Biomedical subjects

Setsuko Komatsu

Publications and source records attributed to Setsuko Komatsu.

At least 55 records · Page 3Linked to original sources

PubMed2004

Chitinase induced by jasmonic acid, methyl jasmonate, ethylene and protein phosphatase inhibitors in rice.

Chitinase is a pathogenesis-related protein that hydrolyzes chitin, a major component of fungal cell walls. Two-week-old rice seedling leaf, leaf sheath and root tissues responded to an exogenous treatment by jasmonic acid (JA) with induction of the chitinases as determined by immunoblot analysis using an anti-endochitinase antibody. Induced accumulation of these chitinases was observed within 24 to 48 h in the leaf sheaths, leaves and roots. Besides, ethylene generator ethephon and abiotic stressor copper could also induce chitinases accumulation among various plant hormones and stress agents examined. Cycloheximide effectively blocked their accumulation by JA, suggesting that de novo protein synthesis is required. Partial blockage of the induced accumulation of chitinases by NADPH oxidase inhibitor and free radical scavengers suggested involvement of reactive oxygen species. Moreover, induced accumulation of these chitinases also by methyl jasmonate and certain protein phosphatase inhibitors indicated their potential importance and wider role in rice seedlings.

Acetates↗

PubMed2004

A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway.

Plant roots have important roles not only in absorption of water and nutrients, but also in stress tolerance such as desiccation, salt, and low temperature. We have investigated stress-response proteins from rice roots using 2-dimensional polyacrylamide-gel electrophoresis and found a rice protein, RO-292, which was induced specifically in roots when 2-week-old rice seedlings were subjected to salt and drought stress. The full-length RO-292 cDNA was cloned, and was determined to encode a protein of 160 amino acid residues (16.9 kDa, pI 4.74). The deduced amino acid sequence showed high similarity to known rice PR10 proteins, OsPR10a/PBZ1 and OsPR10b. RO-292 mRNA accumulated rapidly upon drought, NaCl, jasmonic acid and probenazole, but not by exposure to low temperature or by abscisic acid and salicylic acid. The RO-292 gene was also up-regulated by infection with rice blast fungus. Interestingly, induction was observed almost exclusively in roots, thus we named the gene RSOsPR10 (root specific rice PR10). The present results indicate that RSOsPR10 is a novel rice PR10 protein, which is rapidly induced in roots by salt, drought stresses and blast fungus infection possibly through activation of the jasmonic acid signaling pathway, but not the abscisic acid and salicylic acid signaling pathway.

Abscisic Acid↗

PubMed2004

A novel interaction between calreticulin and ubiquitin-like nuclear protein in rice.

Calreticulin (CRT), a major Ca2+ -sequestering protein, has been implicated in a variety of cellular functions such as Ca2+ storage, signaling and chaperone activity within the cytoplasm and endoplasmic reticulum. To investigate the biological role of CRT in rice, 21 partial cDNAs, encoding proteins that interacted with rice CRT in a yeast two-hybrid interaction-cloning system, were characterized and the nucleotide sequences were found to be identical to each other. A full-length cDNA of 3.5 kb, obtained from rice genomic sequence data and 5' RACE, codes for a novel protein of 966 amino acid residues and was designated as CRTintP (CRT interacting protein). Primary sequence analysis of CRTintP showed no sequence homology with the known functional proteins; however, a potential ubiquitin-like domain at the N-terminal together with a putative leucine zipper, a nuclear localization signal and several sites for serine/threonine kinases were evident. Cellular localization of CRTintP demonstrated its role in directing green fluorescent protein to the nucleus in onion epidermal cells. Northern and immunoblot analysis showed increased expression of CRT and CRTintP in response to cold stress. Co-immunoprecipitation using anti-CRT antibodies confirmed the existence of the CRT-CRTintP complex in vivo in the stressed leaf tissue, suggesting their potential role in regulating stress response.

Active Transport, Cell Nucleus↗

PubMed2004

Characterization of NADPH-dependent oxidoreductase induced by auxin in rice.

An NADPH-dependent oxidoreductase (NADPH-DO) with an isoelectric point of 5.2 and a molecular mass of 35 kDa was isolated from suspension-cultured rice cells. NADPH-DO was inducible by auxin and gibberellin. NADPH-DO mRNA was expressed in roots and leaf sheaths of rice seedlings, but not in leaf blades. The levels of NADPH-DO mRNA and protein in suspension-cultured cells were increased by auxin; they were further raised by the application of zinc. In contrast, NADPH-DO mRNA and protein accumulation in intact roots was stimulated by auxin, but in this case the stimulation was inhibited by zinc. Auxin-induced callus growth and root formation in intact rice plants were further enhanced by zinc. These results indicate that high levels of NADPH-DO are necessary for auxin- and zinc-induced callus formation, and imply that zinc plays a role in the regulation of NADPH-DO induction by auxin.

Journal Article↗

PubMed2003

Cell-cycle-related variation in proteins in suspension-cultured rice cells.

To understand the cell cycle process in plants, we searched for proteins that quantitatively change during the cell cycle in suspension-cultured rice ( Oryza sativa L.) cells. The proteins were analyzed by a two-dimensional polyacrylamide gel electrophoresis image-analysis system. We detected 11 proteins that quantitatively changed during the cell cycle, among which beta-tubulins and a calreticulin-like protein were identified. The amounts of beta-tubulin proteins were low in the M phase and high in the G1 phase. In contrast, mRNAs for two of the three types of beta-tubulin were high in the M phase of the cell cycle. The addition of protease inhibitors MG132 or E64d to the cells decreased the beta-tubulin proteins during 24 h, suggesting that beta-tubulin proteins are degraded in vivo by proteases other than those whose activities are inhibited by MG132 or E64d.

Amino Acid Sequence↗

PubMed2003

A novel brassinolide-enhanced gene identified by cDNA microarray is involved in the growth of rice.

Brassinosteroids (BRs) are growth-promoting natural substances required for normal plant growth and development. To understand the molecular mechanism of BR action, a cDNA microarray containing 1265 rice genes was analyzed for expression differences in rice lamina joint treated with brassinolide (BL). A novel BL-enhanced gene, designated OsBLE2, was identified and cloned. The full-length cDNA is 3243 bp long, encoding a predicted polypeptide of 761 amino acid residues and nine possible transmembrane regions. OsBLE2 expression was most responsive to BL in the lamina joint and leaf sheath in rice seedlings. Besides, auxin and gibberellins also increased its expression. OsBLE2 expressed more, as revealed by in situ hybridization, in vascular bundles and root primordia, where the cells are actively undergoing division, elongation, and differentiation. Transgenic rice expressing antisense OsBLE2 exhibits various degrees of repressed growth. BL could not enhance its expression in transgenic rice expressing antisense BRI1, a BR receptor, indicating that BR signaling to the enhanced expression of OsBLE2 is through BRI1 . BL effect in the d1 mutant rice was much weaker than that in its wild-type control, indicating that heterotrimeric G protein may be a component of BRs signaling. These results suggest that OsBLE2 is involved in BL-regulated growth and development processes in rice.

Amino Acid Sequence↗

PubMed2003

Rice proteomics: a step toward functional analysis of the rice genome.

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.

Agrobacterium tumefaciens↗

PubMed2003

Expression analyses of beta-tubulin isotype genes in rice.

Microtubules play important roles in many cellular processes, such as cell division and cell elongation in plants. beta-tubulins, which are the basic components of microtubules, are encoded by multigene family in eukaryotes and their nucleotide sequences are highly conserved in protein coding regions. A homology search within the rice expressed sequence tag database identified at least eight beta-tubulin (OsTUB) isotypes including three novel OsTUB genes. Northern analysis using specific probes to 3'-UTR of OsTUB isotypes showed differential and tissue-specific expression. Seven out of eight OsTUB genes dominantly expressed in leaf sheath, while OsTUB8 was preferentially expressed in anther including mature pollens. The existence of anther-specific beta-tubulin suggests its unique role in the formation of microtubules during the anther and pollen development or pollen tube growth. Furthermore, transcripts of OsTUB5, 6 and 7 genes were significantly enhanced by gibberellin but all eight OsTUB genes were repressed by abscisic acid. Our results imply that OsTUB genes are differentially regulated by developmental and hormonal signals and different OsTUB isotypes might play special role in the growth and development of specific organs in rice.

Amino Acid Sequence↗

PubMed2003

Characterization of proteins responsive to gibberellin in the leaf-sheath of rice (Oryza sativa L.) seedling using proteome analysis.

In plants gibberellins (GAs) are responsible for triggering stem or internodal elongation. To comprehend the molecular basis of internodal elongation in rice, a proteomics approach using differentially displayed proteins on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was carried out to identify the proteins expressed during the GA controlled leaf-sheath elongation response. Out of 352 protein spots detected on 2-D PAGE, 32 proteins showed modulation in the expression levels in GA3-treated leaf-sheath for 48 h as compared to control. These proteins were analyzed using protein sequencer and/or mass spectrometry in conjunction with the protein database to assign putative identities. The twin protein spots (LS079 and LS083), identified as calreticulin, showed different isoelectric points and expression level in GA3-treated leaf-sheath. The expression level of LS083 (pI 4.0) was down-regulated as compared to the up-regulation of LS079 (pI 4.3). In the presence of GA3 and growth inhibitor, uniconazole and abscisic acid, respectively, no elongation in leaf-sheath occurred and calreticulin did not shift from LS083 to LS079. Over-expression of calreticulin in rice inhibited the callus regeneration and seedling growth. These results suggest that calreticulin is an important component in the GA signaling pathway that regulates rice seedling leaf-sheath elongation.

Amino Acid Sequence↗

PubMed2003

Characterization of calreticulin as a phosphoprotein interacting with cold-induced protein kinase in rice.

Calreticulin is an abundant endo/sarcoplasmic reticulum Ca2+-binding protein. To investigate whether calreticulin (CRO1) is involved in the cold-stress response in rice, a transgenic plant was constructed. The transcriptional level was decreased within 30 min and recovered within 2 h of a cold treatment. The calreticulin protein was shifted from a soluble fraction to an insoluble fraction by cold stress. Endogenous abscisic acid (ABA) is an important factor in cold response, and the synthesis of ABA was strongly induced in CRO1-sense transgenic rice, the same as in cold-sensitive rice. The phosphorylation of calreticulin increased after cold treatment. Over-expression of calreticulin enhanced the activities of 47 kDa Ca2+-dependent protein kinase (CDPK) that had been induced by cold treatment. The 47-kDa CDPK activity increases more in the cold sensitive variety IR36 and the sense transgenic rice than it does in other varieties. The synthesis of ABA, phosphorylation of calreticulin and 47-kDa CDPK activity induced in sense transgenic rice were the same as in cold-sensitive rice and the phosphorylation of antisense transgenic rice was similar to that of cold-tolerant rice. These results suggest that the calreticulin is involved in the signaling pathway leading to response to cold stress.

Calreticulin↗

PubMed2003

A proteomics approach to investigating promotive effects of brassinolide on lamina inclination and root growth in rice seedlings.

The application of brassinolide (BL) to the lamina joint region of rice (Oryza sativa L., cv. Nipponbare) seedlings caused marked bending of laminae, and BL influenced rice root growth under intact conditions. A remarkable increase in lamina inclination at BL 1 microM and an evident increase in root elongation at BL 0.01 microM were observed compared with the control. A total of 786 and 508 proteins extracted from the lamina joint and root, respectively, were detected in images of two-dimensional polyacrylamide gel electrophoresis with isoelectric focusing or immobilized pH gradient tube gel, and 21 proteins changed by BL treatment were analyzed using a gas-phase protein sequencer and mass spectrometer. Proteins related to photosynthesis and stress tolerance were mainly found in the lamina joint and root, respectively. Advanced degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) large subunit was caused by both BL and ethylene. Calmodulin, which decreased in roots treated with BL, increased as a result of ethephon treatment. Proteins that showed homology to glutathione S-transferase (EC 2.5.1.18) increased with BL application in both the lamina joint and root. The results suggest that the physiological functions of these proteins detected using powerful proteome analysis are implicated in rice lamina inclination and/or root elongation triggered by BL.

Brassinosteroids↗

PubMed2003

Modulation of vacuolar H+ -pumps and aquaporin by phytohormones in rice seedling leaf sheaths.

The effects of exogenously applied gibberellic acid (GA(3)) or brassinolide (BL) on the H(+)-pumps and aquaporin in the vacuolar membrane of rice seedling leaf sheath were investigated. Antibodies against mung bean H(+)-PPase, the B subunit of V-ATPase, and radish tonoplast intrinsic protein (TIP) cross-reacted with the vacuolar membrane proteins of rice seedling leaf sheath. The results of immunoblot analysis showed that the amounts of H(+)-PPase and V-ATPase were retained at a high level for two days in the presence of GA(3), although the level gradually decreased without phytohormones, indicating that GA(3) has a promotive effect on the activities of vacuolar H(+)-pumps. However, the levels of V-ATPase and H(+)-PPase were increased on day 2 and then decreased when treated with BL. There were no visible differences in the level of TIP upon treatment with GA(3) and BL. In comparison with the water control, the activity of H(+)-PPase treated with BL was also retained at a relatively high level, suggesting that BL has a stimulative effect on the activities of H(+)-PPase. These results indicate that GA(3) and BL might be involved in the regulation of the quantity and activities of plant vacuolar H(+)-pumps.

Aquaporins↗

PubMed2002

Involvement of a Ca(2+)-dependent protein kinase component downstream to the gibberellin-binding phosphoprotein, RuBisCO activase, in rice.

Previously, we reported the identification of a gibberellin (GA)-binding protein in rice using ligand binding assay that was homologous to RuBisCO activase (Komatsu et al., FEBS Lett. 384, 167-171, 1996). Here, we provide an evidence for the involvement of protein kinases components downstream to the GA-binding phosphoprotein, RuBisCO activase in rice. Ca(2+)-dependent protein kinase activity was studied in subcellular fractions of leaf sheath from transgenic rice containing sense and antisense constructs of RuBisCO activase. In-gel kinase assay using histone III-S as a substrate showed constitutive induction of a 46- and 48-kDa Ca(2+)-dependent protein kinase activity in the sense transgenic plants. Kinase activities of these proteins were significantly reduced in the presence of uniconazole, a potent GA biosynthesis inhibitor, but one of them was strongly promoted by GA(3) treatment in transgenic plants carrying a smaller subunit of RuBisCO activase (OsrcaA1) compared to the larger subunit OsrcaA2. Also, in vitro phosphorylation studies using two-dimensional polyacrylamide gel showed changes in the degree of phosphorylation of several proteins in OsrcaA1- and OsrcaA2-sense transgenic rice. These studies suggest the presence of two independent cytosolic Ca(2+)-dependent protein kinase signaling components downstream to the GA-binding protein in rice suggesting their role in GA signaling.

Calcium↗

PubMed2002

A proteomic analysis of leaf sheaths from rice.

The proteins extracted from the leaf sheaths of rice seedlings were separated by 2-D PAGE, and analyzed by Edman sequencing and mass spectrometry, followed by database searching. Image analysis revealed 352 protein spots on 2-D PAGE after staining with Coomassie Brilliant Blue. The amino acid sequences of 44 of 84 proteins were determined; for 31 of these proteins, a clear function could be assigned, whereas for 12 proteins, no function could be assigned. Forty proteins did not yield amino acid sequence information, because they were N-terminally blocked, or the obtained sequences were too short and/or did not give unambiguous results. Fifty-nine proteins were analyzed by mass spectrometry; all of these proteins were identified by matching to the protein database. The amino acid sequences of 19 of 27 proteins analyzed by mass spectrometry were similar to the results of Edman sequencing. These results suggest that 2-D PAGE combined with Edman sequencing and mass spectrometry analysis can be effectively used to identify plant proteins.

Amino Acid Sequence↗

PubMed2002

Aquaporin isoforms responsive to salt and water stresses and phytohormones in radish seedlings.

Aquaporins in the plasma and vacuolar membranes play a key role in the intercellular and intracellular water transport in plants. First, we quantitated the absolute amounts for mRNAs of eight aquaporin isoforms in hypocotyls of radish seedlings. Then, we investigated the effects of salt and water stresses (150 mM NaCl, 300 mM mannitol and 20% polyethylene glycol) and phytohormones (gibberellic acid, abscisic acid and brassinolide) on the mRNA and protein levels of aquaporins in the plasma membrane (RsPIP1-1, 1-2, 1-3, 2-1, 2-2 and 2-3) and vacuolar membrane (RsTIP1-1 and 2-1). The mRNA and protein levels of RsTIP1-1, RsTIP2-1, RsPIP1-1, RsPIP1-2 and RsPIP1-3 were comparatively constant. In contrast, mannitol treatment altered the mRNA levels of RsPIP2-1, RsPIP2-2 and RsPIP2-3 in roots. Immunoblot analysis showed that the RsPIP2-1 protein level was increased by NaCl treatment and decreased by treatment with mannitol and polyethylene glycol. Gibberellic acid and abscisic acid suppressed the levels of mRNAs of RsPIP2-1, RsPIP2-2 and RsPIP2-3 and the protein level of RsPIP2-1 in roots. On the other hand, the protein levels of RsPIP1-group members and RsTIPs were scarcely changed by these phytohormones. In the case of hypocotyls and cotyledons, the mRNA and protein levels of eight isoforms were not markedly affected by any treatment. These results indicate that aquaporins in the root, especially the RsPIP2 group, may be a stress responsive type of aquaporin at least in the protein level.

Abscisic Acid↗

PubMedDate not supplied

A proteomic approach to analyze auxin- and zinc-responsive protein in rice.

Auxin and zinc are involved in callus and root formation in rice. However, details of the mechanism underlying this process and functional relation between zinc and auxin are unclear. In this study, proteins induced by auxin and zinc in rice were analyzed by a proteomic approach. Root formation on rice seedlings was promoted by 0.45 microM 2,4-D treatment and was further promoted by addition of 260 microM Zn. Microscopic observation revealed that the number of root primodia formed was significantly increased in 2,4-D- and Zn-treated seedlings than that of the control. A total of seven proteins, as analyzed by 2D-PAGE, were increased, and one protein was decreased by 2,4-D and Zn treatment. Expression of elongation factor-1beta' (EF-1beta') both at transcriptional and translational levels was particular abundant in callus and basal parts of young seedlings, and the accumulation of EF-1beta' was consistent with root formation induced by 2,4-D and Zn. Results indicate that higher level of EF-1beta' expression is necessary for auxin- and zinc-induced root formation in rice.

2,4-Dichlorophenoxyacetic Acid↗

PubMedDate not supplied

Characterization of vacuolar membrane proteins changed in rice root treated with gibberellin.

Rice vacuolar membrane proteins changed by gibberellin (GA) were analyzed using a proteome approach. Vacuolar membrane fractions were isolated using a discontinuous sucrose/sorbitol system and 10 proteins increased in vacuolar membrane of the root, treated with GA(3) as compared with control. Fructose-1,6-bisphosphate aldolase C-1 and vacuolar H(+)-ATPase (V-ATPase) increased in root vacuolar membrane by GA(3) interacted in rice roots. It suggests that aldolase C-1 regulates the V-ATPase mediated control of cell elongation that determines root growth.

Adenosine Triphosphatases↗

PubMedDate not supplied

Proteome approach to characterize the methylmalonate-semialdehyde dehydrogenase that is regulated by gibberellin.

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths ofslr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA(3). Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice.

Electrophoresis, Gel, Two-Dimensional↗