Is the Arabidopsis electronic shuttle ADXR-ADX-P450 involved in steroid synthesis and development?

DISTÉFANO AM, BELLIDO AM, FIOL DF, ZABALETA EJ, PAGNUSSAT GC

Mar del Plata

Congreso; 51 Congreso de la Sociedad Argentina de Bioquímica y Biología Molecular (SAIB); 2015

Sociedad Argentina de Bioquímica y Biología Molecular (SAIB)

We have identified Arabidopsis gametophytic mutants with insertions in a nuclear gene encoding for a mitochondrialadrenodoxin reductase (ADXR). In mammals and insects mitochondria, adrenodoxin (ADX) mediates electrontransfer from NADPH via ADXR to the terminal electron acceptor, a cytochrome P450 (P450), which constitutes anessential step for steroid biosynthesis. In this work, we study the occurrence of a similar steroid biosynthetic pathwayin plants. Arabidopsis has an ADRX homologue and 2 ADXs located in mitochondria. However, no mitochondrialP450s have been described in Arabidopsis. We performed an Y2H screening to detect possible ADX redox partners.We found that the P450 CYP711A1 binds to ADX1, which was confirmed by BiFC assays. We also showed thatCYP711A1 has a mitochondrial localization by means of GFP fusions and transient expression in NicotianaBenthamiana. Using the same approach we studied the subcellular localization of different P450s with a predictedsignal peptide targeting mitochondria. Two additional P450s were found localized to mitochondria and showedinteraction with ADX trough BiFC assays. We collected ovules from wild type, adxr/ADXR, and spl (which lacksembryo sacs) mutants plants, to analyzed the female gametophyte steroid profile by UL-MS-MS. Additionally,insertional mutants for CYP711A1 and different P450-encoding genes are being analyzed.