Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.     

Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs.

Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia polymorpha. One to three species of tRNA genes corresponded to each of 20 amino acids including three species for leucine and arginine, two species for serine and glycine, and one for the rest of the amino acids. Interestingly, all tRNA genes were located in the semicircle of the liverwort mitochondrial genome except for the trnY and trnR genes. The region containing these tRNA genes was originally duplicated, and two trnR genes have diverged from each other. On the other hand, trnY and trnfM are present as two identical copies. The G:U and U:N wobbling between the first nucleotide of the anticodon and the third nucleotide of the codon permit the 27 tRNA identified species to translate almost all codons. However, at least two additional tRNA genes, trnl-GAU for AUY codon and trnT-UGU for ACR codon, are required to read all codons used in the liverwort mitochondrial genome. All of the identified tRNA genes are 'native' in liverwort mitochondria, not 'chloroplast-like' tRNAs as are found in the mitochondria of higher plants. This result implies that the tRNA gene transfer from chloroplast to mitochondrial genome in higher plants has occurred after the divergence from bryophytes.     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F_2α, prostaglandin E₂ and prostaglandin D₂ which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.     

A pseudomolecule‐scale genome assembly of the liverwort Marchantia polymorpha

Marchantia polymorpha has recently become a prime model for cellular, evo‐devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule‐scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high‐density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map‐based cloning, feasible.     

Marchantia polymorpha mitochondrial orf identifies transcribed sequence in angiosperm mitochondrial genome

Heterologous hybridizations performed using nine Marchantia polymorpha mitochondrial orfs and the sdh4 gene against angiosperm mtDNA suggested that three of them and the sdh4 gene have been conserved in the mitochondrial genome of different angiosperm species. Solanum tuberosum mtDNA fragments, which hybridized to M. polymorpha orf207 and sdh4 gene, were cloned, sequenced, and their expressions evaluated by Northern and RT-PCR. Hybridizing fragments to sdh4 gene and orf207 from potato mtDNA were shown to be transcribed, but only in the case of sdh4 gene was there homology between the protein encoded by the DNA sequence from M. polymorpha and the potato mitochondrial genome. M. polymorpha orf207 showed little similarity to an open reading frame from potato mtDNA, named here orf78. The putative proteins encoded by both orf207 and orf78 were not related, indicating that these orfs do not constitute homologous sequences.     

Ferredoxin from a liverwort, Marchantia polymorpha. Purification and amino acid sequence

Marchantia polymorpha ferredoxin was purified by DE-52 and Sephadex G-75 column chromatographies to homogeneity. The complete amino acid sequence of the carboxymethylated (Cm) ferredoxin was determined by conventional methods to be as follows. Thr-Phe-Lys- Val-Thr-Leu-Asn-Thr-Pro-Thr-Gly-Gln-Ser-Val-Ile-Asp-Val-Glu-Asp- Asp-Glu-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Ser-Leu-Pro- Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Thr- Ala-Gly-Glu-Val-Asp-Gln-Ser-Asp-Glu-Ser-Phe-Leu-Asp-Asp-Asp-Gln- Met-Asp-Glu-Gly-Tyr-Val-Leu-Thr-Cys-Ile-Ala-Tyr-Pro-Thr-Ser-Asp- Leu-Thr-Ile-Asp-Thr-His-Gln-Glu-Glu-Ala-Leu-Ile. The total number of amino acid residues was 95 and the molecular weight was calculated to be 10,174, excluding iron and sulfur atoms. The distribution of the four cysteine residues chelating the two iron atoms was identical to those of other [2Fe-2S] ferredoxins. The relationship between M. polymorpha and other plants was discussed in terms of plant phylogeny.     

Chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha

Journal of Plant Research - Under low-light conditions, chloroplasts localize along periclinal cell walls at temperatures near 20 °C, but they localize along anticlinal cell walls near...     

Positional cues regulate dorsal organ formation in the liverwort Marchantia polymorpha

Journal of Plant Research - Bryophytes and vascular plants represent the broadest evolutionary divergence in the land plant lineage, and comparative analyses of development spanning this divergence...     

Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L.

Thalli of the haploid liverwort Marchantia polymorpha were successfully used for direct particle bombardment with plasmid pMT, which carries a hygromycin phosphotransferase gene (hpt) controlled by the CaMV 35S promoter and the NOS polyadenylation region. Hygromycin-resistant cell masses arose from the thallus surface and developed directly into hygromycin-resistant thalli. Southern blot analyses indicated that these thalli carried at least 1–4 copies of the hpt gene, which were stably transmitted to their asexual thallus progenies via gemma propagation for three generations. This transformation and direct plant regeneration protocol is expected to be a valuable tool for the molecular analysis of this lower land plant.     

Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha

The plasma membrane H(+)-ATPase generates an electrochemical gradient of H(+) across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H(+)-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H(+)-ATPase in vascular plants. However, there is very little information on the plasma membrane H(+)-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H(+)-ATPase (pT H(+)-ATPase) and non-penultimate threonine-containing H(+)-ATPase (non-pT H(+)-ATPase) as in the green algae and that pT H(+)-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H(+)-ATPase genes, designated MpHA (for M. polymorpha H(+)-ATPase). Four isoforms are the pT H(+)-ATPase; the remaining isoforms are non-pT H(+)-ATPase. An apparent 95-kD protein was recognized by anti-H(+)-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H(+)-ATPase. Furthermore, we found that the pT H(+)-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H(+)-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H(+)-ATPase.     

Antifungal bis[bibenzyls] from the Chinese liverwort Marchantia polymorpha L

Bioassay-guided separation of the antifungal constituents of the Chinese liverwort Marchantia polymorpha L. (Marchantiaceae) led to the isolation of seven bis[bibenzyl]-type macrocycles. On the basis of NMR and MS analyses, the three new compounds plagiochin E (1), 13,13'-O-isoproylidenericcardin D (4), and riccardin H (7) were identified, together with four known compounds: marchantin E (2), neomarchantin A (3), marchantin A (5), and marchantin B (6). Their antifungal activities against Candida albicans were determined by TLC bioautography.     

Glycosylphosphatidylinositol-anchored proteins found in the moss Marchantia polymorpha

A suspension culture of the moss Marchantia polymorpha was incubated with either [3H]ethanolamine or [3H]myristic acid, precursors of a glycosylphosphatidylinositol (GPI) anchor. By both of these a secretory protein of 47 kDa was consistently labelled. Amino acid composition analysis of the chromatographically purified 47-kDa protein exhibited the occurrence of ethanolamine and glucosamine at approximate molar ratios to protein of 1 and 4, respectively. Thus the secretory 47-kDa protein of M. polymorpha was concluded to be a GPI-anchored protein.     

Coordination between growth and stress responses by DELLA in the liverwort Marchantia polymorpha

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Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort Marchantia polymorpha

Sex of the liverwort Marchantia polymorpha is determined by the sex chromosomes Y and X, in male and female plant, respectively. Approximately half of the Y chromosome is made up of unique repeat sequences. Here, we report that part of the Y chromosome, represented by a 90-kb insert of a genomic clone pMM2D3, contains five putative genes in addition to the ORF162 gene, which is present also within the Y chromosome-specific repeat region. One of the five putative genes shows similarity to a male gamete-specific protein of lily and is expressed predominantly in male sex organs, suggesting that this gene has a male reproductive function. Furthermore, Southern blot analysis revealed that these five putative genes are amplified on the Y chromosome, but they also probably have homologs on the X chromosome and/or autosomes. These observations suggest that the Y chromosome evolved by co-amplifying protein-coding genes with unique repeat sequences.