Nucleomorphs as genomic tools for photosynthesis research
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Beside the nucleus, the plastid and mitochondria, cryptomonads possess a fourth DNA-containing semiautonomous organelle, the nucleomorph. This organell was shown to be the remnant of the nucleus of a former free-living rhodophytic alga, which was engulfed and reduced to a complex plastid by a heterotrophic host cell. By sequencing the three minute nucleomorph chromosomes of the cryptomonad Guillardia theta, we present evidence that the purpose of the nucleomorph is to perpetuate itself, the periplastid space it is embedded in and a eukaryotic expression machinery for the synthesis of 30 nucleomorph-encoded chloroplast proteins (Douglas et al. (2001), Nature 410, 1091-1096). Some of these chloroplast located proteins have homologous sequences in the cyanobacterium Synechocystis sp. PCC6803 but differ from those by an amino-terminal extension which functions as a transit peptide for the translocation to the plastid. Beside proteins of known function (FtsZ, Hlip, CbbX), we identified 11 gene products whose homologs in cyanobacteria do not have any known function. These are the genes of interest we have in part characterized. By knock-out experiments in cyanobacteria and physiological investigations we analyzed the functions of the nucleomorph-encoded proteins in respect to the photosynthesis machinery. Major results so far are the detection of components attached to photosystem II and the creation of mutants with a reduced phycocyanin content.Keywords:
Chromoplast
Organelle
Nuclear gene
Transit Peptide
Chlorarachniophytes and cryptophytes possess complex plastids that were acquired by the ingestion of a green and red algal endosymbiont, respectively. The plastids are surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment, which corresponds to the remnant cytoplasm of the endosymbiont. Nucleomorphs contain a greatly reduced genome that possesses only several hundred genes with high evolutionary rates. We examined the relative transcription levels of the genes of all proteins encoded by the nucleomorph genomes of two chlorarachniophytes and three cryptophytes using an RNA-seq transcriptomic approach. The genes of two heat shock proteins, Hsp70 and Hsp90, were highly expressed under normal conditions. It has been shown that molecular chaperone overexpression allows an accumulation of genetic mutations in bacteria. Our results suggest that overexpression of heat shock proteins in nucleomorph genomes may play a role in buffering the mutational destabilization of proteins, which might allow the high evolutionary rates of nucleomorph-encoded proteins.
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MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, Mesostigma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloro- plast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer (LGT) of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coli inhibited cell division and resulted in the filamentous cell formation, clearly demon- strated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred be- fore the evolution of land plants.
Chlamydomonas reinhardtii
Chlamydomonas
Green algae
FtsZ
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ABSTRACT Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans , whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome ( B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans . How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.
Chloroplast membrane
Inner membrane
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Chloroplasts contain proteins that are encoded by different genetic systems, the plastid genome and the nuclear chromosomes. By comparing the gene content of plastid genomes of different taxa, some predictions about nuclear-encoded genes for plastid proteins are possible. However, early in evolution, many genes were transferred from the plastid to the cell nucleus and are therefore missing from all known plastid genomes and escape such predictions. By sequencing the miniaturized chromosomes of the nucleomorph of the cryptophyte Guillardia theta, as well as the plastid genome, we uncovered two genes encoding CbbX which are predicted to be involved in plastid function. Our findings suggest that (1) red-type plastid rbcLS genes evolved together with cbbX, which is related to cbbX genes of purple bacteria; (2) early in rhodoplast evolution, the cbbX gene was duplicated and transferred into the nucleus; (3) the plastid-encoded LysR transcriptional activator gene, rbcR, is homologous to rbcR and cbbR transcriptional activator genes of purple bacteria and cyanobacteria; and (4) the ancestral plastid probably harbored both types of form I RuBisCo.
Nuclear gene
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Organisms that have lost their photosynthetic capabilities are present in a variety of eukaryotic lineages, such as plants and disparate algal groups. Most of such non-photosynthetic eukaryotes still carry plastids, as these organelles retain essential biological functions. Most non-photosynthetic plastids possess genomes with varied protein-coding contents. Such remnant plastids are known to be present in the non-photosynthetic, bacteriovorous alga Pteridomonas danica (Dictyochophyceae, Ochrophyta), which, regardless of its obligatory heterotrophic lifestyle, has been reported to retain the typically plastid-encoded gene for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit ( rbcL ). The presence of rbcL without photosynthetic activity suggests that investigating the function of plastids in Pteridomonas spp. would likely bring unique insights into understanding the reductive evolution of plastids, their genomes, and plastid functions retained after the loss of photosynthesis. In this study, we demonstrate that two newly established strains of the non-photosynthetic genus Pteridomonas possess highly reduced plastid genomes lacking rbcL gene, in contrast to the previous report. Interestingly, we discovered that all plastid-encoded proteins in Pteridomonas spp. are involved only in housekeeping processes (e.g., transcription, translation and protein degradation), indicating that all metabolite synthesis pathways in their plastids are supported fully by nuclear genome-encoded proteins. Moreover, through an in-depth survey of the available transcriptomic data of another strain of the genus, we detected no candidate sequences for nuclear-encoded, plastid-directed Fe–S cluster assembly pathway proteins, suggesting complete loss of this pathway in the organelle, despite its widespread conservation in non-photosynthetic plastids. Instead, the transcriptome contains plastid-targeted components of heme biosynthesis, glycolysis, and pentose phosphate pathways. The retention of the plastid genomes in Pteridomonas spp. is not explained by the Suf-mediated constraint against loss of plastid genomes, previously proposed for Alveolates, as they lack Suf genes. Bearing all these findings in mind, we propose the hypothesis that plastid DNA is retained in Pteridomonas spp. for the purpose of providing glutamyl-tRNA, encoded by trnE gene, as a substrate for the heme biosynthesis pathway.
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Prokaryote
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Endosymbiosis
Nuclear gene
Eukaryote
Cellular compartment
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Abstract The chloroplasts of cryptophytes arose through a secondary endosymbiotic event in which a red algal endosymbiont was integrated into a previously nonphotosynthetic eukaryote. The cryptophytes retain a remnant of the endosymbiont nucleus (nucleomorph) that is replicated once in the cell cycle along with the chloroplast. To understand how the chloroplast, nucleomorph and host cell divide in a coordinated manner, we examined the expression of genes/proteins that are related to nucleomorph replication and chloroplast division as well as the timing of nuclear and nucleomorph DNA synthesis in the cryptophyte Guillardia theta . Nucleus-encoded nucleomorph HISTONE H2A mRNA specifically accumulated during the nuclear S phase. In contrast, nucleomorph-encoded genes/proteins that are related to nucleomorph replication and chloroplast division (FtsZ) are constantly expressed throughout the cell cycle. The results of this study and previous studies on chlorarachniophytes suggest that there was a common evolutionary pattern in which an endosymbiont lost its replication cycle-dependent transcription while cell-cycle-dependent transcriptional regulation of host nuclear genes came to restrict the timing of nucleomorph replication and chloroplast division.
Replication
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Many lines of evidence support the idea that the first chloroplast was the result of an endosymbiotic relationship between a cyanobacterium and a non‐photosynthetic eukaryote. Most of the cyanobacterial genes were lost, but a few remained in the chloroplast genome and as many as a thousand were transferred to the host nucleus. Genes encoding functions required by the chloroplast had to acquire presequences to target the products to the chloroplast. The situation gets more complicated when we consider the algae with chlorophyll c. They are the product of secondary endosymbiosis, where a putative red algal ancestor was engulfed by another non‐photosynthetic eukaryote, which retained the red algal chloroplast but eventually got rid of the rest of the cell. This left the chloroplast surrounded by two additional membranes: one derived from the red algal plasma membrane and the other from the host's phagocytic vacuole. In order for the endosymbiotic relationship to work, there must have been a substantial amount of gene transfer from the red algal nucleus to the host nucleus to support chloroplast functions. In the cryptophytes we even see an intermediate stage in this process, a relict nucleus (nucleomorph) in the periplastidal space between the outer two membranes and the original chloroplast envelope. Now that the draft genome sequence of the diatom Thalassiosira pseudonana (Diatom Genome Consortium) as well as genomes of rhodophyte Cyanidioschyzon merolae and green plants are available, it is possible to investigate the evolutionary history of plastid localized metabolic pathways. Phylogenetic analyses of nuclear‐encoded putatively plastid‐targeted enzymes showed that plastids obviously utilize enzymes not only of expected plastid (cyanobacterial) origin. Within the diatom, apicomplexan, plant and rhodophyte genomes, we have identified several enzymes that originate in α‐proteobacteria (mitochondria) or even in eukaryotic nucleus, but possess N‐terminal plastid‐targeting presequences. Although diatoms are, according to multiprotein phylogeny, related to Alveolates, some plastid‐related metabolic pathways show substantially different evolutionary pattern as well as, in silico, predicted localizations of involved enzymes.
Endosymbiosis
Eukaryote
Thalassiosira pseudonana
Nuclear gene
Green algae
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Wilhelm Gruissem Department of Botany University of California Berkeley, California 94720 Plant and animal cells are fundamentally very similar. Be- sides the organelles found in both cell types, however, plant cells contain a unique class of organelles, the plastids. Since the early discovery by Correns (1909) and Baur (1909) that mutations affecting plastid phenotypes in higher plants frequently exhibit non-Mendelian inheri- tance, research on the DNA of this organelle has now yielded the complete sequence of the plastid genomes from tobacco (Shinozaki et al., 1986) and liverwort (Oh- yama et al., 1986). Plastids exist in a number of different forms with different functions, but the green chloroplast was the first to be discovered, and is the best studied of all plastids. The diversity of plastid types is controlled by the developmental program of the plant, which indicates that there must be a significant flow of information be- tween two separate genetic compartments in the cell. The use of chloroplasts to study photosynthesis and the in- tricacy of photosynthetic complexes has yielded new infor- mation on controls of organelle gene expression and the communication of different genomes in eukaryotic cells. In developing plants, chloroplasts are derived from small proplastids, which are the undifferentiated plastids present in meristematic cells. During the development of chloroplasts in photosynthetic tissues, photosynthetic electron-transfer components are assembled into pho- tosystems I and II, cytochrome bsf, and ATP synthase complexes, each of which consists of up to 20 polypep- tides. Proplastids and chloroplasts can also differentiate into specialized plastid types that assume other functions in nonphotosynthetic plant organs of higher plants, such as amyloplasts in roots and tubers or chromoplasts in many flowers and fruits. Photosynthesis, together with other plastid functions, requires the products of several hundred genes, of which only about 120 are present in the approximately 150 kb chloroplast genome. All other plas- tid proteins are expressed from nuclear genes. The devel- opment and differentiation of photosynthetically compe- tent chloroplasts and other plastid types thus present a challenging opportunity: to decipher how plastid gene ex- pression is controlled temporally and spatially in different plant organs, and also in coordination with the expression of nuclear genes for chloroplast proteins. Initial efforts to analyze the controls of plastid gene expression have con- centrated on the transcription of genes for photosynthetic proteins and tRNAs. Recent progress appears to support a model that places a major emphasis on posttranscrip- tional and translational regulatory mechanisms. In con- trast, known nuclear genes for photosynthetic proteins ap- pear to be regulated primarily at the level of transcription. The purpose of this review is to discuss some of the cen- tral problems and ideas in the field of chloroplast gene ex- pression, not to provide a comprehensive review on all that is known. (For further information on chloroplast ge- nome structures, genes, and transcriptional and transla- tional components, readers should consult Whitfeld and Bottomley, 1983; Ellis, 1984; Sugiura, 1987; Umesono and Ozeki, 1987; Gruissem, 1989; Mullet, 1988; Bonham- Smith and Bourque, 1988.) Linkage of Genes in Many Chloroplast Transcription Units Is Conserved Compared with the small number of genes in animal, fun- gal and plant mitochondria, the chloroplast genome con- tains a substantially larger number of genes, encoding both genetic and photosynthetic functions. The genes identified thus far include a complete set of 30 tRNAs, four ribosomal RNAs (23S, 16S, 5S, and 4.5s) and 20 ribo- somal proteins. Twenty-two genes encode proteins for thylakoid membrane complexes (photosystem I, photosys- tern II, cytochrome bsf complex, and ATP synthase), and the sequences of six other open reading frames share similarities with the mitochondrial genes for the subunits of the human respiratory chain NADH dehydrogenase. Several of the remaining unidentified reading frames are conserved between diverse species, which suggests that they may also encode functional plastid polypeptides. Most plastid genes are organized into polycistronic tran- scription units reminiscent of bacterial operons. The se- quence analysis of the entire tobacco and liverwort chlo- roplast genomes (Shinozaki et al., 1986; Ohyama et al., 1986) together with the partial sequence and mapping data from other plant chloroplast genomes, has revealed that the arrangement of genes within these transcription units is highly conserved, although transcription are extensively rearranged in some plant species (reviewed by Palmer, 1985). Detailed mapping of chloroplast DNAs from pea and geranium, for example, has found that such rearrangements involve primarily inversions of large clus- ters of genes. Most, but not all, the genes linked in these clusters are cotranscribed. It has been possible, at least some cases, to trace the linkage of chloroplast gene sets to the cyanobacterial genome (Cozens et al., 1986) which is the putative ancestral genome of chloroplast genome. However, the conserved arrangement of genes in plant chloroplast genomes is not found algae, for which Chlamydomonas and Euglena are the best studied examples, possibly indicating different endosymbiotic events. Chloroplast RNA Polymerases and Promoter Regions The possibility that transcriptional regulation chlo- roplast genes could be a key control during chloroplast development in plants spurred early investigations into the transcriptional components of this organelle. Applica- tion of different schemes for preparing DNA-dependent RNA polymerase from chloroplasts led to the intriguing idea that chloroplasts of algae and plants may contain at least two different RNA polymerase activities distinguish-
Organelle
Plant cell
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