Barley Protoplasts Research Articles

Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

Template sequence near the initiation nucleotide can modulate brome mosaic virus RNA accumulation in plant protoplasts.

Bromoviral templates for plus-strand RNA synthesis are rich in A or U nucleotides in comparison to templates for minus-strand RNA synthesis. Previous studies demonstrated that plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase is more efficient if the template contains an A/U-rich template sequence near the initiation site (K. Sivakumaran and C. C. Kao, J. Virol. 73:6415-6423, 1999). These observations led us to examine the effects of nucleotide changes near the template's initiation site on the accumulation of BMV RNA3 genomic minus-strand, genomic plus-strand, and subgenomic RNAs in barley protoplasts transfected with wild-type and mutant BMV transcripts. Mutations in the template for minus-strand synthesis had only modest effects on BMV replication in barley protoplasts. Mutants with changes to the +3, +5, and +7 template nucleotides accumulated minus-strand RNA at levels similar to the the wild-type level. However, mutations at positions adjacent to the initiation cytidylate in the templates for genomic and subgenomic plus-strand RNA synthesis significantly decreased RNA accumulation. For example, changes at the third template nucleotide for plus-strand RNA3 synthesis resulted in RNA accumulation at between 18 and 24% of the wild-type level, and mutations in the third template nucleotide for subgenomic RNA4 resulted in accumulations at between 7 and 14% of the wild-type level. The effects of the mutations generally decreased as the mutations occurred further from the initiation nucleotide. These findings demonstrate that there are different requirements of the template sequence near the initiation nucleotide for BMV RNA accumulation in plant cells.

Cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts.

Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3'-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3'-proximal region of the CMV 3a ORF in B3Cmp with the 3'-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3'-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.

Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal.

The three genomic and a single subgenomic RNA of brome mosaic virus (BMV), an RNA virus infecting plants, are packaged by a single-coat protein (CP) into three morphologically indistinguishable icosahedral virions with T = 3 quasi-symmetry. Genomic RNAs 1 and 2 are packaged individually into separate particles whereas genomic RNA3 and subgenomic RNA4 (coat protein mRNA) are copackaged into a single particle. We report here that packaging of dicistronic RNA3 requires a bipartite signal. A highly conserved 3' tRNA-like structure postulated to function as a nucleating element (NE) for CP subunits (Y. G. Choi, T. W. Dreher, and A. L. N. Rao, Proc. Natl. Acad. Sci. USA 99:655-660, 2002) and a cis-acting, position-dependent packaging element (PE) of 187 nt present in the nonstructural movement protein gene are the integral components of the packaging core. Efficient incorporation into BMV virions of nonviral RNA chimeras containing NE and the PE provides confirmatory evidence that these two elements are sufficient to direct packaging. Analysis of virion RNA profiles obtained from barley protoplasts transfected with a RNA3 variant lacking the PE provides the first genetic evidence that de novo synthesized RNA4 is incompetent for autonomous assembly whereas prior packaging of RNA3 is a prerequisite for RNA4 to copackage.

Brome mosaic virus RNA syntheses in vitro and in barley protoplasts.

The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants.

The exposure of detached leaves of C3 plants (pea, barley) and C4 plant (maize) to 5 mM Pb (NO3)2 for 24 h caused a reduction of their photosynthetic activity by 40-60%, whereas the respiratory rate was stimulated by 20-50%. Mitochondria isolated from Pb2+-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb2+ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO2 conditions, indicated that the increased ATP/ADP ratio in Pb2+-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP+-sensitive electrode further showed that mitochondria isolated from Pb2+-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb2+ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.

Immobilization of barley protoplasts on a polyelectrolyte modified electrode for measuring the photoelectric behavior of protoplasts

A novel method to immobilize barley protoplasts on the poly(diallyl dimethyl ammonium chloride) gold/(PDADMAC) electrode was developed for the purpose to measure the photoelectric behavior of barley protoplasts. The electrochemical quartz crystal microbalance (EQCM) results show that the thickness of the adsorbed PDADMAC layer is 2.4 nm. The barley protoplasts are immobilized on the surface of gold/PDADMAC electrode due to the electrostatic adsorption between negatively charged protoplasts and positively charged PDADMAC. The fluorescence image taken by laser scanning confocal microscope shows that the attached barley protoplasts are integrity. For the gold/PDADMAC/barley protoplast electrode an anodic photocurrent was observed under the irradiation of white light (wavelength of 200–800 nm) and its properties are discussed. This novel method may provide a convenient technique for immobilizing cells or other bio-particles on the surface of electrode for studying their electrochemical characters.

Positional Effect of Deletions on Viability, Especially on Encapsidation, of Brome mosaic virus D-RNA in Barley Protoplasts

Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477–500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3′-proximal and the 5′-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs.

INFLUENCE ON ELECTROFUSION BY ADSORPTION OF FLUORESCENT DEXTRAN ON PROTOPLAST MEMBRANES

In this paper, the influence of negatively charged active fluorescent dextran on electrofusion of barley protoplasts was investigated. We discovered that this biopolymer inhibits the electrofusion process. The adsorption of fluorescent dextran on membranes was determined by observing their fluorescence microphotograph in protoplast suspension. A green fluorescent ring appeared around the membrane. The adsorption phenomena were also investigated by measuring the influence of additives on electrophoresis of protoplasts. It was detected that the membrane of barley protoplasts had a negative charge. After being stored in solution containing fluorescent dextran, the protoplasts demonstrated increased relative mobility due to the increase of negative charge on membranes by adsorption. The mobility decreased when the protoplasts were pretreated in the solution containing positively charged substances Bardac 2080. The increase of the membrane's negative charge by adsorption F-DX enhances the repulsion between protoplasts, and therefore decreases the electrofusion yield.

Effect of Heat Shock Treatment on Hordeum Vulgare Protoplast Transformation Mediated by Polyethylene Glycol

The stable transformation of barley protoplasts has been observed in cv. Dissa by PEG-mediated pGL2 plasmid DNA uptake containing hygromycin phosphotransferase (hph) gene under the control of 35S transcript of CaMV. Hygromycin resistant colonies were obtained, when protoplasts were incubated with hygromycin B (25 μg cm−3) at 3 – 4 cell stage. Heat shock treatment, prior to polyethylene glycol (PEG) treatment, markedly increased the protoplast transformation frequency. The presence of active hph gene product, and integration of hph gene was confirmed by hygromycin assay and Southern blotting, respectively. Analysis indicated that rearrangement had possibly occurred during the integration of plasmid DNA into genomic DNA of barley callus. These transformed calli showed no morphogenic response onto the regeneration medium.

Photorespiratory flux and mitochondrial contribution to energy and redox balance of barley leaf protoplasts in the light and during light-dark transitions

The contribution of mitochondrial oxidation of photorespiratory and respiratory substrates to subcellular energy and redox balance was investigated in leaf protoplasts of barley (Hordeum vulgare L.). The ATP/ADP ratios (indicating the energy balance) in chloroplasts and in the extrachloroplast compartment were highest in the light in limiting CO2 (photorespiratory conditions), and they drastically increased after illumination if plants were pre-incubated in darkness for 24 hours. After illumination, the ATP/ADP ratio rapidly decreased in chloroplasts. The NADPH/NADP ratio (as an indicator of redox balance) in chloroplasts declined rapidly during the first seconds of darkness, then slowly increased. In limiting CO2, the ratio decreased more slowly during the first minute of darkness corresponding to post-illumination respiratory burst (PIB). During this period, the activation state of chloroplast NADP-malate dehydrogenase was higher in limiting CO2 than in saturating CO2. However, during the light-enhanced dark respiration (LEDR) period, following PIB, there were no differences in subcellular NADPH/NADP ratios in saturating and limiting CO2. A decline in malate and citrate concentrations in protoplasts and activation of mitochondrial NAD-malic enzyme were revealed during LEDR. The results presented highlight the importance of glycine oxidation in mitochondria in energization of the cytosol and chloroplasts and in maintaining redox balance in the light and during the first minute after illumination. And further, they show non-photorespiratory origin of LEDR.

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

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

INFLUENCE OF POLYPEPTIDES AND PROTEINS ON ELECTROFUSION OF HUMAN CANCER CELLS

The electrofusion of human cancer U937 and K562 cells was studied under the influence of a selected group of polypeptides and proteins adsorbed at their membranes. For poly-L-lysine, we found that the higher the molecular mass (Mm) the higher the relative fusion yield (Fr), whereas poly-L-arginine and poly-L-ornithine showed toxicity. As recently measured for barley protoplast fusion, the same rule of dependence on the isoelectric point (pI) could be verified:which means 0.5 < Fr < 4 in 0.3 M mannitol solution (pH 6). If the pH of the 0.3 M mannitol solution is the same as the pI of the protein, its influence on the membrane nearly disappears. Bridging by Ca ions increases Fr from 0.5 (bovine serum albumin) to Fr ∼ 1 by charge compensation. The value of Fr is commensurate with the pore resealing time τ—determined by trypan blue staining—in such a way that for pI > 8, τ is longer and for pI < 6, τ is shorter than the control without protein content. Qualitatively, plant and animal membranes studied show equal behavior. According to our technique, the adsorption of biopolymers and other substances can be detected by this bioelectrochemical method, which is also a tool for analysis of electric stress mechanisms.

Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded proteins, 1a and 2a. The amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs. In this study, we examined the enzymatic activities of BMV 1a expressed in yeast, where the protein is fully functional in RNA replication. 1a methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-adenosylmethionine (AdoMet) as the methyl donor. Product analysis by nuclear magnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7. Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, and was accompanied by transfer of (3)H-methyl from AdoMet to the covalent 1a-guanylate complex. The covalent complex could be immunoprecipitated by 1a antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further methyltransferase reactions. Mutation of conserved amino acids in the N-terminal half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels. Covalent 1a-guanylate complex formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts. These results show that BMV 1a has activities similar to those of alphavirus nsP1, demonstrating conservation of these putative capping functions across a wide span of sequence divergence within the alphavirus-like superfamily. Conservation of this unusual combination of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.

Infection of barley protoplasts with rice hoja blanca tenuivirus. Brief report.

A barley protoplast system has been established that supports replication of Rice hoja blanca tenuivirus (RHBV). Following polyethylene glycol-mediated RHBV inoculation of barley protoplasts, newly synthesized viral RNAs and proteins could be detected. Time course analyses revealed de novo synthesis of genome length viral RNA4, as well as subgenomic-sized RNA4 molecules of both polarities. Two proteins, N and NS4, encoded by viral complementary RNA3 and viral RNA4 respectively, were detected by Western immunoblot analysis. The barley protoplast system thus constitutes a promising tool for in vivo studies of the sequential steps involved in the multiplication cycle of RHBV.

Brome mosaic virus defective RNAs generated during infection of barley plants.

Brome mosaic virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.

Influence of additives on the protoplasts electrofusion

Various neutral or charged surface active substances were used for testing the influence of additives on the electrofusion of barley protoplasts. It was found that neutral surface active agents DX, TAGB, Span-80 and AEO-9 could promote the electrofusion. The positively charged surface active agents Bardac 2080, Bardac 2280 and amphoteric surface active agents dodecyl-propyl betaine and CAB betaine also promote the electrofusion, but at high concentration the electrofusion efficiency will reduce. The negatively charged polymer agents Cibacron blue DX, Fluoresceinylthiocarbamoyl DX, and active surface substances K 12 and Carsonol TLS − presented negative effect. These phenomena were discussed from the view of adsorption of additives on the membrane and the interactions between protoplasts.

Differential Interactions of Bamboo Mosaic Potexvirus Satellite RNAs, Helper Virus, and Host Plants

Bamboo mosaic potexvirus (BaMV) supports the replication and encapsidation of a single-stranded satellite RNA (satBaMV). Full-length infectious cDNA clones of sat-BaMV variants with a 0.7 to 7.5% difference in nucleotide sequence were constructed, and in vitro transcripts were co-inoculated with BaMV RNA to barley protoplasts. Co-inoculation with BSL6 satBaMV transcripts resulted in a greatly reduced accumulation of genomic and subgenomic RNAs whereas co-inoculation with the prototype BSF4 or other variants did not. satBaMV variant BSL6-mediated interference occurred at the genomic RNA, subgenomic RNA, and capsid protein levels whereas the synthesis of minus strand was not significantly affected. Attenuation of the BaMV symptoms also was observed in Chenopodium quinoa and Nicotiana benthamiana plants co-inoculated with BSL6 satBaMV. In protoplasts of barley and N. benthamiana, BSF4 and BSL6 variants replicated at a similar level whereas in the inoculated leaves of C. quinoa and N. benthamiana BSL6 accumulated to a much lower level than BSF4, and was encapsidated. BSL6 satBaMV was rarely detected in the systemic leaves of N. benthami-ana co-inoculated with BaMV RNA, indicating that BSL6 satBaMV may be a movement-inefficient variant. It is suggested that this variant may provide a potential source for both resistance against BaMV and insights into the interactions of satellite RNA, helper virus, and symptom expression.

The role of mitochondrial electron transport during photosynthetic induction. A study with barley (Hordeum vulgare) protoplasts incubated with rotenone and oligomycin

Mitochondrial contribution to photosynthetic metabolism during photosynthetic induction was investigated in protoplasts from barley leaves (Hordeum vulgare L. cv. Gunilla, Svalöf) by using an inhibitor of mitochondrial Complex I (rotenone) and an inhibitor of the mitochondrial ATPase (oligomycin). Both inhibitors increased the lag phase of photosynthetic induction after the transition of protoplasts from darkness to light. This effect was not observed with broken protoplasts or isolated chloroplasts. Using the method of rapid fractionation of protoplasts it was shown that the delay in photosynthetic induction was accompanied by a decrease in ATP/ADP ratios of the cytosol and mitochondria, whereas the ratio in chloroplasts was not affected. A delay in activation of chloroplastidic NADP‐dependent malate dehydrogenase (EC 1.1.1.82) was observed in the presence of either inhibitor. A delay was also observed in the rise of photochemical quenching of chlorophyll fluorescence in the presence of rotenone or oligomycin during photosynthetic induction. The results indicate that during the transition from dark to light the mitochondrial electron transport chain and its Complex I participate in the reoxidation of excessive redox equivalents from photosynthetic electron transport.

Electropermeabilization and electrofusion of human lymphoma cells modified by proteolytic enzymes

Former results on protein-modified electrofusion of barley protoplasts have been extended by human cell membrane electropermeation and electrofusion after preincubation of cells with proteolytic enzymes. After pulsation, the amount of cells electroporated or broken down increased eight times and also the resealing time was prolonged. That means the lipid bilayer (protein content diminished by lysis) has a lower resistance against permeation. However, the rule of the relative electrofusion yield determination by the protein isoelectric point is still valid. This study of biopolymer interaction with human cell membranes is a prerequisite for drug incorporation (delivery) and electrotransformation by nucleic acids.