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

在拟南芥基因组中许多潜在的铁氧还蛋白还原酶基因中，我们发现AtMFDR与肾上腺素还蛋白还原酶（线粒体铁氧还蛋白还原酶）同源，而AtMFDX1和AtMFDX2是与肾上腺素还蛋白（线粒体铁氧还蛋白）高度相似的铁氧还蛋白基因。推导出的AtMFDR和AtMFDXs的一级结构含有参与电子转移的特征序列，重组的AtMFDX1和AtMFDR蛋白以NAD(P) H为电子给体重建了电子转移活性。蛋白凝胶印迹分析显示AtMFDR蛋白在叶、茎和花中不断积累。澄清它们的亚细胞定位是鉴定植物生理伴侣蛋白的先决条件。亚细胞分离分析显示AtMFDR蛋白与膜相关，通过一系列免疫组织化学分析无法阐明特异性定位。详细的分析正在进行中，以阐明蛋白质发挥其生理功能的膜结构。在对电子传递链进行分子表征的过程中，我们还建立了GC/MS、LC/MS、FT/MS相结合的代谢组学平台。代谢组分析包括植物粗提取物的无偏高通量分析和数据分析程序。建立的方法将为探索由未知功能基因破坏引起的“沉默表型改变”提供可能性。

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.