Publications by authors named "Minoru Nagano"

22 Publications

  • Page 1 of 1

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

J Biol Chem 2021 Jan-Jun;296:100602. Epub 2021 Mar 27.

Laboratoire de Biogènese Membranaire, UMR 5200, CNRS, Université de Bordeaux, Villenave d'Ornon Cedex, France. Electronic address:

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.
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http://dx.doi.org/10.1016/j.jbc.2021.100602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099651PMC
March 2021

Generation of Arabidopsis lines with a red fluorescent marker for endoplasmic reticulum using a tail-anchored protein cytochrome -B.

Plant Signal Behav 2020 09 7;15(9):1790196. Epub 2020 Jul 7.

Faculty of Science and Engineering, Konan University , Kobe, Japan.

The endoplasmic reticulum (ER) is a multifunctional organelle that performs multiple cellular activities in eukaryotes. Visualizing ER using fluorescent proteins is a powerful method of analyzing its dynamics and to understand its functions. However, red fluorescent proteins with both an N-terminal signal peptide (SP) and a C-terminal ER retention tetrapeptide (HDEL) often cause mislocalization to vacuoles or extracellular spaces when they are constitutively expressed in Arabidopsis. To obtain a red fluorescent ER marker, we selected Arabidopsis cytochrome -B (Cb5-B), a tail-anchored (TA) protein on the ER membrane. Its localization is determined by the transmembrane domain (TMD) and tail domain at the C-terminus. We fused the TMD and the tail domain of Cb5-B to the C-terminus of a red fluorescent protein, tdTomato (tdTomato-CTT). When tdTomato-CTT was constitutively expressed under the ubiquitin10 promoter in Arabidopsis, the fluorescent signal was exclusively detected at the ER by means of the reliable ER marker SP-GFP-HDEL. Therefore, tdTomato-CTT can accurately visualize the ER in stable Arabidopsis lines. Additionally, transient assays showed that tdTomato-CTT can also be used as an ER marker in onion, rice, and . We believe that TA proteins could be used to generate various organellar membrane markers in plants.
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http://dx.doi.org/10.1080/15592324.2020.1790196DOI Listing
September 2020

An Arabidopsis NAC domain transcription factor, ATAF2, promotes age-dependent and dark-induced leaf senescence.

Physiol Plant 2020 Oct 28;170(2):299-308. Epub 2020 Jul 28.

Graduate School of Science and Engineering, Saitama University, Saitama, Japan.

Leaf senescence is controlled developmentally and environmentally and is affected by numerous genes, including transcription factors. An Arabidopsis NAC domain transcription factor, ATAF2, is known to regulate biotic stress responses. Recently, we have demonstrated that ATAF2 upregulates ORE1, a key regulator of leaf senescence. Here, to investigate the function of ATAF2 in leaf senescence further, we generated and analyzed overexpressing transgenic and T-DNA inserted mutant lines. Transient expression analysis indicated that ATAF2 upregulates several NAC domain transcription factors that regulate senescence. Indeed, ATAF2 overexpression induced the expression of senescence-related genes, thereby accelerating leaf senescence, whereas the expression of such genes in ataf2 mutants was lower than that of wild-type plants. Furthermore, the ataf2 mutants exhibited significant delays in dark-induced leaf senescence. It was also found that ATAF2 induces the expression of transcription factors, which both promotes and represses leaf senescence. The present study demonstrates that ATAF2 promotes leaf senescence in response to developmental and environmental signals.
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http://dx.doi.org/10.1111/ppl.13156DOI Listing
October 2020

Plant-Unique / Isomerism of Long-Chain Base Unsaturation is Selectively Required for Aluminum Tolerance Resulting from Glucosylceramide-Dependent Plasma Membrane Fluidity.

Plants (Basel) 2019 Dec 23;9(1). Epub 2019 Dec 23.

Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.

/ isomerism of the Δ8 unsaturation of long-chain base (LCB) is found only in plant sphingolipids. This unique geometry is generated by sphingolipid LCB Δ8 desaturase SLD which produces both isomers at various ratios, resulting in diverse / ratios in plants. However, the biological significance of this isomeric diversity remains controversial. Here, we show that the plant-specific unsaturation of LCB selectively contributes to glucosylceramide (GlcCer)-dependent tolerance to aluminum toxicity. We established three transgenic rice lines with altered LCB unsaturation profiles. Overexpression of SLD from rice (OsSLD-OX), which preferentially exhibits -activity, or Arabidopsis (AtSLD-OX), showing preference for -activity, facilitated Δ8 unsaturation in different manners: a slight increase of -unsaturated glycosylinositolphosphoceramide (GIPC) in OsSLD-OX, and a drastic increase of -unsaturated GlcCer and GIPC in AtSLD-OX. Disruption of LCB Δ4 desaturase () significantly decreased the content of GlcCer. Fluorescence imaging analysis revealed that OsSLD-OX and AtSLD-OX showed increased plasma membrane fluidity, whereas had less fluidity, demonstrating that the isomers universally contributed to increasing membrane fluidity. However, the results of a hydroponic assay showed decreased aluminum tolerance in AtSLD-OX and compared to OsSLD-OX and the control plants, which did not correlate with membrane fluidity. These results suggest that -unsaturated GlcCer, not GIPC, selectively serves to maintain the membrane fluidity specifically associated with aluminum tolerance.
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http://dx.doi.org/10.3390/plants9010019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020186PMC
December 2019

One of the NAD kinases, sll1415, is required for the glucose metabolism of Synechocystis sp. PCC 6803.

Plant J 2019 05 14;98(4):654-666. Epub 2019 Mar 14.

Graduate School of Science and Engineering, Saitama University, 225 Shimo-Okubo, Sakura-ku, Saitama-city, Saitama, 338-8570, Japan.

Pyridine nucleotides (NAD(P)(H)) are electron carriers that are the driving forces in various metabolic pathways. Phosphorylation of NAD(H) to NADP(H) is performed by the enzyme NAD kinase (NADK). Synechocystis sp. PCC 6803 harbors two genes (sll1415 and slr0400) that encode proteins with NADK homology. When genetic mutants for sll1415 and slr0400 (Δ1415 and Δ0400, respectively) were cultured under photoheterotrophic growth conditions only the Δ1415 cells showed a growth defect. In wild-type cells, the sll1415 transcript accumulated after the cells were transferred to photoheterotrophic conditions. Furthermore, NAD(P)(H) measurements demonstrated that a dynamic metabolic conversion was implemented during the adaptation from photoautotrophic to photoheterotrophic conditions. Electron microscopy observation and biochemistry quantification demonstrated the accumulation of glycogen in the Δ1415 cells under photoheterotrophic conditions at 96 h. Quantitative real-time reverse transcription PCR (qRT-PCR) demonstrated the accumulation of mRNAs that encoded glycogen biosynthesis-related enzymes in photoheterotrophic Δ1415 cells. At 96 h, enzyme activity measurement in the photoheterotrophic Δ1415 cells demonstrated that the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were decreased, but the activities of glucose dehydrogenase were increased. Furthermore, metabolomics analysis demonstrated that the Δ1415 cells showed increased glucose-6-phosphate and 6-phosphogluconate content at 96 h. Therefore, sll1415 has a significant function in the oxidative pentose phosphate (OPP) pathway for catabolism of glucose under photoheterotrophic conditions. Additionally, it is presumed that the slr0400 had a different role in glucose catabolism during growth. These results suggest that the two Synechocystis sp. PCC 6803 NADKs (Sll1415 and Slr0400) have distinct functions in photoheterotrophic cyanobacterial metabolism.
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http://dx.doi.org/10.1111/tpj.14262DOI Listing
May 2019

Arabidopsis Bax inhibitor-1 interacts with enzymes related to very-long-chain fatty acid synthesis.

J Plant Res 2019 Jan 2;132(1):131-143. Epub 2019 Jan 2.

Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakuraku, Saitama, 338-8570, Japan.

Bax inhibitor-1 (BI-1) is a widely conserved cell death regulator that confers resistance to environmental stress in plants. Previous studies suggest that Arabidopsis thaliana BI-1 (AtBI-1) modifies sphingolipids by interacting with cytochrome b (AtCb5), an electron-transfer protein. To reveal how AtBI-1 regulates sphingolipid synthesis, we screened yeast sphingolipid-deficient mutants and identified yeast ELO2 and ELO3 as novel enzymes that are essential for AtBI-1 function. ELO2 and ELO3 are condensing enzymes that synthesize very-long-chain fatty acids (VLCFAs), major fatty acids in plant sphingolipids. In Arabidopsis, we identified four ELO homologs (AtELO1-AtELO4), localized in the endoplasmic reticulum membrane. Of those AtELOs, AtELO1 and AtELO2 had a characteristic histidine motif and were bound to AtCb5-B. This result suggests that AtBI-1 interacts with AtELO1 and AtELO2 through AtCb5. AtELO2 and AtCb5-B also interact with KCR1, PAS2, and CER10, which are essential for the synthesis of VLCFAs. Therefore, AtELO2 may participate in VLCFA synthesis with AtCb5 in Arabidopsis. In addition, our co-immunoprecipitation/mass spectrometry analysis demonstrated that AtBI-1 forms a complex with AtELO2, KCR1, PAS2, CER10, and AtCb5-D. Furthermore, AtBI-1 contributes to the rapid synthesis of 2-hydroxylated VLCFAs in response to oxidative stress. These results indicate that AtBI-1 regulates VLCFA synthesis by interacting with VLCFA-synthesizing enzymes.
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http://dx.doi.org/10.1007/s10265-018-01081-8DOI Listing
January 2019

An NAC domain transcription factor ATAF2 acts as transcriptional activator or repressor dependent on promoter context.

Plant Biotechnol (Tokyo) 2018 Sep;35(3):285-289

Graduate School of Science and Engineering, Saitama University, Saitama, Saitama 338-8570, Japan.

The ACTIVATION FACTOR 2 (ATAF2) protein has been demonstrated to be involved in various biological processes including biotic stress responses, photo morphogenesis, and auxin catabolism. However, the transcriptional function of ATAF2 currently remains elusive. Therefore, to further understand the molecular function of ATAF2, we evaluated the transcriptional activities of ATAF2 using a transient assay system in this study. We used an effector consisting of a GAL4-DNA binding domain (GAL4-BD) fused to ATAF2, and observed upregulated reporter gene expression, suggesting that ATAF2 potentially has transcriptional activation activity. ATAF2 has been shown to activate reporter gene expression under the control of the promoter. By contrast, ATAF2 significantly repressed reporter gene expression driven by the promoter. These data suggest that ATAF2 is a bifunctional transcription factor that can alter target gene expression depending on the promoter sequences.
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http://dx.doi.org/10.5511/plantbiotechnology.18.0507aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879359PMC
September 2018

Metabolomic analysis of NAD kinase-deficient mutants of the cyanobacterium Synechocystis sp. PCC 6803.

J Plant Physiol 2016 Oct 13;205:105-112. Epub 2016 Sep 13.

Graduate School of Science and Engineering, Saitama University, 225 Shimo-Okubo, Sakura-ku, Saitama-city, Saitama 338-8570, Japan. Electronic address:

NAD kinase (NADK) phosphorylates NAD(H) to NADP(H). The enzyme has a crucial role in the regulation of the NADP(H)/NAD(H) ratio in various organisms. The unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two NADK-encoding genes, sll1415 and slr0400. To elucidate the metabolic change in NADK-deficient mutants growing under photoautotrophic conditions, we conducted metabolomic analysis using capillary electrophoresis mass spectrometry (CE-MS). The growth curves of the wild-type parent (WT) and NADK-deficient mutants (Δ1415 and Δ0400) did not show any differences under photoautotrophic conditions. The NAD(P)(H) balance showed abnormality in both mutants. However, only the metabolite pattern of Δ0400 showed differences compared to WT. These results indicated that the two NADK isoforms have distinct functions in cyanobacterial metabolism.
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http://dx.doi.org/10.1016/j.jplph.2016.09.002DOI Listing
October 2016

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice.

Plant Cell 2016 08 27;28(8):1966-83. Epub 2016 Jul 27.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.

Numerous plant defense-related proteins are thought to congregate in plasma membrane microdomains, which consist mainly of sphingolipids and sterols. However, the extent to which microdomains contribute to defense responses in plants is unclear. To elucidate the relationship between microdomains and innate immunity in rice (Oryza sativa), we established lines in which the levels of sphingolipids containing 2-hydroxy fatty acids were decreased by knocking down two genes encoding fatty acid 2-hydroxylases (FAH1 and FAH2) and demonstrated that microdomains were less abundant in these lines. By testing these lines in a pathogen infection assay, we revealed that microdomains play an important role in the resistance to rice blast fungus infection. To illuminate the mechanism by which microdomains regulate immunity, we evaluated changes in protein composition, revealing that microdomains are required for the dynamics of the Rac/ROP small GTPase Rac1 and respiratory burst oxidase homologs (Rbohs) in response to chitin elicitor. Furthermore, FAHs are essential for the production of reactive oxygen species (ROS) after chitin treatment. Together with the observation that RbohB, a defense-related NADPH oxidase that interacts with Rac1, is localized in microdomains, our data indicate that microdomains are required for chitin-induced immunity through ROS signaling mediated by the Rac1-RbohB pathway.
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http://dx.doi.org/10.1105/tpc.16.00201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006704PMC
August 2016

Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots.

Plant Physiol 2015 Sep 2;169(1):180-93. Epub 2015 Jun 2.

Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan (T.Y., A.F., H.M., Mik.N.);Department of Bioscience, Fukui Prefectural University, Eiheiji-cho, Yoshida, Fukui 910-1195, Japan (K.S.);Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama 338-8570, Japan (Min.N., M.K.-Y.);Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan (M.A., N.K.N., N.T.);Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8589, Japan (I.T.);Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa 921-8836, Japan (N.K.N.); andDepartment of Crop Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan (K.K.)

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.
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http://dx.doi.org/10.1104/pp.15.00106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577372PMC
September 2015

The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice.

PLoS Pathog 2015 Feb 6;11(2):e1004629. Epub 2015 Feb 6.

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Department of Plant Pathology, Ohio State University, Columbus, Ohio, United States of America.

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.
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http://dx.doi.org/10.1371/journal.ppat.1004629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450066PMC
February 2015

The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase.

J Biol Chem 2014 Oct 15;289(41):28569-78. Epub 2014 Aug 15.

From the Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, the Laboratory of Biophysics and

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.
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http://dx.doi.org/10.1074/jbc.M114.603282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192507PMC
October 2014

Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes.

Planta 2014 Jul 1;240(1):77-89. Epub 2014 Apr 1.

Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, 630-0192, Japan.

Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b 5 (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Δ8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.
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http://dx.doi.org/10.1007/s00425-014-2065-7DOI Listing
July 2014

Plastidic protein Cdf1 is essential in Arabidopsis embryogenesis.

Planta 2014 Jan 6;239(1):39-46. Epub 2013 Oct 6.

Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, 338-8570, Japan,

Arabidopsis cell growth defect factor-1 (Cdf1 in yeast, At5g23040) was originally isolated as a cell growth suppressor of yeast from genetic screening. To investigate the in vivo role of Cdf1 in plants, a T-DNA insertion line was analyzed. A homozygous T-DNA insertion mutant (cdf1/cdf1) was embryo lethal and showed arrested embryogenesis at the globular stage. The Cdf1 protein, when fused with green fluorescent protein, was localized to the plastid in stomatal guard cells and mesophyll cells. A promoter-β-glucuronidase assay found expression of Cdf1 in the early heart stage of embryogenesis, suggesting that Cdf1 was essential for Arabidopsis embryogenesis during the transition of the embryo from the globular to heart stage.
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http://dx.doi.org/10.1007/s00425-013-1966-1DOI Listing
January 2014

Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves.

FEBS J 2013 Aug 5;280(16):3797-809. Epub 2013 Jul 5.

Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan.

The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage (Brassica oleracea) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI-TOF MS as an adduct with Phos-tag, a phosphate-capture zinc complex. Various α-hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC-MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4-hydroxysphingenine (dehydrophytosphingosine)-1-phosphate by MALDI-TOF MS with Phos-tag. From these results, lipid X was determined to be phytoceramide-1-phosphate (PC1P) with an α-hydroxy fatty acid. When cabbage homogenates were incubated, PC1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (GIPC). The formation of PC1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin. Arabidopsis thaliana also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC1P in brassicaceous plants.
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http://dx.doi.org/10.1111/febs.12374DOI Listing
August 2013

Plant sphingolipid fatty acid 2-hydroxylases have unique characters unlike their animal and fungus counterparts.

Plant Signal Behav 2012 Nov 23;7(11):1388-92. Epub 2012 Aug 23.

Graduate School of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.

2-Hydroxy fatty acids mainly contained in sphingolipids are synthesized by a sphingolipid fatty acid 2-hydroxylase (FAH). Recently, two FAH homologs in Arabidopsis thaliana (AtFAH1 and AtFAH2), without any cytochrome b₅(Cb5)-like domains, which are essential for the function of Saccharomyces cerevisiae and mammalian FAH, were identified and both AtFAHs were shown to be activated by the interaction with Cb5. In this study, we compared FAHs of various plants, animals and fungi. Interestingly, only plants had two FAH homologs and none of plant FAHs had any Cb5-like domains. In addition, we showed from the interaction and expression analyses that AtFAHs interacted with multiple Cb5s probably in various tissues. Thus, plant FAHs may have evolved unlike animal and fungus FAHs.
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http://dx.doi.org/10.4161/psb.21825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548854PMC
November 2012

Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses.

Plant Physiol 2012 Jul 25;159(3):1138-48. Epub 2012 May 25.

Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.

2-Hydroxy fatty acids (2-HFAs) are predominantly present in sphingolipids and have important physicochemical and physiological functions in eukaryotic cells. Recent studies from our group demonstrated that sphingolipid fatty acid 2-hydroxylase (FAH) is required for the function of Arabidopsis (Arabidopsis thaliana) Bax inhibitor-1 (AtBI-1), which is an endoplasmic reticulum membrane-localized cell death suppressor. However, little is known about the function of two Arabidopsis FAH homologs (AtFAH1 and AtFAH2), and it remains unclear whether 2-HFAs participate in cell death regulation. In this study, we found that both AtFAH1 and AtFAH2 had FAH activity, and the interaction with Arabidopsis cytochrome b₅ was needed for the sufficient activity. 2-HFA analysis of AtFAH1 knockdown lines and atfah2 mutant showed that AtFAH1 mainly 2-hydroxylated very-long-chain fatty acid (VLCFA), whereas AtFAH2 selectively 2-hydroxylated palmitic acid in Arabidopsis. In addition, 2-HFAs were related to resistance to oxidative stress, and AtFAH1 or 2-hydroxy VLCFA showed particularly strong responses to oxidative stress. Furthermore, AtFAH1 interacted with AtBI-1 via cytochrome b₅ more preferentially than AtFAH2. Our results suggest that AtFAH1 and AtFAH2 are functionally different FAHs, and that AtFAH1 or 2-hydroxy VLCFA is a key factor in AtBI-1-mediated cell death suppression.
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http://dx.doi.org/10.1104/pp.112.199547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387700PMC
July 2012

Characterization of glucosylceramides in the Polygonaceae, Rumex obtusifolius L. injurious weed.

Biosci Biotechnol Biochem 2011 20;75(5):877-81. Epub 2011 May 20.

Department of Biology, Graduate School of Natural Science, Konan University, Kobe, Japan.

Rumex obtusifolius L., a member of Polygonaceae, is one of the world's worst weeds. We characterized the glucosylceramide molecular species in leaves of R. obtusifolius by liquid chromatography/tandem mass spectrometry. 4,8-Sphingadienines were principally paired with 2-hydroxy palmitic acids. In contrast, 4-hydroxy-8-sphingenines were chiefly attached to 2-hydroxy fatty acids with 22 to 26 carbon-chain length. A unique characteristic of the 2-hydroxy fatty acid composition of R. obtusifolius was the high content of n-9 monoenoic 2-hydroxy fatty acids with 22 and 24 carbon-chain length. The levels of the Z and E stereoisomers of the 8-unsaturated long-chain bases were reliably distinguished from those in other plant families in ten species of Polygonaceae.
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http://dx.doi.org/10.1271/bbb.100802DOI Listing
September 2011

Nicotinate/nicotinamide mononucleotide adenyltransferase-mediated regulation of NAD biosynthesis protects guard cells from reactive oxygen species in ABA-mediated stomatal movement in Arabidopsis.

J Exp Bot 2010 Aug 30;61(13):3813-25. Epub 2010 Jun 30.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

Nicotinamide adenine dinucleotide (NAD) and its derivative nicotinamide adenine dinucleotide phosphate (NADP) are indispensable co-factors in broad-spectrum metabolic events for the maintenance of cellular homeostasis in all living organisms. In this study, the cellular expression levels of NAD biosynthesis genes in Arabidopsis were investigated. A very high expression of nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) was observed in the differentiated stomatal guard cells of the leaf surface. Transcriptional analysis confirmed that several genes in the biosynthesis pathway were also highly expressed in stomatal guard cells. In fact, NAD and NADP metabolisms were investigated during stomatal movement. Importantly, the generation of phytohormone ABA-induced reactive oxygen species, which acts as a signal for stomatal closure, was accompanied by markedly decreased levels of NAD. The ABA-induced oxidative stress caused stomatal cell death in the nmnat mutant. Furthermore, stomata partially lost their ability to close leading to drought susceptibility. The stomata were less responsive to opening cues as well. These results indicate that NAD biosynthesis is involved in protecting guard cells from ABA-induced local oxidative stress via the regulation of NMNAT activity. In this study, it is demonstrated that NMNAT is essential for the maintenance of NAD homeostasis enabling sustainable stomatal movement.
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http://dx.doi.org/10.1093/jxb/erq190DOI Listing
August 2010

Loss of calmodulin binding to Bax inhibitor-1 affects Pseudomonas-mediated hypersensitive response-associated cell death in Arabidopsis thaliana.

J Biol Chem 2009 Oct 12;284(41):27998-28003. Epub 2009 Aug 12.

Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032; Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003.

Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.
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http://dx.doi.org/10.1074/jbc.M109.037234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788852PMC
October 2009

Functional association of cell death suppressor, Arabidopsis Bax inhibitor-1, with fatty acid 2-hydroxylation through cytochrome b₅.

Plant J 2009 Apr 19;58(1):122-34. Epub 2009 Jan 19.

Institute of Molecular and Cellular Biosciences, the University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.

Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome b(5) (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.
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http://dx.doi.org/10.1111/j.1365-313X.2008.03765.xDOI Listing
April 2009

Cell death suppressor Arabidopsis bax inhibitor-1 is associated with calmodulin binding and ion homeostasis.

Plant Physiol 2007 Feb 1;143(2):650-60. Epub 2006 Dec 1.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.

Cell death suppressor Bax inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein, exists in a wide range of organisms. The split-ubiquitin system, overlay assay, and bimolecular fluorescence complementation analysis demonstrated that Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) interacted with calmodulin in yeast (Saccharomyces cerevisiae) and in plant cells. Furthermore, AtBI-1 failed to rescue yeast mutants lacking Ca2+ ATPase (Pmr1 or Spf1) from Bax-induced cell death. Pmr1 and Spf1, p-type ATPases localized at the inner membrane, are believed to be involved in transmembrane movement of calcium ions in yeast. Thus, the presence of intact Ca2+ ATPases was essential for AtBI-1-mediated cell death suppression in yeast. To investigate the effect of AtBI-1 on calcium homeostasis, we evaluated sensitivity against cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase in AtBI-1-overexpressing or knock-down transgenic Arabidopsis plants. These plants demonstrated altered CPA or ion stress sensitivity. Furthermore, AtBI-1-overexpressing cells demonstrated an attenuated rise in cytosolic calcium following CPA or H2O2 treatment, suggesting that AtBI-1 affects ion homeostasis in plant cell death regulation.
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http://dx.doi.org/10.1104/pp.106.090878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1803746PMC
February 2007
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