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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Nanoparticles have unique properties that make them attractive for use in industrial and medical technology industries but can also be harmful to living organisms, making an understanding of their molecular mechanisms of action essential. We examined the effect of three different sized poly(isobutyl-cyanoacrylate) nanoparticles (iBCA-NPs) on the unicellular green alga Chlamydomonas reinhardtii. We found that exposure to iBCA-NPs immediately caused C. reinhardtii to display abnormal swimming behaviors. Furthermore, after one hour, most of the cells had stopped swimming and 10%-30% of cells were stained with trypan blue, suggesting that these cells had severely impaired plasma membranes. Observation of the cyto-ultrastructure showed that the cell walls had been severely damaged and that many iBCA-NPs were located in the space between the cell wall and plasma membrane, as well as inside the cytosol in some cases. A comparison of three strains of C. reinhardtii with different cell wall conditions further showed that the cell mortality ratio increased more rapidly in the absence of a cell wall. Interestingly, cell mortality over time was essentially identical regardless of iBCA-NP size if the total surface area was the same. Furthermore, direct observation of the trails of iBCA-NPs indicated that the first trigger was their contact with the cell wall, which is most likely accompanied by the inactivation or removal of adsorbed proteins from the cell wall surface. Cell mortality was accompanied by the overproduction of reactive oxygen species, which was detected more readily in cells grown under constant light rather than in the dark.

DOI： 10.1111/jpy.12798

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Authorship：Lead author,　Corresponding author   Language：English   Publisher：UNIV TOKYO CYTOLOGIA  

DOI： 10.1508/cytologia.83.123

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Physical interaction between organelles is a flexible event and essential for cells to adapt rapidly to environmental stimuli. Germinating plants utilize oil bodies and peroxisomes to mobilize storage lipids for the generation of sucrose as the main energy source. Although membrane interaction between oil bodies and peroxisomes has been widely observed, its underlying molecular mechanism is largely unknown. Here we present genetic evidence for control of the physical interaction between oil bodies and peroxisomes. We identified alleles of the sdp1 mutant altered in oil body morphology. This mutant accumulates bigger and more oil body aggregates compared with the wild type and showed defects in lipid mobilization during germination. SUGAR DEPENDENT 1 (SDP1) encodes major triacylglycerol lipase in Arabidopsis. Interestingly, sdp1 seedlings show enhanced physical interaction between oil bodies and peroxisomes compared with the wild type, whereas exogenous sucrose supplementation greatly suppresses the interaction. The same phenomenon occurs in the peroxisomal defective 1 (ped1) mutant, defective in lipid mobilization because of impaired peroxisomal -oxidation, indicating that sucrose production is a key factor for oil body-peroxisomal dissociation. Peroxisomal dissociation and subsequent release from oil bodies is dependent on actin filaments. We also show that a peroxisomal ATP binding cassette transporter, PED3, is the potential anchor protein to the membranes of these organelles. Our results provide novel components linking lipid metabolism and oil body-peroxisome interaction whereby sucrose may act as a negative signal for the interaction of oil bodies and peroxisomes to fine-tune lipolysis.

DOI： 10.1074/jbc.M116.748814

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

We cultured Chlamydomonas reinhardtii cells in a minimal culture medium supplemented with various concentrations of acetate, fatty acids, ethanol, fatty alcohols, or sucrose. The presence of acetate (0.5 or 1.0 %, w/v) was advantageous for cell growth. To determine whether peroxisomes are involved in fatty acid and fatty alcohol metabolism, we investigated the dynamics of peroxisomes, including changes in their number and size, in the presence of acetate, ethanol, and sucrose. The total volume of peroxisomes increased when cells were grown with acetate, but did not change when cells were grown with ethanol or sucrose. We analyzed cell growth on minimal culture medium supplemented with various fatty acids (carbon chain length ranging from one to ten) to investigate which fatty acids are metabolized by C. reinhardtii. Among them, acetate caused the greatest increase in growth when added to minimal culture media. We analyzed the transcript levels of genes encoding putative glyoxysomal enzymes. The transcript levels of genes encoding malate synthase, malate dehydrogenase, isocitrate lyase, and citrate synthase increased when Chlamydomonas cells were grown on minimal culture medium supplemented with acetate. Our results suggest that Chlamydomonas peroxisomes are involved in acetate metabolism via the glyoxylate cycle.

DOI： 10.1007/s10265-014-0681-8

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

In Chlorophycean algal cells, these organelles are generally called microbodies because they lack the enzymes found in the peroxisomes of higher plants. Microbodies in some algae contain fewer enzymes than the peroxisomes of higher plants, and some unicellular green algae in Chlorophyceae such as Chlamydomonas reinhardtii do not possess catalase, an enzyme commonly found in peroxisomes. Thus, whether microbodies in Chlorophycean algae are similar to the peroxisomes of higher plants, and whether they use a similar transport mechanism for the peroxisomal targeting signal (PTS), remain unclear. To determine whether the PTS is present in the microbodies of Chlorophycean algae, and to visualize the microbodies in Chlamydomonas cells, we examined the sub-cellular localization of green fluorescent proteins (GFP) fused to several PTS-like sequences. We detected GFP compartments that were spherical with a diameter of 0.3-1.0 mu m in transgenic Chlamydomonas. Comparative analysis of the character of GFP-compartments observed by fluorescence microscopy and that of microbodies by electron microscopy indicated that the compartments were one and the same. The result also showed that the microbodies in Chlorophycean cells have a similar transport mechanism to that of peroxisomes of higher plants.

DOI： 10.1007/s10265-011-0469-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

The endoplasmic reticulum (ER) is composed of tubules, sheets, and three-way junctions, resulting in a highly conserved polygonal network in all eukaryotes. The molecular mechanisms responsible for the organization of these structures are obscure. To identify novel factors responsible for ER morphology, we employed a forward genetic approach using a transgenic Arabidopsis thaliana plant (GFP-h) with fluorescently labeled ER. We isolated two mutants with defects in ER morphology and designated them endoplasmic reticulum morphology1 (ermo1) and ermo2. The cells of both mutants developed a number of ER-derived spherical bodies, similar to 1 mu m in diameter, in addition to the typical polygonal network of ER. The spherical bodies were distributed throughout the ermo1 cells, while they formed a large aggregate in ermo2 cells. We identified the responsible gene for ermo1 to be GNOM-LIKE1 (GNL1) and the gene for ermo2 to be SEC24a. Homologs of both GNL1 and SEC24a are involved in membrane trafficking between the ER and Golgi in yeast and animal cells. Our findings, however, suggest that GNL1/ERMO1 and SEC24a/ERMO2 have a novel function in ER morphology in higher plants.

DOI： 10.1105/tpc.109.068270

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Peroxisomal enzymatic proteins contain targeting signals (PTS) to enable their import into peroxisomes. These targeting signals have been identified as PTS1 and PTS2 in mammalian, yeast, and higher plant cells; however, no PTS2-like amino acid sequences have been observed in enzymes from the genome database of Cyanidiochyzon merolae (Bangiophyceae), a primitive red algae. In studies on the evolution of PTS, it is important to know when their sequences came to be the peroxisomal targeting signals for all living organisms. To this end, we identified a number of genes in the genome database of the green algae Chlamydomonas reinhardtii, which contains amino acid sequences similar to those found in plant PTS. In order to determine whether these sequences function as PTS in green algae, we expressed modified green fluorescent proteins (GFP) fused to these putative PTS peptides under the cauliflower mosaic virus 35S promoter. To confirm whether granular structures containing GFP-PTS fusion proteins accumulated in the peroxisomes of Closterium ehrenbergii, we observed these cells after the peroxisomes were stained with 3, 3'-diaminobenzidine. Our results confirm that the GFP-PTS fusion proteins indeed accumulated in the peroxisomes of these green algae. These findings suggest that the peroxisomal transport system for PTS1 and PTS2 is conserved in green algal cells and that our fusion proteins can be used to visualize peroxisomes in live cells.

DOI： 10.1007/s00709-009-0031-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Myrosin cells in Capparales plants are idioblasts that accumulate thioglucoside glucohydrolase (TGG, also called myrosinase), which hydrolyzes glucosinolates to produce toxic compounds for repelling pests. Here, we show that AtVAM3 is involved in development of myrosin cells. It has been shown that yeast VAM3 is a Q.-SNARE that is involved in vesicle transport of vacuolar proteins and vacuolar assembly. We found that two Arabidopsis atvam3 alleles, atvam3-3 and atvain3-4/ssin, accumulate large amounts of TGG1 and TGG2 that are enzymatically active. An immunogold analysis revealed that TGGs were specifically localized in the vacuole of myrosin cells in atvam3 mutants. This result indicates that TGGs are normally transported to vacuoles in these mutants and that AtVAM3 is not essential for vacuolar transport of the proteins. We developed a staining method with Coomassie brilliant blue that detects myrosin cells in whole leaves by their high TGG content. This method showed that atvam3 leaves have a larger number of myrosin cells than do wild-type leaves. Myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in atvam3 leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observe independent of leaf veins. The excess of myrosin cells in atvam3 mutants might be responsible for the abnormal abundance of TGGs and the reduction of elongation of inflorescence stems and leaves in these mutants. Our results suggest that AtVAM3 has a plant-specific function in development of myrosin cells.

DOI： 10.1093/pcp/pci232

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Plant cells develop various endoplasmic reticulum (ER)-derived structures with specific functions. The ER body, a novel ER-derived compartment in Arabidopsis, is a spindle-shaped structure (similar to10 mum long and similar to1 mum wide) that is surrounded by ribosomes. Similar structures were found in many Brassicaceae plants in the 1960s and 1970s, but their main components and biological functions have remained unknown. ER bodies can be visualized in transgenic Arabidopsis expressing the green fluorescent protein with an ER-retention signal. A large number of ER bodies are observed in cotyledons, hypocotyls and roots of seedlings, but very few are observed in rosette leaves. Recently nail, a mutant that does not develop ER bodies in whole seedlings, was isolated. Analysis of the nail mutant reveals that a P-glucosidase, called PYK10, is the main component of ER bodies. The putative biological function of PYK10 and the inducibility of ER bodies in rosette leaves by wound stress suggest that the ER body functions in the defense against herbivores.

DOI： 10.1093/pcp/pcg089

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Authorship：Lead author   Language：English   Publisher：OXFORD UNIV PRESS  

DOI： 10.1093/pcp/pce144

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

The endoplasmic reticulum (ER) body is a characteristic structure derived from ER and is referred to as a proteinase-sorting system that assists the plant cell under various stress conditions. Fluorescent ER bodies were observed in transgenic plants of Arabidopsis expressing green fluorescent protein fused with an ER retention signal. ER bodies were widely distributed in the epidermal cells of whole seedlings. In contrast, rosette leaves had no ER bodies. We found that wound stress induced the formation of many ER bodies in rosette leaves. ER bodies were also induced by treatment with methyl jasmonate (MeJA), a plant hormone involved in the defense against wounding and chewing by insects. The induction of ER bodies was suppressed by ethylene. An electron microscopic analysis showed that typical ER bodies were induced in the non-transgenic rosette leaves treated with MeJA. An experiment using coil and etr1-4 mutant plants showed that the induction of ER bodies was strictly coupled with the signal transduction of MeJA and ethylene. These results suggested that the formation of ER bodies is a novel and unique type of endomembrane system in the response of plant cells to environmental stresses. It is possible that the biological function of ER bodies is related to defense systems in higher plants.

DOI： 10.1104/pp.009464

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the beta-oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designated AtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). The AtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that the AtLACS6 is localized on glyoxysomal membranes. AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and beta-oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid beta-oxidation activating the fatty acids.

DOI： 10.1104/pp.012955

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

A novel vesicle, referred to as a precursor-accumulating (PAC) vesicle, mediates the transport of storage protein precursors to protein storage vacuoles in maturing pumpkin seeds. PV72, a type I integral membrane protein with three repeats of epidermal growth factor, was found on the membrane of the PAC vesicles. PV72 had an ability to bind to pro2S albumin, a storage protein precursor, in a Ca2+- dependent manner, via the C-terminal region of pro2S albumin, which was found to function as a vacuolar targeting signal. This implies that PV72 is a vacuolar sorting receptor of the storage protein. PV72 was specifically and transiently accumulated at the middle stage of seed maturation in association with the synthesis of storage proteins. Subcellular fractionation showed that PV72 was also accumulated in the microsomal fraction. A fusion protein consisting of GFP and the transmembrane domain and the cytosolic tail of PV72 was localized in Golgi complex. PV72 in the isolated PAC vesicles had a complex type of oligosaccharide, indicating that PV72 passed though the Golgi complex. These results suggest that PV72 is recycled between PAC vesicles and Golgi complex/post-Golgi compartments. PV72 appears to be responsible for recruiting pro2S albumin molecules from the Golgi complex to the PAC vesicles.

DOI： 10.1093/pcp/pcf152

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

A novel protein, MP73, was specifically found on the membrane of protein storage vacuoles of pumpkin seed. MP73 appeared during seed maturation and disappeared rapidly after seed germination, in association with the morphological changes of the protein storage vacuoles. The MP73 precursor deduced from the isolated cDNA was composed of a signal peptide, a 24-kD domain (P24), and the MP73 domain with a putative long alpha -helix of 13 repeats that are rich in glutamic acid and arginine residues. Immunocytochemistry and immunoblot analysis showed that the precursor-accumulating (PAC) vesicles (endoplasmic reticulum-derived vesicles responsible for the transport of storage proteins) accumulated proMP73, but not MP73, on the membranes. Subcellular fractionation of the pulse-labeled maturing seed demonstrated that the proMP73 form with N-linked oligosaccharides was synthesized on the endoplasmic reticulum and then transported to the protein storage vacuoles via PAC vesicles. Tunicamycin treatment of the seed resulted in the efficient deposition of proMP73 lacking the oligosaccharides (proMP73 Delta Psi) into the PAC vesicles but no accumulation of MP73 in vacuoles. Tunicamycin might impede the transport of proMP73 Delta Psi from the PAC vesicles to the vacuoles or might make the unglycosylated protein unstable in the vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by the action of a vacuolar enzyme to form a 100-kD complex on the vacuolar membranes. These results suggest that PAC vesicles might mediate the delivery of not only storage proteins but also membrane proteins of the vacuoles.

DOI： 10.1105/tpc.13.10.2361

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Plants degrade cellular materials during senescence and under various stresses. We report that the precursors of two stress-inducible cysteine proteinases, RD21 and a vacuolar processing enzyme (VPE), are specifically accumulated in similar to0.5 mum diameter x similar to5 mum long bodies in Arabidopsis thaliana. Such bodies have previously been observed in Arabidopsis but their function was not known. They are surrounded with ribosomes and thus are assumed to be directly derived from the endoplasmic reticulum (ER). Therefore, we propose to call them the ER bodies. The ER bodies are observed specifically in the epidermal cells of healthy seedlings. These cells are easily wounded and stressed by the external environment. When the seedlings are stressed with a concentrated salt solution, leading to death of the epidermal cells, the ER bodies start to fuse with each other and with the vacuoles, thereby mediating the delivery of the precursors directly to the vacuoles. This regulated, direct pathway differs from the usual case in which proteinases are transported constitutively from the ER to the Golgi complex and then to vacuoles, with intervention of vesicle-transport machinery, such as a vacuolar-sorting receptor or a syntaxin of the SNARE family. Thus, the ER bodies appear to be a novel proteinase-storing system that assists in cell death under stressed conditions.

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis thaliana contained polypeptides with 331 amino acids and had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. We expected PMP38 to be localized on peroxisomal membranes, because it had the membrane peroxisomal targeting signal. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein PMP38 in glyoxysomes changes dramatically during the transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid β-oxidation cycle, that are localized in the matrix of glyoxysomes.

DOI： 10.1093/pcp/pce108

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG WIEN  

During germination and subsequent growth of fatty seeds, higher plants obtain energy from the glyconcogenic pathway in which fatty acids are converted to succinate in glyoxysomes, which contain enzymes for fatty acid beta -oxidation and the glyoxylate cycle. The Arabidopsis thaliana peril gene encodes a 3-ketoacyl-CoA thiolase (EC 2.3.1.16) involved in fatty acid beta -oxidation. The peril mutant shows normal germination and seedling growth under white light. However, etiolated cotyledons of the peril mutant grow poorly in the dark and have small cotyledons. To elucidate the mechanisms of lipid degradation during germination in the peril mutant, we examined the morphology Of the peril mutant. The glyoxysomes in etiolated cotyledons of the peril mutant appeared abnormal, having tubular structures that contained many vesicles. Electron microscopic analysis revealed that the tubular structures in glyoxysomes are derived from invagination of the glyoxysomal membrane. By immunoelectron microscopic analysis, acyl-CoA synthetase (EC 6.2.1.3), which was located on the membrane of glyoxysomes in wildtype plants, was located on the membranes of the tubular structures in the glyoxysomes in the ped1 mutant. These invagination sites were always in contact with lipid bodies. The tubular structure had many vesicles containing substances with the same electron density as those in the lipid bodies. From these results, we propose a model in which there is a direct mechanism of transporting lipids from the lipid bodies to glyoxysomes during fatty acid beta -oxidation.

DOI： 10.1007/BF01288364

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press  

Peroxisomes in higher plant cells are known to differentiate into at least three different classes, namely, glyoxysomes, leaf peroxisomes, and unspecialized peroxisomes, depending on the cell types. In germinating fatty seedlings, glyoxysomes that first appear in the etiolated cotyledonary cells are functionally transformed into leaf peroxisomes during greening. Subsequently, the organelles are transformed back into glyoxysomes during senescence of the cotyledons. Flexibility of function is a distinct feature of plant peroxisomes. This article briefly describes recent studies of the regulatory mechanisms involved in the changes of the function of plant peroxisomes.

DOI： 10.1385/CBB:32:1-3:295

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHYCOLOGICAL SOC AMER INC  

The DNA sequence of the cytochrome oxidase subunit I (COXI) gene (1059 bp), was determined in a number of heterokont algae, including five species of the Phaeophyceae [Chorda filum (Linnaeus) Stackhouse, Colpomenia bullosa (Saunders) Yamada, Ectocarpus sp., Pseudochorda nagaii (Tokida) Inagaki, Undaria pinnatifida (Harvey) Suringar], and a member of the Raphidophyceae [Chattonella antiqua (Hada) Ono]. The distribution of a deviant mitochondrial code, the AUA codon for methionine (AUA/Met), which was previously reported in the Xanthophyceae, was inferred from these COXI sequences. Comparative analyses of these sequences revealed that all the algae described above bear the universal genetic code, including the assignment for the AUA codon. A phylogenetic tree was constructed using the obtained sequences along with already-published COXI sequences of various heterokont algae. The clusters of the Xanthophyceae and the Phaeophyceae were resolved as sister groups with high bootstrap support, excluding a bacillariophycean species, a raphidophycean species, and three species of the Eustigratophyceae. Taking the distribution of the deviant code and the COXI phylogenetic tree together, the genetic code change most probably occurred in an ancestor of the Xanthophyceae after it had branched off from the Phaeophyceae.

DOI： 10.1046/j.1529-8817.1998.341005.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The mitochondrial genetic code of those land plants and green algae that have been examined does not deviate from the universal one. A red alga, Chondrus crispus, is the sole reported example throughout the algae that uses a deviant (non-universal) mitochondrial genetic code (UGA = Trp). We have analyzed 366-bp DNA sequences of the gene for mitochondrial cytochrome oxidase subunit I (COXI) from ten chlorophyceaen algae, and detected 3-8 in-frame UAG codons in the sequences of five species. Comparisons of these sequences with those of other algae and land plants have shown that most of the UAG sites in Hydrodictyon reticulatum, Pediastrum boryanum and Tetraedron bitridens correspond to alanine, and those of Coelastrum microporum and Scenedesmus quadricauda to leucine. The three species in which UAG probably codes for alanine are characterized by zoospore formation in asexual reproduction and form a clade in the COXI phylogenetic tree. The two species in which UAG codes for leucine are known to form daughter coenobia and pair in the tree. This is the first report on a deviant mitochondrial genetic code in green algae. Mutational change(s) in the release factor corresponding to UAG would be involved in these code changes. No genetic code deviation has been found in five other species examined.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Plant Physiologists  

The localization of lectin binding sites in the Golgi apparatus, plasma membranes and cell walls of Scenedesmus acuminatus was investigated by cytochemical electron microscopy. The lectins used were concanavalin A (Con A), peanut agglutinin (PNA) and wheat germ agglutinin (WGA), all labeled with gold. Con A-gold particles were deposited not on the Golgi apparatus, but on the outer cell-wall layer. PNA-gold and WGA-gold particles were deposited on distal Golgi cisternae and vesicles derived from the Golgi apparatus. Entire cell-wall layers were evenly labeled by PNA-gold. The plasma membrane and cytoplasmic regions close to the plasma membrane were labeled with WGA-gold. The processing of oligosaccharide in the Golgi apparatus, plasma membranes and cell walls of Scenedesmus acuminatus is discussed in reference to that reported for animal cells.

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DOI： 10.14841/jspp.2010.0.0129.0

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Publisher：The Japanese Society of Plant Physiologists  

The endoplasmic reticulum (ER) forms a dynamic polygonal network composed of tubules, sheets, and three-way junctions. Although the complex ER morphology supports the diverse cellular functions, the molecular mechanisms responsible for the organization of these structures are obscure. Here we report the isolation and characterization of mutants of <i>Arabidopsis thaliana</i> that are defective in ER morphology (<i>ermo1</i>, <i>ermo2</i>, and <i>ermo3</i>). Among three mutants, <i>ermo1</i> and <i>ermo2</i>, which showed partially similar phenotypes, were defective in GNOM-LIKE1 (GNL1) and SEC24a, respectively (1). Both GNL1/ERMO1 and SEC24a/ERMO2 were thought to be involved in the ER-Golgi transport. Our results suggests that the unknown cargo proteins that are specifically transported by GNL1/ERMO1 and SEC24a/ERMO2 may have crucial roles. On the other hand, we identified the responsible gene for <i>ermo3</i> to be a member of GDSL-motif lipase family, which is predicted to be localized within the vacuoles. Our findings suggest a novel pathway that connects ER morphology to lipid metabolic processes.<br>(1) Nakano, R.T. et al., Plant Cell,10.1105/tpc.109.068270 (2009).

DOI： 10.14841/jspp.2010.0.0129.0

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In higher plants, peroxisomes are known to differentiate into several classes. Glyoxysomes that are abundant in cotyledons, contain enzymes of &amp;beta;-oxidation and glyoxylate cycle to degrade storage lipid for postgerminative growth of oilseed plants. Leaf peroxisomes that are found in green leaves, function together with chloroplasts and mitochondria in phororespiration. Malate dehydrogenase (MDH) is an enzyme to catalayze the interconversion of malate and oxaloacetate. Two isoforms of peroxisomal MDH (PMDH) have been identified, the glyoxysome-type isoform, PMDH1 (At2g22780) which is involved in glyoylate cycle and in &amp;beta;-oxidation, and the leaf peroxisome-type isoform, PMDH2 (At5g09660) which is induced by light but those function are not elucidated. We investigated &lt;I&gt;pmdh2&lt;/I&gt; and &lt;I&gt;pmdh1pmdh2&lt;/I&gt; mutants of &lt;I&gt;Arabidopsis&lt;/I&gt; to elucidate physiological function of PMDH2. In this study, we report the phenotypes of these mutants that are grown under high light condition and discuss the function of PMDH2 in leaves.

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The endoplasmic reticulum (ER) has dynamic structures; i.e., the polygonal network composed of sheets, tubules, and "three-way junctions". To identify the factors responsible for ER morphology, we isolated the mutants that showed defects in ER morphology, and designated them <I>endoplasmic reticulum morphology</I> (<I>ermo</I>). <I>ermo1</I> and <I>ermo2</I> developed a number of ER-derived spherical structures, while <I>ermo3</I> developed a large aggregate within the cells. The spherical structures in <I>ermo2</I> also formed a large aggregate. In <I>ermo1</I> and <I>ermo2</I>, the genes involved in COPI and COPII formation, respectively, were mutated. However, we could not find any defects of membrane trafficking in these mutants, suggesting that ERMO1 and ERMO2 have a novel function in maintaining ER morphology. Alternatively, it is possible that ER-Golgi transport plays a crucial role in ER morphology. In addition, ERMO2 and ERMO3 were shown to be required for keeping organelles distributed throughout the cells.

DOI： 10.14841/jspp.2009.0.0035.0

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Publisher：The Japanese Society of Plant Physiologists  

In higher plants, peroxisomes are known to differentiate into several classes. Glyoxysomes that are abundant in cotyledonary cells, contain enzymes of &amp;beta;-oxidation and glyoxylate cycle to degrade storage lipids for postgerminative growth of oil-seed plants. Peroxisomal malate dehydrogenase(PMDH), that is an enzyme of glyoxylate cycle, has two isoforms, At2g22780(PMDH1) and At5g09660(PMDH2). RT-PCR analysis showed that transcription of PMDH1 was activated during germination and that of PMDH2 was induced in response to light. These data suggest that PMDH1 and PMDH2 play different roles, respectively. Therefore, we investigated Arabidopsis &lt;I&gt;pmdh&lt;/I&gt; mutants to elucidate physiological functions of PMDH isoforms. &lt;br&gt;The &lt;I&gt;pmdh1&lt;/I&gt; and &lt;I&gt;pmdh2&lt;/I&gt; display no phenotypic abnormalities during germination. However, &lt;I&gt;pmdh1pmdh2&lt;/I&gt; requires sucrose for postgerminative growth and defects &amp;beta;-oxidation as well as &lt;I&gt;ped1&lt;/I&gt;. These results suggest that PMDHs participate not only in glyoxylate cycle but also in &amp;beta;-oxidation.

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Language：Japanese   Publisher：日本作物学会  

電子顕微鏡用の試料作成法としては,簡便な化学固定法が主流であるが,細胞内のより微細な構造の解析や,瞬間的な動きを捉えるため,また,免疫電子顕微鏡法のための抗原性の保持のためには,凍結固定法が優れている.近年,高圧下で凍結することで,構造を破壊する原因である氷結の形成を防ぐことができる高圧凍結装置(Bal-Tec, HPM010S型)が開発され,組織を材料とした凍結固定法による試料作成が可能となっている.そこで,この装置を用いた高圧凍結および凍結置換法について紹介する.

DOI： 10.1626/jcs.76.128

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Language：Japanese   Publisher：日本作物学会  

電子顕微鏡用の試料作成法としては，簡便な化学固定法が主流であるが，細胞内のより微細な構造の解析や，瞬間的な動きを捉えるため，また，免疫電子顕微鏡法のための抗原性の保持のためには，凍結固定法が優れている．近年，高圧下で凍結することで，構造を破壊する原因である氷結の形成を防ぐことができる高圧凍結装置（Bal-Tec, HPM010S型）が開発され，組織を材料とした凍結固定法による試料作成が可能となっている．そこで，この装置を用いた高圧凍結および凍結置換法について紹介する．

DOI： 10.1626/jcs.76.128

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Language：Japanese  

DOI： 10.14841/jspp.2006.0.369.0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

Yeast VAM3 is a Q&lt;SUB&gt;a&lt;/SUB&gt;-SNARE that is involved in vacuolar transport of proteins and vacuolar assembly. Previously, we showed that &lt;I&gt;atvam3&lt;/I&gt; mutants accumulate large amounts of thioglucoside glucohydrolase (TGG), which hydrolyze glucosinolates to produce toxic compounds for repelling pests. Myrosin cells in &lt;I&gt;Capparales&lt;/I&gt; plants are idioblasts that accumulate TGG. An immunogold revealed TGGs were specifically localized in the vacuole of myrosin cells in &lt;I&gt;atvam3&lt;/I&gt; mutants. This result indicates that TGGs are normally transported to vacuoles in these mutants and that AtVAM3 is not essential for vacuolar transport of TGG. Myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in &lt;I&gt;atvam3&lt;/I&gt; leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observed independent of leaf veins. Our results suggest that AtVAM3 has a plant-specific function in development of myrosin cells.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

Peroxisome is an organelle that exists universally in eukaryotic cells, however the function of it differs among species and type of tissues. In higher plants, peroxisomes are directly transformed from glyoxysomes to leaf peroxisomes in greening cotyledons of fatty seedlings. Glyoxisomes contain enzymes for the fatty acid &amp;beta;-oxidation cycle and the glyoxylate cycle, while leaf peroxisomes contain enzymes for photorespiration pathway. Reverse transformation of leaf peroxisomes to glyoxysomes is observed in the senescing cotyledons. Almost of peroxisomal enzymes bear an element that works as a peroxisomal targeting signal (PTS). We detected a PTS1-like element in malate synthase and glycolate oxidase genes of&lt;I&gt;Chlamydomonas reinhardtii&lt;/I&gt;. To know these enzymes are actually located in the peroxisomes and if higher plant&#039;s PTS acts normally in this unicellular green alga, we expressed a fusion gene of GFP with PTS. We also analyzed expression patterns of these enzymes in photoautotrophic and heterotrophic growth conditions.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

ER body is a compartment that is derived from endoplasmic reticulum in &lt;I&gt;Arabidopsis thaliana&lt;/I&gt;. They are distributed in the epidermal cells of whole seedlings, while rosset leaves have no ER bodies. However, ER bodies are induced in rosset leaves by wounded stress and/or treatment with methyl jasmonate. This indicates that the ER body plays a role in the defense system of plants (Matsushima et al.,2003). Fluorescent ER bodies were observed in transgenic plants of Arabidopsis expressing GFP fused with an ER retention signal (&lt;I&gt;GFP-h&lt;/I&gt;). To know the mechanism of ER body formation, we raised several kinds of morphological ER body mutants by EMS treatment of the epidermal cells of cotyledons from &lt;I&gt;GFP-h&lt;/I&gt; seeds. We will report the morphological features of these mutants obtained through observations by conforcal laser scan microscope and transmission electron microscope. Based on these, we would like to discuss the mechanism of the formation of ER bodies.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

Myrosinases are &amp;beta;-glucosidases that are found mainly in the order Capparales. Myrosinases hydrolyze glucosinolates to produce the toxic compounds for the pests. The abnormal accumulation of myrosinases was found in &lt;I&gt;Arabidopsis&lt;/I&gt; mutants of AtVAM3, which is a syntaxin homolog to yeast VAM3 involved in the vacuolar assembly. In this study, we characterized the abnormal accumulation of myrosinases in two AtVAM3 mutants; one expresses the insertion form of AtVAM3 with an additional peptide and another is a knockout mutant of the &lt;I&gt;AtVAM3&lt;/I&gt; gene. The organ specific expression and the localization to the myrosin cells of the myrosinases in these mutants were the same as in wild-type. Interestingly, the numbers of the myrosin cells were markedly increased in the mutants. The abnormal accumulation of myrosinases in the mutants might be caused by the excessive occurrence of the myrosin cells. These results suggest that AtVAM3 is involved in differentiation of myrosin cells.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publisher：Blackwell Publishing  

We have determined a partial DNA sequence (approximately 1.1 kb) encoding the mitochondrial cytochrome oxidase subunit 1 (cox1) gene from the alga NIES 548, a strain which has been maintained as Tribonema marinum J. Feldmann (Xanthophyceae) at the National Institute of Environmental Study (NIES, Japan). Unexpectedly, phylogenetic analysis of cox1 sequences showed that NIES 548 does not group with Xanthophyceae, but groups strongly with the Phaeophyceae. Furthermore, the cox1 sequence from NIES 548 does not use the codon AUA for methionine (AUA/Met), a genetic marker characteristic for the Xanthophyceae. Given the cox1 results, the molecular phylogenetic position of NIES 548 was thus examined with DNA sequences from genes encoded in the two other subcellular compartments. In the plastid, we analyzed elongation factor tu (tufA) and in the nucleus, the small subunit ribosomal RNA (rrnS). These analyses clearly indicated that NIES 548 is not a member of the Xanthophyceae, but a member of the Phaeophyceae. This result is robust as it was supported by all three genes analyzed, each of which reside in different genomes. The phylogenetic resolutions of these trees were almost the same, proving the usefulness of mitochondrial cox1 gene as a tool for phylogenetic reconstruction in the phycological arena. Our light microscopic observations indicated that NIES 548 is a uniseriate filamentous alga with branches, a clear contradiction of its original descriptions. We conclude that NIES 548 is a phaeophycean alga and is not the same clone of the strain observed by Sartoni and his co-authors.

DOI： 10.1046/j.1440-1835.1999.00166.x

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