Recent transfer of an iron-regulated gene from the plastid to the nuclear genome in an oceanic diatom adapted to chronic iron limitation
© Lommer et al; licensee BioMed Central Ltd. 2010
Received: 1 June 2010
Accepted: 20 December 2010
Published: 20 December 2010
Although the importance and widespread occurrence of iron limitation in the contemporary ocean is well documented, we still know relatively little about genetic adaptation of phytoplankton to these environments. Compared to its coastal relative Thalassiosira pseudonana, the oceanic diatom Thalassiosira oceanica is highly tolerant to iron limitation. The adaptation to low-iron conditions in T. oceanica has been attributed to a decrease in the photosynthetic components that are rich in iron. Genomic information on T. oceanica may shed light on the genetic basis of the physiological differences between the two species.
The complete 141790 bp sequence of the T. oceanica chloroplast genome [GenBank: GU323224], assembled from massively parallel pyrosequencing (454) shotgun reads, revealed that the petF gene encoding for ferredoxin, which is localized in the chloroplast genome in T. pseudonana and other diatoms, has been transferred to the nucleus in T. oceanica. The iron-sulfur protein ferredoxin, a key element of the chloroplast electron transport chain, can be replaced by the iron-free flavodoxin under iron-limited growth conditions thereby contributing to a reduction in the cellular iron requirements. From a comparison to the genomic context of the T. pseudonana petF gene, the T. oceanica ortholog can be traced back to its chloroplast origin. The coding potential of the T. oceanica chloroplast genome is comparable to that of T. pseudonana and Phaeodactylum tricornutum, though a novel expressed ORF appears in the genomic region that has been subjected to rearrangements linked to the petF gene transfer event.
The transfer of the petF from the cp to the nuclear genome in T. oceanica represents a major difference between the two closely related species. The ability of T. oceanica to tolerate iron limitation suggests that the transfer of petF from the chloroplast to the nuclear genome might have contributed to the ecological success of this species.
In contemporary oceans, diatoms account for approximately 40% of the oceanic primary production and play a critical role in the sequestration of atmospheric CO2 into the deep ocean . The high diversity of diatoms and their cosmopolitan distribution in the marine environment reflect the ecological success endured by this group since their first appearance more than 150 Ma ago .
Although diatoms thrive in coastal areas where dissolved nutrients are high, many species of diatoms are prevalent in high-nutrient, low-chlorophyll (HNLC) oceanic regions where primary production is chronically iron-limited . Iron fertilization experiments in HNLC regions have repeatedly demonstrated the ability of opportunistic diatom species to bloom once iron is no longer growth limiting . In contrast, some diatom species such as Thalassiosira oceanica thrive equally well in the presence or absence of iron . A key determinant for the survival and growth of phytoplankton under iron limitation must be the ability to carry out photosynthesis efficiently, despite the high iron requirements of the photosynthetic infrastructure.
The photosynthetic apparatus, largely contained in the chloroplasts, is jointly coordinated by the plastid and nuclear genomes, involving more than 700 genes . The chloroplast genome of most species generally retained less than 200 of the genes contributing to chloroplast function, as the majority of the endosymbiont's chloroplast genes have been lost or incorporated into the host nuclear genome. The plastids of diatoms and other chromalveolates, originated from a secondary endosymbiosis with a red alga, have retained a higher proportion of the symbiont's genes in their genomes relative to their green counterparts, which derived from a primary endosymbiosis with a cyanobacterium. The red origin of the chloroplasts  and the lower cellular iron requirements of the red lineage  may have contributed to the ecological success of diatoms in the marine environment in terms of a putative evolutionary-based pre-adjustment to iron-deplete conditions.
The retention of a core set of chloroplast genes and the factors preventing their transfer to the nucleus are the subject of ongoing debates . The known chloroplast genomes of diatoms are circular with an extended inverted repeat region (IR) and are subject to internal rearrangements such as inversions . Occasional organelle lysis and free release of organellar DNA is considered an important first step in the transfer of organelle-encoded genes to the nuclear genome. Indeed high quantities of chloroplast (cp) and mitochondrial (mt) DNA are frequently transferred and inserted into the nuclear genome, thereafter referred to as nuclear plastid or nuclear mitochondrial DNA (NUPTs, NUMTs) [11–14]. However, stable replacement of a plastid gene by its nuclear copy requires a retargeting of the nuclear gene product back to the chloroplast compartment as well as a functional expression and regulation. Until this multi-step development has been accomplished the chloroplast version cannot be discarded and the gene exists in two copies, which might even overlap in function in a differentially regulated manner . Genes that provide a dual targeting sequence enabling import to both mitochondria and chloroplasts at the same time are also known .
Here, we present the complete T. oceanica CCMP1005 chloroplast genome sequence [GenBank: GU323224], which we assembled from a massively parallel pyrosequencing data set. Assembled genomic shotgun reads show that in the T. oceanica genome, the ferredoxin petF gene has been transferred to the nucleus. Ferredoxin is a photosynthetic redox protein that contains iron, and in diatoms it can be replaced by the iron-free flavodoxin, when iron-limited growth conditions prevail. The petF transfer to the nuclear genome may enable a refined regulation of this gene in response to iron availability in T. oceanica. Through comparative genomics between the coastal Thalassiosira pseudonana and the oceanic T. oceanica, we can trace the T. oceanica PETF gene back to its chloroplast origin and identify elements that may have played a role in the transfer of this important photosynthetic gene.
Characteristics of the T. oceanica chloroplast genome
Chloroplast Genome Features of T. oceanica in Comparison with T. pseudonana and P. tricornutum
Thalassiosira oceanica CCMP 1005
Thalassiosira pseudonana CCMP 1335
Phaeodactylum tricornutum CCAP 1055/1
genome size [bp]
IRa: 18 338
IRb: 18 337
G+C content [%]
A+T content [%]
gene content [#]
in T. oceanica, but not in T. pseudonana
orf127 a , flrn
in T. pseudonana, but not in T. oceanica
in IR of T. oceanica, but not in IR of T. pseudonana
clpC , trnC , trnL
in P. tricornutum only
tsf, syfB, acpP
Transfer of petF gene to the nuclear genome in T. oceanica
Functional expression and differential regulation of PETF
Novel features of the T. oceanica cp genome
As a second novel feature in the T. oceanica chloroplast genome we identified a duplication of the ffs RNA gene referred to as flrn. The ffs RNA adopts a stem-loop structure and is part of a signal recognition particle that might play a role at insertion of proteins into the inner chloroplast membrane . An alignment of the flrn gene with the ffs genes of T. oceanica, T. pseudonana and P. tricornutum (Figure 5b) reveals that the flrn gene is truncated and contains a nucleotide polymorphism in the conserved loop region. Although the similarity between flrn and ffs is high, the truncated nature of flrn suggests that this sequence may represent a pseudogene.
The chloroplast genome is a dynamic structure in which the collinear binding of the inverted repeat regions likely leads to a handle-like structure isolating the LSC and SSC regions in separate domains and serving as a basis for occasional recombinational events that result in an inversion of the SSC region . The weak conservation of synteny between T. oceanica and T. pseudonana cp genomes implies high structural dynamics of the circular genomic molecule, best explained by frequent inversion events. The remarkable lack of genomic rearrangements across the borders of the single copy regions, as observed by comparison between T. oceanica and T. pseudonana, could be explained by the structural separation of the LSC and SSC in a handle-like structure and supports this structural model.
The establishment of the primary endosymbiont as a chloroplast led to the loss of many of the endosymbiont's genes or their transfer to the host's nucleus. Red algal and Glaucophyte chloroplasts retain about 200 protein coding genes, while most members of the green lineage exhibit a further genome reduction to less than 100 genes, indicating functional transfer of several essential genes to the nuclear genome. The substantial retention of coding potential in the primary plastids of the red algal lineage has been hypothesized to have an impact on the chloroplast portability during secondary endosymbiosis, preferentially facilitating further endosymbiotic events of autotrophs from the red lineage over their more reduced green counterparts . Gene content of extant cp genomes derived from the red-algal lineage indicate that further gene loss or transfer proceeded after secondary endosymbiosis events, leading to a gene content of approx. 140 genes in the Stramenopiles and Cryptophytes. Stronger reductions can be found within the Apicomplexa and Dinophytes, referred to as Alveolates, perhaps because the known members of the Apicomplexa and several of the Dinophytes are non-photosynthetic organisms, and sometimes parasitic . All of these subgroups are regarded as descendants of an ancient red algal endosymbiont and its host .
Occasional free release of cp genomic DNA upon chloroplast lysis is considered an important source of chloroplast DNA for integration into the nuclear genome. This mechanism can operate only in organisms containing multiple plastids. Indeed the higher degree of gene retention in P. tricornutum compared to Thalassiosira species might be partially explained by the presence of a single, unique chloroplast in P. tricornutum which would not be available for lysis in the context of the living cell. T. oceanica and T. pseudonana cells on the other hand are equipped with approx. 4 plastids each [25, 26].
As for other evolutionary processes, the transfer of chloroplast-encoded genes to the nuclear genome is difficult, if not impossible, to observe. Experimental approaches with transgenic tobacco have demonstrated that plastid genes can be successfully transferred to the nuclear genome , but most of the evidence for the inferred transfers is derived from phylogenetic and phylogenomic arguments . Why certain genes are preferentially retained or transferred is puzzling and several theories have emerged ranging from a simple economic hypothesis in favour of gene transfer  to the CORR (CO-location for Redox Regulation) hypothesis in favour of gene retention . The ferredoxin petF gene is not essentially retained in the CP genome, as a nuclear location is observed in several phylogenetic groups. So far, with the exception of T. oceanica, the petF gene has been retained in large cp genomes (Figure 6) such as the ones from red algae and descendants (including diatoms), and has generally been transferred to the nucleus in groups with reduced cp genomes such as Chlorophytes and Streptophytes. Several intrinsic features of the petF gene, conserved in the cp genomes of the closely related species T. pseudonana and T. weissflogii, may have facilitated the functional transfer of petF from the chloroplast to the nuclear genome in T. oceanica. Thus, in the coastal species T. pseudonana and T. weissflogii, the petF gene transfer may be waiting to happen as well, given appropriate environmental selection pressures. However, at this point, the establishment of the PETF gene in the nuclear genome of T. oceanica represents an exception within the diatoms for which genome information is available so far.
It has been demonstrated that the photosynthetic architecture in T. oceanica is better adapted to iron-limited areas than its coastal counterpart T. weissflogii. Tolerance to iron limitation most likely arises from a combination of several genetic adaptations that contribute to a better streamlining of the photosynthetic apparatus towards low iron requirements. The low abundance of PSI relative to PSII in T. oceanica is clearly important in reducing the cellular iron requirements . The substitution of the iron-requiring cytochrome c6 by the copper-containing plastocyanin in T. oceanica is an additional strategy to further reduce photosynthetic iron requirements, and, hence, the iron quota of this diatom . Likewise, the flavodoxin expression is up-regulated upon iron limitation in many diatom species  while a s imultaneous down-regulation of the expression of the ferredoxin petF gene is observed (e.g. ). In our study, we could confirm the expression and iron-dependent regulation of the ferredoxin PETF, the plastocyanin PCY and the flavodoxin FLDA gene in T. oceanica (Figure 4). The concerted down-regulation of PETF and PCY upon iron limitation is expected as part of the general down-regulation of the photosynthetic apparatus under iron limitation. In contrast, FLDA is strongly up-regulated in order to substitute ferredoxin with flavodoxin, consequently keeping intact the electron-transport interface between membrane-bound light reactions and dark reactions in the cp stroma.
The contrast in photosynthetic physiology between T. oceanica and its coastal relatives T. pseudonana and T. weissflogii make an attractive case to infer an adaptive significance for the transfer of petF, an iron-regulated cp gene, to the nuclear genome. Whether the transfer of petF to the nuclear genome is simply a byproduct of evolutionary trends towards chloroplast genome reduction or truly confers an ecological advantage with respect to the response to iron remains uncertain, though the observed high tolerance of T. oceanica to severe iron limitation relative to its close relatives T. pseudonana and T. weissflogii suggests that the latter hypothesis is worthy of further investigation.
Single gene transfers are the elemental steps of cp genome reduction and may confer benefits to single species in the context of niche adaptation. It is tempting to speculate that larger phylogenetic groups whose members share a reduced cp genome (as in the green lineage) likewise emerged from a founder species that profited from the benefits of cp genome reduction and improved nuclear control over organelle function. Centralized and synchronized regulation of cp metabolism is assumed to be a potential driving force for intracellular gene transfers. Organisms that already experienced large-scale cp genome reduction should benefit as well from an improved regulation of the transferred genes. The uniform genomic situation in larger phylogenetic groups raises the question whether such competitive advantages might even apply to these groups as a whole. A comparative evaluation of this effect between the red and green lineage is complicated by the interference with different extents of gene losses in both groups as well as the presence of distinct types of photosynthetic physiology in general. However, the terrestrial environment has been conquered exclusively by members of the green lineage, and prerequisite for this achievement might (at least partially) have been the improvement of regulatory capacities linked to cp genome reduction by large scale gene transfer. The settlement of the land represented the occupation of a new ecological niche rich in abiotic stresses of a novel type. It remains to be elucidated to which extent such specific environmental stresses exert a selective pressure favouring gene transfer events , ultimately leading to competitive advantage and enhanced fitness.
Although the chloroplast genomes of some closely related marine phytoplankton species have been sequenced, the differences between species within a genus have been small and restricted to gene reshuffling. Our findings, reporting a traceable single gene transfer from the chloroplast to the nuclear genome, are unique so far, in part because of the availability of both plastid and nuclear genome sequences for T. oceanica and T. pseudonana. The example of petF shows that chloroplast and nuclear genomes are of remarkable plasticity. Whether or not the gene transfer described for T. oceanica confers a competitive advantage still needs to be assessed through experimental approaches. Future analyses of cp genomes from a wider range of ecologically diverse species will likely reveal other surprising patterns of cp gene content, loss and regulation, and further enhance our understanding of their impacts on the evolutionary fitness of species.
Strains and Cultures
T. oceanica Hasle  strain CCMP1005 was grown from an axenic clonal isolate, obtained from serial dilutions of a stock culture to extinction. T. oceanica cells were grown in 8 l batch cultures using iron-free f/2 nutrients  in ASW (artificial seawater medium ) supplied with 10 μM FeCl3 at 100 μE, 25°C and a 14/10 h light/dark cycle. Cells were harvested by filtration on 47 mm 5 μ-PC [polycarbonate]-filters, resuspended into a small volume of media, followed by centrifugation at 4°C for 10 min at 11000 rpm. Cell pellets were frozen in liquid N2 and stored at -80°C. Genome comparison was conducted with the genome data available at JGI and NCBI for T. pseudonana Hasle & Heimdal CCMP1335 [26, 37] and Phaeodactylum tricornutum Bohlin CCAP1055/1 [38, 39].
Nucleic Acid Extraction and Sequencing
Total genomic DNA for sequencing of the T. oceanica genome was extracted from nutrient-replete cells using the QIAGEN DNeasy kits. The quality of nucleic acids was assessed by NanoDrop UV absorption profiles and agarose gel electrophoresis. Next generation 454 sequencing technology  was applied to the gDNA as follows. After mechanical shearing, specific sequencing adaptors were ligated and the genomic DNA fragments were shotgun sequenced using massively parallel pyrosequencing on a 454 gs-flx instrument (Roche, Penzberg, Germany) according to the manufacturer's protocol. The resulting libraries were sequenced on a gs-flx sequencer using the standard manufacturer's protocol.
Cp Genome Sequence Assembly and Gap Closure
1.2 Mio flx pyrosequencing reads were assembled into contigs with the TGICL assembler using the CAP3 algorithm . The quality of the resulting contigs was manually confirmed by inspection using the CLVIEW cluster viewer program. Using the local BLAST package from NCBI , we identified 9 contigs with high sequence coverage as elements of the cp genome. Additional information extracted from the contig ends of the ace-file of the original assembly, enabled the manual assembly of the cp genome to near completeness. The two remaining small gaps were targeted by PCR amplification of the contig ends and demonstrated physical continuity of the gap regions. Bridging fragments were cloned in a TOPO cloning vector and their sequences determined by Sanger sequencing.
Sequence Analysis and Annotation
The assembled chloroplast genome sequence was analyzed by BLAST against the related T. pseudonana cp genome sequence, the NCBI nr (non-redundant) protein database and the NCBI CDD Conserved Domain Database. The BLAST analysis revealed few obvious artificial frameshifts within the original contigs, allowing correction of the chloroplast scaffold for single nucleotide errors placed in low complexity regions of single nucleotide repeats that generally appear to be critical in 454 data. The final chloroplast scaffold was annotated using Artemis  and the submission software Sequin . Inverted repeats at the 3' end of genes representing putative rho-independent transcriptional terminators [45, 46] were identified with the EINVERTED tool from the EMBOSS software package . Ribosomal Binding Sites (RBS) were determined manually from similarity to the AGGAGGT consensus sequence  and close proximity to the respective translation start. The contig containing the nuclear ferredoxin gene PETF was assembled manually from raw genomic 454 reads using local BLAST, database sequence retrieval and BioEdit . Gene modelling was done with the GENSCAN webserver  and confirmed by NCBI blastx against the nr protein database. The derived transcript encoded a protein with high homology to T. pseudonana and P. tricornutum petF protein orthologs.
Circular Map Construction
The circular genomic map was constructed from the primary embl-annotation file using CGVIEW  as follows: The embl-file was converted into an xml-file with the perl-script cgview_xml_builder.pl, which is enclosed in the CGVIEW package; the xml-file containing the formatting details for the circular map was then customized manually by adding functional categorization and appropriate gene symbol shapes and dimensions. The circular map was constructed from the xml-file as png-graphic, using the CGVIEW main function. Gene names were added using the open source graphics program GIMP .
Oligonucleotide Primers used for RT-qPCR Analyses
Forward Primer (5'→3')
Reverse Primer (5'→3')
Nomenclature of Gene Names
The nomenclature of gene names follows the recommendations for Chlamydomonas reinhardtii. Gene names are typed in italic and uppercase for nuclear genes (PETF) or italic and lower case for organelle genes (petF).
We thank Prof. Stefan Rose-John (Department of Biochemistry, Christian-Albrechts-Universität, Kiel, Germany) for advice in the isolation of nuclear genomic DNA from T. oceanica and the access to his laboratory and equipment. This work was supported in part by a DFG grant to JLR (RO2138/6-1) and by the DFG Cluster of Excellence Future Ocean (EXC 80). Prof. T. Bosch and Dr. Georg Hemmrich provided help with the initial cp contig assembly. We thank Tania Klüver for help with the laboratory experiment and culturing of the algae.
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