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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/jpy.12798

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担当区分：筆頭著者,　責任著者   記述言語：英語  

DOI： 10.1508/cytologia.83.123

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Biochemistry & Molecular Biology (ASBMB)  

DOI： 10.1074/jbc.m112.438143

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC PLANT BIOLOGISTS  

We have identified the novel protein Glycine max PEROXISOMAL ADENINE NUCLEOTIDE CARRIER (Gm PNC1) by proteomic analyses of peroxisomal membrane proteins using a blue native/SDS-PAGE technique combined with peptide mass fingerprinting. Gm PNC1, and the Arabidopsis thaliana orthologs At PNC1 and At PNC2, were targeted to peroxisomes. Functional integration of Gm PNC1 and At PNC2 into the cytoplasmic membranes of intact Escherichia coli cells revealed ATP and ADP import activities. The amount of Gm PNC1 in cotyledons increased until 5 d after germination under constant darkness and then decreased very rapidly in response to illumination. We investigated the physiological functions of PNC1 in peroxisomal metabolism by analyzing a transgenic Arabidopsis plant in which At PNC1 and At PNC2 expression was suppressed using RNA interference. The pnc1/2i mutant required sucrose for germination and suppressed the degradation of storage lipids during postgerminative growth. These results suggest that PNC1 contributes to the transport of adenine nucleotides that are consumed by reactions that generate acyl-CoA for peroxisomal fatty acid beta-oxidation during postgerminative growth.

DOI： 10.1105/tpc.108.062877

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Glyoxysomes are present in etiolated cotyledons and contain enzymes for gluconeogenesis, which constitutes the major function of glyoxysomes. However, 281 genes seemingly related to peroxisomal functions occur in the Arabidopsis genome, implying that many unidentified proteins are present in glyoxysomes. To better understand the functions of glyoxysomes, we performed glyoxysomal proteomic analysis of etiolated Arabidopsis cotyledons. Nineteen proteins were identified as glyoxysomal proteins, including 13 novel proteins, one of which is glyoxysomal protein kinase 1 (GPK1). We cloned GPK1 cDNA by RT-PCR and characterized GPK1. The amino acid sequence deduced from GPK1 cDNA has a hydrophobic region, a putative protein kinase domain, and a possible PTS1 motif. Immunoblot analysis using fractions collected on a Percoll density gradient confirmed that GPK1 is localized in glyoxysomes. Analysis of suborganellar localization and protease sensitivity showed that GPK1 is localized on glyoxysomal membranes as a peripheral membrane protein and that the putative kinase domain is located inside the glyoxysomes. Glyoxysomal proteins are phosphorylated well in the presence of various metal ions and [γ- 32P]ATP, and one of them is identified as thiolase by immunoprecipitation. Immunoinhibition of phosphorylation in glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in glyoxysomes.

DOI： 10.1093/pcp/pcg145

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：筆頭著者   記述言語：英語   出版者・発行元：OXFORD UNIV PRESS  

DOI： 10.1093/pcp/pce144

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

DOI： 10.1093/pcp/pcf101

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Biochemistry and Molecular Biology Inc.  

Short-chain acyl-CoA oxidases are β-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete β-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform β-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid β-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid β-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete β-oxidation of acyl-CoA.

DOI： 10.1074/jbc.274.18.12715

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=180561

総ページ数：275  

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記述言語：日本語  

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DOI： 10.14841/jspp.2010.0.0129.0

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記述言語：英語  

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出版者・発行元：日本植物生理学会  

小胞体は，非常に複雑なネットワーク状の構造をとりつつ，ダイナミックに運動するオルガネラである．このような形態の複雑性は小胞体機能の多様性に関与しているが，これらの構造を形成・維持する分子機構は未解明の部分が多い．我々はこれまでに小胞体の形態異常を示す3種類のシロイヌナズナ変異体を単離している (<i>ermo1</i>, <i>ermo2</i>, および<i>ermo3</i>)．このうち<i>ermo1</i>と<i>ermo2</i>の表現型は部分的に類似しており，GNOM-LIKE1 (GNL1) とSEC24aをコードする遺伝子に変異がみつかった (1)．これらの因子はともに小胞体―ゴルジ体間の輸送に関与すると考えられている．我々は，ERMO1およびERMO2によって特異的に輸送される未知の因子が小胞体の形態において重要な働きを担っていると考えている．一方，<i>ermo3</i>ではGDSL-motif lipaseと呼ばれるリパーゼをコードする遺伝子に変異を同定した．この酵素は液胞内腔に局在されると予測されている．我々の解析結果は，小胞体の形態と脂質代謝経路とをつなぐ全く新しい経路の存在を示唆している．<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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出版者・発行元：日本植物生理学会  

高等植物のペルオキシソームは，脂肪代謝や光呼吸など様々な代謝系を担う細胞小器官である。ペルオキシソームに局在するリンゴ酸脱水素酵素PMDHはグリオキシル酸回路を構成する酵素の一つであり，シロイヌナズナには2つのアイソフォームが存在する。我々はこれまでに，発芽初期に発現するPMDH1 (At2g22780)が，発芽時の脂肪代謝に関与することを明らかにした（第49回日本植物生理学会年会）。本研究では，緑化組織において発現するPMDH2 (At5g09660)の機能に注目し，シロイヌナズナ欠損変異体&lt;I&gt;pmdh2&lt;/I&gt;及び&lt;I&gt;pmdh1pmdh2&lt;/I&gt;の解析を行った。定量PCRによって光呼吸系酵素群の遺伝子発現を解析した結果，野生型と&lt;I&gt;pmdh1pmdh2&lt;/I&gt;の間に大きな違いは認められなかった。しかし，150μmol m&lt;SUP&gt;-2&lt;/SUP&gt;s&lt;SUP&gt;-1&lt;/SUP&gt;の白色光下で2週間生育した&lt;I&gt;pmdh1pmdh2&lt;/I&gt;の生重量は，野生型の約50％であった。50μmol m&lt;SUP&gt;-2&lt;/SUP&gt;s&lt;SUP&gt;-1&lt;/SUP&gt;の弱光下で生育した場合，生重量に差はなかったが，&lt;I&gt;pmdh1pmdh2&lt;/I&gt;の緑葉にはH&lt;SUB&gt;2&lt;/SUB&gt;O&lt;SUB&gt;2&lt;/SUB&gt;の蓄積が観察された。さらに450μmol m&lt;SUP&gt;-2&lt;/SUP&gt;s&lt;SUP&gt;-1&lt;/SUP&gt;の白色光を照射した&lt;I&gt;pmdh2&lt;/I&gt;及び&lt;I&gt;pmdh1pmdh2&lt;/I&gt;変異体の緑葉では，光化学系IIの最大電子伝達効率(&lt;I&gt;Fv/Fm&lt;/I&gt;) の著しい低下や，細胞死が観察された。以上の結果は，PMDH2が緑葉における光ストレス耐性に不可欠であることを示唆している。

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出版者・発行元：日本植物生理学会  

小胞体はチューブ状構造とシート状構造が three-way junctionと呼ばれる三叉の接続を介して複雑に組み合わさったネットワーク状の構造をとっている．この形態の形成・維持に寄与する因子を同定するため，小胞体の形態に異常を示す変異体を3系統単離し，<i>endoplasmic reticulum morphology</i>（<i>ermo</i>）と命名した．<i>ermo1</i>と<i>ermo2</i>では，小胞体に由来する1 &mu;m前後の球状構造体が数多く観察され，<i>ermo2</i>ではそれらが細胞内の一カ所に凝集していた．一方<i>ermo3</i>では球状の構造体は観察されず，小胞体が一カ所に凝集していた．これらの変異体では異常な構造・凝集体の他に正常なネットワークも観察された．マッピングの結果，<i>ermo1</i>と<i>ermo2</i>は小胞体ーゴルジ体間の輸送においてそれぞれCOPI小胞とCOPII小胞の出芽に関わると考えられている遺伝子の変異であることがわかった．しかし，これらの変異体において細胞内輸送に明らかな異常は観察できていない．このことから，ERMO1およびERMO2は小胞体の形態維持に関わる新奇機能をもっている，あるいは変異体における輸送異常は検出できないレベルだが，小胞体ーゴルジ体間の輸送が小胞体形態に重要であることが考えられた．また，ERMO2およびERMO3はオルガネラを細胞内に分布させるのに必要であることが示された．

DOI： 10.14841/jspp.2009.0.0035.0

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出版者・発行元：日本植物生理学会  

【目的】高等植物のペルオキシソームは組織や成長段階によって、その名称や機能が異なる。脂肪性種子植物の胚乳や黄化子葉に存在するペルオキシソームの一種、グリオキシソームはβ酸化系やグリオキシル酸回路が局在し、貯蔵脂肪の分解を行って発芽に必要なエネルギーを供給する。ペルオキシソーム局在型のリンゴ酸脱水素酵素であるPMDHはグリオキシル酸回路を構成する酵素の一つであり2つのアイソフォーム、At2g22780(PMDH1)、At5g09660(PMDH2)が存在する。両者の遺伝子発現解析から、PMDH1は発芽の初期に、PMDH2は光に応答して発現が上昇することが明らかになった。これは2つのPMDHアイソフォームがそれぞれ異なる役割を担っていることを示唆する。そこで本研究では、PMDHの生理機能を明らかにする為、シロイヌナズナの遺伝子欠損変異体を用いた解析を行った。&lt;br&gt;【結果と考察】&lt;I&gt;pmdh1&lt;/I&gt;、&lt;I&gt;pmdh2&lt;/I&gt;変異体には際だった表現型は観察されなかった、しかし両遺伝子を欠損した&lt;I&gt;pmdh1pmdh2&lt;/I&gt;二重変異体では種子貯蔵脂肪の分解が抑制され、芽生えの成長にはスクロースを必要とした。さらにβ酸化系欠損変異体である&lt;I&gt;ped1&lt;/I&gt;と同様に、β酸化系活性の低下が見られた。以上の結果よりPMDHはグリオキシル酸回路だけでなく、β酸化系の活性化にも関与しているのではないかと考えられる。現在、緑葉におけるPMDHの機能について、ストレス応答との関連性に注目して解析中である。

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記述言語：日本語   出版者・発行元：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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記述言語：日本語   出版者・発行元：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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記述言語：日本語  

DOI： 10.14841/jspp.2006.0.369.0

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：日本植物生理学会  

われわれは，&lt;I&gt;atvam3&lt;/I&gt;変異体（AtVAM3のペプチド挿入変異体および欠損変異体）においてミロシン細胞数が増大し，ミロシナーゼ（TGG）が大量蓄積されることを報告した．TGGはグルコシノレート（カラシ油配糖体）を分解し害虫に対する忌避物質を生成するチオグルコシダーゼであり，特徴的な異型細胞（ミロシン細胞）の液胞中に蓄積されている．ミロシン細胞は，野生型の葉では維管束周辺に点在していたが，&lt;I&gt;atvam3&lt;/I&gt;変異体では葉全体にわたって連続的なネットワークを形成していた．免疫電顕において，&lt;I&gt;atvam3&lt;/I&gt;変異体のTGGもミロシン細胞の液胞中に特異的に検出され，輸送異常は観察されなかった．&lt;I&gt;atvam3&lt;/I&gt;変異体における液胞タンパク質の蓄積パターンを野生型植物と比較したところ，TGG以外のタンパク質（RD21，アリューレイン，2Sアルブミン，12Sグロブリン，γ-TIP）に変化は見られなかった．ペプチド挿入型AtVAM3の細胞内局在は正常なAtVAM3と一致し，液胞膜および液胞前区画で検出された．また，&lt;I&gt;atvam3&lt;/I&gt;変異体においてもその局在は変化せず，液胞も正常に発達していた．酵母VAM3は液胞融合や液胞タンパク質の輸送に必須であることが報告されているが，AtVAM3は液胞融合およびTGGの液胞輸送には必須ではなく，秩序だった細胞分化という多細胞生物ならではの機能を持つことが示唆された．&lt;br&gt;Ueda et al. &lt;I&gt;Plant Cell Physiol.&lt;/I&gt; (2005) &lt;I&gt;in press&lt;/I&gt;

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：日本植物生理学会  

ペルオキシソームは、真核細胞に普遍的に存在するオルガネラである。生物種や組織・器官、成長段階の違いによって機能は異なる。光合成をおこなう高等植物では、高等動物や菌類とは異なり、黄化子葉が光を受けて緑化する過程で脂肪酸代謝に関わるグリオキシソームから光呼吸の役割を担う緑葉ペルオキシソームへと機能転換する。この時、内部の酵素は入れ替わる。この現象は、カボチャやシロイヌナズナなどの高等植物について詳しく調べられているが、同じく光合成をおこなう細胞である緑藻に関しては、内部に存在する酵素が同じであるかどうか、機能転換機構が存在するかどうかなど、まだ明らかにされていない。高等植物で機能転換時に入れ替わるとされている酵素遺伝子のホモログをクラミドモナスにおいて調べると、リンゴ酸合成酵素（MS）とグリコール酸酸化酵素（GO）は、ペルオキシソーム輸送シグナルとされているPTS1を、リンゴ酸脱水素酵素（MD）はPTS2を持つことが明らかとなった。そこで、単細胞緑藻クラミドモナスにおいて、これらのペルオキシソーム輸送シグナル（PTS）が機能しているかどうかを調べるために、PTSを付加したGFPをクラミドモナス細胞内で発現させ、その局在を解析している。また、これらの酵素について、従属栄養状態時と、独立栄養状態時とで、高等植物の子葉で見られるような発現量の変動を示すかどうかについても解析したので報告する。

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：日本植物生理学会  

ER bodyは、シロイヌナズナの子葉や胚軸、根の表皮細胞に特異的に存在する構造体である。しかし、ER bodyをもたないロゼット葉にも、傷害や食害などのストレス処理、ジャスモン酸メチル処理によって、ER body形成が誘導される。現在までに、ER bodyは小胞体由来であり、小胞体から液胞へ液胞プロセシング酵素（VPE）や液胞プロテアーゼ（RD21）の前駆体およびβグルコシダーゼの一つであるPYK10を輸送していることを明らかにした（2001、2003年度大会にて報告）。これらの結果から、ER body形成は、防御機構と関連していることが示唆されている。ER bodyを可視化することができる形質転換シロイヌナズナ（小胞体残留シグナルを付加したGFP-HDELを発現する）を、EMS処理した後、子葉表皮細胞中のER bodyの形態が異常になったと思われる株を選別した（A2：凝集体を形成する変異体、A28：凝集体と小胞状の構造体を多数形成する変異体、B23：小胞状の構造体を多数形成する変異体）。これらの突然変異体について、GFP蓄積部位とER bodyとの関係を調べるために、共焦点レーザー顕微鏡と電子顕微鏡を用いた形態学的な解析を行ったので報告する。また、子葉の老化に伴うER bodyの挙動についても詳しい解析を行った。これらの結果から、ER body形成のメカニズムについて考察する。

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：日本植物生理学会  

VAM3は輸送小胞と液胞の融合に関与するSNAREである．われわれは，AtVAM3のペプチド挿入変異体がミロシナーゼ（TGG1, TGG2）を大量に蓄積することを見い出した．ミロシナーゼはアブラナ科を含むフウチョウソウ目に特徴的な&amp;beta;-グルコシダーゼであり，グルコシノレート（カラシ油配糖体）を分解して害虫に対する忌避物質を生成する．そこで，AtVAM3の挿入変異体ならびにノックアウト変異体におけるミロシナーゼの蓄積について解析を行った．その結果，AtVAM3変異体におけるミロシナーゼの発現器官は野生型と変わらないが，個々の器官における蓄積量が著しく増加していることが分かった． AtVAM3変異体では，ミロシナーゼ活性も蓄積量に比例して上昇していた．ミロシナーゼはミロシン細胞と呼ばれる特殊な細胞に局在することが知られている．ミロシナーゼの特異抗体を用いた蛍光抗体法では，AtVAM3変異体においてもミロシナーゼは維管束周辺のミロシン細胞に局在していた．しかし，興味深いことに，AtVAM3変異体ではミロシン細胞の数が増えていることが明らかになった．ミロシン細胞の増加とミロシナーゼの蓄積量は，ノックアウト変異体よりも挿入変異体でより顕著に観察された．以上の結果から，AtVAM3がミロシン細胞の分化に関与している可能性が示唆された．

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：日本語   出版者・発行元：日本電子顕微鏡学会  

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   出版者・発行元：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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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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