Evolution of plant phage-type RNA polymerases: the genome of the basal angiosperm <Emphasis Type="Italic">Nuphar advena</Emphasis> encodes two mitochondrial and one plastid phage-type RNA polymerases

Background  

In mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we searched for the respective genes in the basal angiosperm Nuphar advena.     

Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids

Angiosperms possess a small family of phage-type RNA polymerase genes that arose by gene duplication from an ancestral gene encoding the mitochondrial RNA polymerase. We have isolated and sequenced the genes and cDNAs encoding two phage-type RNA polymerases, PpRpoT1 and PpRpoT2, from the moss Physcomitrella patens. PpRpoT1 comprises 19 exons and 18 introns, PpRpoT2 contains two additional introns. The N-terminal transit peptides of both polymerases are shown to confer dual-targeting of green fluorescent protein fusions to mitochondria and plastids. In vitro translation of the cDNAs revealed initiation of translation at two in-frame AUG start codons. Translation from the first methionine gives rise to a plastid-targeted polymerase, whereas initiation from the second methionine results in exclusively mitochondrial-targeted protein. Thus, dual-targeting of Physcomitrella RpoT is caused by and might be regulated by multiple translational starts. In phylogenetic analyses, the Physcomitrella RpoT polymerases form a sister group to all other phage-type polymerases of land plants. The two genes result from a gene duplication event that occurred independently from the one which led to the organellar polymerases with mitochondrial or plastid targeting properties in angiosperms. Yet, according to their conserved exon-intron structures they are representatives of the molecular evolutionary line leading to the RpoT genes of higher land plants.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> arabinogalactan proteins contain abundant terminal 3-<Emphasis Type="Italic">O</Emphasis>-methyl-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosyl residues not found in angiosperms

A biochemical investigation of arabinogalactan proteins (AGPs) in Physcomitrella patens was undertaken with particular emphasis on the glycan chains. Following homogenization and differential centrifugation of moss gametophytes, AGPs were obtained by Yariv phenylglycoside-induced precipitation from the soluble, microsomal membrane, and cell wall fractions. Crossed-electrophoresis indicated that each of these three AGP fractions was a mixture of several AGPs. The soluble AGP fraction was selected for further separation by anion-exchange and gel-permeation chromatography. The latter indicated molecular masses of ∼100 and 224 kDa for the two major soluble AGP subfractions. The AGPs in both of these subfractions contained the abundant (1,3,6)-linked galactopyranosyl residues, terminal arabinofuranosyl residues, and (1,4)-linked glucuronopyranosyl residues that are typical of many angiosperm AGPs. Unexpectedly, however, the moss AGP glycan chains contained about 15 mol% terminal 3-O-methyl-l-rhamnosyl residues, which have not been found in angiosperm AGPs. This unusual and relatively nonpolar sugar, also called l-acofriose, is likely to have considerable effects on the overall polarity of Physcomitrella AGPs. A review of the literature indicates that the capacity to synthesize polymers containing 3-O-methyl-l-rhamnosyl residues is present in a variety of bacteria, algae and lower land plants but became less common through evolution to the extent that this sugar has been found in only a few species of angiosperms where it occurs as a single residue on steroidal glycosides.     

Evolution of the Cinnamyl/Sinapyl Alcohol Dehydrogenase (CAD/SAD) Gene Family: The Emergence of Real Lignin is Associated with the Origin of Bona Fide CAD

Lignin plays a vital role in plant adaptation to terrestrial environments. The cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and might have contributed to the lignin diversity in plants. To investigate the evolutionary history and functional differentiation of the CAD gene family, we made a comprehensive evolutionary analysis of this gene family from 52 species, including bacteria, early eukaryotes and green plants. The phylogenetic analysis, together with gene structure and function, indicates that all members of land plants, except two of moss, could be divided into three classes. Members of Class I (bona fide CAD), generally accepted as the primary genes involved in the monolignol biosynthesis, are all from vascular plants, and form a robustly supported monophyletic group with the lycophyte CADs at the basal position. This class is also conserved in the predicted three-dimensional structure and the residues constituting the substrate-binding pocket of the proteins. Given that Selaginella has real lignin, the above evidence strongly suggests that the earliest occurrence of the bona fide CAD in the lycophyte could be directly correlated with the origin of lignin. Class II comprises members more similar to the aspen sinapyl alcohol dehydrogenase gene, and includes three groups corresponding to lycophyte, gymnosperm, and angiosperm. Class III is conserved in land plants. The three classes differ in patterns of evolution and expression, implying that functional divergence has occurred among them. Our study also supports the hypothesis of convergent evolution of lignin biosynthesis between red algae and vascular plants.     

One RNA polymerase serving two genomes

The land plant Arabidopsis thaliana contains three closely related nuclear genes encoding phage-type RNA polymerases (RpoT;1, RpoT;2 and RpoT;3). The gene products of RpoT;1 and RpoT;3 have previously been shown to be imported into mitochondria and chloroplasts, respectively. Here we show that the transit peptide of RpoT;2 possesses dual targeting properties. Transient expression assays in tobacco protoplasts as well as stable transformation of Arabidopsis plants demonstrate efficient targeting of fusion peptides consisting of the N-terminus of RpoT;2 joined to green fluorescent protein to both organelles. Thus, RpoT;2 might be the first RNA polymerase shown to transcribe genes in two different genomes. RNA polymerase activity of recombinant RpoT;2 is uneffected by the inhibitor tagetin, qualifying the gene product of RpoT;2 as a phage-type polymerase.     

Removal of Noisy Characters from Chloroplast Genome-Scale Data Suggests Revision of Phylogenetic Placements of <Emphasis Type="Italic">Amborella</Emphasis> and <Emphasis Type="Italic">Ceratophyllum</Emphasis>

It is widely appreciated that noisy, highly variable data can impede phylogeney reconstruction. Researchers have for a long time omitted problematic data from phylogenetic analyses, such as the third-codon positions and variable regions. In the analyses of the phylogenetic relations of the angiosperms; however, inclusion of complete gene sequences into genomic-scale alignments has become a common practice. Here we demonstrate that this practice can be misleading. We show that support of the basal-most position of Amborella trichopoda among the angiosperms in the chloroplast genomic data is based only on a tiny subset (< 1% of the total alignment length) of the most variable positions in alignment, exhibiting mean maximum likelihood (ML) distance among the angiosperm operational taxonomic units (OTUs) approximately 36 substitutions/site. Exclusion of these positions leads to disappearance of the basal Amborella branch. Likewise, the recently reported sister-group relationship of Ceratophyllum to the eudicots is based on the presence of 2% of the most variable positions in the genomic alignment, exhibiting, on average, 20 substitutions/site in comparison among the angiosperm OTUs. These observations highlight a need for excluding a certain proportion of saturated positions in alignment from phylogenomic analyses.     

Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants

Stomata play significant roles in plant evolution. A trio of closely related basic Helix-Loop-Helix (bHLH) subgroup Ia genes, SPCH, MUTE and FAMA, mediate sequential steps of stomatal development, and their functions may be conserved in land plants. However, the evolutionary history of the putative SPCH/MUTE/FAMA genes is still greatly controversial, especially the phylogenetic positions of the bHLH Ia members from basal land plants. To better understand the evolutionary pattern and functional diversity of the bHLH genes involved in stomatal development, we made a comprehensive evolutionary analysis of the homologous genes from 54 species representing the major lineages of green plants. The phylogenetic analysis indicated: (1) All bHLH Ia genes from the two basal land plants Physcomitrella and Selaginella were closely related to the FAMA genes of seed plants; and (2) the gymnosperm ‘SPCH’ genes were sister to a clade comprising the angiosperm SPCH and MUTE genes, while the FAMA genes of gymnosperms and angiosperms had a sister relationship. The revealed phylogenetic relationships are also supported by the distribution of gene structures and previous functional studies. Therefore, we deduce that the function of FAMA might be ancestral in the bHLH Ia subgroup. In addition, the gymnosperm “SPCH” genes may represent an ancestral state and have a dual function of SPCH and MUTE, two genes that could have originated from a duplication event in the common ancestor of angiosperms. Moreover, in angiosperms, SPCHs have experienced more duplications and harbor more copies than MUTEs and FAMAs, which, together with variation of the stomatal development in the entry division, implies that SPCH might have contributed greatly to the diversity of stomatal development. Based on the above, we proposed a model for the correlation between the evolution of stomatal development and the genes involved in this developmental process in land plants.     

Phylogeny and divergence of basal angiosperms inferred from<Emphasis Type="Italic"> APETALA3</Emphasis>- and<Emphasis Type="Italic"> PISTILLATA</Emphasis>-like MADS-box genes

The B-class MADS-box genes composed of APETALA3 (AP3) and PISTILLATA (PI) lineages play an important role in petal and stamen identity in previously studied flowering plants. We investigated the diversification of the AP3-like and PI-like MADS-box genes of eight species in five basal angiosperm families: Amborella trichopoda (Amborellaceae); Brasenia schreberi and Cabomba caroliniana (Cabombaceae); Euryale ferox, Nuphar japonicum, and Nymphaea tetragona (Nymphaeaceae); Illicium anisatum (Illiciaceae); and Kadsura japonica (Schisandraceae). Sequence analysis showed that a four amino acid deletion in the K domain, which was found in all previously reported angiosperm PI genes, exists in a PI homologue of Schisandraceae, but not in six PI homologues of the Amborellaceae, Cabombaceae, and Nymphaeaceae, suggesting that the Amborellaceae, Cabombaceae, and Nymphaeaceae are basalmost lineages in angiosperms. The results of molecular phylogenetic analyses were not inconsistent with this hypothesis. The AP3 and PI homologues from Amborella share a sequence of five amino acids in the 5 region of exon 7. Using the linearized tree and likelihood methods, the divergence time between the AP3 and PI lineages was estimated as somewhere between immediately after to several tens of millions of years after the split between angiosperms and extant gymnosperms. Estimates of the age of the most recent common ancestor of all extant angiosperms range from ~140–210 Ma, depending on the trees used and assumptions made.     

Mitochondrial <Emphasis Type="Italic">matR</Emphasis> sequences help to resolve deep phylogenetic relationships in rosids

Background  

Rosids are a major clade in the angiosperms containing 13 orders and about one-third of angiosperm species. Recent molecular analyses recognized two major groups (i.e., fabids with seven orders and malvids with three orders). However, phylogenetic relationships within the two groups and among fabids, malvids, and potentially basal rosids including Geraniales, Myrtales, and Crossosomatales remain to be resolved with more data and a broader taxon sampling. In this study, we obtained DNA sequences of the mitochondrial matR gene from 174 species representing 72 families of putative rosids and examined phylogenetic relationships and phylogenetic utility of matR in rosids. We also inferred phylogenetic relationships within the "rosid clade" based on a combined data set of 91 taxa and four genes including matR, two plastid genes (rbcL, atpB), and one nuclear gene (18S rDNA).     

Phylogenetic studies in <Emphasis Type="Italic">Ravenelia esculenta</Emphasis> and related rust fungi

Ravenelia esculenta Naras. and Thium. is a rust fungus, which infects mostly thorns, inflorescences, flowers and fruits of Acacia eburnea Willd. Aecial stages of the rust produce hypertrophy in infected parts. DNA of the rust fungus was isolated from aeciospores by ‘freeze thaw’ method. 18S rDNA was amplified and sequenced by automated DNA sequencer. BLAST of the sequence at NCBI retrieved 96 sequences producing significant alignments. Multiple sequence alignment of these sequences was done by ClustalW. Phylogenetic analysis was done by using MEGA 3.1. UPGMA Minimum Evolution tree with bootstrap value of 1000 replicates was constructed using these sequences. From phylogenetic tree it is observed that Ravenelia esculenta and the genus Gymnosporangium share a common ancestry, though Ravenelia esculenta is autoecious on angiosperm and the genus Gymnosporangium is heteroecious with pycnia, aecia on angiosperm and uredia, telia on gymnosperm. Two major clades are recognized which are based on the nature of aecial host (gymnosperm or angiosperm). These clades were also showing shift from pteridophytes to angiosperms as telial hosts. The tree can be interpreted in the other way also where there is separation of 14 families of Uredinales depending upon nature of teliospores, nature of aeciospores and structure of pycnia. These studies determine the phylogenetic position of Ravenelia esculenta among other rust fungi besides broad separation of Uredinales into two clades. These studies also show that there is phylogenetic correlation between molecular and morphological data. This is first report of DNA sequencing and phylogenetic positioning in genus Ravenelia from India.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Phytochrome of the green alga Mougeotia: cDNA sequence, autoregulation and phylogenetic position

A cDNA clone encoding phytochrome (apoprotein) of the zygnematophycean green alga Mougeotia scalaris has been isolated and sequenced. The clone consisted of 3372 bp, encoded 1124 amino acids, and showed strain-specific nucleotide exchanges for M. scalaris, originating from different habitats. No indication was found of multiple phytochrome genes in Mougeotia. The 5 non-coding region of the Mougeotia PHY cDNA harbours a striking stem-loop structure. Homologies with higher-plant phytochromes were 52–53% for PHYA and 57–59% for PHYB. Highest homology scores were found with lower-plant phytochromes, for example 67% for Selaginella (Lycopodiopsida), 64% for Physcomitrella (Bryopsida) and 73% for Mesotaenium (Zygnematophyceae). In an unrooted phylogenetic tree, the position of Mougeotia PHY appeared most distant to all other known PHYs. The amino acids Gly-Val in the chromophore-binding domain (-Arg-Gly-Val-His-Gly-Cys-) were characteristic of the zygnematophycean PHYs known to date. There was no indication of a transmembrane region in Mougeotia phytochrome in particular, but a carboxyl-terminal 16-mer three-fold repeat in both, Mougeotia and Mesotaenium PHYs may represent a microtubule-binding domain. Unexpected for a non-angiosperm phytochrome, its expression was autoregulated in Mougeotia in a red/far-red reversible manner: under P_r conditions, phytochrome mRNA levels were tenfold higher than under P_fr conditions.     

Evolution of seed storage protein genes: Legumin genes of Ginkgo biloba

Legumin-like seed storage proteins have been intensively studied in crop plants. However, little is known about the molecular evolution of these proteins and their genes and it was assumed that they originated from an ancestral gene that already existed at the beginning of angiosperm evolution. We have evidence for the ubiquitous occurrence of homologous proteins in gymnosperms as well. We have characterized the major seed storage globulin from Ginkgo biloba by amino acid sequencing, which reveals clear homology to legumin-like proteins from angiosperms. The Ginkgo legumin is encoded by a gene family; we describe two of its members. The promoter regions contain sequence motifs which are known to function as regulatory elements involved in seed-specific expression of angiosperm legumins, although the tissues concerned are different in gymnosperms and angiosperms. The Ginkgo legumin gene structure is divergent from that of angiosperms and suggests that the evolution of legumin genes implicated loss of introns. From our data and from functional approaches recently described it becomes obvious that the posttranslational processing site of legumin precursors is less conserved than hitherto assumed. Finally, we present a phylogenetic analysis of legumin encoding sequences and discuss their utility as molecular markers for the reconstruction of seed plant evolution.Correspondence to: K.-P. Häger     

Identification of shared single copy nuclear genes in Arabidopsis, Populus, Vitis and Oryza and their phylogenetic utility across various taxonomic levels

Background

Although the overwhelming majority of genes found in angiosperms are members of gene families, and both gene- and genome-duplication are pervasive forces in plant genomes, some genes are sufficiently distinct from all other genes in a genome that they can be operationally defined as 'single copy'. Using the gene clustering algorithm MCL-tribe, we have identified a set of 959 single copy genes that are shared single copy genes in the genomes of Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa. To characterize these genes, we have performed a number of analyses examining GO annotations, coding sequence length, number of exons, number of domains, presence in distant lineages, such as Selaginella and Physcomitrella, and phylogenetic analysis to estimate copy number in other seed plants and to demonstrate their phylogenetic utility. We then provide examples of how these genes may be used in phylogenetic analyses to reconstruct organismal history, both by using extant coverage in EST databases for seed plants and de novo amplification via RT-PCR in the family Brassicaceae.

Results

There are 959 single copy nuclear genes shared in Arabidopsis, Populus, Vitis and Oryza ["APVO SSC genes"]. The majority of these genes are also present in the Selaginella and Physcomitrella genomes. Public EST sets for 197 species suggest that most of these genes are present across a diverse collection of seed plants, and appear to exist as single or very low copy genes, though exceptions are seen in recently polyploid taxa and in lineages where there is significant evidence for a shared large-scale duplication event. Genes encoding proteins localized in organelles are more commonly single copy than expected by chance, but the evolutionary forces responsible for this bias are unknown. Regardless of the evolutionary mechanisms responsible for the large number of shared single copy genes in diverse flowering plant lineages, these genes are valuable for phylogenetic and comparative analyses. Eighteen of the APVO SSC single copy genes were amplified in the Brassicaceae using RT-PCR and directly sequenced. Alignments of these sequences provide improved resolution of Brassicaceae phylogeny compared to recent studies using plastid and ITS sequences. An analysis of sequences from 13 APVO SSC genes from 69 species of seed plants, derived mainly from public EST databases, yielded a phylogeny that was largely congruent with prior hypotheses based on multiple plastid sequences. Whereas single gene phylogenies that rely on EST sequences have limited bootstrap support as the result of limited sequence information, concatenated alignments result in phylogenetic trees with strong bootstrap support for already established relationships. Overall, these single copy nuclear genes are promising markers for phylogenetics, and contain a greater proportion of phylogenetically-informative sites than commonly used protein-coding sequences from the plastid or mitochondrial genomes.

Conclusions

Putatively orthologous, shared single copy nuclear genes provide a vast source of new evidence for plant phylogenetics, genome mapping, and other applications, as well as a substantial class of genes for which functional characterization is needed. Preliminary evidence indicates that many of the shared single copy nuclear genes identified in this study may be well suited as markers for addressing phylogenetic hypotheses at a variety of taxonomic levels.     

The chloroplast genome of the “basal” angiosperm Calycanthus fertilis – structural and phylogenetic analyses

The nucleotide sequence of the complete chloroplast genome of a basal angiosperm, Calycanthus fertilis, has been determined. The circular 153337 bp long cpDNA is colinear with those of tobacco, Arabidopsis and spinach. A total of 133 predicted genes (115 individual gene species, 18 genes duplicated in the inverted repeats) including 88 potential protein-coding genes (81 gene species), 8 ribosomal RNA genes (4 gene species) and 37 tRNA genes (30 gene species) representing 20 amino acids were identified based on similarity to their homologs from other chloroplast genomes. This is the highest gene number ever registered in an angiosperm plastome. Calycanthus fertilis cpDNA also contains a homolog of the recently discovered mitochondrial ACRS gene. Since no gene transfer from mitochondria to the chloroplast has ever been documented, we investigated the evolutionary affinity of this gene in detail. Phylogenetic analysis of the protein-coding subset of the plastome suggests that the ancient line of Laurales emerged after the split of the angiosperms into monocots and dicots. Calycanthus fertilis Walter var. ferax (Michy.) Rehder is a synonym of C. floridus L. var. glaucus (Willd.) Torr. & A. Gray.Data deposition: The sequence reported in this paper has been deposited in the EMBL database (accession no. AJ428413).