Plant Metabolomics towards understanding of the molecular diversity and functional specificity of the P450 containing electron trnasfer chain

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Among a number of potential ferredoxin reductase genes in Arabidopsis genome, we have identified AtMFDR to correspond to a homologue of adrenodoxin reductase (mitochondrial ferredoxin reductase), and AtMFDX1 and AtMFDX2 as the genes for ferredoxins highly similar to adrenodoxin (mitochondrial ferredoxin). The deduced primary structures of AtMFDR and AtMFDXs contained the signature sequences involved in the electron transfer, and the recombinant AtMFDX1 and AtMFDR proteins were fully active to reconstitute the electron-transfer activity with NAD(P) H as the electron donor. Protein-gel blot analysis demonstrated constant accumulation of AtMFDR protein in leaf, stem, and flower. Clarification of their subcellular localization is prerequisite to identify physiological partner proteins in planta. A subcellular fractionation analysis demonstrated membrane association of AtMFDR protein, and specific localization could not be elucidated through a series of immunohistochemical analyses. Detailed analysis is in progress to clarify the membrane structure where the proteins exert their physiological functions.During the course of molecular characterization of the electron transfer chain, we have also established a metabolomics platform on a basis of combination of GC/MS, LC/MS, and FT/MS. The metabolome analysis includes the non-biased high throughput analysis of plant crude extracts and data analysis program. The established method will open the possibilities to explore "silent phenotypic alterations" caused by disruption of genes of unknown functions.