Abstract
Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. The activation of NR in green leaves takes place after dark/light shifts, and is dependent on photosynthesis. Previous studies using various inhibitors pointed to protein phosphatases sensitive to okadaic acid, including protein phosphatase 2A (PP2A), as candidates for activation of NR. PP2As are heterotrimeric enzymes consisting of a catalytic (C), structural (A), and regulatory (B) subunit. In Arabidopsis there are five, three, and 18 of these subunits, respectively. By using inducible artificial microRNA to simultaneously knock down the three structural subunits we show that PP2A is necessary for NR activation. The structural subunits revealed overlapping functions in the activation process of NR. Bimolecular fluorescence complementation was used to identify PP2A regulatory subunits interacting with NR, and the two B55 subunits were positive. Interactions of NR and B55 were further confirmed by the yeast two-hybrid assay. In Arabidopsis the B55 group consists of the close homologs B55α and B55β. Interestingly, the homozygous double mutant (b55α × b55β) appeared to be lethal, which shows that the B55 group has essential functions that cannot be replaced by other regulatory subunits. Mutants homozygous for mutation in Bβ and heterozygous for mutation in Bα revealed a slower activation rate for NR than wild-type plants, pointing to these subunits as part of a PP2A complex responsible for NR dephosphorylation.
Highlights
Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains
At the posttranslational level NR is rapidly activated by dephosphorylation, and active photosynthesis is necessary for this activation
One of the A subunits, ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), was suggested to have a cardinal role in regulation of phosphatase activity, whereas functions of the other two, PP2AA2 and PP2AA3, were only unmasked when RCN1 was absent (Zhou et al, 2004). By using these different loss-of-function mutants and a transgenic line with tamoxifen-inducible expression of artificial microRNAs targeting all three A subunits of phosphatase 2A (PP2A) (Michniewicz et al, 2007), we here investigated the importance of A subunits for activation of NR
Summary
Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. One of the A subunits, ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), was suggested to have a cardinal role in regulation of phosphatase activity, whereas functions of the other two, PP2AA2 and PP2AA3, were only unmasked when RCN1 was absent (Zhou et al, 2004) By using these different loss-of-function mutants and a transgenic line with tamoxifen-inducible expression of artificial microRNAs (amiRNAs) targeting all three A subunits of PP2A (Michniewicz et al, 2007), we here investigated the importance of A subunits for activation of NR. A clear effect on NR by inducible knockdown of all three A subunits established the involvement of PP2A for activation of NR in vivo
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