Photosystem II cyclic heterogeneity and photoactivation in the diazotrophic, unicellular cyanobacterium cyanothece species ATCC 51142

Abstract

The unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 demonstrated important modifications to photosystem II (PSII) centers when grown under light/dark N2-fixing conditions. The properties of PSII were studied throughout the diurnal cycle using O2-flash-yield and pulse-amplitude-modulated fluorescence techniques. Nonphotochemical quenching (qN) of PSII increased during N2 fixation and persisted after treatments known to induce transitions to state 1. The qN was high in cells grown in the dark, and then disappeared progressively during the first 4 h of light growth. The photoactivation probability, epsilon, demonstrated interesting oscillations, with peaks near 3 h of darkness and 4 and 10 h of light. Experiments and calculations of the S-state distribution indicated that PSII displays a high level of heterogeneity, especially as the cells prepare for N2 fixation. We conclude that the oxidizing side of PSII is strongly affected during the period before and after the peak of nitrogenase activity; changes include a lowered capacity for O2 evolution, altered dark stability of PSII centers, and substantial changes in qN.

Figure 4

O₂ yields from Cyanothece sp. ATCC 51142 using cells harvested at L12 to D6 during growth under N₂-fixing conditions in 12-h light/12-h dark conditions. The samples were stimulated by Xe flashes given at 3 Hz starting 0.1 s after the start of recording. Since the L12 to D0 boundary involved a change in culture flasks, the synchronicity of the two cultures at that point was verified by performing both recordings (12 h apart). The two recordings are superimposed on the graph and are indistinguishable. An arbitrary constant was added to the recordings for readability (a.u., arbitrary unit).

Figure 1

O₂ yields from Cyanothece sp. ATCC 51142 using cells harvested at L0 to L4 during growth under N₂-fixing conditions in 12-h light/12-h dark conditions. The samples were stimulated by Xe flashes given at 3 Hz starting 0.1 s after the start of recording. An arbitrary constant was added to the recordings for readability (a.u., arbitrary units).

Figure 6

PAM fluorescence monitoring of the induction of state 1 in Cyanothece sp. ATCC 51142 at D2 and D3. The fluorescence levels used in calculations are tagged as described in Methods. R, D, F, A, and 1, resting state in the dark, in the presence of DCMU, measurements with a saturating flash, measurements under actinic light, and state 1, respectively. Samples of 1 mL at 2 μg chlorophyll/mL were treated with 10 μm DCMU and the maximum intensity of the red actinic light of the PAM fluorimeter to induce state 1, as per the protocol in Meunier et al. (1997). The actinic light went off just before the flash.

Figure 2

O₂ yields from Cyanothece sp. ATCC 51142 using cells harvested at L6 to L12 during growth under N₂-fixing conditions in 12-h light/12-h dark conditions. The samples were stimulated by Xe flashes given at 3 Hz starting 0.1 s after the start of recording. An arbitrary constant was added to the recordings for readability (a.u., arbitrary units).

Figure 3

O₂ yields from Cyanothece sp. ATCC 51142 at L10 after a dark adaptation (D) and a photoactivation treatment (P). The dark period necessary to prepare the sample and centrifuge the cells to the electrode (about 15 min) was sufficient to deactivate most Mn centers (D). The same cells were left on the electrode and photoactivated by 3 min of flashes given at 10 Hz. After 10 s of darkness, flashes were given at 3 Hz and the amplitude of the O₂ yields were recorded (P). An arbitrary constant was added to the recordings for readability (a.u., arbitrary unit).

Figure 8

Amplitudes of O₂ production under flashing light from Cyanothece sp. ATCC 51142 cells harvested at L8 (A) and D8 (B). A, Curve 1, after controlling light leakages during centrifugation and sample manipulation (⋄); curve 2, after 30 s of darkness following A (▵); curves 3, 4, and 5, each after an additional 30 s of darkness following the previous experiment (○, □, and ▿); curve P, after a 3-min, 10-Hz photoactivation treatment following the experiment (). B, After controlling light leakages during centrifugation and sample manipulation (⋄); after a subsequent 30 s of darkness (▵); after an additional 30 s of darkness following the previous experiment (□ and ○).

Figure 5

Average amplitudes of O₂ production under flashing light in Cyanothece sp. ATCC 51142 after a photoactivation treatment and over a 3-d period starting 108 h after subculture. Black bars, Dark periods; white bars, light periods. The dashed line represents a sine wave with a fixed 24-h period, which had the phase and amplitude fitted to the data using the DeltaGraph program (Delta Point, Inc., Monterey, CA). a.u., Arbitrary units.

Figure 7

Calculations of q_N in 1 mL of Cyanothece sp. ATCC 51142 grown under 12-h light/12-h dark conditions using the levels of fluorescence depicted in Figure 6. Black bar, dark period; white bar, light period. A, Remaining q_N after the induction of state 1 measured with flashes (▪) or with the actinic light (•). B, Total q_N in the dark in the presence of DCMU measured with flashes (□) or with the actinic light (○).

Figure 9

Photoactivation probability, ε, in dark-adapted cells of Cyanothece sp. ATCC 51142 for the last 48 h of the experiment shown in Figure 5 (○). Black bars, dark periods; white bars, light periods. P1, P2, and P3, Significant repeating periods of high photoactivation; the location of the peaks should be taken as the center of mass of the area under the curve (which is effectively spaced 24 h apart, whereas the maximum measured photoactivation probability is not necessarily spaced this way). The modeling errors were represented by the standard deviations calculated from the error matrix returned by the multivariable linear regression over a generic five-step equation. They were found to be smaller than or equal to 0.1% + 0.1 × ε, which are the values shown by the error bars. As many O₂ yields as possible were used for the regression (in most cases, 14); however, the results were insensitive to the number of flashes used and to the inclusion of the first flash. The results were essentially identical to a generic four-step equation ignoring the first flash.