Divergent roles for the two PolI-like organelle DNA polymerases of Arabidopsis

DNA polymerases play a central role in the process of DNA replication. Yet, the proteins in charge of the replication of plant organelle DNA have not been unambiguously identified. There are however many indications that a family of proteins homologous to bacterial DNA polymerase I (PolI) is implicated in organelle DNA replication. Here, we have isolated mutant lines of the PolIA and PolIB genes of Arabidopsis (Arabidopsis thaliana) to test this hypothesis. We find that mutation of both genes is lethal, thus confirming an essential and redundant role for these two proteins. However, the mutation of a single gene is sufficient to cause a reduction in the levels of DNA in both mitochondria and plastids. We also demonstrate that polIb, but not polIa mutant lines, are hypersensitive to ciprofloxacin, a small molecule that specifically induces DNA double-strand breaks in plant organelles, suggesting a function for PolIB in DNA repair. In agreement with this result, a cross between polIb and a plastid Whirly mutant line yielded plants with high levels of DNA rearrangements and severe growth defects, indicating impairments in plastid DNA repair pathways. Taken together, this work provides further evidences for the involvement of the plant PolI-like genes in organelle DNA replication and suggests an additional role for PolIB in DNA repair.     

Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species

The fates of mitochondrial and plastid nucleoids during pollen development in six angiosperm species (Antirrhinum majus, Glycine max, Medicago sativa, Nicotiana tabacum, Pisum sativum, and Trifolium pratense) were examined using epifluorescence microscopy after double staining with 4',6-diamidino-2- phenylindole (DAPI) to stain DNA and with a potentiometric dye (either DiOC7 or rhodamine 123) for visualization of metabolically active mitochondria. From the pollen mother cell stage to the microspore stage of pollen development, mitochondria and plastids both contained DNA detectable by DAPI staining. However, during the further maturation preceding anthesis, mitochondrial DNA became undetectable cytologically in either the generative or the vegetative cell of mature pollen; even in germinated pollen tubes containing hundreds of metabolically active mitochondria undergoing cytoplasmic streaming, vital staining with DAPI failed to reveal mitochondrial DNA. By the mature pollen stage, plastid DNA also became undetectable by DAPI staining in the vegetative cell. However, in the generative cell of mature pollen the timing of plastid DNA disappearance as detected by DAPI varied with the species. Plastid DNA remained detectable only in the generative cells of pollen grains from species known or suspected to have biparental transmission of plastids. The apparent absence of cytologically detectable organelle genomes in living pollen was further examined using molecular methods by hybridizing organelle DNA-specific probes to digests of total DNA from mature pollen and from other organs of A. majus and N. tabacum, both known to be maternal for organelle inheritance. Mitochondrial DNA was detected in pollen of both species; thus the cytological alteration of mitochondrial genomes during pollen development does not correspond with total mtDNA loss from the pollen. Plastid DNA was detectable with molecular probes in N. tabacum pollen but not in A. majus pollen. Since the organelle DNA detected by molecular methods in mature pollen may lie solely in the vegetative cell, further study of the basis of maternal inheritance of mitochondria and plastids will require molecular methods which distinguish vegetative cell from reproductive cell organelle genomes. The biological effect of the striking morphological alteration of organelle genomes during later stages of pollen development, which leaves them detectable by molecular methods but not by DAPI staining, is as yet unknown.     

Bi-parental cytoplasmic DNA inheritance in Wisteria (Fabaceae): evidence from a natural experiment

Cytoplasmic inheritance was investigated in interspecific hybrids of Wisteria sinensis and W. floribunda. Species-specific nuclear, mitochondrial and plastid DNA markers were identified from wild-collected plants of each species in its native range. These markers provide evidence for the bi-parental transmission of plastids in hybrid swarms of these two species in the southeastern USA. These population level molecular data corroborate previous cytological evidence of this phenomenon in Wisteria.     

Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids

Angiosperms possess a small family of phage-type RNA polymerase genes that arose by gene duplication from an ancestral gene encoding the mitochondrial RNA polymerase. We have isolated and sequenced the genes and cDNAs encoding two phage-type RNA polymerases, PpRpoT1 and PpRpoT2, from the moss Physcomitrella patens. PpRpoT1 comprises 19 exons and 18 introns, PpRpoT2 contains two additional introns. The N-terminal transit peptides of both polymerases are shown to confer dual-targeting of green fluorescent protein fusions to mitochondria and plastids. In vitro translation of the cDNAs revealed initiation of translation at two in-frame AUG start codons. Translation from the first methionine gives rise to a plastid-targeted polymerase, whereas initiation from the second methionine results in exclusively mitochondrial-targeted protein. Thus, dual-targeting of Physcomitrella RpoT is caused by and might be regulated by multiple translational starts. In phylogenetic analyses, the Physcomitrella RpoT polymerases form a sister group to all other phage-type polymerases of land plants. The two genes result from a gene duplication event that occurred independently from the one which led to the organellar polymerases with mitochondrial or plastid targeting properties in angiosperms. Yet, according to their conserved exon-intron structures they are representatives of the molecular evolutionary line leading to the RpoT genes of higher land plants.     

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.     

Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.     

Origin, evolution and genetic effects of nuclear insertions of organelle DNA

In eukaryotes, nuclear genomes are subject to an influx of DNA from mitochondria and plastids. The nuclear insertion of organellar sequences can occur during the illegitimate repair of double-stranded breaks. After integration, nuclear organelle DNA is modified by point mutations, and by deletions. Insertion of organelle DNA into nuclear genes is not rare and can potentially have harmful effects. In humans, some insertions of nuclear mitochondrial DNA are associated with heritable diseases. It remains to be determined whether nuclear organelle DNA can contribute beneficially to gene evolution.     

Nuclear genome diversity in somatic cells is accelerated by environmental stress

DNA transfer to the nucleus from prokaryotic ancestors of the cytoplasmic organelles (mitochondria and plastids) has occurred during endosymbiotic evolution in eukaryotes. In most eukaryotes, organelle DNA transfer to nucleus is a continuing process. The frequency of DNA transposition from plastid (chloroplast) to nucleus has been measured in tobacco plants (Nicotiana tabacum) experimentally. We have monitored the effects of environmental stress on the rate of DNA transfer from plastid to nucleus by exploiting nucleus-specific reporter genes in two transplastomic tobacco lines. DNA migration from plastids to the nucleus is markedly increased by mild heat stress. In addition, insertions of mitochondrial DNA into induced double-strand breaks are observed after heat treatment. These results show that movement of organelle DNA to the nucleus is remarkably increased by heat stress.     

Cytological Basis of Plastid Inheritance in Phaseolus vulgaris-Investigations of Plastids,Mitochondria and Their DNA Nucleoids During Pollen Development

Electron microscopic and DNA fluorescence microscopic observations of the plastids, mitochondria and their DNA in the developing pollen of Phaseolus vulgaris L. have demonstrated that the male plastids were excluded during microspore mitosis. The formed generative cell was free of plastids because of regional localization of plastids in early developing microspore and the extremely unequal distribution during division. The fluorescence observations of DNA showed that cytoplasmic (plastid and mitochondria) nucleoids degenerated and disappeared during the development of microspore/pollen, and were never presented in the generative cell at different development stages. These results provided precise cytological evidence of maternal plastid inheritance in Phaseolus vulgaris, which was not in accord with the biparental plastid inheritance identified from early genetic analysis. Based on authors' previous observations in a variety of common bean that the organelle DNA of male gamete was completely degenerated, the early genetic finding of the biparental plastid inheritance was unlikely to be effected by genotypic difference. Thus those biparental plastid inheritance might be caused by occational male plastid transmission, and plastid uniparental maternal inheritance was the species character of Phaseolus vulgaris.     

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

菜豆花粉发育中质体和线粒体及其DNA存在的状况——着重阐明质体遗传的细胞学基础

应用电镜和DNA的DAPI荧光检测技术研究了菜豆(Phaseolus vulgaris L.)小孢子/花粉发育中质体和线粒体及其DNA存在的状况。观察表明:在小孢子分裂时质体全部分配到营养细胞中,初形成的生殖细胞已不含质体。线粒体和质体的DNA在花粉发育中也先后降解,生殖细胞从刚形成时发育至成熟花粉时期这两种细胞器DNA均不存在。研究结果为菜豆质体母系遗传提供了确切的细胞学证据。遗传分析的研究曾确定菜豆质体为双亲遗传,对与本研究结论不同的原因进行了讨论。     

Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth

Summary Albino rice plants derived from pollen contain plastid genomes that have suffered large-scale deletions. From the roots of albino plants, we obtained several calli containing homogeneous plastid DNA differing in the size and position of the deletion. Southern blotting and pulsed field gel electrophoresis experiments revealed that the DNAs were linear molecules having a hairpin structure at both termini, existing as monomers (19 kb) or dimers, trimers and tetramers linked to form head-to-head and tail-to-tail multimers. This characteristic form is similar to that of the vaccinia virus, in which the replication origin is thought to lie at or near the hairpin termini. Furthermore, polymerase chain reaction experiments revealed complete loss of the ribosomal RNA genes of the plastid DNA. The results suggest that plant cells can grow without translation occurring in plastids. All of the deleted plastid DNAs commonly retained the region containing the tRNA^Glu gene (trnE), which is essential for biosynthesis of porphyrin. As porphyrin is the precursor of heme for mitochondria and other organelles, it is considered thattrnE on the remnant plastid genome may be transcribed by an RNA polymerase encoded on nuclear DNA.