Protein phosphatase 2A B55 and A regulatory subunits interact with nitrate reductase and are essential for nitrate reductase activation

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.

Figure 1.

The scaffolding A subunits of PP2A are required for light activation of NR. A, Relative transcript levels for the PP2AA subunit in tamoxifen-treated wild-type and amiRNA-1 plants, and mock-treated plants. Expression levels were tested with RT-PCR using SYBR green with plant samples also used for testing NR activation. se for the RT-PCR assay is given, n = 3. B, Activity state of NR following a dark-to-light transition of wild-type (dots) and amiRNA-1 (squares) plants treated with tamoxifen (black symbols), and mock-treated plants (white symbols). Plants were grown in 12-h light/12-h darkness for 3 weeks. Two hours into the photoperiod, when NR activity was at a high level, plants were transferred to darkness and samples harvested after 1 h of darkness (time zero), then after 20 and 40 min of white light (150 μmol m⁻² s⁻¹). Data are means of three samples, se is given.

Figure 2.

NR activity state in light or darkness in different A subunit mutant plants. Wild-type Col, the double-mutant pp2aa2 pp2aa3, WS, and rcn1 mutant were tested. Leaves of rosette stage plants were harvested daily during 6 d, 1.5 to 3 h into the light period, and after approximately 12 h of darkness. A, Light samples. B, Dark samples. n = 13, se is given. To test changes in activity stage during light/dark transitions samples were harvested from rosette stage plants 3 h into the daily light period (time 0), light was turned off, and samples were harvested after 2 h of darkness. Light was then switched on, and samples taken after 20 and 40 min of light. C, Double-mutant pp2aa2 pp2aa3 and control wild-type Col. D, rcn1 mutant and control WS. The data are means of four independent experiments, se is given.

Figure 3.

Fusion proteins of NR and B55 subunits localize to the cytoplasm in Arabidopsis protoplasts. A, NR1 (At1g77760) and B, NR2 (At1g37130) were fused at their C terminus to EYFP (NR-EYFP) and transiently expressed in protoplasts from Arabidopsis cell suspension culture. C, B55α (At1g51690) protein fused at its N terminus to EYFP and transiently expressed in protoplasts localized to cytosol. D, B55β (At1g17720) protein fused at its N terminus to EYFP and transiently expressed in protoplasts localized to cytosol.

Figure 4.

Full-length and truncated forms of NR interact with Bα as tested by BiFC. A and B, Interaction of full-length NR1 and Bα in cytosol of Arabidopsis mesophyll protoplasts. C and D, Interaction of the NR1 fragment comprising the dimerization domain, hinge1, and heme domain with Bα in Arabidopsis mesophyll protoplasts. Scale bar = 10 μm.

Figure 5.

NR1 and B55α interact in yeast. HF7c cells were cotransformed with the indicated combination of bait (BD) and prey (AD) vectors and grown on selection plates at 30°C for 48 h. The growth on −HTL medium and −TL medium was assayed and the ratio calculated as an indicator of the strength of protein interaction. n = 3.

Figure 6.

NR activation rate is reduced in low B55 dosage plants. Plants were grown in 16-h light/8-h darkness for 3 weeks. Three hours into the photoperiod when NR activity was at a high level samples were harvested before subjected to darkness. Samples were then harvested after 45 min of darkness. Plants were transferred back to light for activation of NR, and sampled after 15 and 30 min of light (150 μmol m⁻² s⁻¹). Data are means of five samples at 0, 45, and 60 min, and three samples at 75 min. se is given. The plants used were first determined by PCR to be heterozygous for the mutation T-DNA insert in Bα and homozygous for insert in Bβ (homozygosis for insert in both genes appeared lethal, see text).