Light and benzylaminopurine induce changes in ultrastructure and gene expression in plastids of Petunia hybrida cell cultures

Summary The regulatory effect of light and the cytokinin 6-benzylaminopurine (BA) on the plastid ultrastructure and plastid DNA gene expression is studied in white and mutant green cell suspension cultures of Petunia hybrida. By electron microscopy we show that both light and 6-benzylaminopurine induce the formation of thylakoid membranes and grana structures in plastids of the green cultures. For membrane formation in plastids of white cultures, light in combination with BA is required. Light and benzylaminopurine also influence the plastid DNA gene expression. By in-organello protein synthesis with isolated plastids we show that light as well as benzylaminopurine affects the synthesis of plastid DNA encoded proteins. A characteristic effect of benzylaminopurine on plastids from white and green cultures is the reduction in the synthesis of the CFI subunits of 55,000 and 57,000 D, and the reduction in the synthesis of large polypeptides with a molecular weight higher than 67,000 D. In contrast to benzylaminopurine, light only affects the DNA gene expression of plastids from white cell cultures, that are in a very early stage of plastid development. Light stimulates the synthesis of polypeptides with a molecular weight of 84,000, 70,000 and 46,000 D which are encoded by cpDNA in these white culture plastids. In green cell cultures both plastids with a etioplast-like phenotype and with a chloroplast like morphology synthesize similar polypeptides, resulting in the same polypeptide pattern. Our results indicate that qualitative differences in plastid DNA gene expression as an effect of light do occur but only in plastids at very early stages of chloroplast development. We observe a gradual reduction in the number of high molecular weight polypeptides at later stages of chloroplast development. This suggests that these large polypeptides are characteristic for plastids at an early developmental stage.Abbreviations LSU of RuBPCase large subunit of Ribulose-1, 5-bisphosphate carboxylase - CF₁ coupling factor of the ATPase complex - LCH chlorophyll a/b protein - BA 6-benzylaminopurine - cpDNA chloroplast DNA     

A uniparental mutant of Chlamydomonas reinhardtii resistant to chloramphenicol

Summary A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed.     

Characterization of cytoplasmic mutants of Nicotiana tabacum with altered photosynthetic function

Summary A series of cytoplasmic mutants of tobacco (Nicotiana tabacum) were generated and characterized. Compared to wild type tobacco, they were found to have diminished levels of photosynthetic pigments and a range of functional impairments including modified chlorophyll fluorescence properties, loss of Photosystem I and/or II electron transport activity, and aberrant ultrastructure. Although the loss of defined functional activities was correlated with the depletion of specific thylakoid membrane proteins, no simple rules governed the relationship between structural defects and photosynthetic deficiencies. All of these mutants exhibited pleiotropic losses of polypeptides, including those known to be nuclear-encoded; this is consistent with the concept that loss of one component of a multi-subunit membrane protein complex results in unstable complex assembly. The phenotype of two mutants was developmentally regulated, in one case with slow chloroplast developments and in the other by premature senescence of Photosystem II centers as a function of leaf development. These mutants should be especially useful in studying membrane protein assembly.Abbreviations CF₁ coupling factor (chloroplast ATPase) - Chl chlorophyll - CP chlorophyll protein - Diuron 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DPIP 2,6-dichlorophenolindolphenol - EDTA ethylenediaminetetraacetic acid - F₀ initial fluorescence - F_p peak fluorescence - F_t terminal fluorescence - F_v variable fluorescence - kDa kilodalton(s) - LDS lithium dodecyl sulfate - LHC light harvesting complex - MV methyl viologen - OEC oxygen evolving complex - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PS photosystem - Q_A the primary quinone acceptor of Photosystem II - RC reaction center - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TMQH₂ tetramethyl-p-benzohydroquinone - TP tobacco plastome - Tricine N-tris (hydroxymethyl) methylglycine - WT wild type     

Cocksfoot mottle sobemovirus coat protein contains two nuclear localization signals

Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized to the cytoplasm and to the nucleus in plant cells indicating the involvement of the motif in nuclear localization. In mammalian cells, mutations in the arginine-rich region were sufficient to completely abolish nuclear transport. The analysis of deletions of amino acid residues 1–11, 1–22, and 22–33 of CP demonstrated that there were two separate nuclear localization signals (NLS) within the N-terminus—a strong NLS1 in the arginine-rich region (residues 22–33) and a weaker NLS2 within residues 1–22. Analysis of point mutants revealed that the basic amino acid residues in the region of the two NLSs were individually not sufficient to direct CP to the nucleus. Additional microinjection studies with fluorescently labeled RNA and CP purified from CfMV particles demonstrated that the wild-type CP was capable of transporting the RNA to the nucleus. This feature was not sequence-specific in transient assays since both CfMV and GFP mRNA were transported to the cell nucleus by CfMV CP. Together the results suggest that the nucleus may be involved in CfMV infection.     

Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation

Coat proteins (CPs) of geminiviruses are multifunctional proteins. Using transient expression experiments, we have recently identified putative sequence motifs of African cassava mosaic virus (ACMV) CP involved in nuclear import (NLS) and export (NES) (Virology 286 (2001) 373). Here, we report on the effect of corresponding deletion mutants in the context of infecting viruses. Since NLS and NES may overlap with DNA binding and multimerisation domains, we have investigated their effect on viral infection, particularly, on particle formation. All deletion mutants were infectious in Nicotiana benthamiana when co-inoculated with DNA B, but poorly sap-transmissible. Some of the mutants showed reduced levels of viral single-stranded DNA (ssDNA), whereas the amount of double-stranded DNA (dsDNA) was not greatly affected. None of these CP mutants was able to produce stable virus particles. In contrast, viruses with CP fused to Flag epitopes at the N- or C-terminus (CP:Flag or Flag:CP) were readily sap-transmissible and formed amorphous nucleoprotein particles but only few geminate structures. The relevance of the identified sequences in replicating viruses with reference to nuclear import and export as well as to particle stability and DNA binding is discussed.     

Characterization of host-range and temperature-sensitive mutants of adenovirus type 5 with particular regard to transformation of a hamster embryo cell line (Nil)

Seventeen conditional lethal mutants (7 host-range and 10 temperature-sensitive) of adenovirus type 5 (Ad5) were classified by complementation test and characterized physiologically in viral-DNA synthesis, induction of cell DNA synthesis (in hamster kidney cells), capsid polypeptides production, and transformation of Nil cells (a hamster embryo cell line) under the restrictive conditions.Seven host-range (hr) mutants were divided into six groups by complementation test and into three classes by phenotypic characterization. Mutants assigned to class III (complementation groups D, E, F) were positive in viral-DNA synthesis and capsid polypeptides (hexon, penton base, fiber) production, and showed some degree of leakiness. Class II mutants (complementation groups B, C) were positive in viral-DNA synthesis with a small amount of capsid polypeptides production. Class I mutant (complementation group A) was an early mutant defective in viral-DNA synthesis but positive in induction of host-DNA synthesis. Transformation of Nil cells was observed with classes I and II mutants and not with class III mutants.Ten temperature-sensitive (ts) mutants were divided into seven complementation groups and into five classes by the available phenotypic criteria. Class V mutant (complementation group G) was positive in viral-DNA synthesis and capsid polypeptides production with extreme leakiness. Class IV mutants (complementation groups E, F) were positive in viral-DNA synthesis and capsid polypeptides production. Class III mutants (complementation groups C, D) were quite similar to class IV except for reduced hexon production. Class II mutants (complementation group B) were early mutants defective in viral-DNA synthesis but positive in induction of host-DNA synthesis. Class I mutants (complementation group A) were similar to class II but with a reduced degree of induction of host-DNA synthesis. Transformation of Nil cells was observed with classes II, III, and IV mutants and not with I and V mutants.In brief, the phenotypic characterization of hr and ts mutants in infection of hamster cells showed a good correlation between complementation grouping and the defective function. Transformation of Nil cells was observed with most groups of the mutants except for the apparently leaky late groups and one group of early mutants under the restrictive conditions.     

Construction of a novel transposon mutagenesis system useful in the isolation of Streptococcus parasanguis mutants defective in Fap1 glycosylation

Streptococcus parasanguis, a primary colonizer of the tooth surface, has long, peritrichous fimbriae. A fimbria-associated protein, Fap1, is identified as an adhesin of S. parasanguis FW213. The mature Fap1 protein is glycosylated, and the glycosylation is required for fimbria biogenesis and bacterial adhesion. Little is known about the mechanism of Fap1 glycosylation due to the lack of identifiable mutants. A novel transposon mutagenesis system was established and used to generate a mutant library. Screening of the library with a monoclonal antibody specific for a glycan epitope of Fap1 yielded six mutants with decreased expression levels of surface-associated glycosylated Fap1 protein. Southern blot analyses revealed that three of the mutants had the transposon inserted in the fap1 locus, whereas the other three mutants had insertions in other genes. Among the latter three mutants, two expressed Fap1 polypeptides on which no glycosylation was detected by glycan-specific antibodies; the other mutant expressed a partially glycosylated Fap1 polypeptide. These data suggest that three mutants were isolated with defects in genes implicated in Fap1 glycosylation.     

Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17.

The polypeptides induced in cells infected with a Glasgow isolate of HSV-I (17 syn+) have been characterized by SDS polyacrylamide gel electrophoresis. Study of the kinetics of synthesis in three cell lines has detected a total of 52 polypeptides, 33 of which can be identified in polypeptide profiles of purified virions. These include six low mol. wt. polypeptides that have not been previously reported. Several polypeptides were labelled with glucosamine in infected BHK cells. The different polypeptide patterns obtained at permissive (31 degrees C) and nonpermissive (38 degrees C) temperature in cells infected with 16 temperature-sensitive (ts) mutants are reported. The effect of multiplicity of infection (m.o.i.) on the polypeptide profile has been examined for two of the DNA -ve mutants: below ten, the profile varied with the m.o.i. whereas above ten it was constant. All mutants were therefore examined at an m.o.i. of approx. 20. Mutants from the same complementation group showed very similar profiles. A number of general conclusions concerning control of protein synthesis in HSV infected cells can be made: (I) As most of the 16 ts mutants affected the synthesis of several or many polypeptides it follows that a large proportion of genome specifies controlling functions. (2) The high frequency with which some polypeptides were affected suggests they are at or near the terminus of biosynthetic pathways which are under multiple control. (3) Conversely, some polypeptides were affected with a low frequency suggesting that their synthesis is not dependent on the expression of many virus functions. (4) Several individual ts mutations lead to the synthesis of increased amounts of different large polypeptides. (5) Analysis of every band detectably affected by at least one ts mutation has disclosed nine classes of dependence relationship between polypeptide synthesis and the DNA phenotype of the mutants, illustrating that this relationship is complex and different for different polypeptides. (6) The inhibition of host protein synthesis by the virus may not be a simple single step process.     

Characterization of a chloroplast mutation in the psaA2 gene of Chlamydomonas reinhardtii

Summary The synthesis of polypeptides related to the CPI chlorophyll-protein complex of photosystem I has been studied by pulse-labeling experiments in twenty chloroplast mutants of Chlamydomonas reinhardtii. Three mutations of the same locus (Girard-Bascou 1987) result in the absence of these CPI-related polypeptides. Among these mutations one, (FUD26) leads to the synthesis of a new polypeptide presumed to be a truncated CPI apoprotein. The molecular characterization of this mutation in the psaA2 gene has been achieved by DNA sequencing the 3 end of this gene. The FUD26 mutation is a 4 base pair deletion resulting in frameshift and premature termination of the protein.     

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

Caenorhabditis elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear genes that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD⁺-dependent ketoacid oxidation occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations both among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease.     

Cooperative effect of two amino acid mutations in the coat protein of Pepper mild mottle virus overcomes L3-mediated resistance in Capsicum plants

We found that an L ³ resistance-breaking field isolate of Pepper mild mottle virus (PMMoV), designated PMMoV-Is, had two amino acid changes in its coat protein (CP), namely leucine to phenylalanine at position 13 (L13F) and glycine to valine at position 66 (G66V), as compared with PMMoV-J, which induces a resistance response in L ³ -harboring Capsicum plants. The mutations were located to a CP domain corresponding to the outer surface of PMMoV particles in computational molecular modeling. Analyses of PMMoV CP mutants containing either or both of these amino acid changes revealed that both changes were required to efficiently overcome L ³ -mediated resistance with systemic necrosis induction. Although CP mutants containing either L13F or G66V could not efficiently overcome L ³ -mediated resistance, these amino acid changes had different effects on the elicitor activity of PMMoV CP. L13F caused a slight reduction in the elicitor activity, resulting in virus restriction to necrotic local lesions that were apparently larger than those induced by wild-type PMMoV, while G66V rendered wild-type PMMoV the ability to overcome L ³ -mediated resistance, albeit with a lower efficiency than PMMoV with both changes. These results suggest that a cooperative effect of the L13F and G66V mutations on the elicitor activity of CP is responsible for overcoming the L ³ -mediated resistance.     

Translational accuracy and sexual differentiation in Chlamydomonas reinhardtii

Summary A renewal of ribosomes has been previously reported to occur during gametogenesis in C. reinhardtii. In order to further characterize these new ribosomes, we performed pulse-labelling experiments on whole cells of C. reinhardtii, during gametogenesis and in the presence of various aminoglycosides known to alter translational accuracy: Hygromycin and Paromomycin are assumed to increase the rate of translational errors at the level of 80S and 70S ribosomes whereas Kasugamycin is assumed to induce the opposite effect. Three lines of evidence support an increased inaccuracy in protein translation during gametogenesis: (1) gamete cells displayed a higher sensitivity than vegetative cells to Hygromycin and Paromomycin; 4 g/ml Hygromycin cancelled cytoplasmic protein synthesis in gametes but not in vegetative cells; Paromomycin induced the synthesis of new polypeptides of high molecular weight and of nuclear origin in gametes but not in vegetative cells. In addition, chloroplast protein synthesis was more sensitive to Hygromycin and Paromomycin in gametes than in vegetative cells. (2) Kasugamycin-sensitive alterations of thylakoid membranes were detected during gametogenesis. (3) ³⁵S-misincorporation in the OEE3 polypeptide, of nuclear origin and normally devoid of sulphur containing amino acids, was more than three times higher in gametes than in vegetative cells. This increase was prevented by Kasugamycin, suggesting that 80S translation in gametes was more inaccurate than in vegetative cells. The possible significance of these changes occurring during gametogenic differentiation is discussed in light of the importance of a modulation of translational accuracy at particular stages of the life cycle in other lower eukaryotes.Abbreviations Hm Hygromycin B - Pm Paromomycin - Ks Kasugamycin - CAP Chloramphenicol - OEE Oxygen evolving enhancer     

Plastid inheritance in Oenothera: organelle genome modifies the extent of biparental plastid transmission

Summary The transmission abilities of four out of the five major plastome types of Oenothera (I–V) were analyzed in a constant nuclear background by assessing both the frequency of biparental inheritance and the extent of variegation in the progeny. Reciprocal crosses were performed between plants carrying one of four wild-type plastomes and plants carrying one of seven white plastid mutants. The frequency of biparental plastid transmission ranged from 0 to 56% depending on the plastid types involved in the crosses. The transmission abilities of the four representative wild-type plastids appear to be in the order of I > III > II > IV in the nuclear background of O. hookeri str. Johansen. In general, variegated seedlings from crosses that produced a higher frequency of biparental plastid transmission also had an increased abundance of tissue containing plastids of paternal origin. Although the transmission abilities of most Oenothera plastid mutants are comparable to the wild-type plastids, three mutant plastids derived from species having different type I plastids show three distinguishable transmission patterns. This study confirms the significant role of the plastome in the process of plastid transmission and possibly in plastid multiplication. However, the hypothesis of differential plastid multiplication rates suggested by earlier studies can explain the results only partially. The initiation of plastid multiplication within the newly formed zygote also seems to be plastome-dependent.     

Chloroplast thylakoid membrane polypeptides and cytoplasmic male sterility

Summary Comparative studies on the chloroplast thylakoid membrane polypeptides between cytoplasmic male sterile lines and their respective maintainers from sugar beets, maize and sorghum were performed by applying two dimensional electrophoretic separation techniques. Differences in the size, number and distribution of resolved peptide spots near 33 kd were observed between CMS and their maintainers. This indicates that variation of composition of the thylakoid membrane proteins is correlated with the pollen sterility.     

Fine structure of OXI1, the mitochondrial gene coding for subunit II of yeast cytochrome c oxidase

Summary Genetic and biochemical studies have been performed with 110 mutants which are defective in cytochrome a·a₃ and map in the regions on mit DNA previously designated OXI1 and OXI2. With 88 mutations allocated to OXI1 fine structure mapping was achieved by the analysis of rho ^– deletions. The order of six groups of mutational sites (A 1, A2, B 1, B2, C 1, C2) thus determined was confirmed by oxi _i x oxi _j recombination analysis.Analysis of mitochondrially translated polypeptides of oxil mutants by SDS-polyacrylamide electrophoresis reveals three classes of mutant patterns: i) similar to wild-tpye (19 mutants); ii) lacking SU II of cytochrome c oxidase (53 mutants); iii) lacking this subunit and exhibiting a single new polypeptide of lower M_r (16 mutants). Mutations of each of these classes are scattered over the OXI1 region without any detectable clustering; this is consistent with the assumption that all oxil mutations studied are within the same gene.New polypeptides observed in oxil mutants of class iii) vary in Mr in the range from 10,500 to 33,000. Those of M_r 17,000 to 33,000 are shown to be antigenically related to subunit II of cytochrome c oxidase. Colinearity is established between the series of new polypeptides of M_r values increasing from 10,500 to 31,500 and the order of the respective mutational sites on the map, e.g. mutations mapping in A 1 generate the smallest and mutations mapping in C2 the largest mutant fragments.From these data we conclude that i) all mutations allocated to the OXI1 region are in the same gene; ii) this gene codes for subunit II of cytochrome c oxidase; iii) the direction of translation is from CAP to 0X12. Out of 19 mutants allocated to OXI2 three exhibit a new polypeptide; these and all the other oxi2 mutants lack subunit III of cytochrome oxidase. This result provides preliminary evidence that the OXI2 region harbours the structural gene for this subunit III.Abbreviations mitDNA mitochondrial DNA - SU I, SU II, SU III subunits of cytochrome c oxidase - SDS sodium dodecyl sulfate - M_r Molecular weight - d Dalton     

Protein synthesis in cells infected by Autographa californica nuclear polyhedrosis virus (Ac-NPV): the effect of cytosine arabinoside

Twenty-two virion polypeptides (VP) were detected reproducibly when the occluded form (OV) of [35S]methionine-labeled Ac-NPV, purified from polyhedral inclusion bodies (PIB), was analyzed by polyacrylamide gel electrophoresis and autoradiography. Ten of the VPs and the polyhedral protein (PP) were phosphorylated and 6 VPs were glycoproteins. Purified, nonoccluded virus (NOV) revealed 25 polypeptides. During a single cycle of virus replication, the synthesis of 33 infected cell polypeptides (1CP) was detected in different relative proportions at different times. They were arbitrarily designated as early (0-12hr), middle (12-18 hr), and late 18-24 hr) polypeptides. Most of the middle and late proteins seemed to be viral structural proteins. Rapid post-translational cleavage of IPCs was not observed; however, pulse-chase experiments revealed post-translational modifications of at least four polypeptides. Inhibition of DNA synthesis at the time of infection did not prevent the synthesis of ICPs, VPs, or the formation of PIBs. Progeny PIB from both control and cytosine arabinoside (Ara C)-treated cells that were infected with radiochemically pure [3H]thymidine-labeled NOV contained parental virus DNA. Electron micrographs of thin sections showed that, whereas PIBs from control cultures contained complete virions, those from Ara (C-treated cells contained both full and empty virus.