Brent L. Nielsen

The mystery of the rings: structure and replication of mitochondrial genomes from higher plants

Higher plant mitochondrial genomes are unique in their size, complexity and evolutionary dynamics. However, in spite of the existence of physical maps, the structural organization of the mitochondrial genome in vivo is not fully understood. The molecules observed are mainly linear or complex, and some of them are much larger than predicted from the genome size. In addition, circular DNA species are found in a continuous size distribution up to >100 kb — although this is heavily skewed towards small molecules. There is a high single-stranded DNA content, and both theta- and sigma-modes of replication have been reported. Recombination between large and small repeats as well as amplification by replication appear to contribute considerably to mitochondrial genome diversity.

Photosynthesis in Salt-Adapted Heterotrophic Tobacco Cells and Regenerated Plants.

Tobacco (Nicotiana tabacum L.) cells growing heterotrophically in the light on supplied sucrose (S0) have previously been adapted to grow in 428 mM NaCl (S25). Among the changes occurring in salinity-adapted cell cultures are (a) elevated levels of chlorophyll compared to unadapted cells; (b) decreased levels of starch; (c) alterations in chloroplast ultrastructure, including loss of starch grains, increased thylakoid membrane structure, and the presence of plastoglobules; and (d) increased rates of O2 evolution, CO2 fixation, and photophosphorylation relative to S0 cells. These latter changes apparently derive from the fact that thylakoid membranes in S25 cells contain higher levels of photosystem I- and II-associated proteins as well as thylakoid ATPase components. S25 chloroplasts contain immunologically detectable levels of ribulose-1,5-bisphosphate carboxylase/oxygenase, whereas S0 completely lack the enzyme. These changes taken together suggest that even in the presence of sucrose, S25 cells have acquired a significant degree of salt-tolerant photosynthetic competence. This salt-tolerant photoysynthetic capability manifests itself in plants backcrossed with normal plants for three generations. These plants contain chloroplasts that demonstrate in vitro more salt-tolerant CO2 fixation, O2 evolution, and photophosphorylation than do backcross progeny of plants regenerated from S0 cultures.

An Arabidopsis homologue of bacterial RecA that complements an E. coli recA deletion is targeted to plant mitochondria

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mtrecA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mtrecA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mtrecA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mtrecA in homologous recombination and/or repair in Arabidopsis mitochondria.

Chloroplast DNA Replication : Mechanism, Enzymes and Replication Origins

Chloroplasts contain circular DNA molecules which are found in low copy number in proplastids but are amplified to very high copy number in actively dividing leaf cells. A double displacement loop (D-loop) mechanism for chloroplast DNA (ctDNA) replication has been proposed, and pairs of replication origins which fit this model have been identified in some species. It appears that ctDNA replication is under the control of at least some nuclear gene products, as genes for DNA polymerase, topoisomerases, DNA primase and other accessory replication proteins have not been reported in the sequenced chloroplast genomes, and ctDNA replication remains active in the absence of active chloroplast transcription or translation. Only a few chloroplast replication proteins have been isolated, and to date most have not been characterized in detail. The mechanism by which ctDNA copy number is regulated during plant development is not known. In this review we summarize the current understanding of ctDNA replication.

CHARACTERIZATION OF REPLICATION ORIGINS FLANKING THE 23S RRNA GENE IN TOBACCO CHLOROPLAST DNA

Using 5′ end-labeled nascent strands of tobacco chloroplast DNA (ctDNA) as a probe, replication displacement loop (D-loop) regions were identified. The strongest hybridization was observed with restriction fragments containing the rRNA genes from the inverted repeat region. Two-dimensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb BamHI fragment containing part of the rRNA operon. Analysis of in vitro replication products indicated that templates from either of the origin regions supported replication, while the vector alone or ctDNA clones from other regions of the genome did not support in vitro replication. Sequences from both sides of the BamHI site in the rRNA spacer region were required for optimal in vitro DNA replication activity. Primer extension was used for the first time to identify the start site of DNA synthesis for the D-loop in the rRNA spacer region. The major 5′ end of the D-loop was localized to the base of a stem-loop structure which contains the rRNA spacer BamHI site. Primer extension products were insensitive to both alkali and RNase treatment, suggesting that RNA primers had already been removed from the 5′ end of nascent DNA. Location of an origin in the rRNA spacer region of ctDNA from tobacco, pea and Oenothera suggests that ctDNA replication origins may be conserved in higher plants.

Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana

A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.

The Arabidopsis At1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities

BackgroundThe Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is also homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. However, Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. The Arabidopsis Twinkle homologue has been shown by others to be dual targeted to mitochondria and chloroplasts.ResultsTo determine the functional activity of the Arabidopsis protein we obtained the gene for the full-length Arabidopsis protein and expressed it in bacteria. The purified protein was shown to have both DNA primase and DNA helicase activities. Western blot and qRT-PCR analysis indicated that the Arabidopsis gene is expressed most abundantly in young leaves and shoot apex tissue, as expected if this protein plays a role in organelle DNA replication. This expression is closely correlated with the expression of organelle-localized DNA polymerase in the same tissues. Homologues from other plant species show close similarity by phylogenetic analysis.ConclusionsThe results presented here indicate that the Arabidopsis phage T7 gp4/Twinkle homologue has both DNA primase and DNA helicase activities and may provide these functions for organelle DNA replication.

Molecular characterization of avian reoviruses using nested PCR and nucleotide sequence analysis.

A nested polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis identified and differentiated avian reoviruses (ARVs). PCR products amplified from the S1 gene segment of ARV of USA isolates were 738 and 342 bp, respectively. PCR products were conformed by Southern and dot blot hybridizations. The amplified cDNA fragments were cloned into the pUC18 vector and subjected to DNA sequencing. The nucleotide and deduced amino acid sequences of four USA (S1133, 1733, 2408, and CO8) and two Australian isolates (RAM-1 and SOM-4) were compared. Results of paired difference analysis and a predicted dendrogram revealed that USA isolates were closely related, but different from, Australian isolates. The deduced amino acid sequences of the N-terminal region of ARV sigma C showed a heptapeptide repeat of hydrophobic residues in all ARV isolates.

Transcriptome assembly, profiling and differential gene expression analysis of the halophyte Suaeda fruticosa provides insights into salt tolerance

BackgroundImprovement of crop production is needed to feed the growing world population as the amount and quality of agricultural land decreases and soil salinity increases. This has stimulated research on salt tolerance in plants. Most crops tolerate a limited amount of salt to survive and produce biomass, while halophytes (salt-tolerant plants) have the ability to grow with saline water utilizing specific biochemical mechanisms. However, little is known about the genes involved in salt tolerance. We have characterized the transcriptome of Suaeda fruticosa, a halophyte that has the ability to sequester salts in its leaves. Suaeda fruticosa is an annual shrub in the family Chenopodiaceae found in coastal and inland regions of Pakistan and Mediterranean shores. This plant is an obligate halophyte that grows optimally from 200–400 mM NaCl and can grow at up to 1000 mM NaCl. High throughput sequencing technology was performed to provide understanding of genes involved in the salt tolerance mechanism. De novo assembly of the transcriptome and analysis has allowed identification of differentially expressed and unique genes present in this non-conventional crop.ResultsTwelve sequencing libraries prepared from control (0 mM NaCl treated) and optimum (300 mM NaCl treated) plants were sequenced using Illumina Hiseq 2000 to investigate differential gene expression between shoots and roots of Suaeda fruticosa. The transcriptome was assembled de novo using Velvet and Oases k-45 and clustered using CDHIT-EST. There are 54,526 unigenes; among these 475 genes are downregulated and 44 are upregulated when samples from plants grown under optimal salt are compared with those grown without salt. BLAST analysis identified the differentially expressed genes, which were categorized in gene ontology terms and their pathways.ConclusionsThis work has identified potential genes involved in salt tolerance in Suaeda fruticosa, and has provided an outline of tools to use for de novo transcriptome analysis. The assemblies that were used provide coverage of a considerable proportion of the transcriptome, which allows analysis of differential gene expression and identification of genes that may be involved in salt tolerance. The transcriptome may serve as a reference sequence for study of other succulent halophytes.

Mechanisms for maintenance, replication and repair of the chloroplast genome in plants

Photosynthesis is a complex process that occurs in chloroplasts of higher plants, and requires a large number of proteins to assemble the photosynthetic machinery. Many chloroplast-localized proteins are nuclear-encoded and must be imported into the chloroplasts from the cytoplasm. A considerable number of genes for photosynthesis and other chloroplast functions, including transcription and translation, are encoded in the chloroplast genome (ctDNA), which ranges in size from about 130–160 kbp in most higher plants. CtDNA replication is not linked with the plant cell cycle and the chloroplast genome can be amplified to a very high copy number per cell in rapidly dividing leaf tissue. Later in leaf development and plant growth, the ctDNA levels reduce to very low levels (Oldenburg and Bendich, 2004b). The controls that regulate ctDNA replication initiation, replication, and copy number are not understood. From earlier publications on a number of plant species it appears that ctDNA may replicate by more than one mechanism, including a recombination-dependent replication mechanism (Rowan et al., 2010, this issue; Oldenburg and Bendich, 2004b; Marechal and Brisson, 2010), a double D-loop mechanism (Chiu and Sears, 1992; Kunnimalaiyaan and Nielsen, 1997a, b), and rolling circle replication (Kolodner and Tewari, 1975). In this issue, Rowan et al. (2010) report on the role of chloroplast-targeted RecA (cpRecA) in the maintenance of ctDNA in Arabidopsis. Previously published reports provide evidence that some ctDNA molecules may be recombination intermediates as shown by the presence of branched DNA molecules in some DNA preparations (Oldenburg and Bendich, 2004a, b; Scharff and Koop, 2007). As summarized in a review by Marechal and Brisson (2010), recombination has been shown to be involved in the repair of double-strand breaks and point mutations in ctDNA. It has been known for some time that a plant homologue of bacterial RecA is localized in chloroplasts (Cerutti et al., 1992), but, to date, little is known about the role of DNA recombination in the maintenance of ctDNA. Rowan et al. (2010) show clear evidence that cpRecA is involved in the maintenance of the chloroplast genome copy number in plants, as T-DNA insertions (from the Agrobacterium Ti plasmid) in the nuclear gene encoding this protein led to a reduction in ctDNA copy number in the mutant plants relative to wild-type plants and to a change in the structure of the ctDNA. The levels of detectable single-stranded DNA increased in the mutants, which is compatible with the decreased amount of cpRecA which would normally coat the single-stranded DNA regions and thus block its detection. After a few generations the mutants began to show significant signs of distress and reduced chloroplast function, including variegation and necrosis. These findings represent a significant advance in our understanding of the mechanisms involved in the maintenance of ctDNA integrity. The authors suggest that the role of cpRecA is primarily in DNA repair, as supported by the analysis of wild-type plants that have been treated with ciprofloxacin, which induces double-strand DNA breaks. In these plants, altered ctDNA structures were observed as in the cpRecA plants. Similar experiments with insertions in the DRT 100 homologue, which has only very weak homology to bacterial RecA but can partially complement E. coli recA mutants showed no effect, suggesting that DRT 100 may not be directly involved in the repair of ctDNA. The role of cpRecA in DNA repair is clearly supported by these experiments; it is also possible that cpRecA may be involved in recombination-mediated replication of the chloroplast genome. CpRecA and DRT 100 are not the only RecA homologues localized to chloroplasts. A dual-targeted (to both chloroplasts and mitochondria) RecA (distinguished from the others as RecA2) has been identified in the Arabidopsis nuclear genome (Christensen et al., 2005). T-DNA insertions in this gene lead to non-viable plants (BL Nielsen, JD Cupp, unpublished observations; Shedge et al., 2007), suggesting that RecA2 may be essential for ctDNA and/or mtDNA maintenance and plant development. However, at this point in time there are no data to determine whether the lethal phenotype is due to the disruption of chloroplast or mitochondrial DNA maintenance mechanisms, or both. The RecA2 gene was not included in the current study by Rowan et al. (2010, this issue) but its role in ctDNA replication should be evaluated. The observation that T-DNA insertions in cpRecA were not lethal may be due to functional (at least partial) complementation by RecA2.