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. 2023 Mar 15;12(6):907.
doi: 10.3390/cells12060907.

Action of 2,6-Dichloro-1,4-benzoquinone on the O2-Evolving Activity of Photosystem II in Chlamydomonas reinhardtii Cells with and without Cell Wall: Inhibitory Effect of Its Oxidized Form

Vasily V Terentyev  1 , Anna K Shukshina  1 , Angelina A Chetverkina  1
Affiliations

Action of 2,6-Dichloro-1,4-benzoquinone on the O2-Evolving Activity of Photosystem II in Chlamydomonas reinhardtii Cells with and without Cell Wall: Inhibitory Effect of Its Oxidized Form

Vasily V Terentyev et al. Cells. .

Abstract

Chlamydomonas reinhardtii is a widely used object in studies on green algae concerning both photosynthesis aspects and possible biotechnological approaches. The measurement of the maximum O2 evolution by photosystem II (PSII) in living algal cells in the presence of artificial acceptors is one of the commonly used methods for determining the photosynthetic apparatus state or its change as compared to a control, parent strain, etc., because PSII is the most sensitive component of the thylakoid membrane. The present study shows the need to use low concentrations of 2,6-dichloro-1,4-benzoquinone (DCBQ) paired with potassium ferricyanide (FeCy) for achieving the maximum O2 evolution rate, while a DCBQ concentration above certain threshold results in strong suppression of O2 evolution. The required DCBQ concentration depends on the presence of the cell wall and should be exactly ~0.1 mM or in the range of 0.2-0.4 mM for cells with and without a cell wall, respectively. The inhibition effect is caused, probably, by a higher content of DCBQ in the oxidized form inside cells; this depends on the presence of the cell wall, which influences the efficiency of DCBQ diffusion into and out of the cell, where it is maintained by FeCy in the oxidized state. The possible mechanism of DCBQ inhibition action is discussed.

Keywords: 2,6-dichloro-1,4-benzoquinone (DCBQ); Chlamydomonas; O2 evolution rate; cell wall; photosystem II.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Transmitted images of algal cells obtained with confocal microscopy for determination of Chl autofluorescence (Chl) and fluorescence of Calcofluor white (CW). CC-503 and 137c are strains of С. reinhardtii without and with a well-formed cell wall, respectively. The scale bar is 10 µm.
Figure 2
Figure 2
Dependence of O2 evolution rate on DCBQ concentration in the presence of 0.5 mM FeCy observed in living cells of CC-503 (blue squares) and 137c (green circles) strains of C. reinhardtii. The results are presented as normal (A) and double-reciprocal (B) plots. The average values of A were used to calculate the values of B. SDs (n = 5) are shown as bars in A.
Figure 3
Figure 3
Curves of O2 evolution observed in the suspension of living cells of C. reinhardtii strains: CC-503 (A) and 137c (B). Blue and green (CC-503 and 137c, respectively) lines correspond to cells without additions, orange lines refer to cells in the presence of 0.5 mM FeCy, red lines refer to cells in the presence of 0.5 mM FeCy and 0.2 mM or 0.1 mM DCBQ in the case of CC-503 or 137c cells, respectively. ↑ and ↓ indicate approximate moments when the actinic light was switched on and switched off.
Figure 4
Figure 4
Dependence of the O2 evolution rate on DCBQ concentration in the absence of FeCy (solid lines) and after the addition of 0.5 mM FeCy to the same sample with repeated measurement of O2 evolution (dashed lines) in living cells of CC-503 (blue squares) and 137c (green circles) strains of C. reinhardtii. Arrows indicate the direction of changes that occurred after FeCy addition in repeated measurements of O2 evolution. SDs (n = 5) are shown as bars.
Figure 5
Figure 5
Light-induced reduction in DCBQ in the presence of living algal cells of C. reinhardtii. Panels (A,B), referring to the CC-503 and 137c, respectively, show the absorption changes in cell suspensions (green curve) under the addition of 30 µM DCBQ (red curve) and following illumination of the samples. The disappearance of the peak at 272–273 nm indicates DCBQ reduction over time. Panel (C) represents the absorption decrease at 272 nm under illumination in the case of CC-503 (blue-filled squares) and 137c (green-filled circles) cells over time. The unfilled blue square and green circle show the values after the addition of 0.2 mM FeCy. The gray unfilled squares and circles represent the CC-503 and 137c cells, respectively, after the addition of 30 µM DCBQ but without illumination. SDs (n = 3–4) are shown as bars.
Figure 6
Figure 6
A scheme of the proposed cycle of DCBQ molecules diffusion in living cells of C. reinhardtii without (CC-503) and with (137c) a cell wall. Actions of PSII in the chloroplast and extracellular FeCy in the cycle are indicated by green and yellow arrows, respectively. The thickness of the straight blue arrows reflects the difference in diffusion efficiency of DCBQ molecules moving inside the cell and going back. The sizes of the “DCBQ” writing indicate possible differences between the contents of the oxidized (ox) and reduced (red) forms of DCBQ, respectively, inside and outside the algal cell.
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