SummaryCoumarins are derived via the phenylpropanoid pathway in plants. The 2H-1-benzopyran-2-one core structure of coumarins is formed via the ortho-hydroxylation of cinnamates, trans/cis isomerization of the side chain, and lactonization. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however, ortho-hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored using Arabidopsis thaliana, which accumulates scopoletin and its b-glucopyranoside scopolin in its roots. T-DNA insertion mutants of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3¢-O-methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis. Ortho-hydroxylases of cinnamates were explored in the oxygenase families in A. thaliana, and one of the candidate genes in the Fe(II)-and 2-oxoglutaratedependent dioxygenase (2OGD) family was designated as F6¢H1. T-DNA insertion mutants of F6¢H1 showed severe reductions in scopoletin and scopolin levels in the roots. The pattern of F6¢H1 expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6¢H1 protein exhibited orthohydroxylase activity for feruloyl CoA (K m = 36.0 AE 4.27 lM; k cat = 11.0 AE 0.45 sec )1 ) to form 6¢-hydroxyferuloylCoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)-and 2-oxoglutarate-dependent dioxygenase is the pivotal enzyme in the ortho-hydroxylation of feruloyl CoA in scopoletin biosynthesis.

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Camalexin is the major phytoalexin in Arabidopsis. An almost complete set of camalexin biosynthetic enzymes have been elucidated but only limited information is available regarding molecular mechanisms regulating camalexin biosynthesis. Here, we demonstrate that ANAC042, a member of the NAM, ATAF1/2, and CUC2 (NAC) transcription factor family genes, is involved in camalexin biosynthesis induction. T-DNA insertion mutants of ANAC042 failed to accumulate camalexin at the levels achieved in the wild type, and were highly susceptible to Alternaria brassicicola infection. The camalexin biosynthetic genes CYP71A12, CYP71A13, and CYP71B15/PAD3 were not fully induced in the mutants, indicating that the camalexin defects were at least partly a result of reduced expression levels of these P450 genes. β-Glucuronidase (GUS)-reporter assays demonstrated tissue-specific induction of ANAC042 in response to differential pathogen infections. Bacterial flagellin (Flg22) induced ANAC042 expression in the root-elongation zone, the camalexin biosynthetic site, and the induction was abolished in the presence of either a general kinase inhibitor (K252a), a Ca(2+)-chelator (BAPTA), or methyl jasmonate. The GUS-reporter assay revealed repression of the Flg22-dependent ANAC042 expression in the ethylene-insensitive ein2-1 background but not in sid2-2 plants defective for salicylic acid biosynthesis. We discuss ANAC042 as a key transcription factor involved in previously unknown regulatory mechanisms to induce phytoalexin biosynthesis in Arabidopsis.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) is the best MS technology for obtaining exact mass measurements owing to its great resolution and accuracy, and several outstanding FT-ICR/ MS-based metabolomics approaches have been reported. A reliable annotation scheme is needed to deal with directinfusion FT-ICR/MS metabolic profiling. Correlation analyses can help us not only uncover relations between the ions but also annotate the ions originated from identical metabolites (metabolite derivative ions). In the present study, we propose a procedure for metabolite annotation on directinfusion FT-ICR/MS by taking into consideration the classification of metabolite-derived ions using correlation analyses. Integrated analysis based on information of isotope relations, fragmentation patterns by MS/MS analysis, co-occurring metabolites, and database searches (KNApSAcK and KEGG) can make it possible to annotate ions as metabolites and estimate cellular conditions based on metabolite composition.A total of 220 detected ions were classified into 174 metabolite derivative groups and 72 ions were assigned to candidate metabolites in the present work. Finally, metabolic profiling has been able to distinguish between the growth stages with the aid of PCA. The constructed model using PLS regression for OD 600 values as a function of metabolic profiles is very useful for identifying to what degree the ions contribute to the growth stages. Ten phospholipids which largely influence the constructed model are highly abundant in the cells. Our analyses reveal that global modification of those phospholipids occurs as E. coli enters the stationary phase. Thus, the integrated approach involving correlation analyses, metabolic profiling, and database searching is efficient for high-throughput metabolomics.

The cytochromes P450 (P450s) of higher plants play crucial roles in both primary metabolism and a wide variety of secondary metabolic processes, such as in the phenylpropanoid, terpenoid, and alkaloid pathways, which produce, for example, lignin monomers, flavonoids, anthocyanins, and species-specific phytoalexins (Schuler and Werck-Reichhart 2003). Plant P450s are also involved in the detoxification of xenobiotic chemicals such as herbicides (Schuler and Werck-Reichhart 2003;Nelson et al. 2004). Both the biosynthesis and degradation of plant growth regulators cannot be completed without the involvement of specific P450 protein families (Schuler and Werck-Reichhart 2003). In general, serious phenotypic changes including dwarfism, abnormal morphology, and differential hormonal responses have been the major driving forces of studies to clarify these P450 functions. A good background of natural product chemistry is also indispensable for the functional characterization of P450s. However, irrespective of the importance of plant P450 functions in both understanding plant biological processes and their possible applications as biocatalysts, no general key technology has been established to clarify the diverse enzymatic properties of P450s of unknown functions. For example, even in Arabidopsis thaliana the physiological functions of more than 70% of the P450 proteins remain unknown (Nelson et al. 2004).To develop a versatile experimental platform for P450 characterization, we streamlined a metabolic profiling system based on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Full-length cDNA clones of several Arabidopsis P450 genes encoding unknown functions were selected for the preparation of recombinant enzymes. Those P450s were constitutively expressed in parallel in a cultured cell system and whole plants. Reverse genetics approaches with T-DNA knockout mutants and RNA interference were also incorporated into this metabolic profiling scheme. Changes in the metabolic profiles of T87 cells resulting from P450 overexpression, if any, were evaluated with a comprehensive metabolomics approach Nakamura et al. 2007;Ohta et al. 2007).Here, we report examples of our metabolic profiling study of the CYP78A and CYP86C genes in Arabidopsis. There are six genes for CYP78A subfamily proteins in Arabidopsis, the catalytic properties of which are unknown. It has been reported that the CYP78A proteins are involved in plant developmental processes. Thus, CYP78A11 has been shown to be the PLASTOCHRON1 in rice, which controls the timing of lateral organ formation from the apical meristem (Miyoshi et al. Abstract Genome sequence analysis has revealed the presence of almost infinite numbers of cytochrome P450 genes in a variety of organisms. To establish a robust experimental platform from which to explore the catalytic potential of those putative P450 proteins, we have developed a comprehensive metabolic profiling system based on Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR/MS) analysis, to...