Photosynthetic modulation during the diurnal cycle in a unicellular diazotrophic cyanobacterium grown under nitrogen-replete and nitrogen-fixing conditions

Abstract

Cyanobacteria are the only oxygenic photosynthetic organisms that can fix nitrogen. In diazotrophic cyanobacteria, the regulation of photosynthesis during the diurnal cycle is hypothesized to be linked with nitrogen fixation and involve the D1 protein isoform PsbA4. The amount of bioavailable nitrogen has a major impact on productivity in aqueous environments. In contrast to low- or nitrogen-fixing (-N) conditions, little data on photosynthetic regulation under nitrogen-replete (+ N) conditions are available. We compared the regulation of photosynthesis under -N and + N conditions during the diurnal cycle in wild type and a psbA4 deletion strain of the unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. We observed common changes to light harvesting and photosynthetic electron transport during the dark in + N and -N conditions and found that these modifications occur in both diazotrophic and non-diazotrophic cyanobacteria. Nitrogen availability increased PSII titer when cells transitioned from dark to light and promoted growth. Under -N conditions, deletion of PsbA4 modified charge recombination in dark and regulation of PSII titer during dark to light transition. We conclude that darkness impacts the acceptor-side modifications to PSII and photosynthetic electron transport in cyanobacteria independently of the nitrogen-fixing status and the presence of PsbA4.

Figure 1

Growth and absorbance of WT Cyanothece sp. ATCC 51142 cultures. (a) Growth was measured for 7 days under 12:12 h, light:dark conditions in ASP2 + N (blue) or ASP2 − N (orange) media. Traces shown are the average of four independent growth experiments. Bar shows alternating 12 h light:dark periods. (b) Representative whole-cell absorption spectra collected at the D10 (solid lines) and L2 (dashed lines) time points from cultures grown in 12 h LD in + N or − N media. Data are normalized to the absorbance at 440 nm.

Figure 2

Fluorescence emission spectra at 77 K collected from WT Cyanothece sp. ATCC 51142 cultures. Spectra were collected at D10 (solid lines) and L2 (dashed lines) time points from cultures grown under 12:12 h, light:dark conditions in ASP2 + N (blue) or ASP2 − N (orange) media. Excitation of Chla at 435 nm (a,c) and phycobilin at 580 nm (b,d). Traces shown are the average of two independent biological replicates. Traces in (a,b) were normalized to PSI fluorescence at 720 nm and (c,d) were normalized to the area under the curve.

Figure 3

Photosystem II quantification and activity measured by following Q_A⁻ reoxidation kinetics in WT Cyanothece sp. ATCC 51142. Data were collected from cultures grown under 12:12 h, light:dark conditions in + N (blue) or − N (orange) media. (a) Fv and (b) Fv/Fm measurements. Fo and Fm values were measured by exposing cells treated with 40 µM DCMU. Error bars indicate standard error of the mean from three independent biological replicates. (c,d) Q_A⁻ reoxidation kinetics to examine acceptor-side events at D10 (solid lines) and L2 (dotted lines) time points were taken by exposing dark-adapted cells to a single blue actinic flash and blue measuring flashes at regular intervals during the measurement in the absence (c) and presence (d) of DCMU. Only 1 s of measurement is presented in (c) to emphasize the fast and intermediate relaxation phases. To further display the initial fast phase, the first 4 ms of measurement is enlarged in the inset in (c). Total 2 ml of culture adjusted to 3 µg ml⁻¹ Chla concentration was used for each measurement. Data are the average of three independent biological replicates.

Figure 4

Photosystem I quantification and redox kinetics in WT Cyanothece sp. ATCC 51142. Data were collected from cultures grown under 12:12 h, light:dark conditions in + N (blue) or − N (orange) media. (a) Absorbance change at 705 nm in dark-adapted cells exposed to actinic light at 630 nm for 5 s in cells adjusted to 3 µg ml⁻¹ Chla in the presence of 40 μM DCMU and 1 μM DBMIB. Data are presented as absorbance change per million cells for D10 and L2 time points. Error bars indicate standard error of the mean from two independent biological replicates. (b) P700 redox kinetics collected from cells at D10 (solid lines) and L2 (dashed lines) time points. Data shown are the average of two independent biological repeats.

Figure 5

Growth of WT and ∆psbA4 strains of Cyanothece sp. ATCC 51142. Growth was measured for 7 days under 12:12 h, light:dark conditions in ASP2 + N (blue) or ASP2 − N (orange) media. Traces shown are the average of four independent growth experiments. Bar shows alternating 12 h light:dark periods.

Figure 6

Photosystem II quantification and activity measured by following Q_A⁻ reoxidation kinetics in the ∆psbA4 strain of Cyanothece sp. ATCC 51142. Data were collected from ∆psbA4 cultures grown under 12:12 h, light:dark conditions in + N (blue) or − N (orange) media. Fv (a) and Fv/Fm (b) measurements. Fo and Fm values were measured by exposing cells treated with 40 µM DCMU. Error bars indicate standard error of the mean from three independent biological replicates. (c,d) Q_A⁻ reoxidation kinetics to examine acceptor-side events at D10 (solid lines) and L2 (dotted lines) time points were taken by exposing dark-adapted cells to a single blue actinic flash and blue measuring flashes at regular intervals during the measurement in the absence (c) and presence (d) of DCMU. Only 1 s of measurement is presented in (c) to emphasize the fast relaxation phase. To further display the initial fast phase, the first 4 ms of measurement is enlarged in the inset in (c). Total 2 ml of culture adjusted to 3 µg ml⁻¹ Chla concentration was used. Data are the average of three independent biological replicates.