Shigeru Tamogami

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.

Integrated transcriptomics, proteomics, and metabolomics analyses to survey ozone responses in the leaves of rice seedling.

Ozone (O(3)), a serious air pollutant, is known to significantly reduce photosynthesis, growth, and yield and to cause foliar injury and senescence. Here, integrated transcriptomics, proteomics, and metabolomics approaches were applied to investigate the molecular responses of O(3) in the leaves of 2-week-old rice (cv. Nipponbare) seedlings exposed to 0.2 ppm O(3) for a period of 24 h. On the basis of the morphological alteration of O(3)-exposed rice leaves, transcript profiling of rice genes was performed in leaves exposed for 1, 12, and 24 h using rice DNA microarray chip. A total of 1535 nonredundant genes showed altered expression of more than 5-fold over the control, representing 8 main functional categories. Genes involved in information storage and processing (10%) and cellular processing and signaling categories (24%) were highly represented within 1 h of O(3) treatment; transcriptional factor and signal transduction, respectively, were the main subcategories. Genes categorized into information storage and processing (17, 23%), cellular processing and signaling (20, 16%) and metabolism (18, 19%) were mainly regulated at 12 and 24 h; their main subcategories were ribosomal protein, post-translational modification, and signal transduction and secondary metabolites biosynthesis, respectively. Two-dimensional gel electrophoresis-based proteomics analyses in combination with tandem mass spectrometer identified 23 differentially expressed protein spots (21 nonredundant proteins) in leaves exposed to O(3) for 24 h compared to respective control. Identified proteins were found to be involved in cellular processing and signaling (32%), photosynthesis (19%), and defense (14%). Capillary electrophoresis-mass spectrometry-based metabolomic profiling revealed accumulation of amino acids, gamma-aminobutyric acid, and glutathione in O(3) exposed leaves until 24 h over control. This systematic survey showed that O(3) triggers a chain reaction of altered gene, protein and metabolite expressions involved in multiple cellular processes in rice.

Interplant communication: Airborne methyl jasmonate is essentially converted into JA and JA-Ile activating jasmonate signaling pathway and VOCs emission

Methyl jasmonate (MeJA) was identified as an airborne signal involved in mediating interplant defense response communications over a decade ago. However, how MeJA activates plant defense systems and what becomes of the compound after it has done so has, thus far, remained unknown. To investigate this, Achyranthes bidentata plants were exposed to deuterated methyl jasmonate (d(2)MeJA) followed by absolute quantification of metabolic products of d(2)MeJA, and emissions of volatile organic compound (VOC) as defensive markers. We found that d(2)MeJA was metabolized mainly into deuterated jasmonic acid (d(2)JA) and jasmonoyl isoleucine (d(2)JA-Ile), and to a much lesser extent, deuterated jasmonoyl leucine (d(2)JA-Leu). Increases in d(2)JA-Ile/Leu and also endogenous JA-Ile/Leu were tightly co-related with, and significantly influenced the pattern and amount of, VOC emissions. The amount of accumulated d(2)JA-IIe was 13.1-fold higher than d(2)JA-Leu, whereas the amounts of JA-IIe and JA-Leu accumulated were almost identical. This study demonstrates that exogenous MeJA activates defensive systems (such as VOC emissions) in receiver plants by essentially converting itself into JA and JA-IIe and initiating a signal transduction leading to VOC emissions and induction of endogenous JA-IIe and JA-Leu, which in turn cause further amplification of VOC emissions.

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.

Quantification of amino acid conjugates of jasmonic acid in rice leaves by high-performance liquid chromatography–turboionspray tandem mass spectrometry

Abstract HPLC–turboionspray MS–MS (TIS-MS–MS) was shown to be useful in the quantification of the amino acid conjugates of jasmonic acid (JA) as well as free JA in rice leaves. With the TIS-MS–MS system, high sensitivity was obtained under multiple reaction monitoring conditions. Using this quantification method, rapid increase and subsequent decrease in the leucine conjugate and valine conjugate of JA were observed in rice leaves stressed by wounding. These amino acid conjugates of JA may work as endogenous signalling compounds as well as free JA.

Momilactones A and B in Rice Straw Harvested at Different Growth Stages.

Momilactones A and B in rice straw harvested at different growth stages were quantified by HPLC-MS-MS. They increased to their maximal levels at the heading stage and then gradually decreased. In addition, these phytotoxins were found to be relatively easily extracted with water.

Rice OsACDR1 ( Oryza sativa accelerated cell death and resistance 1) is a potential positive regulator of fungal disease resistance

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 pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-offunction 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 localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

Cloning of novel rice allene oxide cyclase (OsAOC): mRNA expression and comparative analysis with allene oxide synthase (OsAOS) gene provides insight into the transcriptional regulation of octadecanoid pathway biosynthetic genes in rice

Abstract Here we cloned a novel rice (japonica-type cv. Nipponbare) cDNA highly homologous to allene oxide cyclase (AOC, EC 5.3.99.6; jasmonate biosynthetic pathway), named OsAOC . OsAOC showed significant similarity at amino acid level with the available AOCs only from the dicotyledonous tomato and Arabidopsis . Examination of the steady-state mRNA level in an early time course experiment revealed that OsAOC expressed very weakly in healthy leaves, and showed responsiveness to wounding by cut within 30 min, followed by an increase with time till 120 min. Interestingly, jasmonic acid (JA) treatment itself potently enhanced its expression over the control (wounded leaves), suggesting feedback activation of OsAOC . Other signalling components, salicylic acid (SA), ethylene (using ethephon, ET), abscisic acid (ABA), and hydrogen peroxide (H 2 O 2 ), also strongly and transiently up-regulated the OsAOC transcript. Moreover, heavy metals and protein phosphatase (PP) inhibitors were potent in inducing the OsAOC transcript. A protein synthesis inhibitor, cycloheximide (CHX), alone or in combination with JA and SA further enhanced its expression, suggesting the involvement of a de novo synthesized negative regulator in its regulation. A long-term time course experiment revealed that wounding by cut was the most effective in inducing the expression of OsAOC starting at 3 h after treatment, whereas all the above pharmacological agents showed induction at 3–12 h, after which the OsAOC expression declined under light, whereas this expression was substantially suppressed under darkness. Additionally, chitosan (CT), a fungal elicitor also transiently up-regulated the OsAOC expression. Intriguingly, OsAOC mRNA expression manifested a clear rhythmicity under light/dark cycle. Finally, parallel investigations of allene oxide synthase ( OsAOS , accession number AY062258) and its comparison with OsAOC indicated their differential response to various environmental cues. These results strongly suggest a role for OsAOC (and OsAOS ) in rice plant defense/stress response pathway(s).

Reduction of polar auxin transport in tobacco by the tumorigenic Agrobacterium tumefaciens AK-6b gene

The plant-tumorigenic 6b (AK-6b) gene of Agrobacterium tumefaciens strain AKE10 induces morphological alterations to tobacco plants, Nicotiana tabacum. To investigate the molecular mechanisms underlying these processes, we generated transgenic tobacco harboring the AK-6b gene under the control of a dexamethazone-inducible promoter. Upon induction, transgenic tobacco seedlings exhibited distinct classes of aberrant morphologies, most notably adventitious outgrowths and stunted epicotyls. Histological analysis revealed massive proliferation and altered venation in the newly established outgrowths. Prominent vascular development suggested that auxin metabolism or signaling had been altered. Indeed, basipetal auxin transport in the hypocotyls of the transgenic seedlings was reduced by 50–80%, whereas intracellular auxin contents were only slightly reduced. Analysis of cell extracts by HPLC revealed a large accumulation of phenolic compounds, including the flavonoid kaempferol-3-rutinoside, in transgenic plants compared with wild-type seedlings. As some naturally occurring flavonoids have been shown to affect auxin transport, we suggest that the AK-6b gene expression impairs auxin transport via modulation of phenylpropanoid metabolism, and ultimately results in the observed morphological alterations.

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

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.