Chloroplast Polymerase Research Articles

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform.

Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5' and 3' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5'UTRs, 10 3'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

Genetic evidence for the origins of range disjunctions in the Australian dry sclerophyll plant Hardenbergia violacea

Aim The distribution of genetic variation in the Australian dry sclerophyll plant Hardenbergia violacea (Fabaceae) is examined in the context of Pleistocene climate change in order to identify likely refugia. Particular consideration is given to the origin of range disjunctions in South Australia and Tasmania, and to determining whether the Tasmanian population is indigenous or recently introduced from mainland Australia. Location Southeastern Australian mainland and Tasmania. Methods A combination of chloroplast polymerase chain reaction–restriction fragment length polymorphism and genomic amplified fragment length polymorphism (AFLP) marker systems was used to examine the genetic structure of 292 individuals from 13 populations across the range of H. violacea in southeastern Australia. Results Hardenbergia violacea populations in Tasmania and southern Victoria were characterized by low, almost monotypic chloroplast diversity. New South Wales showed higher haplotype diversity and haplotype sharing among widely distributed populations. Principal coordinates analysis (PCoA) of the AFLP data found a strong latitudinal cline in AFLP variation from northern New South Wales south to Tasmania. The Tasmanian population formed an isolated and somewhat disjunct genetic cluster at one end of this cline. However, the South Australian population was an exception to the clinal variation shown by all other populations, forming a highly disjunct cluster in the PCoA. Within-population genetic diversity was low in both disjunct populations. Main conclusions The genetic evidence indicates that the Tasmanian population is likely to be indigenous and probably the product of vicariance, which was followed by range contraction at the Last Glacial Maximum or an earlier glacial event. The deep phylogenetic disjunction in South Australia is evidence of a much earlier separation on mainland Australia. The chloroplast structure indicates that, during the Pleistocene, H. violacea underwent broad-scale recolonization in southern Victoria and Tasmania, possibly from a large continental refugium in eastern New South Wales. We conclude that H. violacea, and presumably the sclerophyll communities in which it occurs, have undergone multiple range contractions to large continental refugia during different Pleistocene glaciations in southeastern Australia.

Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe.

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.

Green life: Control of chloroplast gene transcription

AbstractChloroplasts and other plastids are plant cell organelles that account for major biochemical functions. They contain their own gene expression system but are integrated into the signaling network of the entire cell. Both nuclear and plastid genes are involved in chloroplast biogenesis, and the gene expression pathways both inside and outside the organelle are subject to developmental and environmental control. The plastid transcription apparatus reflects this general scheme, with a basic organelle‐encoded enzymatic machinery surrounded by factors that may be encoded by nuclear genes. Among the transcription regulatory mechanisms thought to play a role during plastid development are: (1) differential usage of promoter elements; (2) phosphorylation of transcription factors by a protein kinase, which is itself subject to phosphorylation and redox control; (3) dynamic changes in the composition of the transcription apparatus. In etioplasts, the dominating polymerase ‘B’ is a bacterial‐type enzyme, whereas the major chloroplast polymerase ‘A’ is a much larger enzyme reminiscent of those in the nucleus. These two enzyme forms may share common components and recruit others during development.

DNA-dependent RNA polymerase of spinach chloroplasts: Characterization of α-like and σ-like polypeptides

The 80 000 x g supernatants of spinach leaf homogenates contain polypeptides that are immunologically related to the ββ′, α and σ subunits of Escherichia coli RNA polymerase. The σ-like (90 kDa and 33 kDa) and α-like (38 kDa) polypeptides are separated from ββ′-like ones by chromatography on Heparin-Sepharose or Servacel-PEI. If a protein fraction containing the α- and σ-like polypeptides is added to transcriptionally active purified chloroplast polymerase (consisting of the ββ′-like 150/145 kDa and 80 kDa polypeptides and the 110/102 kDa polypeptides the latter two being not homologous to polypeptides of E. coli polymerase) a correct initiation and transcription of the spinach chloroplast rbcL gene is obtained. Chloroplast protein fractions containing the σ-like polypeptide(s) also enhance correct transcription initiation when they are combined with E. coli RNA polymerase.

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

1. 1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves. 2. 2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography. 3. 3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA. 4. 4. The three enzymes require either Mg 2+ or Mn 2+ for activity. 5. 5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase. 6. 6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

A γ‐like DNA Polymerase in Spinach Chloroplasts

A DNA polymerase has been extracted from spinach chloroplasts and purified by chromatography on DEAE-cellulose and hydroxyapatite. A great similarity between the purified chloroplast polymerase and the mammalian mitochondrial DNA polymerase gamma was found by several criteria: preference for the synthetic primer-template (dT)12-18 . poly(rA), optimal requirement for Mn2+ (0.1-1.0 mM), KCl (100 mM) and pH (8-9), high relative molecular mass (approximately 105,000), resistance to aphidicolin and inhibition by N-ethylmaleimide. Some peculiar features of the chloroplast DNA polymerase have, however, been noticed. The mammalian DNA polymerase gamma has been suggested to be responsible for the replication of mitochondrial DNA. Thus, both the presence of a gamma-like DNA polymerase in chloroplasts and the similarities between the chloroplast and the mitochondrial DNA (absence of a nucleosomal structure an presence of displacement loops) lead to the suggestion that chloroplast DNA is also replicated by a gamma-like DNA polymerase and that the gamma polymerases present in eukaryotes are, therefore, involved in a strand-displacement DNA synthesis. An alpha-like DNA polymerase activity, present and predominant in crude leaf extracts, was practically absent from purified chloroplast preparations.

The polypeptide subunit structure of the DNA-dependent RNA polymerase of Zea mays chloroplasts.

Analysis of the maize chloroplast DNA-dependent RNA polymerase by electrophoresis on polyacrylamide gels that contain sodium dodecyl sulfate shows that the enzyme is multimeric. It contains at least two polypeptides of 180,000 and 140,000 daltons. Polypeptides of lower molecular mass that are associated with the enzyme at several stages of purification may also be subunits of the enzyme. Electrophoresis of a mixture of purified maize nuclear DNA-dependent RNA polymerase IIA and chloroplast polymerase on sodium dodecyl sulfate-polyacrylamide gels resolves the 160,000 dalton polypeptide of IIA from the 140,000 dalton polypeptide of the chloroplast enzyme, but does not resolve the 180,000 dalton components found in both enzymes. The polypeptides of less than 40,000 daltons in the highly purified maize nuclear IIA preparations are absent from preparations of purified maize chloroplast RNA polymerase.

Poly(A) Polymerase and Poly(G) Polymerase in Wheat Chloroplasts

Extracts of wheat chloroplasts contain a poly(A) polymerase which can polymerize AMP residues from ATP onto an RNA primer. Whole extracts of wheat leaves also contain another poly(A) polymerase which is present in much larger amount and is probably derived from the nuclei. Both polymerases can utilize as primer poly(A), poly(C), transfer RNA, and ribosomal RNA, but only the chloroplast polymerase can utilize poly(U) and poly(G). Both enzymes have a specific requirement for ATP. Extracts of wheat chloroplasts contain, in addition to the poly(A) polymerase, a poly(G) polymerase which can polymerize GMP residues from GTP onto primers such as poly(G), poly(A), or ribosomal RNA. The poly(G) polymerase cannot utilize ATP but can slowly polymerize CMP from CTP. When the two chloroplast polymerases are present together in an in vitro incubation with ATP plus GTP and poly(A), the polymerization product is a mixed poly(A,G) tract.

Ribonucleic acid synthesizing activity of spinach chloroplasts and nuclei

Purified chloroplast and nuclei preparations from spinach leaves possess RNA polymerase activity, as measured by the incorporation of 3H-ATP into an acid-insoluble product. In both types of organelle the polymerase reaction is dependent on the presence of all four nucleoside triphosphates, Mg ++, and an ATP-generating system. The reaction is inhibited by RNase, DNase, and actinomycin D. No stimulation was found with spermidine, with ammonium sulfate, or with the plant growth regulators kinetin, indoleacetic acid, and gibberellic acid. RNA polymerase activity was higher in leaf extracts from young plants. The product of the in vitro RNA polymerase reaction was isolated by phenol extraction and sucrose density gradient centrifugation. The chloroplast product had a major peak at about 11S, and was somewhat polydisperse in the range 11–23S. The product of the in vitro nuclear reaction had a maximum at about 8S and was less polydisperse than the chloroplast product. Short-term incorporation of 3H-uridine by intact spinach plants revealed the presence of a rapidly labelled RNA fraction in the chloroplasts with sedimentation properties similar to the product of the in vitro chloroplast polymerase reaction. The corresponding rapidly labelled RNA fraction in the nuclei resembled the in vitro nuclear product but was more polydisperse in the high molecular weight regions of the gradient. In long-term, in vivo incorporation experiments the labelled RNA pattern of both chloroplasts and nuclei corresponded closely to the ribosomal and transfer RNA fractions.