Photochemistry of <i>Acetabularia</i> RhodopsinII from a Marine Plant, <i>Acetabularia acetabulum</i>

Kikukawa, Takashi,Shimono, Kazumi,Tamogami, Jun,Miyauchi, Seiji,Kim, So Young,Kimura-Someya, Tomomi,Shirouzu, Mikako,Jung, Kwang-Hwan,Yokoyama, Shigeyuki,Kamo, Naoki American ChemicalSociety 2011 Biochemistry Vol.50 No.41

<P><I>Acetabularia</I> rhodopsins are the firstmicrobialrhodopsins discovered in a marine plant organism, <I>Acetabulariaacetabulum</I>. Previously, we expressed <I>Acetabularia</I> rhodopsin II (ARII) by a cell-free system from one of two opsingenes in <I>A. acetabulum</I> cDNA and showed that ARIIis a light-driven proton pump [Wada, T., et al. (2011) <I>J.Mol. Biol.</I><I>411</I>, 986–998]. In thisstudy, the photochemistry of ARII was examined using the flash-photolysistechnique, and data were analyzed using a sequential irreversiblemodel. Five photochemically defined intermediates (P<SUB><I>i</I></SUB>) were sufficient to simulate the data. Noticeably, both P<SUB>3</SUB> and P<SUB>4</SUB> contain an equilibrium mixture of M, N,and O. Using a transparent indium tin oxide electrode, the photoinducedproton transfer was measured over a wide pH range. Analysis of thepH-dependent proton transfer allowed estimation of the p<I>K</I><SUB>a</SUB> values of some amino acid residues. The estimated valueswere 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values wereassigned as the p<I>K</I><SUB>a</SUB> of Asp81 (Asp85<SUP>BR</SUP>) in the dark, Asp92 (Asp96<SUP>BR</SUP>) at N, Glu199 (Glu204<SUP>BR</SUP>) at M, Glu199 in the dark, an undetermined proton-releasingresidue at the release, and the pH to start denaturation, respectively.Following this analysis, the proton transfer of ARII is discussed.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2011/bichaw.2011.50.issue-41/bi2009932/production/images/medium/bi-2011-009932_0006.gif'></P>

Rice OsOPRs: Transcriptional Profiling Responses to Diverse Environmental Stimuli and Biochemical Analysis of OsOPR1

장성국,조경원,Junko Shibato,Oksoo Han,Hitoshi Iwahashi,Shigeru Tamogami,Sajad Majeed Zargar,Akihiro Kubo,Yoshinori Masuo,Ganesh Kumar Agrawal,Randeep Rakwal 한국식물학회 2009 Journal of Plant Biology Vol.52 No.3

The 12-oxo-phytodienoic acid reductase (OPR) is a key enzyme in jasmonic acid (JA) biosynthesis. Previously, we reported the presence of 13 OPR isogenes (OsOPR1-13) in rice. OsOPRs phylogenetically belong to two subgroups, OPRI and OPRII. OsOPR13 is assigned to the second subgroup, which is involved in JA biosynthesis, while the others are found in the first subgroup. Here, we systematically investigated transcript levels of OsOPRs in various tissues and against diverse environmental stresses. Each gene was differentially involved in flower maturation, showing a tissue-specific response. OsOPR1, OsOPR2, and OsOPR13 were also active in responses to wounding, a fungal elicitor (chitosan), salt, UV-C irradiation, H2O2, and ozone exposure. In the case of JA-responsive OsOPRs (OsOPR1, OsOPR2, OsOPR6, OsOPR10, and OsOPR13), co-application of JA and SA suppressed jasmonate-induced transcript levels and delayed OsOPR10 expression. We also investigated the biochemical properties of OsOPR1 and found a flavin cofactor with optimal activity at pH7.8 and values of 0.048 min−1 kcat and 8.33 μM Km for (9S,13R)- 12-oxo-phytodienoic acid. Here, we discuss the role of OsOPRs in stress responses and floral development.

Light/Dark Responsiveness of Kinetin-Inducible Secondary Metabolites and Stress Proteins in Rice Leaf

Cho, Kyoung-Won,Kim, Dea-Wook,Jung, Young-Ho,Shibato, Junko,Tamogami, Shigeru,Yonekura, Masami,Jwa, Nam-Soo,Kubo, Akihiro,Agrawal, Ganesh Kumar,Rakwal, Randeep The Korean Society of Crop Science 2007 Journal of crop science and biotechnology Vol.10 No.2

Kinetin(KN) is an inducer of rice(Oryza sativa L.) defense/stress responses, as evidenced by the induction of inducible secondary metabolite and defense/stress protein markers in leaf. We show a novel light-dependent effect of KN-triggered defense stress responses in rice leaf. Leaf segments treated with KN(100 ${\mu}M$) show hypersensitive-like necrotic lesion formation only under continuous light illumination. Potent accumulation of two phytoalexins, sakuranetin and momilactone A(MoA) by KN that peaks at 48 h after treatment under continuous light is completely suppressed by incubation under continuous dark. Using two-dimensional gel electrophoresis we identified KN-induced changes in ribulose-1, 5-bisphosphate carboxylase/oxygenase, energy- and pathogenesis-related proteins(OsPR class 5 and 10 members) by N-terminal amino acid sequencing and mass spectrometry. These changes were light-inducible and could not be observed in the dark(and control). Present results provide a new dimension(light modulation/regulation) to our finding that KN has a potential role in the rice plant self-defense mechanism.

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Wang, Yiming,Kwon, Soon Jae,Wu, Jingni,Choi, Jaeyoung,Lee, Yong-Hwan,Agrawal, Ganesh Kumar,Tamogami, Shigeru,Rakwal, Randeep,Park, Sang-Ryeol,Kim, Beom-Gi,Jung, Ki-Hong,Kang, Kyu Young,Kim, Sang Gon,K The Korean Society of Plant Pathology 2014 Plant Pathology Journal Vol.30 No.4

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Yiming Wang,Soon Jae Kwon,Jingni Wu,최재영,이용환,Ganesh Kumar Agrawal,Shigeru Tamogami,Randeep Rakwal,Sang Ryeol Park,김범기,정기홍,강규영,김상곤,김선태 한국식물병리학회 2014 Plant Pathology Journal Vol.30 No.4

Rice blast disease caused by Magnaporthe oryzae is oneof the most serious diseases of cultivated rice (Oryzasativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, weutilized the transcriptome analysis approach using a300 K tilling microarray to rice leaves infected withcompatible and incompatible M. oryzae strains. Priorto the microarray experiment, total RNA was validatedby measuring the differential expression of rice defenserelatedmarker genes (chitinase 2, barwin, PBZ1, andPR-10) by RT-PCR, and phytoalexins (sakuranetinand momilactone A) with HPLC. Microarray analysisrevealed that 231 genes were up-regulated (>2 foldchange, p < 0.05) in the incompatible interaction comparedto the compatible one. Highly expressed genes were functionally characterized into metabolic processesand oxidation-reduction categories. The oxidativestress response was induced in both early and laterinfection stages. Biotic stress overview from MapMananalysis revealed that the phytohormone ethylene aswell as signaling molecules jasmonic acid and salicylicacid is important for defense gene regulation. WRKYand Myb transcription factors were also involved in signaltransduction processes. Additionally, receptor-likekinases were more likely associated with the defenseresponse, and their expression patterns were validatedby RT-PCR. Our results suggest that candidate genes,including receptor-like protein kinases, may play a keyrole in disease resistance against M. oryzae attack

Rice OsACDR1 (Oryza sativa Accelerated Cell Death and Resistance 1) Is a Potential Positive Regulator of Fungal Disease Resistance

김정아,조경원,Raksha Singh,Young-Ho Jung,Seung-Hee Jeong,So-Hee Kim,Jae-eun Lee,Yoon-Seong Cho,Ganesh K. Agrawal,Randeep Rakwal,Shigeru Tamogami,Birgit Kersten,전종성,Gynheung An,Nam-Soo Jwa 한국분자세포생물학회 2009 Molecules and cells Vol.28 No.5

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal patho-gen (Magnaporthe grisea) and showed inhibition of appres-sorial penetration on the leaf surface. In contrast, loss-of-function and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its local-ization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.