ATR3 encodes a diflavin reductase essential for Arabidopsis embryo development

Abstract

Summary• The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable.• Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the Nterminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2.• Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein.• This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

“…replication [24]. These observations are now understood because replicative DNA polymerases have been shown to require an Fe-S cluster [50].…”

“…Because Dre2 and Tah18 proteins in yeast, human and Arabidopsis are known to form a protein-protein interaction [23,24,41], we tested whether coexpression of AtTAH18 and AtDRE2 could complement dre2 yeast. When expressed from either the NP or MET25 promoter for AtTAH18, the combined expression of the plant genes almost fully complemented growth of a Gal-DRE2 single-and Gal-TAH18/Gal-DRE2 double mutant.…”

“…Correct promoter insertion was confirmed by PCR of chromosomal DNA with primers hybridizing to flanking regions and the inserted cassette. The AtTAH18 coding region was previously isolated [24] and the AtDRE2 coding region was PCR amplified from Arabidopsis thaliana cDNA with gene-specific primers. AtTAH18 and AtDRE2 were cloned into p414 and p416 yeast vectors, respectively [30].…”

“…In this paper, we use the yeast gene names for the Arabidopsis homologues, with the prefix At for Arabidopsis thaliana. Varadarajan et al [24] found that AtTAH18 interacted specifically with AtDRE2 identified in a yeast two-hybrid screen. Moreover, cell fractions of yeast or Nicotiana benthamiana overexpressing AtTAH18::GFP were shown to have NADPHdependent cytochrome c reductase activity.…”

“…In the yeast cytosol, the diflavin reductase Tah18 and its partner Dre2 form an electron input module for the CIA pathway [23]. The Tah18 and Dre2 proteins were recently described in Arabidopsis, as ATR3, a homologue of ATR1 and ATR2 (Arabidopsis thaliana P450 reductases), and AtCIAPIN1 (Arabidopsis thaliana homologue of the human cytokine-induced apoptosis inhibitor 1) [24]. In this paper, we use the yeast gene names for the Arabidopsis homologues, with the prefix At for Arabidopsis thaliana.…”