Structure and organization of Marchantia polymorpha chloroplast genome. IV. Inverted repeat and small single copy regions

We characterized the genes in the regions of large inverted repeats (IRA and IRB, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA(GAC), isoleucine tRNA(GAU), alanine tRNA(UGC), arginine tRNA(ACG) and asparagine tRNA(GUU)). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA(UAG) and a proline tRNA(GGG)-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).     

Structure and organization of Marchantia polymorpha chloroplast genome. II. Gene organization of the large single copy region from rps'12 to atpB

The nucleotide sequence (56,410 base-pairs) of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha has been determined. The sequence starts from one end (JLA) of the large single-copy region and encompasses genes for 21 tRNAs, six ATPase subunits (atpA, atpB, atpE, atpF, atpH and atpI), two photosystem I polypeptides (psaA and psaB), four photosystem II polypeptides (psbA, psbC, psbD and psbG), five ribosomal proteins (rps2, rps4, rps7, rps'12 and rps14), and three RNA polymerase subunits (rpoB, rpoC1 and rpoC2). In addition, we detected 18 open reading frames ranging from 29 to 2136 amino acid residues long, four of which share significant amino acid sequence homology to those of an Escherichia coli malK protein (designated mbpX), human mitochondrial ND2 (ndh2) and ND3 (ndh3) of a respiratory chain NADH dehydrogenase, or a bacterial antenna protein of a light-harvesting complex (lhcA). Sequence analysis suggests that four tRNA genes and six protein genes might be split by introns; they are trnG(UCC), trnK(UUU), trnL(UAA), trnV(UAC), atpF, ndh2, rpoC1, rps'12, ORF135 and ORF167. In the large single-copy region described here, the gene organization deduced is highly conserved with respect to that of higher plants, but an inversion of some 30,000 base-pairs flanked by trnL(CAA) and trnD(GUC) was seen between the liverwort and tobacco chloroplast genomes.     

Structure and organization of Marchantia polymorpha chloroplast genome. III. Gene organization of the large single copy region from rbcL to trnI(CAU)

The nucleotide sequence (25,320 base-pairs) of a part of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha was determined. This region encodes putative genes for four tRNAs, isoleucine tRNA(CAU), arginine tRNA(CCG), proline tRNA(UGG) and tryptophan tRNA(CCA); eight photosynthetic polypeptides, the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL), 51,000 Mr photosystem II chlorophyll alpha apoprotein (psbB), apocytochrome b-559 polypeptides (psbE and psbF), 10,000 Mr phosphoprotein (psbH), cytochrome f preprotein (petA), cytochrome b6 polypeptide (petB), and cytochrome b6/f complex subunit 4 polypeptide (petD); 13 ribosomal proteins (L2, L14, L16, L20, L22, L23, L33, S3, S8, S11, S12, S18 and S19); initiation factor 1 (infA); ribosome-associating polypeptide (secX); and alpha subunit of RNA polymerase (rpoA). Functionally related genes were located in several clusters in this region of the genome. There were two ribosomal protein gene clusters: rpl23-rpl2-rps19-rpl22-rps3-rpl16-+ ++rpl14-rps8-infA-secX-rps11-rpoA, with a gene arrangement similar to that of the Escherichia coli S10-spc-alpha operons, and the rps12'-rpl20-rps18-rpl33 cluster. There were gene clusters encoding photosynthesis components such as the psbB-psbH-petB-petD and the psbE-psbF clusters. Thirteen open reading frames, ranging in length from 31 to 434 amino acid residues, remain to be identified.     

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.     

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.     

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.     

地钱、拳卷地钱和粗裂地钱的紫外谱线组法鉴别

本文建立了紫外谱线组法鉴别地钱、拳卷地钱和粗裂地钱的方法。通过这三种药材的石油醚、氯仿、乙醇和水提取液的紫外谱线比较,发现地钱、拳卷地钱和粗裂地钱的紫外谱线图、最大吸收峰数目及峰位值具有明显差异。该法简单、准确,可用来鉴别地钱、拳卷地钱和粗裂地钱的原药材。     

Composition and physiological function of the chloroplast NADH dehydrogenase‐like complex in Marchantia polymorpha

The chloroplast NADH dehydrogenase‐like (NDH) complex mediates cyclic electron transport and chloro‐respiration and consists of five sub‐omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super‐complex. In Marchantia polymorpha, 11 plastid‐encoded subunits and all the nuclear‐encoded subunits of the A, B, membrane and ferredoxin‐binding sub‐complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDH–PSI super‐complex formation. It is also unlikely that the subunits of the lumen sub‐complex, PnsL1–L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue‐native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDH–PSI super‐complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild‐type genome copies were completely segregated out, the ΔndhB lines grew like the wild‐type photoautotrophically. A post‐illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the ΔndhB lines. In ruptured chloroplasts, antimycin A‐insensitive, and ferredoxin‐dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the ΔndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions.     

Gene clusters for ribosomal proteins in the mitochondrial genome of a liverwort, Marchantia polymorpha.

We detected 16 genes for ribosomal proteins in the complete sequence of the mitochondrial DNA from a liverwort, Marchantia polymorpha. The genes formed two major clusters, rps12-rps7 and rps10-rpl2-rps19-rps3-rpl16-rpl5- rps14-rps8- rpl6-rps13-rps11-rps1, very similar in organization to Escherichia coli ribosomal protein operons (str and S10-spc-alpha operons, respectively). In contrast, rps2 and rps4 genes were located separately in the liverwort mitochondrial genome (the latter was part of the alpha operon in E. coli). Furthermore, several ribosomal proteins encoded by the liverwort mitochondrial genome differed substantially in size from their counterparts in E. coli and liverwort chloroplast.     

Protoplasts of Marchantia polymorpha and its Development

For the first time protoplasts from normal differentiated thalliof male and female Marchantia polymorpha were prepared by aone step cell wall digestion with 2% Driselase. Cell wall formationonly takes place in light with a carbohydrate source. Furtherconditions for growth and differentiation were studied. Growthalways starts with a primary callus. This can be maintainedby a higher osmolarity of the medium. After reduction of theosmolarity, differentiation appears in three forms: gemmae formationat the callus surface, production of limited growing "cauloids"and transition to an indefinite growing thallus with one orseveral apical regions. (Received December 21, 1987; Accepted February 12, 1988)     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura     

In vitro Propagation,Dedifferentiation and redifferentiation of Marchantia polymorpha L.

The gemma and gametophyte of Marchantia #olymorpha were propagated in vitro. Dedifferentiation and redifferentiation as well as the media used and cultural conditions reguired were described. Since the differentiation of bryophytes was very difficult, it was necessary to culture the tissue through initiation of partial dedifferentiation on MS agar medium supplemented with 1 mg/1 2,4-D and 3% sucrose, and then subsequently the tissue was transplanted onto 1/2 Knop agar medium with addition of 4–8 mg/1 2,4-D, 0.25–0.5 mg/1 BA and Fe salt of MS medium. The formed calli were visual but still contained rhizoid, in this stage. The small calli finally were subcultured in white agar medium supplemented with mixture of pyruvic acid, citric acid and fumaric acid (5 mmol/1); 1 mg/1 2,4-D and 4% glucose. They could be differentiated thoroughly into normal tissue. The time of the total process for differentiation requied as long as 10 months. The redifferentiation and regeneration of thalli were far easier than those of higher plants even if they were transplanted onto MS phytohormone-free medium.     

Resistance and capacity in the thallus of Marchantia polymorpha

Calculations of the resistance r and capacity c of cell membrances and the resistancer ₁ of cell interiors of a community of cells in Marchantia polymorpha L. thalli are presented. These parameters of a multicellular system were determined by the adaptation of methods employed for the calculation of the resistance and capacity of single cells. The obtained results indicate that such a procedure is justified. A generally accepted resistance-capacity model of the cell was used as a basis for the determination of r, c, and r₁ (representing membrane resistance, membrane capacity, and resistance of cell interior, respectively). The calculations were based on measurements of impedance and phase shift within the frequency range of 5 Hz-1000 Hz. Stainless steel plates were employed as the measuring electrodes; polarization resistance and capacity were determined by separate measurements. The calculations confirmed the assumption that the parameters r, c, and r₁ were constant within the investigated frequency range.
The calculations of resistance and capacity for 25 plants were constant within the investigated frequency range. The calculations of resistance and capacity for 25 plants were carried out by four different methods and they yielded results of the order of : r = 0.45 kΩ± 0.15 kΩ, r₁= 1.0 kΩ± 0.45 kΩ, c = 11 μF ± 3.5 μF. Circular diagrams of impedance also confirmed the validity of the accepted model within the frequency range of 25–300 Hz.     

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture

A cell line of M. polymorpha was grown photoautotrophically in liquid suspension culture using 1% CO₂ in air as sole carbon source. The growth rate in terms of cell dry-weight during the exponential phase was 0.171 and the doubling time was 1.76 d. The rate of increase in chlorophyll was 1.6 times higher than the growth rate. The highest content of chlorophyll was 24 mg g^-1 dry weight, and the photosynthetic activity of the cells in the exponential phase, as calculated from the growth rate, was at least 60 mol mg^-1 chlorophyll h^-1.     

Photomorphogenic and Chlorophyll Studies in the Bryophyte Marchantia polymorpha

Besides the standard rod (R) and far-red (FK) irradiations, a graded series of different R/FR ratios were tested as 10 min terminal exposures at the end of the daily 8-hour photoperiod of white fluorescent light. Water filtered incandescent light of 3780 lux during 10 min caused A rather weak hut reproducible effect. A superposition to the water layer of different filter combinations shifting the initial transmittance more towards the FR region, and thus gradually lowering the R/FB ratio, resulted in a parallel increase in orthotropic growth and a decrease in chlorophyll content. Our data show growth similarities with the results of other authors on light grown seedlings of higher plants. Rather high levels of the P_FR form of phytochrome seem to he required to maintain horizontal growth and optimal chlorophyll content in Marchantia thalli.     

Nucleotide sequences of chloroplast 5S ribosomal RNA from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata

The nucleotide sequences of chloroplast 5S rRNAs from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata were determined. Their nucleotide sequences, 119 nucleotides long, were highly homologous to each other (96% identity) and had high homology with those from chloroplast 5S rRNAs of two higher plants, tobacco (92% identity) and spinach (92-91% identity), but less homology (87-85% identity) with that from a lower plant, the fern Dryopteris acuminata.