The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants

The photosynthetic gene rbcL has been lost or dramatically altered in some lineages of nonphotosynthetic parasitic plants, but the dynamics of these events following loss of photosynthesis and whether rbcL has sustained functionally significant changes in photosynthetic parasitic plants are unknown. To assess the changes to rbcL associated with the loss of functional constraints for photosynthesis, nucleotide sequences from nonparasitic and parasitic plants of Scrophulariales were used for phylogeny reconstruction and character analysis. Plants in this group display a broad range of parasitic abilities, from photosynthetic ("hemiparasites") to nonphotosynthetic ("holoparasites"). With the exception of Conopholis (Orobanchaceae), the rbcL locus is present in all parasitic plants of Scrophulariales examined. Several holoparasitic genera included in this study, including Boschniakia, Epifagus, Orobanche, and Hyobanche, have rbcL pseudogenes. However, the holoparasites Alectra orobanchoides, Harveya capensis, Harveya purpurea, Lathraea clandestina, Orobanche corymbosa, O. fasciculata, and Striga gesnerioides have intact open reading frames (ORFs) for the rbcL gene. Phylogenetic hypotheses based on rbcL are largely in agreement with those based on sequences of the nonphotosynthetic genes rps2 and matK and show a single origin of parasitism, and loss of photosynthesis and pseudogene formation have been independently derived several times in Scrophulariales. The mutations in rbcL in nonparasitic and hemiparasitic plants would result in largely conservative amino acid substitutions, supporting the hypothesis that functional proteins can experience only a limited range of changes, even in minimally photosynthetic plants. In contrast, ORFs in some holoparasites had many previously unobserved missense substitutions at functionally important amino acid residues, suggesting that rbcL genes in these plants have evolved under relaxed or altered functional constraints.      

Plastid genome structure and loss of photosynthetic ability in the parasitic genus Cuscuta

The genus Cuscuta (dodder) is composed of parasitic plants, some species of which appear to be losing the ability to photosynthesize. A molecular phylogeny was constructed using 15 species of Cuscuta in order to assess whether changes in photosynthetic ability and alterations in structure of the plastid genome relate to phylogenetic position within the genus. The molecular phylogeny provides evidence for four major clades within Cuscuta. Although DNA blot analysis showed that Cuscuta species have smaller plastid genomes than tobacco, and that plastome size varied significantly even within one Cuscuta clade, dot blot analysis indicated that the dodders possess homologous sequence to 101 genes from the tobacco plastome. Evidence is provided for significant rates of DNA transfer from plastid to nucleus in Cuscuta. Size and structure of Cuscuta plastid genomes, as well as photosynthetic ability, appear to vary independently of position within the phylogeny, thus supporting the hypothesis that within Cuscuta photosynthetic ability and organization of the plastid genome are changing in an unco-ordinated manner.     

Mechanisms of Functional and Physical Genome Reduction in Photosynthetic and Nonphotosynthetic Parasitic Plants of the Broomrape Family

Nonphotosynthetic plants possess strongly reconfigured plastomes attributable to convergent losses of photosynthesis and housekeeping genes, making them excellent systems for studying genome evolution under relaxed selective pressures. We report the complete plastomes of 10 photosynthetic and nonphotosynthetic parasites plus their nonparasitic sister from the broomrape family (Orobanchaceae). By reconstructing the history of gene losses and genome reconfigurations, we find that the establishment of obligate parasitism triggers the relaxation of selective constraints. Partly because of independent losses of one inverted repeat region, Orobanchaceae plastomes vary 3.5-fold in size, with 45 kb in American squawroot (Conopholis americana) representing the smallest plastome reported from land plants. Of the 42 to 74 retained unique genes, only 16 protein genes, 15 tRNAs, and four rRNAs are commonly found. Several holoparasites retain ATP synthase genes with intact open reading frames, suggesting a prolonged function in these plants. The loss of photosynthesis alters the chromosomal architecture in that recombinogenic factors accumulate, fostering large-scale chromosomal rearrangements as functional reduction proceeds. The retention of DNA fragments is strongly influenced by both their proximity to genes under selection and the co-occurrence with those in operons, indicating complex constraints beyond gene function that determine the evolutionary survival time of plastid regions in nonphotosynthetic plants.     

Phylogeny, rate variation, and genome size evolution of Pelargonium (Geraniaceae)

The phylogeny of 58 Pelargonium species was estimated using five plastid markers (rbcL, matK, ndhF, rpoC1, trnL-F) and one mitochondrial gene (nad5). The results confirmed the monophyly of three major clades and four subclades within Pelargonium but also indicate the need to revise some sectional classifications. This phylogeny was used to examine karyotype evolution in the genus: plotting chromosome sizes, numbers and 2C-values indicates that genome size is significantly correlated with chromosome size but not number. Accelerated rates of nucleotide substitution have been previously detected in both plastid and mitochondrial genes in Pelargonium, but sparse taxon sampling did not enable identification of the phylogenetic distribution of these elevated rates. Using the multigene phylogeny as a constraint, we investigated lineage- and locus-specific heterogeneity of substitution rates in Pelargonium for an expanded number of taxa and demonstrated that both plastid and mitochondrial genes have had accelerated substitution rates but with markedly disparate patterns. In the plastid, the exons of rpoC1 have significantly accelerated substitution rates compared to its intron and the acceleration was mainly due to nonsynonymous substitutions. In contrast, the mitochondrial gene, nad5, experienced substantial acceleration of synonymous substitution rates in three internal branches of Pelargonium, but this acceleration ceased in all terminal branches. Several lineages also have dN/dS ratios significantly greater than one for rpoC1, indicating that positive selection is acting on this gene, whereas the accelerated synonymous substitutions in the mitochondrial gene are the result of elevated mutation rates.     

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.     

A subset of conserved tRNA genes in plastid DNA of nongreen plants.

The plastid genome of the nonphotosynthetic parasitic plant Epifagus virginiana contains only 17 of the 30 tRNA genes normally found in angiosperm plastid DNA. Although this is insufficient for translation, the genome is functional, so import of cytosolic tRNAs into plastids has been suggested. This raises the question of whether the tRNA genes that remain in E. virginiana plastid DNA are active or have just fortuitously escaped deletion. We report the sequences of 20 plastid tRNA loci from Orobanche minor, which shares a nonphotosynthetic ancestor with E. virginiana. The two species have 9 intact tRNA genes in common, the others being defunct in one or both species. The intron-containing trnLUAA gene is absent from E. virginiana, but it is intact, transcribed, and spliced in O. minor. The shared intact genes are better conserved than intergenic sequences, which indicates that these genes are being maintained by natural selection and, therefore, must be functional. For the most part, the tRNA species conserved in nonphotosynthetic plastids are also those that have never been found to be imported in plant mitochondria, which suggests that the same rules may govern tRNA import in the two organelles. A small photosynthesis gene, psbI, is still intact in O. minor, and computer simulations show that some small nonessential genes have an appreciable chance of escaping deletion.     

姜科砂仁属植物DNA条形码序列的筛选

砂仁属（Amomum）隶属于姜科,全属约150种,我国有39种。该属多种植物可作药物或香料,但目前砂仁属的分类还不清楚,准确鉴定物种有很大难度。本研究利用DNA barcoding技术,对砂仁属50种121个个体的matK、rbcL-a、trnH-psbA序列及其不同组合进行比较,用Taxon DNA计算种间、种内bar-coding gap,运用相似法的BLASTn计算条码的正确鉴定率,筛选适合砂仁属的条码片段。结果显示：所有条码的barcoding gap均不存在;matK的正确鉴定率高于trnH-psbA和rbcL-a,联合片段的条码正确鉴定率高于单片段条码,三个片段联合条码的正确鉴定率最高。因此,推荐matK＋rbcL-a＋trnH-psbA作为砂仁属物种鉴定的候选条码。     

Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes

The non-photosynthetic, parasitic flowering plant Epifagus virginiana has recently been shown to contain a grossly reduced plastid genome that has lost many photosynthetic and chloro-respiratory genes. We have cloned and sequenced a 3.9 kb domain of plastid DNA from Epifagus to investigate the patterns of evolutionary change in such a reduced genome and to determine which genes are still present and likely to be functional. This 3.9 kb domain is colinear with a 35.4 kb region of tobacco chloroplast DNA, differing from it by a minimum of 11 large deletions varying in length from 354 bp to 11.5 kb, as well as by a number of small deletions and insertions. The nine genes retained in Epifagus encode seven tRNAs and two ribosomal proteins and are coextensive and highly conserved in sequence with homologs in photosynthetic plants. This suggests that these genes are functional in Epifagus and, together with evidence that the Epifagus plastid genome is transcribed, implies that plastid gene products play a role in processes other than photosynthesis and gene expression. Genes that are completely absent include not only photosynthetic genes, but surprisingly, genes encoding three subunits of RNA polymerase, four tRNAs and one ribosomal protein. In addition, only pseudogenes are found for two other tRNAs. Despite these defunct tRNA genes, codon and amino acid usage in Epifagus protein genes is normal. We therefore hypothesize that the expression of plastid genes in Epifagus relies on the import of nuclear encoded tRNAs and RNA polymerase from the cytoplasm.     

NADPH thioredoxin reductase C is localized in plastids of photosynthetic and nonphotosynthetic tissues and is involved in lateral root formation in Arabidopsis

Plastids are organelles present in photosynthetic and nonphotosynthetic plant tissues. While it is well known that thioredoxin-dependent redox regulation is essential for leaf chloroplast function, little is known of the redox regulation in plastids of nonphotosynthetic tissues, which cannot use light as a direct source of reducing power. Thus, the question remains whether redox regulation operates in nonphotosynthetic plastid function and how it is integrated with chloroplasts for plant growth. Here, we show that NADPH-thioredoxin reductase C (NTRC), previously reported as exclusive to green tissues, is also expressed in nonphotosynthetic tissues of Arabidopsis thaliana, where it is localized to plastids. Moreover, we show that NTRC is involved in maintaining the redox homeostasis of plastids also in nonphotosynthetic organs. To test the relationship between plastids of photosynthetic and nonphotosynthetic tissues, transgenic plants were obtained with redox homeostasis restituted exclusively in leaves or in roots, through the expression of NTRC under the control of organ-specific promoters in the ntrc mutant. Our results show that fully functional root amyloplasts are not sufficient for root, or leaf, growth, but fully functional chloroplasts are necessary and sufficient to support wild-type rates of root growth and lateral root formation.     

Twenty-fold difference in evolutionary rates between the mitochondrial and plastid genomes of species with secondary red plastids

Within plastid-bearing species, the relative rates of evolution between mitochondrial and plastid genomes are poorly studied, but for the few lineages in which they have been explored, including land plants and green algae, the mitochondrial DNA mutation rate is nearly always estimated to be lower than or equal to that of the plastid DNA. Here, we show that in protists from three distinct lineages with secondary, red algal-derived plastids, the opposite is true: their mitochondrial genomes are evolving 5-30 times faster than their plastid genomes, even when the plastid is nonphotosynthetic. These findings have implications for understanding the origins and evolution of organelle genome architecture and the genes they encode.     

A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus

Plastids are widespread in plant and algal lineages. They are also exploited by some nonphotosynthetic protists, including malarial parasites, to support their diverse modes of life. However, cryptic plastids may exist in other nonphotosynthetic protists, which could be important in studies on the diversity and evolution of plastids. The parasite Perkinsus marinus, which causes mass mortality in oyster farms, is a nonphotosynthetic protist that is phylogenetically related to plastid-bearing dinoflagellates and apicomplexans. In this study, we searched for P. marinus methylerythritol phosphate (MEP) pathway genes, responsible for de novo isoprenoid synthesis in plastids, and determined the full-length gene sequences for 6 of 7 of these genes. Phylogenetic analyses revealed that each P. marinus gene clusters with orthologs from plastid-bearing eukaryotes, which have MEP pathway genes with essentially the same mosaic pattern of evolutionary origin. A new analytical method called sliding-window iteration of TargetP was developed to examine the distribution of targeting preferences. This analysis revealed that the sequenced genes encode bipartite targeting peptides that are characteristic of proteins targeted to secondary plastids originating from endosymbiosis of eukaryotic algae. These results support our idea that Perkinsus is a cryptic algal group containing nonphotosynthetic secondary plastids. In fact, immunofluorescent microscopy indicated that 1 of the MEP pathway enzymes, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, was localized to small compartments near mitochondrion, which are possibly plastids. This tiny organelle seems to contain very low quantities of DNA or may even lack DNA entirely. The MEP pathway genes are a useful tool for investigating plastid evolution in both of the photosynthetic and nonphotosynthetic eukaryotes and led us to propose the hypothesis that ancestral "chromalveolates" harbored plastids before a secondary endosymbiotic event.     

Characterization of Mitochondrial Small-Subunit Ribosomal RNAs from Holoparasitic Plants

Mitochondrial small-subunit (19S) rDNA sequences were obtained from 10 angiosperms to further characterize sequence divergence levels and structural variation in this molecule. These sequences were derived from seven holoparasitic (nonphotosynthetic) angiosperms as well as three photosynthetic plants. 19S rRNA is composed of a conservative core region (ca. 1450 nucleotides) as well as two variable regions (V1 and V7). In pairwise comparisons of photosynthetic angiosperms to Glycine, the core 19S rDNA sequences differed by less than 1.4%, thus supporting the observation that variation in mitochondrial rDNA is 3–4 times lower than seen in protein coding and rDNA genes of other subcellular organelles. Sequences representing four distinct lineages of nonasterid holoparasites showed significantly increased numbers of substitutions in their core 19S rDNA sequences (2.3–7.6%), thus paralleling previous findings that showed accelerated rates in nuclear (18S) and plastid (16S) rDNA from the same plants. Relative rate tests confirmed the accelerated nucleotide substitution rates in the holoparasites whereas rates in nonparasitic plants were not significantly increased. Among comparisons of both parasitic and nonparasitic plants, transversions outnumbered transitions, in many cases more than two to one. The core 19S rRNA is conserved in sequence and structure among all nonparasitic angiosperms whereas 19S rRNA from members of holoparasitic Balanophoraceae have unique extensions to the V5 and V6 variable domains. Substitution and insertion/deletion mutations characterized the V1 and V7 regions of the nonasterid holoparasites. The V7 sequence of one holoparasite (Scybalium) contained repeat motifs. The cause of substitution rate increases in the holoparasites does not appear to be a result of RNA editing, hence the underlying molecular mechanism remains to be fully documented. Received: 18 May 1997 / Accepted: 11 July 1997     

Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes

Heme biosynthesis represents one of the most essential metabolic pathways in living organisms, providing the precursors for cytochrome prosthetic groups, photosynthetic pigments, and vitamin B(12). Using genomic data, we have compared the heme pathway in the diatom Thalassiosira pseudonana and the red alga Cyanidioschyzon merolae to those of green algae and higher plants, as well as to those of heterotrophic eukaryotes (fungi, apicomplexans, and animals). Phylogenetic analyses showed the mosaic character of this pathway in photosynthetic eukaryotes. Although most of the algal and plant enzymes showed the expected plastid (cyanobacterial) origin, at least one of them (porphobilinogen deaminase) appears to have a mitochondrial (alpha-proteobacterial) origin. Another enzyme, glutamyl-tRNA synthase, obviously originated in the eukaryotic nucleus. Because all the plastid-targeted sequences consistently form a well-supported cluster, this suggests that genes were either transferred from the primary endosymbiont (cyanobacteria) to the primary host nucleus shortly after the primary endosymbiotic event or replaced with genes from other sources at an equally early time, i.e., before the formation of three primary plastid lineages. The one striking exception to this pattern is ferrochelatase, the enzyme catalyzing the first committed step to heme and bilin pigments. In this case, two red algal sequences do not cluster either with the other plastid sequences or with cyanobacterial sequences and appear to have a proteobacterial origin like that of the apicomplexan parasites Plasmodium and Toxoplasma. Although the heterokonts also acquired their plastid via secondary endosymbiosis from a red alga, the diatom has a typical plastid-cyanobacterial ferrochelatase. We have not found any remnants of the plastidlike heme pathway in the nonphotosynthetic heterokonts Phytophthora ramorum and Phytophthora sojae.     

Rapid evolution of the plastid translational apparatus in a nonphotosynthetic plant: Loss or accelerated sequence evolution of tRNA and ribosomal protein genes

Summary The vestigial plastid genome of Epifagus virginiana (beechdrops), a nonphotosynthetic parasitic flowering plant, is functional but lacks six ribosomal protein and 13 tRNA genes found in the chloroplast DNAs of photosynthetic flowering plants. Import of nuclear gene products is hypothesized to compensate for many of these losses. Codon usage and amino acid usage patterns in Epifagus plastic genes have not been affected by the tRNA gene losses, though a small shift in the base composition of the whole genome (toward A + T -richness) is apparent. The ribosomal protein and tRNA genes that remain have had a high rate of molecular evolution, perhaps due to relaxation of constraints on the translational apparatus. Despite the compactness and extensive gene loss, one translational gene (infA, encoding initiation factor 1) that is a pseudogene in tobacco has been maintained intact in Epifagus.Offprint requests to: J.D. Palmer     

Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae

This study provides a phylogenetic/comparative approach to deciphering the processes underlying the evolution of plastid rRNA genes in genomes under relaxed functional constraints. Nonphotosynthetic green algal taxa that belong to two distinct classes, Chlorophyceae (Polytoma) and Trebouxiophyceae (Prototheca), were investigated. Similar to the situation described previously for plastid 16S rRNA genes in nonphotosynthetic land plants, nucleotide substitution levels, extent of structural variations, and percentage AT values are increased in nonphotosynthetic green algae compared to their closest photosynthetic relatives. However, the mutational processes appear to be different in many respects. First, with the increase in AT content, more transversions are noted in Polytoma and holoparasite angiosperms, while more transitions characterize the evolution of the 16S rDNA sequences in Prototheca. Second, although structural variations do accumulate in both Polytoma and Prototheca (as well as holoparasitic plastid 16S rRNAs), insertions as large as 1.6 kb characterize the plastid 16S rRNA genes in the former, whereas significantly smaller indels (not exceeding 24 bp) seem to be more prevalent in the latter group. The differences in evolutionary rates and patterns within and between lineages might be due to mutations in replication/repair-related genes; slipped-strand mispairing is likely the mechanism responsible for the expansion of insertions in Polytoma plastid 16S rRNA genes. Received: 29 December 2000 / Accepted: 18 May 2001