Evidence that sigma factors are components of chloroplast RNA polymerase.

Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae.     

The Mitochondrial Genome of Protists

The data on the structure and functions of the mitochondrial genomes of protists (Protozoa and unicellular red and green algae) are reviewed. It is emphasized that mitochondrial gene structure and composition, as well as organization of mitochondrial genomes in protists are more diverse than in multicellular eukaryotes. The gene content of mitochondrial genomes of protists are closer to those of plants than animals or fungi. In the protist mitochondrial DNA, both the universal (as in higher plants) and modified (as in animals and fungi) genetic codes are used. In the overwhelming majority of cases, protist mitochondrial genomes code for the major and minor rRNA components, some tRNAs, and about 30 proteins of the respiratory chain and ribosomes. Based on comparison of the mitochondrial genomes of various protists, the origin and evolution of mitochondria are briefly discussed.     

The mitochondrial genome of protists

The data on the structure and functions of the mitochondrial genomes of protists (Protozoa and unicellular red and green algae) are reviewed. It is emphasized that mitochondrial gene structure and composition, as well as organization of mitochondrial genomes in protists are more diverse than in multicellular eukaryotes. The gene content of mitochondrial genomes of protists are closer to those of plants than animals or fungi. In the protist mitochondrial DNA, both the universal (as in higher plants) and modified (as in animals and fungi) genetic codes are used. In the overwhelming majority of cases, protist mitochondrial genomes code for the major and minor rRNA components, some tRNAs, and about 30 proteins of the respiratory chain and ribosomes. Based on comparison of the mitochondrial genomes of various protists, the origin and evolution of mitochondria are briefly discussed.     

Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.     

Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta)

The 22 published chloroplast genomes of green algae, representing sparse taxonomic sampling of diverse lineages that span over one billion years of evolution, each possess a unique gene arrangement. In contrast, many of the >190 published embryophyte (land plant) chloroplast genomes have relatively conserved architectures. To determine the phylogenetic depth at which chloroplast gene rearrangements occur in green algae, a 1.5-4 kb segment of the chloroplast genome was compared across nine species in three closely related genera of Trebouxiophyceae (Chlorophyta). In total, four distinct gene arrangements were obtained for the three genera Elliptochloris, Hemichloris, and Coccomyxa. In Elliptochloris, three distinct chloroplast gene arrangements were detected, one of which is shared with members of its sister genus Hemichloris. Both species of Coccomyxa examined share the fourth arrangement of this genome region, one characterized by very long spacers. Next, the order of genes found in this segment of the chloroplast genome was compared across green algae and land plants. As taxonomic ranks are not equivalent among different groups of organisms, the maximum molecular divergence among taxa sharing a common gene arrangement in this genome segment was compared. Well-supported clades possessing a single gene order had similar phylogenetic depth in green algae and embryophytes. When the dominant gene order of this chloroplast segment in embryophytes was assumed to be ancestral for land plants, the maximum molecular divergence was found to be over two times greater in embryophytes than in trebouxiophyte green algae. This study greatly expands information about chloroplast genome variation in green algae, is the first to demonstrate such variation among congeneric green algae, and further illustrates the fluidity of green algal chloroplast genome architecture in comparison to that of many embryophytes.     

Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages

In the plant chloroplast genome the codon usage of the highly expressed psbA gene is unique and is adapted to the tRNA population, probably due to selection for translation efficiency. In this study the role of selection on codon usage in each of the fully sequenced chloroplast genomes, in addition to Chlamydomonas reinhardtii, is investigated by measuring adaptation to this pattern of codon usage. A method is developed which tests selection on each gene individually by constructing sequences with the same amino acid composition as the gene and randomly assigning codons based on the nucleotide composition of noncoding regions of that genome. The codon bias of the actual gene is then compared to a distribution of random sequences. The data indicate that within the algae selection is strong in Cyanophora paradoxa, affecting a majority of genes, of intermediate intensity in Odontella sinensis, and weaker in Porphyra purpurea and Euglena gracilis. In the plants, selection is found to be quite weak in Pinus thunbergii and the angiosperms but there is evidence that an intermediate level of selection exists in the liverwort Marchantia polymorpha. The role of selection is then further investigated in two comparative studies. It is shown that average relative codon bias is correlated with expression level and that, despite saturation levels of substitution, there is a strong correlation among the algae genomes in the degree of codon bias of homologous genes. All of these data indicate that selection for translation efficiency plays a significant role in determining the codon bias of chloroplast genes but that it acts with different intensities in different lineages. In general it is stronger in the algae than the higher plants, but within the algae Euglena is found to have several unusual features which are noted. The factors that might be responsible for this variation in intensity among the various genomes are discussed. Received: 6 June 1997 / Accepted: 24 July 1997     

Plastid signals remodel light signaling networks and are essential for efficient chloroplast biogenesis in Arabidopsis

Plastid signals are among the most potent regulators of genes that encode proteins active in photosynthesis. Plastid signals help coordinate the expression of the nuclear and chloroplast genomes and the expression of genes with the functional state of the chloroplast. Here, we report the isolation of new cryptochrome1 (cry1) alleles from a screen for Arabidopsis thaliana genomes uncoupled mutants, which have defects in plastid-to-nucleus signaling. We also report genetic experiments showing that a previously unidentified plastid signal converts multiple light signaling pathways that perceive distinct qualities of light from positive to negative regulators of some but not all photosynthesis-associated nuclear genes (PhANGs) and change the fluence rate response of PhANGs. At least part of this remodeling of light signaling networks involves converting HY5, a positive regulator of PhANGs, into a negative regulator of PhANGs. We also observed that mutants with defects in both plastid-to-nucleus and cry1 signaling exhibited severe chlorophyll deficiencies. These data show that the remodeling of light signaling networks by plastid signals is a mechanism that plants use to integrate signals describing the functional and developmental state of plastids with signals describing particular light environments when regulating PhANG expression and performing chloroplast biogenesis.     

藻类植物叶绿体基因组研究进展

藻类植物的cpDNA结构复杂,普遍缺失反向重复序列IR,且存在IR的藻类植物种类的cpDNA也有IR变短退化迹象.藻类植物的cpDNA包含的基因一般比高等植物要多,编码能力更强.藻类植物cpDNA全序列的测定方法主要是Fosmid文库构建,配合使用Long-PCR技术.该文对国内外有关藻类植物叶绿体基因组结构、叶绿体编码基因、叶绿体基因组在藻类系统发育中的应用以及藻类植物叶绿体基因组的提取和序列测定方法等进行综述,为藻类植物的系统发育和叶绿体起源以及功能基因组学的研究提供理论依据.     

Analysis of mitochondrial and chloroplast genomes in two volvocine algae: Eudorina elegans and Eudorina cylindrica (Volvocaceae,Chlorophyta)

The colonial volvocine algae span the full range of organizational complexity, from four-celled species to multicellular species, and this group of algae is often used for the study of evolution. In recent years, many organelle genomes have been sequenced using the application of next generation sequencing technology; however, only a few organelle genomes have been reported in colonial volvocine algae. In this study, we determined the organelle genomes of Eudorina elegans and Eudorina cylindrica and analysed the organelle genome size, structure and gene content between these volvocine species. This provided useful information to help us understand the composition of colonial volvocine organelle genomes. Based on the chloroplast genome protein-coding genes, we conducted a phylogenomics analysis of the volvocine algae. The result revealed an unexpected phylogenetic relationship, namely, E. elegans is more closely related to Pleodorina starrii than to E. cylindrica. The substitution rate of volvocine algae was then calculated based on organelle genome protein-coding genes; our analysis suggested the possibility that the two Eudorina species may be under similar evolutionary pressure. Lastly, the synteny analysis of the mitochondrial genome showed that gene arrangements and contents are highly conserved in the family Volvocaceae, and the synteny analysis of the chloroplast genome indicated that the genus Eudorina may have experienced genomic changes.     

Identification of chloroplast signal recognition particle RNA genes

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.     

Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns

Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.     

Structural diversity of signal recognition particle RNAs in plastids

One of the pathways for protein targeting to the plasma membrane in bacteria utilizes the co-translationally acting signal recognition particle (SRP), a universally conserved ribonucleoprotein complex consisting of a 54 kDa protein and a functional RNA. An interesting exception is the higher plant chloroplast SRP, which lacks the otherwise essential RNA component. Furthermore, green plant chloroplasts have an additional post-translational SRP-dependent transport system in which the chloroplast-specific cpSRP43 protein binds to imported substrate proteins and to the conserved 54 kDa SRP subunit (cpSRP54). While homologs to the bacterial SRP protein and RNA component previously have been identified in genome sequences of red algae and diatoms, a recent study investigated the evolution of the green plant SRP system.¹ Analysis of hundreds of plastid and nuclear genomes showed a surprising pattern of multiple losses of the plastid SRP RNA during evolution and a widespread presence in all non-spermatophyte plants and green algae. Contrary to expectations, all green organisms that have an identified cpSRP RNA also contain a cpSRP43. Notably, the structure of the plastid SRP RNAs is much more diverse than that of bacterial SRP RNAs. The apical GNRA tetraloop is only conserved in organisms of the red lineage and basal organisms of the green lineage, whereas further chloroplast SRP RNAs are characterized by atypical, mostly enlarged apical loops.     

Origin and phylogeny of chloroplasts revealed by a simple correlation analysis of complete genomes

The complete sequenced genomes of chloroplast have provided much information on the origin and evolution of this organelle. In this paper we attempt to use these sequences to test a novel approach for phylogenetic analysis of complete genomes based on correlation analysis of compositional vectors. All protein sequences from 21 complete chloroplast genomes are analyzed in comparison with selected archaea, eubacteria, and eukaryotes. The distance-based analysis shows that the chloroplast genomes are most closely related to cyanobacteria, consistent with the endosymbiotic origin of chloroplasts. The chloroplast genomes are separated to two major clades corresponding to chlorophytes (green plants) s.l. and rhodophytes (red algae) s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution. For instance, the analysis places the chloroplasts of two chromophytes (Guillardia and Odontella) within the rhodophyte lineage, supporting secondary endosymbiosis as the source of these chloroplasts. The relationships among the green algae and land plants in our tree also agree with results from traditional phylogenetic analyses. Thus, this study establishes the value of our simple correlation analysis in elucidating the evolutionary relationships among genomes. It is hoped that this approach will provide insights on comparative genome analysis.     

Contrasting modes and tempos of genome evolution in land plant organelles

Despite inhabiting the same cell lineage for roughly a billion years and being dependent on the same nucleus for most of their gene products and genetic control, the two organelle genomes of land plants exhibit remarkably different tempos and patterns of evolutionary change. With a few notable exceptions, chloroplast genomes are highly conserved in size and gene arrangement, whereas mitochondrial genomes vary enormously in size and organization. Conversely, nucleotide substitution rates are on average several times higher in chloroplast DNA than in mitochondrial DNA. Mechanistic and selective forces underlying these differences are only poorly understood.     

The chloroplast genome sequence of Chara vulgaris sheds new light into the closest green algal relatives of land plants

The phylum Streptophyta comprises all land plants and six monophyletic groups of charophycean green algae (Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales, and Charales). Phylogenetic analyses of four genes encoded in three cellular compartments suggest that the Charales are sister to land plants and that charophycean green algae evolved progressively toward an increasing cellular complexity. To validate this phylogenetic hypothesis and to understand how and when the highly conservative pattern displayed by land plant chloroplast DNAs (cpDNAs) originated in the Streptophyta, we have determined the complete chloroplast genome sequence (184,933 bp) of a representative of the Charales, Chara vulgaris, and compared this genome to those of Mesostigma (Mesostigmatales), Chlorokybus (Chlorokybales), Staurastrum and Zygnema (Zygnematales), Chaetosphaeridium (Coleochaetales), and selected land plants. The phylogenies we inferred from 76 cpDNA-encoded proteins and genes using various methods favor the hypothesis that the Charales diverged before the Coleochaetales and Zygnematales. The Zygnematales were identified as sister to land plants in the best tree topology (T1), whereas Chaetosphaeridium (T2) or a clade uniting the Zygnematales and Chaetosphaeridium (T3) occupied this position in alternative topologies. Chara remained at the same basal position in trees including more land plant taxa and inferred from 56 proteins/genes. Phylogenetic inference from gene order data yielded two most parsimonious trees displaying the T1 and T3 topologies. Analyses of additional structural cpDNA features (gene order, gene content, intron content, and indels in coding regions) provided better support for T1 than for the topology of the above-mentioned four-gene tree. Our structural analyses also revealed that many of the features conserved in land plant cpDNAs were inherited from their green algal ancestors. The intron content data predicted that at least 15 of the 21 land plant group II introns were gained early during the evolution of streptophytes and that a single intron was acquired during the transition from charophycean green algae to land plants. Analyses of genome rearrangements based on inversions predicted no alteration in gene order during the transition from charophycean green algae to land plants.