In Vitro DNA Synthesis by Chloroplasts Isolated from Marchantia polymorpha L. Cell Suspension Cultures

Lysate of chloroplasts prepared from liverwort Marchantia polymorpha L. cell suspension cultures incorporated [³H]-dTTP into acid insoluble materials when DNA was added exogenously as a template. The incorporation was highly dependent on the addition of template DNA, four deoxynucleoside triphosphates and magnesium ions (maximum incorporation at 5mM). Magnesium ions could be replaced by manganese ions. DNA synthesis inhibitors, N-ethylmaleimide (NEM) and ethidium bromide (EtBr), strongly inhibited the incorporation. Dideoxythymidine triphosphate (ddTTP), an inhibitor of DNA polymerases β and γ, inhibited the incorporation at the concentration of 50 μM (molar ratio of ddTTP/dTTP = 17). On the other hand, the incorporation by the chloroplast lysate was resistant to arabinofuranosyl cytosine triphosphate (araCTP) and aphidicolin as well as the RNA polymerase inhibitors, rifampicin and α-amanitin. The chloroplast lysate highly utilized denatured calf thymus DNA and bacteriophage ?X174 single-stranded DNA as templates when added exogenously, while a synthetic homopolymer, poly(rA)-oligo(dT)₁₂ ~ ₁₈, did not stimulate the incorporation at all. Autoradiographic analysis of DNA synthesized in isolated chloroplasts showed that the chloroplast DNA synthesis took place at several specific sites on the chloroplast DNA from cells of the liverwort, Marchantia polymorpha.     

A Myosin XI Tail Domain Homologous to the Yeast Myosin Vacuole-Binding Domain Interacts with Plastids and Stromules in Nicotiana benthamiana

The actin cytoskeleton plays a role in mobility of many different organelles in plant cells, including chloroplasts, mitochondria, Golgi, and peroxisomes. While progress has been made in identifying the myosin motors involved in trafficking of various plant organelles, not all of the cargoes mobilized by different members of the myosin XI family have yet been identified. The involvement of myosins in chloroplast positioning and mitochondrial movement was demonstrated by expression of a virus-induced gene silencing （VIGS） construct in tobacco. When VIGS with two different conserved sequences from a myosin Xl motor was performed in plants with either GFP-labeled plastids or mitochondria, chloroplast positioning in the dark was abnormal, and mitochondrial movement ceased. Because these and prior obser- vations have implicated a role for myosins and the actin cytoskeleton in plastid and stromule movement, we searched for myosin tail domains that could associate with plastids and stromules. While a yellow fluorescent protein （YFP） fusion with the entire tail region of myosin XI-F was usually found only in the cytoplasm, we observed that an Arabidopsis or Nicotiana benthamiana YFP：：myosin XI-F tail domain homologous to the yeast myo2p vacuole-binding domain associated with plastids and stromules after transient expression in N. benthamiana. Taken together, these observations implicate myosin motor proteins in dynamics of plastids and stromules.     

Chloroplast Phosphoglycerate Kinase Is Involved in the Targeting of Bamboo mosaic virus to Chloroplasts in Nicotiana benthamiana Plants

The Bamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus. Previously, we identified that the chloroplast phosphoglycerate kinase (chl-PGK) from Nicotiana benthamiana is one of the viral RNA binding proteins involved in the BaMV infection cycle. Because chl-PGK is transported to the chloroplast, we hypothesized that chl-PGK might be involved in viral RNA localization in the chloroplasts. To test this hypothesis, we constructed two green fluorescent protein (GFP)-fused mislocalized PGK mutants, the transit peptide deletion mutant (NO TRANSIT PEPTIDE [NOTP]-PGK-GFP) and the nucleus location mutant (nuclear location signal [NLS]-PGK-GFP). Using confocal microscopy, we demonstrated that NOTP-PGK-GFP and NLS-PGK-GFP are localized in the cytoplasm and nucleus, respectively, in N. benthamiana plants. When NOTP-PGK-GFP and NLS-PGK-GFP are transiently expressed, we observed a reduction in BaMV coat protein accumulation to 47% and 27% that of the wild-type PGK-GFP, respectively. To localize viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence. Using confocal microscopy, we observed that BaMV viral RNA localizes to chloroplasts. Furthermore, elongation factor1a fused with the transit peptide derived from chl-PGK or with a Rubisco small subunit can partially restore BaMV accumulation in NbPGK1-knockdown plants by helping BaMV target chloroplasts.Bamboo mosaic virus (BaMV) is a single-stranded, positive-sense RNA virus. The genomic RNA of BaMV contains five open reading frames (ORFs) and is 6,366 nucleotides in length with a 5′ cap and a 3′ poly(A) tail (Lin et al., 1994; Yang et al., 1997). ORF1 encodes a 155-kD replicase comprised of a capping enzyme domain that exhibits S-adenosylmethionine-dependent guanylyltransferase activity (Li et al., 2001a; Huang et al., 2004), a helicase-like domain with RNA 5′-triphosphatase activity (Li et al., 2001b), and an RNA-dependent RNA polymerase domain (Li et al., 1998; Cheng et al., 2001). The three overlapping ORFs (i.e. ORF2, ORF3, and ORF4) are known as the triple gene block. They encode for proteins involved in viral movement (Lin et al., 2004, 2006; Vijaya Palani et al., 2006). ORF5 encodes the viral capsid protein (CP), required for virion assembly and viral movement (Cruz et al., 1998).The genomes of positive-strand RNA viruses are templates for both translation and replication. Viral replication complexes are likely to be assembled using host factors to synthesize the minus-strand RNA and then the plus-strand progeny RNA. Recent studies have shown that host factors play important roles in assembling the viral RNA replication complex, selecting and recruiting viral replication templates, activating the complex for RNA synthesis, and other steps (Ahlquist et al., 2003; Patarroyo et al., 2012). The translation and the minus-strand RNA synthesis of poliovirus are regulated by host poly(C) and poly(A) binding proteins and viral polymerase precursor 3CD (Waggoner and Sarnow, 1998; Herold and Andino, 2001; Walter et al., 2002). A number of host genes required for Brome mosaic virus replication have been identified systemically by the yeast (Saccharomyces cerevisiae) genetic approach (Ishikawa et al., 1997; Kushner et al., 2003; Mas et al., 2006; Gancarz et al., 2011). The same approach was used to identify the host factors involved in the replication of Tomato bushy stunt virus (TBSV; Panavas et al., 2005; Li et al., 2009b). A heat shock protein90 homolog (Huang et al., 2012) and the Nicotiana benthamiana glutathione transferase U4 (NbGSTU4; Chen et al., 2013), were identified to interact with the 3′ untranslated region (UTR) of BaMV RNA and enhanced the minus-strand RNA synthesis at the early replication step. The Ser/Thr kinase-like protein localized on cell membrane facilitates the BaMV intercellular movement (Cheng et al., 2013).Previously, we have identified two host proteins (i.e. p51 and p43) interacting specifically with the 3′ UTRs of BaMV by using electrophoretic mobility shift assay (EMSA) and the UV cross-linking competition technique. The results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and BLAST indicate that the protein sequences of p43 and p51 match the chloroplast phosphoglycerate kinase (chl-PGK) and elongation factor1a (EF1a) of Nicotiana benthamiana, respectively (Lin et al., 2007). Phosphoglycerate kinase is an ATP-generating enzyme that acts in the glycolytic, gluconeogenic, and photosynthetic pathways (Banks et al., 1979; McHarg et al., 1999). chl-PGK is encoded in the nucleus and translated to produce a 50-kD precursor protein and is then processed into mature 43 kD in the chloroplast. In a knockdown experiment through virus-induced gene silencing, the reduction of PGK decreased the accumulation of BaMV coat protein (Lin et al., 2007).Eukaryotic EF1a has been shown to play a role in binding to the tRNA-like structure and upstream pseudoknot in the 3′ UTR of Tobacco mosaic virus to regulate the gene expression and viral replication (Pathak et al., 2008). EF1a has also been involved in the recruitment of viral RNA and has facilitated the replicase complex assembly of TBSV (Pogany et al., 2008). The 3′ UTR of BaMV cannot only bind its replicase but also the EF1a and has been proposed to regulate viral RNA replication (Lin et al., 2007).In this study, we transiently expressed two mislocalized PGK mutants to study the possible functions of chl-PGK that is involved in viral RNA replication. In addition, we used confocal microscopy to investigate the localization of BaMV RNA. Finally, we provided evidence that the down-regulation of BaMV accumulation in PGK-knockdown plants can be restored by the expression of the BaMV RNA binding protein EF1a that is fused to a chloroplast transit peptide.     

The Rate of Photosynthesis in Isolated Chloroplasts

Chloroplasts were isolated using aqueous and nonaqueous procedures.Aqueous chloroplasts lost approximately 50 per cent, of theirsoluble proteins during isolation. Nonaqueous chloroplasts retainedall their soluble enzymes, but lost their ability to performthe light reactions of photosynthesis. It was possible to reconstitutea chloroplast system of higher activity by adding soluble enzymesfrom nonaqueous chloroplasts to protein-deficient aqueous chloroplasts.The properties of the reconstituted chloroplast system wereas follows: 1. The CO₂ fixation rate of the reconstituted chloroplast system( 4 µM./. chlorophyll/hr.) was 3–4 times that ofthe aqueous chloroplasts ( I µM./. chlorophyll/hr.). Thefixation of aqueous chloroplasts isapparently limited in partby lack of soluble enzymes. 2. During light-fixation, the reconstituted chloroplast systemaccumulated PGA. This indicates that the reduction of PGA totriosephosphate is a rate-limiting step in this system. 3. It was possible to increase the CO₂ fixation to 12 µM.CO₂/mg. chlorophyll/ hr. by addition of ATP and TPNH to thesystem, but the reduction of PGA was still rate-limiting. 4. Further increase in the fixation rate was obtained by concentratingthe reaction mixture. Part of the striking differences of theCO₂-fixing capabilities of chloroplasts in vivo and in vitrois caused by dilution effects. Extrapolation of the dilutioneffect to the protein concentration which exists in chloroplastsyields a CO₂ fixation rate of approximately 30 µM./mg.chlorophyll/hr. 5. Inhibitors which are located in vivo outside the chloroplastsaffect the CO₂ fixation in vitro. 6. Under consideration of the examined factors which influencethe CO₂ fixation of isolated chloroplasts, it is possible toraise the fixation from approximately 1 per cent, to at least15 per cent, of the fixation in vivo.     

Photochemical Activity of Chloroplasts Isolated from Etiolated Plants

The rates of phosphorylation, ferricyanide, and dye reductionwere determined with chloroplasts isolated from Linum usitatissimumgrown in darkness and subjected to periods of light of differentduration. With an increased period of illumination, the chlorophyllcontent increased as did also the rate of the three processesmeasured, but no correlation between these two factors was observed.Neither was there any correlation between the rate of any photochemicalreaction and the plastoquinone content. It was concluded thatsome unspecified factor, possibly structural, which developsduring illumination must control the rate of the photochemicalreactions.     

Novel Glycerolipids and Glycolipids from the Surface Lipids of Nicotiana benthamiana

The surface lipids of Nicotiana benthamiana contained novel glycerolipids and several varieties of glycolipids. As glycerolipids, the triacylglycerol, 1,3-diacylglycerol, and 1,2-diacylglycerol types of glycerolipids were isolated and identified. Each lipid contained acetyl, 16–methylheptadecanoyl, and 18–methylnonadecanoyl moieties. The acetylated position of each lipid was determined by 2D-NMR, using the HMBC technique. The structures were 1,3-di-O-acetyl-2-O-acylglycerol, 1-O-acetyl-3-O-acylglycerol, and 1-O-acetyl-2-O-acylglycerol. As glycolipids, one glucose ester and four types of sucrose esters were isolated and identified. These glycolipids contained acetic acid and such branched short-chain fatty acids as 5-methylhexanoic, 4-methylhexanoic, 6-methylheptanoic, and 5-methylheptanoic acids. The structure of the glucose ester was 3,4-di-O-acyl-α-D-glucopyranose. The structures of the sucrose esters were 6-O-acetyl-4-O-acyl-α-D-glucopyranosyl-(3-O-acyl)-β-D-fructofuranoside, 4-O-acyl-α-D-glucopyranosyl-(3-O-acyl)-β-D-fructofuranoside, 3,4-di-O-acyl-α-D-glucopyranosyl-β-D-fructofuranoside, and 6-O-acetyl-α-D-glucopyranosyl-β-D-fructofuranoside.     

Formation of In Vitro Mixed-Species Biofilms by Lactobacillus pentosus and Yeasts Isolated from Spanish-Style Green Table Olive Fermentations

The present work details the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain cocultures. However, biofilm formation was inhibited in the presence of d-(+)-mannose. In addition, biofilm formation by C. boidinii monoculture was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of “sticky” material formed by the yeasts contributed to substrate adherence. Hence, the data obtained in this work suggest that yeast-lactobacilli biofilms may be favored by the presence of a specific mate of yeast and L. pentosus, and that more than one mechanism might be implicated in the biofilm formation. This knowledge will help in the design of appropriate mixed starter cultures of L. pentosus-yeast species pairs that are able to improve the quality and safety of Spanish-style green table olive processing.     

In Vitro Action of Carbenicillin Against Bacteria Isolated from Clinical Material

More than 500 bacteria isolated from patient material were tested against carbenicillin (disodium alpha-carboxybenzylpenicillin) by diffusion and dilution modalities. The same bacteria, which included Pseudomonas aeruginosa, Escherichia coli, Klebsiella-Aerobacter-Enterobacter group, various species of Proteus, Staphylococcus aureus and epiddermidis, enterococci, pneumococci, Streptococcus pyogenes, etc., were examined for susceptibility to other antibiotics commonly used with special emphasis on ampicillin and cephalothin. The responses of pyocine-typed P. aeruginosa were the most remarkable. The majority of these bacteria displayed susceptibility to carbenicillin by both the dilution and the diffusion techniques. The concentrations of this antibiotic used in the laboratory were of the same order of magnitude as that of the other drugs. The laboratory behavior of the other bacteria, toward this new semisynthetic penicillin derivative approximated their response to ampicillin and cephalothin.     

Parental origin of the allotetraploid tobacco Nicotiana benthamiana

Nicotiana section Suaveolentes is an almost all‐Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4–5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long‐term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.     

The Formation Of Parallel RNA-RNA Duplexes In Vitro

Abstract

Over the years the structural properties of nucleic acids have been of interest, providing data that may be of importance for DNA and RNA organization and function in the cell. We have attempted to look for the formation of parallel RNA-RNA duplexes in vitro. RNA molecules comprising complementary in the same polarity alternating stretches of A and U of increasing length were enzymatically synthesized and annealed in physiological conditions. The fractionation in the denaturing Polyacrylamide gels revealed the formation of two types of full-length parallel RNase A - stable duplexes established either by A-U or by A-A and U-U self pairs. These results suggest novel structural properties of versatile RNA molecules that potentially may be realized in vivo.     

Flower Formation from Citrus unshiu Buds Cultured In Vitro

Flowers were formed in vitro when buds of Satsuma mandarin (Citrusunshiu Mare) from the summer flush of growth harvested duringthe winter rest period before the onset of flower initiation,were grown on a solid Murashige and Skoog medium supplementedwith sucrose and a cytokinin. Flower development was dependentupon illumination, and was enhanced when a piece of stem wasattached to the bud. The percentage of flowering explants wasalways lower than the percentage of naturally flowering budsin spring, but treatments such as ringing which increase floweringin vitro, increased the number of explants flowering int vitroas well. Citrus unshiu Marc, ‘Satsuma’ mandarin, in vitro flowering, ringing     

利用病毒载体在烟草中瞬时表达融合HBsAg基因

利用马铃薯PVX病毒载体构建了外源人工融合乙肝表面抗原HBsAg基因的表达载体,在烟草中利用农杆菌介导进行瞬时表达,以快速鉴定外源基因瞬时表达的状况以及重组蛋白的免疫活性。利用PCR技术从含有人工融合HBsAg基因的表达载体中分别扩增出LP PreS1 PreS2 S、PreS1 PreS2 S、PreS2 S序列,将其分别与PVX病毒载体pgR106连接,构建成PVX-LP、PVX-S1和PVX-S2等3个转化载体,并将此载体导入农杆菌菌株GV3101中用于侵染烟草植株叶片。感染植株经RT-PCR、RNA Dot blotting和HBsAg蛋白的ELISA检测显示,3个人工融合的HBsAg基因均可在植物体内得到转录,翻译成具有活性的蛋白。结果表明,外源融合HB-sAg基因经过植物病毒载体瞬时表达系统可以在植物系统中正常转录和翻译。     

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Plant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.     

Physiological function of NbRanBP1 in Nicotiana benthamiana

This study addresses the physiological functions of the Ran-binding protein homolog NbRanBP1 in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbRanBP1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. The NbRanBP1 gene was constitutively expressed in diverse tissues and an NbRanBP1:GFP fusion protein was primarily localized to the nuclear rim and the cytosol. BiFC analysis revealed in vivo interaction between NbRanBP1 and NbRan1 in the nuclear envelope and the cytosol. Depletion of NbRanBP1 or NbRan1 reduced nuclear accumulation of a NbBTF3:GFP marker protein. In the later stages of development, NbRanBP1 VIGS plants showed stress responses such as reduced mitochondrial membrane potential, excessive production of reactive oxygen species, and induction of defense-related genes. The molecular role of RanBP1 in plants is discussed in comparison with RanBP1 function in yeast and mammals.     

The Purity of Chloroplasts Isolated in Non-aqueous Media

By measuring the relative amounts of high-molecular-weight ribonueleicacids in chloroplasta and in cytoplasm reliable values wereobtained for the purity of chloroplasts isolated in non-aqueousmedia from leaves of tobacco (Nicotiana tabacum, var. WhiteBarley), broad bean (Viciafaba, var. White Fan), and tomatoplants (Lycopersicon esculenium, var. Money Maker). Measurementsof pyruvate kinase activity, previously used to test chloroplastpurity, agreed well with the results of ribosomal ribonucleic-acidanalysis for the bean and tomato leaves. The purest chloroplastfractions from tobacco leaves always contained more pyruvatekinase than could be accounted for on the basis of the cytoplasmiccontamination measured by the nucleic-acid analysis. Some pyruvatekinase may therefore be present in the chloroplasts in tobaccoleaves. The purest chloroplasts obtained from any of the three speciesstill contained 11 per cent of the cytoplasm even after severemechanical treatments designed to remove cytoplasm adheringto the surface of the plastids. Chloroplast fractions obtainedby the usual non-aqueous techniques always contained at least15 per cent of the cytoplasm.     

Microbial Formation of Nitrosamines In Vitro

Mortierella parvispora and an unidentified bacterium converted trimethylamine to dimethylamine, and the bacterium (but not the fungus) formed dimethylnitrosamine in the presence of nitrite. Dimethylnitrosamine also appeared in cell suspensions of Escherichia coli and Streptococcus epidermidis and in hyphal mats of Aspergillus oryzae incubated with dimethylamine and nitrate. Suspensions of a number of microorganisms produced N-nitrosodiphenylamine from diphenylamine and nitrite at pH 7.5, and soluble enzymes catalyzing the N-nitrosation of diphenylamine were obtained from two of these organisms. In the presence of these enzymes, several dialkylamines were converted to the corresponding N-nitroso compounds.