Figure 5. Photoinhibition is strongly correlated with Δψ but not ΔpH in minira lines.

Photoinhibition, estimated by the q_I fluorescence parameter, is plotted against either the ΔpH or Δψ components of pmf, estimated by the ECS_ss (A) and ECS_inv (B) parameters, as described in Materials and methods. Measurements shown were taken during exposure to 500 μmol photons m⁻²s⁻¹ actinic light (mean ± s.d., n = 3). Two-way analysis of variance (ANOVA) of all combined data, 15 minira lines and wild type, showed a stronger correlation between q_I and Δψ (F = 9.5, p=0.003) than ΔpH (F = 4.05, p=0.05). This correlation is also seen with the expected pH-dependent alterations of P₇₀₀⁺ reduction for the observed partitioning differences from wild type (Figure 5—figure supplement 1) and an increase in the fraction of total pmf stored as Δψ at the expense of ΔpH over multiple light intensities (Figure 5—figure supplement 2). Increased storage of pmf as Δψ is also observed in tobacco ATP synthase knock-down plants (Figure 5—figure supplement 3). ECS units were defined as the deconvoluted ΔA₅₂₀  μg chlorophyll⁻¹ cm². The influence of Δψ on the rate of PSII recombination was estimated based on the change in the equilibrium constant for the sharing of electrons between pheophytin and Q_A (described in Materials and methods) (C). The influence of Δψ on the calculated recombination rate taking into account the fraction of reduced Q_A using the equations described in Materials and methods and described in the main text. Data were obtained at 100 (solid symbols), 300 (half filled symbols), and 500 μmol photons m⁻²s⁻¹ (open symbols) (mean ± s.d., n = 3).

DOI: http://dx.doi.org/10.7554/eLife.16921.031

Figure 5—figure supplement 1. Reduction kinetics of P₇₀₀+.

Light-dependent P₇₀₀⁺ reduction half-times (mean ± s.d., n = 3) of wild type, minira 3–1 and minira 14–1 (A). Quenching of the 810 nm absorbance signal was followed during a brief dark interval and the half time of the first order decay determined at each light intensity.

DOI: http://dx.doi.org/10.7554/eLife.16921.032

Figure 5—figure supplement 2. The electric field component of the pmf dominates under high pmf conditions.

ECS measurements were performed to determine the partitioning of the light-driven pmf between ΔpH and Δψ. Measurements were performed at 100 (solid symbols), 300 (half filled symbols), and 500 μmol photons m⁻²s−¹ (open symbols) and the total pmf (ECS_t) as well as the composition of the pmf determined as in Figure 5. As the total pmf increases, the fraction of pmf stored as ECS_inv (proportional to ΔpH) (A) decreases linearly and is observed in all lines regardless of mutation, while the electric field (ECS_ss) (proportional to Δψ) (B) increases linearly with total pmf, becoming a large fraction of the total pmf under high pmf conditions. Symbols represent the same plant for each light intensity (mean ± s.d., n = 3). ECS units were defined as the deconvoluted ΔA₅₂₀ μg chlorophyll⁻¹ cm².

DOI: http://dx.doi.org/10.7554/eLife.16921.033

Figure 5—figure supplement 3. Tobacco ATPC1 antisense knockdown increase Δψ partitioning under high pmf conditions.

The partitioning of the pmf in wild type Samsun (black) and ATPC1 (red) γ-subunit antisense line were determined from the deconvoluted ECS signal at ΔA_520 nm (A). Following the light-dark transition (time 0 s), the ECS amplitude drop, which is proportional to the total pmf, is larger in the ATPC1 line due to the substantial knockdown of ATP synthase complexes and an inability to efflux protons from the lumen. The inversion of the ECS signal during the dark period, proportional to the ΔpH, represents a larger fraction of the pmf in the wild type than in ATPC1, indicating an increase in Δψ storage in the higher pmf ATPC1. Light conditions are represented above the traces and indicate the light (yellow) and dark (black) intervals. Leaf discs from another ATPC1 plant and Samsun were subjected to 600 μmol photons m⁻²s⁻¹ light for the indicated times in the presence of water (open symbols) or lincomycin (closed symbols). Increases in q_I are more rapid in the higher pmf ATPC1 line when PSII repair is blocked with lincomycin.

DOI: http://dx.doi.org/10.7554/eLife.16921.034