.) cell suspension cultures: Establishment, characterization, and application

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10⁶ protoplasts/1.0 g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research.     

Plasmalemma hyperpolarity and culture of sense and antisenseabp transgenic tobacco protoplasts

The relationship among transfer and expression of auxin binding protein gene (abp), auxin (NAA)-induced plasmalemma hyperpolarity and sensibility to auxin during protoplast culture was studied by measuring transmembrane potential difference (Em) and culturing the protoplasts of sense and antisenseabp transgenic tobacco. The concentration of NAA inducing the highest degree of hyperpolarity of senseabp transgenic tobacco protoplasts was lower than the control, and in protoplast culture, their sensibility to auxin increased. The concentration of antisenseabp transgenic tobacco protoplasts was higher than the control, and in protoplast culture, their sensibility to auxin decreased. These results demonstrated that ABP synthesized in endoplasmic reticulum needed to transport to cell membrane and functioned there.     

The ectopic expression of apple <Emphasis Type="Italic">MYB1</Emphasis> and <Emphasis Type="Italic">bHLH3</Emphasis> differentially activates anthocyanin biosynthesis in tobacco

Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula?×?P. alba), are applied to poplars and compared. Both the in vitro culture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the first time, the stable integration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plantlets can be shown. For biolistic transformation, stem segments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efficient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% macerozyme, 1?×?10⁷ protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then complemented by an efficient protoplast transformation protocol with 40% PEG₁₅₀₀. The results of this study confirm that both the biolistic and the protoplast transformation methods can be considered suitable for transferring cisgenes directly into poplar.     

Dual Mechanisms in Polyamine-mediated Control of Ribonuclease Activity in Oat Leaf Protoplasts

Dibasic amino acids and polyamines added to oat (Avena sativa L.) leaf protoplast isolation media decrease the RNase activity of extracted protoplasts relative to controls. This effect, which is manifested even when the added polyamine is removed by exhaustive dialysis prior to assay, is due to a prevention of the rise in RNase activity which usually follows protoplast isolation. Polyamines, but not dibasic amino acids, also decrease RNase activity in vitro. This in vitro effect seems to result from electrovalent attachment of the polyamine to the RNA, because the greater the net positive charge on the polyamine, the greater is its inhibitory effect in vitro. The activity of dibasic amino acids when added during protoplast isolation probably results from their conversion to polyamines.     

Characterisation of Escherichia coli cell surface by isoelectric equilibrium analysis

A characterisation of the lipopolysaccharide (outermost) layer of Escherichia coli cells has been made by isoelectric equilibrium analysis. Unmodified E. coli cells show a surface isoelectric point (pI) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxyl groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a pI of 3.85. An analysis of these results suggests that the ionisable groups occurring in the isoelectric zone i.e. the zone amenable to investigation by the isoelectric equilibrium method are: carboxyl groups and amino groups of polysaccharide and protein components. The carboxyl groups have a pK between 3.2 and 4.5 and the amino groups have a pK of 7.5. ε-Amino groups, phenolic hydroxyl groups and guanidyl groups do not occur, and phosphate and amino groups of the phospholipid complex are not detected. The number of thiol groups in the isoelectric zone has been determined using 6,6′-dithiodinicotinic acid. The number of anionogenic and cationogenic groups has been determined. From the density of the negative charges on the surface it is estimated that the isoelectric zone might extend up to 60 Å below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharide-phospholipid-protein complex. Since reactive groups of the phosphilipid component of the complex have not been detected in the isoelectric zone, it is suggested that the arrangement of lipopolysaccharide phospholipid protein complex is such that the phospholipids are located at a depth of more than 60 Å from the bacterial surface.     

Electrofusion of biochemically well characterized nitrate reductase deficient Nicotiana plumbagnifolia mutants. Studies of optimization and complementation

Electrofusion was carried out using biochemically well characterized NR⁻Nicotiana plumbaginifolia mutants (Cnx 20, Nia 26, NA 36). In analytical experiments, optimal conditions for mesophyll-mesophyll and callus-callus protoplast fusions were assessed. Subsequently, in large scale experiments NR⁺ somatic hybrids were obtained after mesophyll protoplast fusions between Cnx 20 + Nia 26 as well as after callus protoplast fusions between Cnx 20 + Nia 26 and between Cnx 20 + NA 36. In addition, complementation of the nia mutants Nia 26 and NA 36, each characterized by a distinct biochemical phenotype, was studied using electrofusion. In these experiments no completing NR⁺ somatic hybrid callus was obtained. As fusion conditions were optimal and fusion products were observed to be formed it was concluded that the nia mutations, although leading to distinct biochemical phenotypes, are allelic. We also studied complementation in short term experiments. NR activity in vivo was assayed 3–4 weeks after fusion. Plants could be regenerated from the majority of the NR⁺ somatic hybrid calli, resulting from the fusions between Cnx 20 + Nia 26 and Cnx 20 + NA 36. Chromosome numbers of shoot tip cells of glass house grown plants varied between 32–58, the majority having the normal tetraploid number (2n = 40). Most of the plants appeared to be sterile.     

Correlation between the Maintenance of Photosynthesis and in Situ Protoplast Volume at Low Water Potentials in Droughted Wheat

Studies were undertaken to examine the relationship between water deficit effects on photosynthesis and the extent of protoplast volume reduction which occurs in leaves at low water potential (Ψ_w). This relationship was monitored in two cultivars (`Condor' and `Capelle Desprez') of cultivated wheat (Triticum aestivum) that differed in sensitivity to drought, and in a wild relative of cultivated wheat (Triticum kotschyi) that has been previously found to be `drought resistant.' When subjected to periods of water stress, Condor and T. kotschyi plants underwent osmotic adjustment; Capelle plants did not. Photosynthetic capacity was maintained to different extents in the three genotypes as leaf Ψ_w declined during stress; Capelle plants were most severely affected. Calculations of internal leaf [CO₂] and stomatal conductance from gas exchange measurements indicated that differences in photosynthetic inhibition at low Ψ_w among the genotypes were primarily due to nonstomatal effects. The extent of protoplast volume reduction that occurred in leaves at low Ψ_w was also found to be different in the three genotypes; maintenance of protoplast volume and photosynthetic capacity in stressed plants of the genotypes appeared to be correlated. When the extent of water stress-induced inhibition of photosynthesis was plotted as a function of declining protoplast volume, this relationship appeared identical for the three genotypes. It was concluded that there is a correlative association between protoplast volume and photosynthetic capacity in leaves of wheat plants subjected to periods of water stress.