Thioredoxin targets are regulated in heterocysts of cyanobacterium Anabaena sp. PCC 7120 in a light-independent manner

Abstract

In the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, glucose 6-phosphate dehydrogenase (G6PDH) plays an important role in producing the power for reducing nitrogenase under light conditions. Our previous study showed that thioredoxin suppresses G6PDH by reducing its activator protein OpcA, implying that G6PDH is inactivated under light conditions because thioredoxins are reduced by the photosynthetic electron transport system in cyanobacteria. To address how Anabaena sp. PCC 7120 maintains G6PDH activity even under light conditions when nitrogen fixation occurs, we investigated the redox regulation system in vegetative cells and specific nitrogen-fixing cells named heterocysts, individually. We found that thioredoxin target proteins were more oxidized in heterocysts than in vegetative cells under light conditions. Alterations in the redox regulation mechanism of heterocysts may affect the redox states of thioredoxin target proteins, including OpcA, so that G6PDH is activated in heterocysts even under light conditions.

Keywords: Anabaena; heterocyst; nitrogen fixation; oxidative pentose phosphate pathway; photosynthesis; redox regulation; thioredoxin.

Fig. 1.

Trx reduction by FTR (A), NTR, or NTRC (B). The oxidized Trxs (2 µM) were incubated with 0.5 mM NADPH, 0.2 µM FNR, 1 µM Fd, and 1 µM FTR for FTR-dependent reduction. The mixture of NADPH, FNR, and Fd was designated as an FTR reduction system. The oxidized Trxs (2 µM) were incubated with 0.5 mM NADPH and 1 µM NTR or NTRC. Proteins were precipitated with 10% (w/v) TCA, resuspended in SDS sample buffer containing 2 mM AMS, subjected to non-reducing SDS–PAGE, and stained with Coomassie brilliant blue. Red, reduced form; Ox, oxidized form.

Fig. 2.

Determination of the E_m values of Trxs. (A) Trxs were equilibrated with redox buffer containing various concentrations of reduced DTT and 50 mM oxidized DTT. Proteins were precipitated with 10% (w/v) TCA, resuspended in SDS sample buffer containing 2 mM AMS, loaded on non-reducing SDS–PAGE gels, and stained with Coomassie brilliant blue. Red, reduced form; Ox, oxidized form. (B) The reduction levels of Trxs were plotted against the redox potential of DTT buffer. The E_m values of Trxs were calculated by fitting the data to the Nernst equation. These values are presented as means ±SD (n=3). (C) The E_m values of Trxs were compared.

Fig. 3.

Estimation of the protein levels of Trxs and Trx reductases in Anabaena 7120. (A) Dilution series of recombinant proteins and the proteins extracted from Anabaena 7120 grown in the presence of nitrate (+N) or in the absence of combined nitrogen (–N) were subjected to SDS–PAGE and detected by immunoblotting. (B) In vivo levels of proteins were estimated by image analysis. Data are presented as means ±SD (n=3). N.D., not detected.

Fig. 4.

Redox states of Trxs in Anabaena 7120. Proteins were extracted from Anabaena 7120 grown in the presence of nitrate (+N) or in the absence of combined nitrogen (–N) under dark (D), low light (30 µmol photons m⁻² s⁻¹; LL), or high light (200 µmol photons m⁻² s⁻¹; HL) conditions. For the preparation of reduced proteins, cells were treated with 25 mM DTT for 10 min before adding TCA. In vivo redox states of Trxs were analyzed as described in the Materials and methods. Red, reduced form; Ox, oxidized form. Protein samples (20–40 µg) were loaded in each lane.

Fig. 5.

Redox states of Trx target protein in vegetative cells and heterocysts. (A) Protein expression of GFP-tagged CP12ΔC in the V-CP12 or H-CP12 strain was observed under a fluorescence microscope. Heterocysts are indicated by arrows. (B) In vivo redox states of OpcA in V-CP12 and H-CP12 strains were analyzed. Proteins were extracted from a 3 d culture of the V-CP12 or H-CP12 strain grown in the presence of nitrate (+N) or in the absence of combined nitrogen (–N) under light conditions (30 µmol photons m⁻² s⁻¹). Red, reduced form; Ox, oxidized form. Protein samples (20 µg) were loaded per lane. (C) In vivo redox states of GFP-tagged CP12ΔC in V-CP12 and H-CP12 strains were analyzed. Proteins were extracted from a 3 d culture of V-CP12 or H-CP12 strains grown in the presence of nitrate (+N) or in the absence of combined nitrogen (–N) under dark (D) or light (L) conditions. The proteins extracted from DTT-treated cells were also loaded. Red, reduced form; Ox, oxidized form.

Fig. 6.

Time-course analysis of in vivo redox states of OpcA or GFP-tagged CP12ΔC in response to nitrogen deprivation. Proteins were extracted from Anabaena 7120, V-CP12, or H-CP12 strains grown in the absence of combined nitrogen under light conditions (30 µmol photons m⁻² s⁻¹). Red, reduced form; Ox, oxidized form. Protein samples (20–40 µg) were loaded in each lane.

Fig. 7.

Western blot analysis of Trx-m1 and FTR-C in vegetative cells (V), whole cells (W), and heterocysts (H). (A) The purity of heterocysts was checked by immunoblotting using RbcL and NifH antibodies. Proteins extracted from vegetative cells and enriched heterocysts (1 µg) were loaded in each lane. (B) Trx-m1 and FTR-C were detected using each specific antibody. Protein extracts (20 µg) from vegetative cells, whole cells, or heterocysts were loaded per lane.

Fig. 8.

Proposed model of redox cascades in vegetative cells and heterocysts. Our results showed that Trx target proteins are reduced in vegetative cells but oxidized in heterocysts under light conditions. The differences in photosynthetic electron flow and protein expression levels of redox cascade components may affect their redox state.