Plants are sessile organisms and are unable to avoid environmental biotic and abiotic stress by changing their habitat.　Therefore, plants have developed unique defensive systems.　Plants use hormones not only to control growth and development but also to initiate defense responses to these stresses.　Jasmonic acid （JA） is categorized as one of plant hormones, and we focused on metabolism, transportation, and biological activities of JA.　The unique point of this study is preparation of various kinds of labeled compounds for a target compound, and analyze them using MS/MS system, which afford following experimental results; 1】de novo synthesis of jasmonoyl isoleucine （JA-Ile）in damaged leaves and its transportation into non-damaged leaves to induce JA depended wound response, 2】metabolism of JA-Ile via CYP94b3 to give 12-hydroxyJA-Ile, 3】isolation of cis-jasmone form Lasiodiplodia theobromae and investigation of its synthetic pathway to give cis-jasmone.

Auxin is the first discovered plant hormone that plays various physiological roles in plants.　Indole-3-acetic acid （IAA） is the most studied naturally occurring auxin and its action mechanism has been extensively investigated.　Recently, the main IAA biosynthetic pathway in plants has been settled by genetic and biochemical approaches.　IAA is mainly produced from tryptophan through the indole-3-pyruvate pathway in plants.　Phenylacetic acid （PAA） is another naturally occurring auxin of which physiological roles still remain unknown.　More recently, we demonstrated that PAA functions as auxin through the TIR1/AFB pathway, but it does not possess polar transport characteristics in plants unlike IAA.　These facts suggest that two types of auxins with distinct transport characteristics may cooperatively regulate plant growth and development, thus providing new insights into auxin biology.

Cultivated rice （Oryza sativa） produces many types of diterpene phytoalexins, such as momilactones and phytocassanes as antimicrobial substances.　It is known that the biosynthetic genes are clustered on rice chromosomes and co-expressed in response to environmental stimuli. We demonstrated that expressions of the clustered genes and the upstream MEP pathway genes are coordinately regulated by transcription factors inductively expressed under stress conditions.　Comparative genomics and biochemical studies using wild rice species not only have shown when and how rice plants acquired the ability to co-ordinately produce phytoalexins, but also how evolutionary events led to the clustering of the biosynthetic genes.　Interestingly, bryophyte moss and paddy weeds barnyardgrass were also shown to inductively produce momilactones upon environmental stresses and that they possess the gene cluster.　The results imply that a chemical defense system using momilactones appears to be maintained in diverse plant species as a consequence of convergent evolution.

Strigolactones （SLs）are plant hormones that regulate shoot branching as well as being known as rhizosphere signals for parasitic and symbiotic interactions.　In 2012, an SL-like molecule called carlactone （CL） was identified through the biochemical functional analysis of three enzymes, CAROTENOID CLEAVAGE DIOXYGENASE7 （CCD7）, CCD8, and DWARF27 （D27）, yet its natural occurrence in plant tissue was not proved.　Using a chemically synthesized stable isotope labeled CL as an internal standard we successfully identified CL as an endogenous metabolite both in rice and Arabidopsis by LC-MS/MS analysis.　Moreover, we proved bioconversion of CL into some SL molecules in rice, conclusively demonstrating that CL is an endogenous biosynthetic precursor for SLs.　In addition, we found that CL is converted into its carboxylated analog, carlactonic acid （CLA）, in a manner dependent on a cytochrome P450, MAX1.　We also identified a methyl esterified derivative of CLA, methyl carlactonoate （MeCLA）, as an endogenous SL-like molecule in Arabidopsis.　Intriguingly, among three CL-related molecules （CL, CLA, MeCLA）, only MeCLA could physically interact with the SL receptor protein, AtD14, suggesting that the methyl esterification step of CLA has a critical role to produce bioactive hormone compounds in the shoot branching inhibition pathway.

Jasmonate （JA） regulates many physiological processes of plant stress responses as well as plant development.　We have studied physiological roles of JA in rice defense responses.　The cpm2 and hebiba are rice mutants defective in a gene encoding allene oxide cyclase, a key enzyme of JA biosynthesis.　As endogenous levels of JA were extremely low in both mutants, we treated the mutants with Pyricularia oryzae to investigate the function of JA in defense responses.　The inoculation assays revealed that JA is required for defense against P. oryzae.　We also demonstrated that JA mediates stress-induced accumulation of a flavonoid phytoalexin, sakuranetin.　Additionally, we identified rice naringenin 7-O-methyltransferase （OsNOMT） as a key enzyme of sakuranetin biosynthesis and demonstrated that the expression of OsNOMT was induced by JA.　Studies using osjar1 mutant defective in jasmonoyl-isoleucine （JA-Ile） biosynthesis suggested that JA-Ile is a major signaling molecule in the JA-mediated defense responses.　Additionally, we found that JA response was induced in narrow region around stressed area by using JA-inducible reporter system, suggesting that endogenous JA level is regulated very locally.　Here we summaries our studies focusing on physiological roles of JA in rice defense responses.

Plants are in nature challenged and colonized by a rich diversity of microbes, ranging from pathogens to mutualists.　Plants have evolved an elaborate, multilayered immune system to recognize and manage these plant-infecting microbes.　An important immune layer is provided by pattern recognition receptors （PRRs） that detect microbe- and host damage-associated molecular patterns （MAMPs and DAMPs, respectively） at the cell surfaces.　PRRs have emerged as a promising toolset to confer durable, cost-effective and broad-spectrum disease resistance on crop plants.　Here, we discuss the functional significance of PRRs in plant-microbe interactions and their exploitation to develop a more sustainable setting of agriculture.

Salicylic acid （SA） is a phenolic compound that has been recognized as a plant hormone, playing a key role in the activation of defense responses after pathogen infection.　Two SA biosynthesis pathways have been proposed in plants; one via phenylalanine and the other via isochorismic acid.　Once plants synthesize SA to activate defense response, most of SA can be converted to SA metabolites.　These SA metabolites may have distinct roles in plants, such as inactivation of SA signaling and long distance propagation of defense signaling.　In addition, some plant pathogens can modulate host SA biosynthesis pathway by enzymatic effectors that metabolize SA precursors leading to less SA accumulation.　As SA is a critical component in plant immunity, disrupting SA biosynthesis can be a common infection strategy for plant pathogens.　Here we review our current knowledge and recent progress on the biosynthesis and metabolism of SA during plant-microbe interactions.

Oriental hybrid lily flowers have a nice fragrance.　Especially in a confined space, however, the scent can be perceived as too strong and therefore unpleasant.　Lilium cv. ‘Casa Blanca’, a typical oriental hybrid cultivar, has a large pure-white flower with strong smell.　We clarified that the compounds responsible for the strong fragrance of ‘Casa Blanca’ are aromatics.　We succeeded in decreasing the emissions of scent compounds and made the fragrance milder throughout the period of use as an ornamental by application of an inhibitor of PAL.　Here I introduce the characteristics of floral scents, the scent suppression method of lily and the outline of development of fragrance suppressor for ornamental flower.

Imaging MS is a new tool to map the spatial distribution of molecules including secondary metabolites on the biological materials.　By using combinations of scanning system and various methods for ionization, matrices, and mass apparatuses, we can detect localization of biomolecules in tissues.　Recently, we applied Imaging MS to intact plant tissues by the in house-developed apparatus which has the refinement of Matrix Associated Laser Desorption/Ionization-Fourier transform ion cyclotron resonance （MALDI-FTICR） MS incorporating ultra-high mass resolution, ultra-high molecular mass accuracy and high spatial resolution.　This Imaging MS analysis succeeded in visualizing of cellular localization of secondary metabolites of Catharanthus roseus stem tissues glued to a small conductive glass.

To understand the mechanisms by which plant hormones regulate physiological processes, it is crucial to exactly know their endogenous concentrations at cellular levels.　We recently established a method to determine the levels of abscisic acid （ABA） and jasmonoyl-isoleucine （JA-Ile） from single cells by employing “live-single cell mass spectrometry （MS）”.　Single cell contents were taken from Vicia faba leaves by using nano-electrospray ionization tips under a stereomicroscope.　Stable isotope-labeled hormones were then added to the samples as internal standards to normalize ionization efficiencies and quantify the hormone levels.　The samples were directly introduced into a mass spectrometer and subjected to MS/MS analysis.　With careful validation of the data obtained, we succeed in the detection of stress-induced accumulation of ABA and JA-Ile from living single cells.　In this article, we summarize our challenge to establish a method to quantify plant hormones by single cell MS analysis.

Our aim is to establish “Organellar Reaction Cluster” as a novel field of science along with its related foundational techniques.　We will establish techniques for simultaneously modifying the genomes of nuclei, plastids, and mitochondria with transportation of proteins and bioactive molecules into specific organelles.　Using these foundational techniques, the effect of organellar interactions and subcellular localization on metabolism will be quantified.　The designed metabolic pathways with high efficiency will be implemented with transgenic plants to substantiate “Organellar Reaction Cluster”.