Cytochrome c oxidase subunit I gene in Thalassiosira nordenskioeldii strains inhabiting in cold and warm sea waters.

From the biota beneath the sea ice in Lake Saroma, which is adjacent to Sea of Okhotsk, a diatom culture of Saroma 16 was isolated. Strutted processes and a labiate process in Saroma 16 were characteristic of those in Thalassiosira nordenskioeldii. Similarity search analysis showed that the 826-bp rbcL-3P region sequence of this strain was 100% identical to multiple sequences registered as T. nordenskioeldii in a public database. The 4305-bp PCR-amplified mitochondrial cytochrome c oxidase subunit I (COI) gene (COI)-5P region of Saroma 16 included a 1060-bp open reading frame (ORF), which was interrupted by 934-bp and 2311-bp introns that included frame-shifted ORFs encoding reverse-transcriptase (RTase)-like proteins. Previous reports showed that a strain of the same species, CNS00052, originating from the East China Sea included no introns in the COI, whereas North Atlantic Ocean strains of the same species, such as CCMP992, CCMP993, and CCMP997, included a 2.3-kb intron in the same position as Saroma 16.

The Mitochondrial Endonuclease M20 Participates in the Down-Regulation of Mitochondrial DNA in Pollen Cells.

Maintaining the appropriate number of mitochondrial DNA (mtDNA) molecules is crucial for supporting mitochondrial metabolism and function in both plant and animal cells. For example, a substantial decrease in mtDNA levels occurs as a key part of pollen development. The molecular mechanisms regulating mtDNA copy number are largely unclear, particularly with regard to those that reduce mtDNA levels. Here, we identified and purified a 20-kD endonuclease, M20, from maize (Zea mays) pollen mitochondria. We found M20 to be an His-Asn-His/Asn (H-N-H/N) nuclease that degrades linear and circular DNA in the presence of Mg2+ or Mn2+ Arabidopsis (Arabidopsis thaliana) AtM20, which shared high sequence similarity with maize M20, localized to the mitochondria, had a similar H-N-H/N structure, and degraded both linear and circular DNA. AtM20 transcript levels increased during pollen development, in parallel with a rapid reduction in mtDNA. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing techniques were used to generate knockout lines of AtM20 (atm20), which exhibited a significant delay in the reduction in mtDNA levels in pollen vegetative cells but normal mtDNA levels in somatic cells. The delayed reduction in pollen mtDNA levels was rescued by the transgenic expression of AtM20 in atm20 plants. This study thus uncovers an endonucleolytic DNase in plant mitochondria and its crucial role in reducing mtDNA levels, pointing to the complex mechanism regulating mtDNA levels in plants.

Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B.

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.

Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from Botryococcus braunii, race B.

The green microalga Botryococcus braunii Showa, which produces large amounts of triterpene hydrocarbons, exclusively uses the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosyntheses, and the terminal enzyme in this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), is regarded as a light-dependent key regulatory enzyme. In order to investigate the possible association of HDR and ferredoxin in this organism, we constructed tertiary structure models of B. braunii HDR (BbHDR) and one of ferredoxin families in the alga, a photosynthetic electron transport F (BbPETF)-like protein, by using counterparts from E. coli and Chlamydomonas reinhardtii as templates, respectively, and performed docking analysis of these two proteins. After docked models are superimposed onto their counterpart proteins in a non-photosynthetic organism, Plasmodium falciparum, the BbPETF-like protein comes in contact with the backside of BbHDR, which was defined in a previous report (Rekittke et al. 2013), and the distance of the two Fe-S centers is 14.7 Å. This distance is in almost the same level as that for P. falicarum, 12.6 Å. To our knowledge, this is the first model suggesting the possible association of HDR with a ferredoxin in O2-evolving photosynthetic organisms.

Isolation and characterization of two squalene epoxidase genes from Botryococcus braunii, race B.

The B race of the green microalga Botryococcus braunii produces triterpene hydrocarbons, botryococcenes and methylsqualenes that can be processed into jet fuels with high heating values. In this alga, squalene is also converted into membrane sterols after 2,3-epoxidation. In the present study, cDNA clones of two distinct squalene epoxidases (BbSQE-I and -II) were isolated. Predicted amino acid sequences encoded on these genes are 45% identical with each other. Introduction of BbSQE-I or -II into Saccharomyces cerevisie erg1 mutants resulted in the complementation of ergosterol auxotrophy. The relative expression level of SQE-II increased 3.5-fold from the early stage to the middle phase of a culture period of 42 days, while that of SQE-I was almost constant throughout the culture period. Southern blot analyses suggested that these genes are single-copied genes. This is the first report on the isolation of functional SQEs that are encoded in duplicated loci in the algal genome.

THE ROLE OF ZINC FINGER PROTEIN IN RNAi INTERFERENCE IN A UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE).

In our previous study, we generated a strain of 19-P (1030) in which artificial RNA interference (RNAi) was induced by transcribing a hairpin RNA of ~780-bp stem. We utilized this RNAi-induced strain to uncover RNAi-related genes. Random insertional mutagenesis was performed to generate tag-mutants that show a RNAi deficient phenotype. The 92-12C is one such tag-mutant, which bears a 14-kb deletion in chromosome 1. Complementation of 92-12C revealed that a protein gene, including a Cys-Cys-Cys-His-type zinc finger motif and an ankyrin repeat motif, is essential for effective RNAi in Chlamydomonas reinhardtii (Dangeard). BLAST analysis revealed that the zinc finger protein is homologous to an mRNA splicing-related protein of other species. Therefore, one of the probable scenarios is that mRNA coding for RNAi-related proteins cannot be properly spliced, which causes RNAi deficiency in the 92-12C tag-mutant.

Agrobacterium-mediated transformation of Euphorbia tirucalli callus.

In order to establish a basis for transformation technology in the petroleum plant Euphorbia tirucalli, the callus of the plant was infected with Agrobacterium, washed with distilled water, sterilized with distilled water containing 100 mg/l of carbenicillin, selected on solidified B5 medium containing 13 mg/l of G418 and 100 mg/l of carbenicillin, and then on solidified B5 medium containing 25 mg/l of G418 and 100 mg/l of carbenicillin for the transgenic calli, and then the callus lines were subcultured successively on solidified B5 medium containing 50 mg/l of G418. We performed PCR analysis of sterilized G418-resistant callus line DNA and concluded that the gene introduced was integrated into the callus genome.

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.

Cloning and characterization of a cDNA encoding beta-amyrin synthase from petroleum plant Euphorbia tirucalli L.

Euphorbia tirucalli L., known as the petroleum plant, produces a large amount of triterpenes, such as beta-amyrin. Degenerate RT-PCR based on the sequences conserved among known beta-amyrin synthases led to cloning of a putative triterpene synthase cDNA, EtAS, from leaves of E. tirucalli. The deduced amino acid sequence of the EtAS cDNA showed the highest identity of 82% to the Panax ginseng beta-amyrin synthase. Heterologous expression of the EtAS ORF in the methylotrophic yeast, Pichia pastoris, resulted in production of beta-amyrin, revealing that the EtAS cDNA codes for a beta-amyrin synthase. This is the first report of a gene involved in the triterpene synthetic pathway from Euphorbiaceae plants.

A ZYGOTE-SPECIFIC PROTEIN WITH HYDROXYPROLINE-RICH GLYCOPROTEIN DOMAINS AND LECTIN-LIKE DOMAINS INVOLVED IN THE ASSEMBLY OF THE CELL WALL OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA).

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall-free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline-rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP-2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)x repeats, establishing it as a member of the cell wall hydroxyproline-rich glycoproteins family. It also contains a 4-fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell-free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP-2 also blocks pellicle formation. Fourth, ZSP-2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys-rich (glutathione-sensitive) lectin-like domains of ZSP-2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.