Miho Takemura

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.

Characterization of a Novel Gene Encoding a Putative Single Zinc-finger Protein, ZIM, Expressed during the Reproductive Phase in Arabidopsis thaliana

By differential screening of an arrayed normalized cDNA library from the inflorescence apex in Arabidopsis, a cDNA clone having a deduced amino acid sequence with a motif for a zinc finger was isolated as one of the genes expressed specifically in the reproductive phase. The deduced protein has a modular structure with a putative single C2-C2 zinc-finger motif distantly related to a GATA-1-type finger, a basic region with a sequence resembling a nuclear localization signal, and an acidic region. The gene seemed to have been formed by the exon-shuffling during its molecular evolution, since individual domains are encoded by discrete exons. RNA gel blot analysis showed its expression in shoot apex and flowers in the reproductive phase. The gene was named ZIM for Zinc-finger protein expressed in Inflorescence Meristem. The nuclear localization of ZIM was detected using GFP as a reporter. These results suggest that ZIM is a putative transcription factor involved in inflorescence and flower development.

Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L.

Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5′- and 3′-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.

Expression of the gene for sterol-biosynthesis enzyme squalene epoxidase in parenchyma cells of the oil plant, Euphorbia tirucalli

In plants, phytosterols and triterpenes are major secondary metabolites. In an attempt to reveal the mechanism for synthesis and storage of these compounds, we isolated and characterized cDNA clones for squalene epoxidase (SE), from a succulent shrub, Euphorbia tirucalli. Southern-blot analysis of total DNA using cDNA fragment as a probe showed that the E. tirucalli squalene epoxidase gene (EtSE) is single-copy type in terms of restriction fragment length polymorphism (RFLP). Deduced amino-acid sequence of the cDNA showed 83 and 75% identity to those of rice and ginseng, respectively, in an area excluding a less homologous putative transmembrane region in the N-terminal end. Functional characterization with heterologous expression using an erg1-disrupted yeast mutant KLN1 indicated that the EtSE recovered ergosterol auxotrophy of the mutant, and gave rise to an ergosterol accumulation in the EtSE transformant. RT-PCR analysis showed the EtSE transcripts in leaves and stem internodes accumulated in almost equal amounts, which were more abundant than those in roots. In situ hybridization using EtSE antisense probe revealed prominent EtSE expression on a parenchyma cell adjacent to primary laticifers that were located in a rosary orientation in the inner region of cortex. This is the first report of expression of a gene for a rate-limiting enzyme in mevalonate pathway in organs and tissues of a plant.

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.

Arabidopsis ZIM, a Plant-specific GATA Factor, Can Function as a Transcriptional Activator

Arabidopsis ZIM is a putative transcription factor containing an atypical GATA-type zinc-finger motif. Transcriptional activation by ZIM was tested using a transient GAL4 fusion assay and measuring the expression of a luciferase reporter in tobacco BY-2 cells. ZIM functioned as a transcriptional activator, and the transactivation domain was found to occur in its N-terminal acidic region.

Molecular Characterization of the Cytoplasmic Interacting Protein of the Receptor Kinase IRK Expressed in the Inflorescence and Root Apices of Arabidopsis

Meristem maintenance and differentiation is regulated by intercellular communication through receptor-like kinases (RLKs) in plants, but the underlying molecular mechanisms of RLK signaling remain largely unknown. A cytoplasmic interactor for inflorescence and root apices receptor-like kinase (IRK), which is a typical meristematic RLK with leucine-rich repeats in Arabidopsis, was identified using a yeast two-hybrid assay and named IRK-interacting protein (IRKI). IRKI is a novel but highly conserved protein found in higher plants. The interaction between IRK and IRKI was confirmed by an in vitro pull-down assay and supported by their simultaneous expression in actively dividing cells in meristems. In the root tip, IRKI expression and localization visualized by green fluorescence protein (GFP) were observed in the quiescent center, initial cells, and immature stele cells. IRKI expression was expanded by exogenous auxin treatment and repressed by inhibitor treatment of polar auxin transport.

Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria

A pseudogene, ψnad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)+ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this ψnad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.

Gene content, organization and molecular evolution of plant organellar genomes and sex chromosomes: insights from the case of the liverwort Marchantia polymorpha.

The complete nucleotide sequence of chloroplast DNA (121,025 base pairs, bp) from a liverwort, Marchantia polymorpha, has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. We also determined the complete nucleotide sequence of the liverwort mitochondrial DNA and deduced 96 possible genes in the sequence of 186,608 bp. The complete chloroplast genome encodes twenty introns (19 group II and 1 group I) in 18 different genes. One of the chloroplast group II introns separates a ribosomal protein gene in a trans-position. The mitochondrial genome contains thirty-two introns (25 group II and 7 group I) in the coding regions of 17 genes. From the evolutionary point of view, we describe the origin of organellar introns and give evidence for vertical and horizontal intron transfers and their intragenomic propagation. Furthermore, we describe the gene organization of the Y chromosome in the dioecious liverwort M. polymorpha, the first detailed view of a Y chromosome in a haploid organism. On the 10 megabase (Mb) Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome. These 14 genes are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.

Molecular evolution of mitochondrial introns in the liverwort Marchantia polymorpha

We here describe in detail the characterization and molecular evolution of group II introns in the mitochondrial genome of the liverwort Marchantia polymorpha. We find that 18 introns of the 25 group II introns can be assigned by their similarities to six clusters, indicating an intra-genomic propagation of one ancestral intron each into the respective clusters in the liverwort mitochondrial genome. Interestingly, the intra-genomic propagation of some of these introns occurred only after the evolutionary separation of the bryophytes from the other clades of plants. Finally we report that the maturase-like sequences in the liverwort group II introns have further evolved by horizontal and independent transposition and substitution by analogous sequences from other fungal introns.