Abstract
Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3, encoding the ATP-binding cassette Transporter of the Mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in CNX2, Cofactor of Nitrate reductase and Xanthine dehydrogenase 2, encoding GTP 3′,8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 knockout mutant, which could only survive with ammonia as nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both cnx2-1 and cnx2-2, and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5. Interestingly, atm3-1 mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterise CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.
Abbreviations
- ABA3
- abscisic acid protein 3
- ABC
- ATP-binding cassette
- ATM3
- ABC transporter of the mitochondria 3
- AldOx
- aldehyde oxidase
- cPMP
- cyclic pyranopterin monophosphate
- CNX
- cofactor of nitrate reductase and xanthine dehydrogenase
- Col-0
- Columbia-0 ecotype
- EMS
- ethyl methanesulfonate
- F1
- filial 1
- FeS
- iron-sulfur
- GSSG
- glutathione disulfide
- GS-S-SG
- glutathione trisulfide
- ISU/ISCU
- iron-sulfur cluster assembly protein 1
- Ler
- Landsberg ecotype
- mARC
- mitochondrial amidoxime-reducing component
- Moco
- molybdenum cofactor
- MOCS
- molybdenum cofactor synthesis protein
- NFS1
- nitrogen fixation S (NIFS)-like 1
- SAM
- S-adenosylmethionine
- SNP
- single nucleotide polymorphism
- SSLP
- simple sequence length polymorphism
- T-DNA
- transfer DNA
- TMH
- transmembrane helix
- TOM40
- translocator of the outer membrane 40
- URM
- ubiquitin-related modifier
- XDH
- xanthine dehydrogenase