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Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.

Suspension-cultured cells of rice secrete alpha-amylase into the culture medium. It has been shown that the mature form of the alpha-amylase contains xylose-bearing N-linked oligosaccharide: (formula; see text) We demonstrate that suspension-cultured cells of rice secrete alpha-amylase containing oligomannose-type oligosaccharides in the presence of 1-deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, alpha-amylase purified from germinated rice seedlings contains several kinds of oligomannose-type and N-acetyllactosamine-type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.

Loop L5 of kinesin is located near the ATPase site, in common with kinesins of various animal species. The rice plant-specific kinesin K16 also has a corresponding loop that is slightly shorter than that of mouse brain kinesin. The present study was designed to monitor conformational changes in loop L5 during ATP hydrolysis. For this purpose, we introduced one reactive cysteine into the L5 of rice kinesin and modified it with fluorescent probes. The cysteine in L5 was labeled with a fluorescent probe 2-(4'(iodoacetamide) anilino-naphthalene-6-sulufonic acid sodium salt) [IAANS]. IAANS was incorporated into L5 at an almost equimolar ratio in the absence of nucleotides. In contrast, the incorporated amount was reduced to 0.62 and 0.32 mol IAANS/mol motor domain in the presence of ATP and ADP, respectively. Upon nucleotide addition, the fluorescent intensity of IAANS incorporated into L5 was significantly reduced to 63% and 51% for ATP and ADP, respectively. These results suggest that L5 of rice kinesin significantly changes its conformation during ATP hydrolysis.

A fluorescent photoreactive ATP derivative, 2'(3')-O-(4-benzoylbenzoyl)-1,N(6)-etheno-ATP (Bz(2)-epsilonATP), was synthesized and reacted with the rice kinesin K16 motor domain (K16MD). In the presence of ADP or ATP, UV irradiation of the K16MD solution containing Bz(2)-epsilonATP resulted in a new 100 kDa band, which was an intermolecular cross-linked product of motor domains. In contrast, no cross-linking was observed in the absence of nucleotides. For the motor domain of mouse brain kinesin and skeletal muscle myosin subfragment-1, no such intermolecular photo cross-linking by Bz(2)-epsilonATP was observed. Our results indicate that Bz(2)-epsilonATP acts unusually as a photoreactive crosslinker to detect conformational changes in K16MD induced by nucleotide binding resulting in the formation of dimers.

This paper presents a 5-axis motion sensor which can detect 3-axis acceleration and 2-axis angular rate using resonant electrostatic driving and non-resonant capacitive detection mode. This sensor is fabricated by silicon on insulator (SOI) bulk-micromachining, and is vacuum sealed by anodic bonding and activation of the non-evaporated getters (NEG). The sensor chip size is 5.0 × 5.0 × 1.7 mm 3 . By applying electrostatic reference vibration of a proof mass along Z -axis at resonant frequency of 3.42 kHz, 3-axis acceleration and 2-axis angular rate can be detected capacitively at non-resonant detection mode. Measured acceleration sensitivities along X -, Y - and Z -axis are 1.9, 1.1 and 4.7 V/G, respectively. Angular rate sensitivities around X - and Y -axis are 6.7 and 12 mV/(° s), respectively.

To determine the role of alpha-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed expression or overexpression of alpha-amylase I-1. In the lines with suppressed expression of alpha-amylase I-1 at both the mRNA and protein levels, seed germination and seedling growth were markedly delayed in comparison with those in the wild-type plants. However, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in the plants overexpressing alpha-amylase I-1. In immunocytochemical analysis with specific anti-alpha-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts and extracellular space in young leaf cells. We further examined the expression and targeting of alpha-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of the expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of alpha-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, we concluded that alpha-amylase I-1 targets the chloroplasts through the endoplasmic reticulum-Golgi system and plays a significant role in the starch degradation in rice leaves.

Fecha de presentación internacional: 15.07.2003. - Titulares: Consejo Superior de Investigaciones Científicas (CSIC). - Universidad Pública de Navarra. - Niigata University., NPPase is an enzyme that catalyses the hydrolysis of a wide range of small molecules with phosphodiester and phosphosulphate bonds, in particular ADPG (adenosine diphosphate glucose) and APS (adenosine 5'-phosphosulphate). The enzyme obtained from plant extracts is used in assay devices for determining levels of nucleoside diphosphate sugars, based either on the sugar-1-phosphate released, or on the nucleoside monophosphate, both of which are products formed by the reaction catalysed by NPPase, as well as the detection of sulphonucleotides such as 3'-phosphoadenosine 5'-phosphosulphate (PAPS) and APS. The amino acid sequence of the enzyme is also described, as well as the nucleotide sequence of a complete cDNA and another incomplete cDNA. Finally, it describes the production of transgenic plants that overexpress NPPase and that have a high content of sugars, low content of starch and cell-wall polysaccharides and high resistance to high concentrations of salts and high temperature.

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori . P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K d constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.

In this study, we present data showing that two members of the GRAS family of genes from rice, CIGR1 and CIGR2 (chitin-inducible gibberellin-responsive), inducible by the potent elicitor N -acetylchitooligosaccharide (GN), are rapidly induced by exogenous gibberellins. The pattern of mRNA accumulation was dependent on the dose and biological activity of the gibberellins, suggesting that the induction of the genes by gibberellin is mediated by a biological receptor capable of specific recognition and signal transduction upon perception of the phytoactive compounds. Further pharmacological analysis revealed that the CIGR1 and CIGR2 mRNA accumulation by treatment with gibberellin is dependent upon protein phosphorylation/dephosphorylation events. In rice calli derived from slender rice 1, a constitutive gibberellin-responsive mutant, or d1, a mutant deficient in the alpha -subunit of the heterotrimeric G-protein, CIGR1 and CIGR2 were induced by a GN elicitor, yet not by gibberellin. Neither gibberellin nor GN showed related activities in defense or development, respectively. These results strongly suggested that the signal transduction cascade from gibberellin is independent of that from GN, and further implied that CIGR1 and CIGR2 have dual, distinct roles in defense and development.

Phosphoinositol kinase was purified from suspension-cultured rice (Oryza sativa L.) cells. The apparent molecular mass of the rice enzyme was estimated to be 58 kDa by SDS-PAGE and 234 kDa by Toyop...

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

Despite recent progress in sequencing the complete genome of rice ( Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60-98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.

Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.

(1993). Purification of β-Glucan Synthase II from Suspension-cultured Rice Cells. Bioscience, Biotechnology, and Biochemistry: Vol. 57, No. 4, pp. 682-684.

Plutella xylostella (PX) that were 130000-fold more resistant to Cry1Ac were selected from the susceptible strain and maintained in the laboratory. The LC50 of the susceptible strain (PXS) was 0.38 microg toxin/g diet, whereas that of the resistant strain (PXR) was 4800 microg toxin/g diet. Brush border membrane vesicles (BBMV) were prepared from both PXS and PXR. In ligand blot analysis, Cry1Ac bound to a 120-kDa protein of BBMV; however, the intensity of the band was almost equal in both strains of insect. Hence, we analyzed the lipid components of BBMV from PXS and PXR. BBMV lipids were fractionated into non-polar lipid, phospholipid, neutral glycolipid and acidic glycolipid. Neutral glycolipid content was substantially lower in the BBMV of PXR than of PXS. The same trend was observed when lipids were extracted from whole midgut instead of BBMV. Thin layer chromatography of midgut neutral glycolipids revealed the presence of more than seven components. Among the midgut neutral glycolipids, a possible hexasaccharylceramide and a possible trisaccharylceramide of PXR were less than half the level found in PXS. The other lipid fractions in PXR and PXS were similar to each other.

alpha-Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and Km for pNP-alpha-Man hydrolysis were pH 4.3-4.5 and 1.04 mM, respectively. The enzyme digested mannobioses such as Manalpha-1,2Man, Manalpha-1,6Man, Manalpha-1,3Man but Manalpha-1,4Man. Zn2+ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose-type sugar chains by two-dimensional mapping using NH2-P and C18 silica columns. They were Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2 and Man5GlcNAc2. All these oligosaccharides were digested by the purified alpha-mannosidase, and Man-GlcNAc2 and mannose were formed. Glycopeptides, having these high mannose-type sugar chains, could also be digested by the alpha-mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the alpha-mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre-treated with the alpha-mannosidase, was rapidly and completely digested by V8 protease.

The novel rice plant specific kinesin K16 has several unique enzymatic characteristics comparing with conventional kinesin. The most interesting property is that the ADP-free K16 motor domain is very stable, contrast to conventional kinesin that is very labile in ADP-free state. Recently, we have determined crystal structure of the novel rice kinesin K16 motor domain (K16MD) in complex with MgADP at 2.5A resolution. The overall structure of the K16MD is similar to that of conventional kinesin motor domains, as expected from the high similarity of amino acid sequence (43.2 %). However, the neck-linker of the ADP bound K16 motor domain showed an ordered conformation in a position quite different from that observed in conventional kinesin, which may reflect the unique enzymatic characteristics of rice kinesin K16. In the present study, we analyzed the inner structure of the K16 motor domain in detail and compared the structure with Eg5 and other related kinesins. It has been revealed that K16MD does not have interaction of amino acids side chains, which stabilizes the docking conformation of neck-linker. We have also analyzed the conformation of neck-linker in the solution using the K16 by FRET. Motor domain mutant G220C chimera protein fused with GFP at the neck-linker has been prepared and labeled IAE-DANS. The FRET efficiencies between the fluorescent probe DANS at C220 and GFP in the presence and absence of nucleotides were analyzed.

Characterization was done on the sugar chains of glutelin, a major storage protein in rice seeds. The basic subunits of glutelin were shown to be glycoproteins by staining with PAS and the lectins concanavalin A (Con A) and peanut agglutinin (PNA). The binding of PNA to the basic subunits was substantially reduced by treatment of the glutelin with NaIO4, NaOH, or O-glycanase. The sugar chains of the subunits, obtained by hydrazinolysis and O-glycanase, were pyridylaminated and subjected to 2-dimensional HPLC analysis using C(18) and acrylamide-derivatized columns. It was found that the Galbeta-1,3GalNAc disaccharide, which was previously identified as a core 1 structure of mucin-type sugar chains, is conjugated to the glutelin subunits. Furthermore, amino acid sequencing of an 11-kDa peptide of the subunits, recognized by both PNA and Con A, suggested that both N-linked and O-linked glycosylation occurs in the carboxy-terminal region of these subunits.

It is widely accepted that the smooth muscle contractile apparatus is primarily regulated by reversible phosphorylation and dephosphorylation of the 20 kDa light chain of myosin (Hartshorne, 1987). The enzyme which is responsible for the phosphorylation is the Ca2+/calmodulin dependent myosin light chain kinase (MLCK) and this is the key enzyme to confer Ca2+ sensitivity to the smooth muscle contractile apparatus (Hartshorne, 1987). Another critical component of the phosphorylation hypothesis is the dephosphorylation of the myosin light chain which is catalyzed by a protein phosphatase. Several protein phosphatases have been purified from the soluble fraction of smooth muscle cells. Pato and collaborators (Pato and Adelstein, 1983a, 1983b; Pato and Kerc, 1985) reported four protein phosphatases from gizzard smooth muscle soluble fraction and three of which (SMP I, II and IV) have been purified. Among them, SMP III and IV can dephosphorylate intact myosin. The purified SMP-IV has a molecular weight of 150 kDa and is composed of two subunits of MW 58 kDa and 40 kDa. SMP-IV preferentially dephosphory-lates the α-subunit of Phosphorylase kinase (a known substrate of Type 1 phosphatases) but is insensitive to Inhibitor-2 which is known to inhibit Type 1 phosphatases specifically. Phosphatases have also been purified from the soluble fraction of aortic smooth muscle (Werth et al., 1982; DiSalvo and Gifford 1983; Erdodi et al., 1989). Erdodi et al. (1989) recently purified two types of phosphatases from dog aortic smooth muscle which dephosphorylate native actomyosin. A 260 kDa phosphatase was similar in its properties to the Type 2A phosphatases. On the other hand, a 150 kDa phosphatase dephosphorylated the α-subunit of Phosphorylase kinase although it was not inhibited by Inhibitor 1 or Inhibitor 2, which are known to be specific inhibitors of Type 1 protein phosphatase. These reports suggest that smooth muscle phosphatases purified from the soluble fraction, which dephosphorylate myosin, may be different from both Type 1 and Type 2 phosphatases. However, it is still obscure whether or not these phosphatases are responsible for the regulation of contraction in vivo. Furthermore, it is not known if phosphatase activity is physiologically regulated, and this is one of the most important areas to be elucidated.

We report the isolation of Golgi membranes from rice cells by linear glycerol density gradient centrifugation and the characterization of the glycoproteins in the Golgi membrane obtained in this way by lectin-binding analysis

Substitution of CO in closo-[Cp*2Fe3S2(CO)3] (1) by PPh3 was accelerated upon photolysis or by treatment with reductant or oxidant to give mono(PPh3)-substituted clusters. In the redoxinduced reaction, [1]− and [1]+ were proposed as labile intermediates to substitution of CO.