Experimental reconstruction of double-stranded break repair-mediated plastid DNA insertion into the tobacco nucleus.

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we introduced double-stranded DNA breaks into the nuclear genome of tobacco through inducible expression of I-SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through double-stranded DNA break repair. Microhomology-mediated linking of disparate segments of chloroplast DNA occurs frequently during healing of induced nuclear double-stranded breaks (DSB) but the resulting nuclear integrants are often immediately unstable. Non-Mendelian inheritance of a selectable marker (neo), used to identify plastid DNA transfer, was observed in the progeny of about 50% of lines emerging from the screen. The instability of these de novo nuclear insertions of plastid DNA (nupts) was shown to be associated with deletion not only of the nupt itself but also of flanking nuclear DNA within one generation of transfer. This deletion of pre-existing nuclear DNA suggests that the genetic impact of organellar DNA transfer to the nucleus is potentially far greater than previously thought.

Transcription of nuclear organellar DNA in a model plant system.

Endosymbiotic gene transfer from cytoplasmic organelles (chloroplasts and mitochondria) to the nucleus is an ongoing process in land plants. Although the frequency of organelle DNA migration is high, functional gene transfer is rare because a nuclear promoter is thought necessary for activity in the nucleus. Here we show that a chloroplast promoter, 16S rrn, drives nuclear transcription, suggesting that a transferred organellar gene may become active without obtaining a nuclear promoter. Examining the chromatin status of a known de novo chloroplast integrant indicates that plastid DNA inserts into open chromatin and that this relaxed condition is maintained after integration. Transcription of nuclear organelle DNA integrants was explored at the whole genome level by analyzing RNA-seq data of Oryza sativa subsp. japonica, and utilizing sequence polymorphisms to unequivocally discriminate nuclear organelle DNA transcripts from those of bona fide cytoplasmic organelle DNA. Nuclear copies of organelle DNA that are transcribed show a spectrum of transcriptional activity but at comparatively low levels compared with the majority of other nuclear genes.

Evolution and biology of supernumerary B chromosomes.

B chromosomes (Bs) are dispensable components of the genome exhibiting non-Mendelian inheritance and have been widely reported on over several thousand eukaryotes, but still remain an evolutionary mystery ever since their first discovery over a century ago [1]. Recent advances in genome analysis have significantly improved our knowledge on the origin and composition of Bs in the last few years. In contrast to the prevalent view that Bs do not harbor genes, recent analysis revealed that Bs of sequenced species are rich in gene-derived sequences. We summarize the latest findings on supernumerary chromosomes with a special focus on the origin, DNA composition, and the non-Mendelian accumulation mechanism of Bs.

Cytoplasmic organelle DNA preferentially inserts into open chromatin.

DNA transfer from chloroplasts and mitochondria to the nucleus is ongoing in eukaryotes but the mechanisms involved are poorly understood. Mitochondrial DNA was observed to integrate into the nuclear genome through DNA double-strand break repair in Nicotiana tabacum. Here, 14 nuclear insertions of chloroplast DNA (nupts) that are unique to Oryza sativa subsp. indica were identified. Comparisons with the preinsertion nuclear loci identified in the related subspecies, O. sativa subsp. japonica, which lacked these nupts, indicated that chloroplast DNA had integrated by nonhomologous end joining. Analyzing public DNase-seq data revealed that nupts were significantly more frequent in open chromatin regions of the nucleus. This preference was tested further in the chimpanzee genome by comparing nuclear loci containing integrants of mitochondrial DNA (numts) with their corresponding numt-lacking preinsertion sites in the human genome. Mitochondrial DNAs also tended to insert more frequently into regions of open chromatin revealed by human DNase-seq and Formaldehyde-Assisted Isolation of Regulatory Elements-seq databases.

Potential functional replacement of the plastidic acetyl-CoA carboxylase subunit (accD) gene by recent transfers to the nucleus in some angiosperm lineages.

Eukaryotic cells originated when an ancestor of the nucleated cell engulfed bacterial endosymbionts that gradually evolved into the mitochondrion and the chloroplast. Soon after these endosymbiotic events, thousands of ancestral prokaryotic genes were functionally transferred from the endosymbionts to the nucleus. This process of functional gene relocation, now rare in eukaryotes, continues in angiosperms. In this article, we show that the chloroplastic acetyl-CoA carboxylase subunit (accD) gene that is present in the plastome of most angiosperms has been functionally relocated to the nucleus in the Campanulaceae. Surprisingly, the nucleus-encoded accD transcript is considerably smaller than the plastidic version, consisting of little more than the carboxylase domain of the plastidic accD gene fused to a coding region encoding a plastid targeting peptide. We verified experimentally the presence of a chloroplastic transit peptide by showing that the product of the nuclear accD fused to green fluorescent protein was imported in the chloroplasts. The nuclear gene regulatory elements that enabled the erstwhile plastidic gene to become functional in the nuclear genome were identified, and the evolution of the intronic and exonic sequences in the nucleus is described. Relocation and truncation of the accD gene is a remarkable example of the processes underpinning endosymbiotic evolution.

Endosymbiotic evolution: the totalitarian nucleus is foiled again.

DNA transfer between host cells and their endosymbionts has had a profound effect on the evolution of eukaryotic cells. A new sequencing study suggests that other forces may be equally important.

Endosymbiotic evolution: RNA intermediates in endosymbiotic gene transfer.

More than a billion years of endosymbiotic evolution has resulted in extensive gene relocation between the genetic compartments of eukaryotic cells. A new study uses chloroplast genome transformation to shed light on the mechanisms involved.

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.

Plastid DNA in the nucleus: new genes for old.

Nuclear genomes of eukaryotes are bombarded by a continuous deluge of organellar DNA which contributes significantly to eukaryote evolution. Here, we present a new PCR-based method that allows the specific amplification of nuclear integrants of organellar DNA (norgs) by exploiting recent deletions present in organellar genome sequences. We have used this method to amplify nuclear integrants of plastid DNA (nupts) from the nuclear genomes of several Nicotiana species and to study the evolutionary forces acting upon these sequences. The role of nupts in endosymbiotic evolution and the different genetic factors influencing the time available for a chloroplastic gene to be functionally relocated in the nucleus are discussed.

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants.

Mitochondria and chloroplasts (photosynthetic members of the plastid family of cytoplasmic organelles) in eukaryotic cells originated more than a billion years ago when an ancestor of the nucleated cell engulfed two different prokaryotes in separate sequential events. Extant cytoplasmic organellar genomes contain very few genes compared with their candidate free-living ancestors, as most have functionally relocated to the nucleus. The first step in functional relocation involves the integration of inactive DNA fragments into nuclear chromosomes, and this process continues at high frequency with attendant genetic, genomic, and evolutionary consequences. Using two different transplastomic tobacco lines, we show that DNA migration from chloroplasts to the nucleus is markedly increased by mild heat stress. In addition, we show that insertion of mitochondrial DNA fragments during the repair of induced double-strand breaks is increased by heat stress. The experiments demonstrate that the nuclear influx of organellar DNA is a potentially a source of mutation for nuclear genomes that is highly susceptible to temperature fluctuations that are well within the range experienced naturally.

Plastid sequences contribute to some plant mitochondrial genes.

DNA of plastid (chloroplast) origin comprises between 1% and 10% of the mitochondrial genomes of higher plants, but functions are currently considered to be limited to rare instances where plastid tRNA genes have replaced their mitochondrial counterparts, where short patches of mitochondrial genes evolved using their homologous plastidic copies by gene conversion or where a new promoter region is created. Here, we show that, in some angiosperms, plastid-derived DNA in mitochondrial genomes (also called mtpt for mitochondrial plastid DNA) contributes codons to unrelated mitochondrial protein-coding sequences and may also have a role in posttranscriptional RNA processing. We determined that these transfers of plastid DNA occurred a few to 150 Ma and that mtpts can sometimes remain dormant many millions of years before contributing to the mitochondrial proteome.

Conservation of plastid sequences in the plant nuclear genome for millions of years facilitates endosymbiotic evolution.

The nuclear genome of eukaryotes contains large amounts of cytoplasmic organelle DNA (nuclear integrants of organelle DNA [norgs]). The recent sequencing of many mitochondrial and chloroplast genomes has enabled investigation of the potential role of norgs in endosymbiotic evolution. In this article, we describe a new polymerase chain reaction-based method that allows the identification and evolutionary study of recent and older norgs in a range of eukaryotes. We tested this method in the genus Nicotiana and obtained sequences from seven nuclear integrants of plastid DNA (nupts) totaling 25 kb in length. These nupts were estimated to have been transferred 0.033 to 5.81 million years ago. The spectrum of mutations present in the potential protein-coding sequences compared with the noncoding sequences of each nupt revealed that nupts evolve in a nuclear-specific manner and are under neutral evolution. Indels were more frequent in noncoding regions than in potential coding sequences of former chloroplastic DNA, most probably due to the presence of a higher number of homopolymeric sequences. Unexpectedly, some potential protein-coding sequences within the nupts still contained intact open reading frames for up to 5.81 million years. These results suggest that chloroplast genes transferred to the nucleus have in some cases several millions of years to acquire nuclear regulatory elements and become functional. The different factors influencing this time frame and the potential role of nupts in endosymbiotic gene transfer are discussed.

Introducing an RNA editing requirement into a plastid-localised transgene reduces but does not eliminate functional gene transfer to the nucleus.

In higher plants, DNA transfer from the plastid (chloroplast) genome to the nucleus is a frequent, ongoing process. However, there has been uncertainty over whether this transfer occurs by a direct DNA mechanism or whether RNA intermediates are involved. Previous experiments utilising transplastomic Nicotiana tabacum (tp7 and tp17) enabled the detection of plastid-to-nucleus transfer in real time. To determine whether RNA intermediates are involved in this transfer, transplastomic lines (tpneoACG) were generated containing, in their plastid genomes, a nuclear promoter-driven kanamycin resistance gene (neo) with a start codon that required plastid RNA editing but otherwise identical to tp7 and tp17. Therefore it was expected that kanamycin resistance would only be acquired following RNA-mediated transfer of neo to the nucleus. Screening of tpneoACG progeny revealed several kanamycin-resistant plants, each of which contained the neo gene located in the nucleus. Surprisingly, neo was unedited in all these plants, indicating that neoACG was active in the absence of an edited start codon and suggesting that RNA intermediates were not involved in the transfers. However, analysis of tpneoACG revealed that only a low proportion of transcripts potentially able to mediate neo transfer were edited, thus precluding unequivocal conclusions regarding the role of RNA in plastid-to-nucleus transfer. The low proportion of edited transcripts was found to be due to predominant antisense neo transcripts, rather than to low editing efficiency of the sense transcripts. This study highlights a number of important considerations in the design of experiments utilising plastid RNA editing. The results also suggest that RNA editing sites reduce but do not eliminate functional plastid-to-nucleus gene transfer. This is relevant both in an evolutionary context and in placing RNA editing-dependent genes in the plastid genome as a means of transgene containment.

The origin and characterization of new nuclear genes originating from a cytoplasmic organellar genome.

Endosymbiotic transfer of DNA and functional genes from the cytoplasmic organelles (mitochondria and chloroplasts) to the nucleus has been a major factor driving the origin of new nuclear genes, a process central to eukaryote evolution. Although organelle DNA transfers very frequently to the nucleus, most is quickly deleted, decays, or is alternatively scrapped. However, a very small proportion of it gives rise, immediately or eventually, to functional genes. To simulate the process of functional transfer, we screened for nuclear activation of a chloroplast reporter gene aadA, which had been transferred from the chloroplast to independent nuclear loci in 16 different plant lines. Cryptic nuclear activity of the chloroplast promoter was revealed, which became conspicuous when present in multiple nuclear copies. We screened ∼50 million cells of each line and retrieved three plants in which aadA showed strong nuclear activation. Activation occurred by acquisition of the CaMV 35S nuclear promoter or by nuclear activation of the native chloroplast promoter. Two fortuitous sites within the 3' UTR of aadA mRNA both promoted polyadenylation without any sequence change. Complete characterization of one nuclear sequence before and after gene transfer demonstrated integration by nonhomologous end joining involving simultaneous insertion of multiple chloroplast DNA fragments. The real-time observation of three different means by which a chloroplast gene can become expressed in the nucleus suggests that the process, though rare, may be more readily achieved than previously envisaged.

Endosybiotic evolution in action: Real-time observations of chloroplast to nucleus gene transfer.

The origin of new genes has long been considered a fundamental question in evolutionary biology. In eukaryotes, a major pathway for the 'birth' of new nuclear genes has been transfer of genes from the cytoplasmic organelles (mitochondria and plastids) to the nucleus. While the vast majority of gene transfer occurred shortly after endosymbiosis, the process continues today and is still driving the evolution of nuclear genomes. In tobacco (Nicotiana tabacum) a number of studies have indicated that DNA can transfer from the chloroplast to the nucleus at relatively high frequency. Less has been known, however, about how a newly transferred organelle gene can become activated in this new genetic environment. In a recent report we observed, in real-time, the activation of a plastid reporter gene newly transferred to the nucleus. A key observation from this study was that non-homologous repair is an important generator of novel sequence combinations which, in rare instances, can result in the nuclear activation of plastid genes. In addition, the activation of relocated genes can be aided by the fortuitous presence of plastid sequences able to promote nuclear expression.

Instability of plastid DNA in the nuclear genome.

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.

Transfer of plastid DNA to the nucleus is elevated during male gametogenesis in tobacco.

In eukaryotes, many genes were transferred to the nucleus from prokaryotic ancestors of the cytoplasmic organelles during endosymbiotic evolution. In plants, the transfer of genetic material from the plastid (chloroplast) and mitochondrion to the nucleus is a continuing process. The cellular location of a kanamycin resistance gene tailored for nuclear expression (35SneoSTLS2) was monitored in the progeny of reciprocal crosses of tobacco (Nicotiana tabacum) in which, at the start of the experiments, the reporter gene was confined either to the male or the female parental plastid genome. Among 146,000 progeny from crosses where the transplastomic parent was male, 13 transposition events were identified, whereas only one atypical transposition was identified in a screen of 273,000 transplastomic ovules. In a second experiment, a transplastomic beta-glucuronidase reporter gene, tailored to be expressed only in the nucleus, showed frequent stochastic expression that was confined to the cytoplasm in the somatic cells of several plant tissues. This gene was stably transferred in two out of 98,000 seedlings derived from a male transplastomic line crossed with a female wild type. These data demonstrate relocation of plastid DNA to the nucleus in both somatic and gametophytic tissue and reveal a large elevation of the frequency of transposition in the male germline. The results suggest a new explanation for the occurrence of uniparental inheritance in eukaryotes.

Reconstructing evolution: gene transfer from plastids to the nucleus.

During evolution, the genomes of eukaryotic cells have undergone major restructuring to meet the new regulatory challenges associated with compartmentalization of the genetic material in the nucleus and the organelles acquired by endosymbiosis (mitochondria and plastids). Restructuring involved the loss of dispensable or redundant genes and the massive translocation of genes from the ancestral organelles to the nucleus. Genomics and bioinformatic data suggest that the process of DNA transfer from organelles to the nucleus still continues, providing raw material for evolutionary tinkering in the nuclear genome. Recent reconstruction of these events in the laboratory has provided a unique tool to observe genome evolution in real time and to study the molecular mechanisms by which plastid genes are converted into functional nuclear genes. Here, we summarize current knowledge about plastid-to-nuclear gene transfer in the context of genome evolution and discuss new insights gained from experiments that recapitulate endosymbiotic gene transfer in the laboratory.

Evidence for alternative splicing of MADS-box transcripts in developing cotton fibre cells.

The MADS-box family of genes encodes transcription factors that have widely ranging roles in diverse aspects of plant development. In this study, four cotton MADS-box cDNA clones of the type II (MIKC) class were isolated, with phylogenetic analysis indicating that the cotton sequences are of the AGAMOUS subclass. The corresponding transcripts were detected in developing cotton fibre cells as well as in whole ovule and flower tissue, with differential expression in stems, leaves and roots. Reverse transcription PCR showed extensive alternative splicing in one of the reactions, and 11 mRNAs of different intron/exon composition and length were characterised. Sequence differences between the transcripts indicated that they could not be derived from the same pre-mRNA and that the sequenced transcript pool was derived from two distinct MADS-box genes. Several of the alternatively spliced transcripts potentially encoded proteins with altered K-domains and/or C-terminal regions and the variant proteins may have altered cellular roles. This work is the first that describes MADS-box gene expression in elongating cotton fibres and adds to a growing body of evidence for the prevalence of alternative splicing in the expression of MADS-box and other genes.