Masami Yonekura

Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings

Employing classical two‐dimensional electrophoresis (2‐DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2‐DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N‐terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N‐terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose‐1, 5‐bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post‐treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium‐binding protein, calreticulin, a novel ATP‐dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.

High-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: drastic reductions/fragmentation of ribulose-1,5-bisphosphate carboxylase/oxygenase and induction of stress-related proteins.

Employing high‐resolution two‐dimensional electrophoresis (2‐DE), we studied changes in the rice leaf protein patterns, in response to applied heavy and alkaline metals, important environmental pollutants in our surroundings. Drastic changes in 2‐DE protein patterns after treatment with copper, cadmium, and mercury, over control were found, including changes in the morphology of the leaf segments. Changes in the major leaf photosynthetic protein, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO, both suppression and fragmentation), and induction of proteins are reported. A total of 33 proteins, which were highly reproducible in repeated experiments, were visually identified as changed over the control, and taken for N‐terminal or internal amino acid sequencing. Among these, nine proteins were N‐terminally blocked, and six proteins could not be sequenced. Most of the proteins showed homology to RuBisCO protein, and some to defense/stress‐related proteins, like the pathogenesis related class 5 protein (OsPR5), the probenazole‐inducible protein (referred to as the OsPR10), superoxide dismutase, and the oxygen evolving protein. Results presented here strongly indicate a highly specific action of some of these metals in disturbing the photosynthetic machinery, as evidenced by prominent reductions/fragmentation of the major photosynthetic protein, RuBisCO, and resulting in stress.

Chitosan activates defense/stress response(s) in the leaves of Oryza sativa seedlings

Abstract In this study, we examined the response(s) of rice (Oryza sativa L. japonica-type cv. Nipponbare) seedling leaves treated with a fungal elicitor chitosan (CT). Small brownish necrotic spots (streaks) appeared in the interveinal regions on the leaf surface after treatment by 0.1% CT, over the cut control. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis further revealed strong induction of ascorbate peroxidase, and changes in “phytocystatins” (cysteine proteinase inhibitors). Using two-dimensional polyacrylamide gel electrophoresis, evidence is provided for the accumulation of two major classes of pathogenesis-related (PR) proteins, namely OsPR5 and OsPR10 in the leaves. In parallel, northern analyses revealed potent accumulation of the OsPR5 and OsPR10 mRNAs; a time- and dose-dependent expression, and a requirement for de novo protein synthesis was observed. Furthermore, CT-elicited changes were also accompanied by production of anti-fungal phytoalexins, the flavonoid sakuranetin and the diterpenoid lactone momilactone A, as determined by liquid chromatography-mass spectrometry/mass spectrometry analysis. Present results reveal for the first time the potency of CT in initiating multiple events linked with defense/stress response(s) in the leaves of whole rice plants.

Defense/stress responses elicited in rice seedlings exposed to the gaseous air pollutant sulfur dioxide

Abstract This report examines for the first time, the response of rice ( Oryza sativa japonica-type cv. Nipponbare) seedlings exposed to the air pollutant sulfur dioxide (SO 2 ). Distinctive reddish-brown necrotic spots and interveinal browning appeared on the leaf surface after exposure to SO 2 , over control, partly reminiscent of the hypersensitive reaction lesions. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting analysis revealed strong induction of ascorbate peroxidase(s), and changes in cysteine proteinase inhibitors (‘phytocystatins’)-like proteins. Employing classical two-dimensional electrophoresis (2-DE) followed by amino acid sequencing, we identified several changes in the 2-DE protein profiles of SO 2 -fumigated leaves. Most prominent changes in leaves were the induced accumulation of a pathogenesis-related (PR) class 5 (OsPR5) protein, three PR 10 class proteins (OsPR10s), a novel ATP-dependent CLP protease, and an unknown protein. Subsequent Northern analysis showed potent accumulation of OsPR5 and OsPR10 transcripts in leaves. Finally, mass spectrometry analysis revealed a strong production of phytoalexins, sakuranetin and momilactone A in SO 2 stressed leaves. Our results not only demonstrate the highly damaging effect of SO 2 , but also identify SO 2 triggered multiple events linked with defense/stress response in the leaves of rice seedlings.

Comprehensive Royal Jelly (RJ) Proteomics Using One- and Two-Dimensional Proteomics Platforms Reveals Novel RJ Proteins and Potential Phospho/Glycoproteins

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).

Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein

Royal jelly was fractionated by ion-exchange chromatography and a protein (DIII protein) that had growth stimulating activity to the U-937 human myeloid cell line was obtained. The molecular weight of the DIII protein was 58 kDa on SDS-PAGE. The growth stimulating activity of the DIII protein was shown to be relatively heat and pH stable.

Storage-dependent Degradation of 57-kDa Protein in Royal Jelly: a Possible Marker for Freshness

In order to find a maker for freshness of royal jelly (RJ), the composition change of RJ during storage was investigated. The contents of 10-hydroxy-2-decenoic acid, a bioactive component of RJ, and several vitamins did not change during storage at 40°C for 7 days. However, a specific protein, designated royal jelly protein-1 (RJP-1), was gradually degraded during storage under various conditions (from 4°C to 50°C for up to 7 days). The specific degradation of RJP-1 was proportional to storage temperature and storage period. RJP-1 was purified to homogeneity and characterized as a monomeric glycoprotein with a molecular mass of 57 kDa. These results suggest that 57-kDa protein in RJ can be used as a marker for freshness of RJ, reflecting the conditions under which RJ has been stored.

Light-dependent induction of OsPR10 in rice (Oryza sativa L.) seedlings by the global stress signaling molecule jasmonic acid and protein phosphatase 2A inhibitors

Abstract The OsPR10 gene was first shown to be weakly induced only 3 days after treatment with a herbicide, probenazole (N. Midoh, M. Iwata, Cloning and characterization of a probenazole-inducible gene for an intracellular pathogenesis-related protein in rice, Plant Cell Physiol. 37 (1996) 9–18), and remains uncharacterized till date. We provide first evidence for the induction of OsPR10 by the global signaling molecule, jasmonic acid (JA), and two protein phosphatase (PP) 2A inhibitors, cantharidin (CN) and endothall (EN). These inductions were light- and time-dependent, and also specific to the dose of applied JA, CN and EN. OsPR10 was not responsive to cut, and was expressed mainly in leaves and leaf sheaths, but not in roots. Furthermore, OsPR10 induction was blocked completely by cycloheximide, indicating requirement of de novo protein synthesis in its induction. Two signal transducers, salicylic acid (SA) and abscisic acid (ABA) induced OsPR10 , and a cytokinin, kinetin (KN) also strongly up-regulated its expression, under light. Simultaneously applied staurosporine, a serine/threonine protein kinase inhibitor, strongly enhanced the JA inducible OsPR10 expression. The inducible nature of OsPR10 was affected significantly by a combination of applied JA, with varying concentrations of SA, ABA and KN. Induction of OsPR10 was followed by the appearance of necrotic lesions/coalescing necrotic regions clearly visible at 48 h under light, with CN and EN, but not with JA at a similar time period. Finally, using two-dimensional polyacrylamide gel electrophoresis, we identified a new member of the OsPR10 protein family, the OsPR10-3 protein. This represents the first systematic report in rice demonstrating that the inducible OsPR10 expression is light dependent, and may be regulated via a kinase-signaling cascade.

350-kDa Royal Jelly Glycoprotein (Apisin), Which Stimulates Proliferation of Human Monocytes, Bears the β1-3Galactosylated N-Glycan: Analysis of the N-Glycosylation Site

While doing a structural analysis of minor component N-glycans linked to 350-kDa royal jelly glycoprotein (RJGP), which stimulates the proliferation of human monocytes, we found that a Galβ1-3GlcNAcβ1-4Man unit occurs on the insect glycoprotein. The structure of the fluorescence-labeled N-glycan was analyzed by sugar component analysis, IS-MS, and 1H-NMR. The structural analysis showed that the 350-kDa RJGP bears Galβ1-3GlcNAcβ1-4(GlcNAcβ1-2)Manα1-3 (Manα1-3Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc, suggesting this insect glycoprotein is one of the substrates for both β1-3 galactosyl and β1-4 N-acetylglucosamininyl transferases. To our knowledge, this is the first report that succeeded in identifing an insect glycoprotein bearing the β1-3 galactosylated N-glycan.

Naringenin 7-O-methyltransferase involved in the biosynthesis of the flavanone phytoalexin sakuranetin from rice (Oryza sativa L.).

An inducible S-adenosyl-L-methionine:naringenin 7-O-methyltransferase (NOMT) catalyzing the methylation of naringenin to sakuranetin, a major rice phytoalexin was purified approximately 985-fold from ultraviolet (UV)-irradiated rice leaves. The enzyme is not found in healthy tissues and was purified to a nearly homogeneous preparation in one step using adenosine-agarose affinity chromatography, with 1 g rice leaves (UV-irradiated) as starting material. Gel filtration chromatography resulted in an almost pure enzyme, as evidenced by a major band migrating to a position corresponding to a molecular mass of approximately 41 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified NOMT was strongly inhibited by Mn(2+), Ni(2+), Cu(2+), Zn(2+), Hg(2+), and Cd(2+), and to a low degree by Co(2+), Mg(2+), Ba(2+), Ca(2+) and ethylenediamine tetraacetic acid. The amino acid sequence of a NOMT cyanogen bromide (CNBr)-cleavage peptide was highly homologous to that of a caffeic acid 3-O-methyltransferase from maize, and about 70% of the amino acid sequence was obtained after sequencing the peptides generated by CNBr and/or formic acid hydrolysis. NOMT was also shown to be induced in a time-dependent manner, and purified from rice leaves treated with jasmonic acid and copper chloride.