Figure 5.

In Vitro Reconstitution of Active NDH in crr31-1 Ruptured Chloroplasts.

(A) Schematic model of Fd-dependent PQ reduction in ruptured chloroplasts. AA inhibits PGR5-PGRL1 complex-dependent PQ reduction.

(B) to (H) Fd-dependent PQ reduction activities were measured in the presence of 5 μM AA. a.u., arbitrary units.

(B) Fd-dependent PQ reduction activity in ruptured chloroplasts isolated from various genotypes as indicated. Increase in chlorophyll fluorescence level (Fo′) was monitored after consecutive addition of 0.25 mM NADPH and 5 μM Fd under weak illumination (1 μmol photons m⁻² s⁻¹) in osmotically ruptured chloroplasts (20 μg chlorophyll mL⁻¹). The increase in fluorescence reflects NDH-dependent PQ reduction in the presence of AA. WT, wild type.

(C) Restoration of Fd-dependent PQ reduction activity in ruptured chloroplasts isolated from crr31-1 by the addition of GST-CRR31. Ruptured chloroplasts from crr31-1 were incubated with 5 μM GST or GST-CRR31 for 5 min prior to the consecutive addition of NADPH and Fd.

(D) and (E) Restoration of Fd-dependent PQ reduction activity with GST-CRR31 depends on NDH. Ruptured chloroplasts from crr31-1 and crr23/ndhl (D) and from crrj-1 and crrl-1 (E) were preincubated with 1 μM GST-CRR31 for 5 min, and the reaction was started as in (B).

(F) to (H) Fd concentration dependence of PQ reduction in ruptured chloroplasts isolated from the wild type (F), crr31-1 (G), and crr31-1 in the presence of 1 μM GST-CRR31 (H). Fd concentrations added to the reaction are indicated to the right of each curve.