FIGURE 3.

Phosphorylation and assembly states of thylakoid membrane protein complexes after BN-PAGE. (A) Thylakoid protein samples corresponding to 20 μg total chlorophyll were separated by BN-PAGE. Material for the first two wells was isolated from plants grown under LD conditions for 2 weeks. Dark-WL 50: 50 min illumination with WL after dark period. Dark: Sample harvested at the end of the night period. All other plants were grown for 5 days under LD conditions, afterward shifted for 3 days to continuous white light and subsequently grown for 6 days under the light-quality regimes indicated on top (matching in total 2 weeks of growth). These samples served as control allowing for comparison with results published earlier (Dietzel et al., 2011). PSI: 6 days PSI-light; PSI-PSII 30: 6 days PSI-light followed by 30 min PSII-light; PSII: 6 days PSII-light; PSII-PSI 30: 6 days PSII-light followed by 30 min PSI-light. The protein complexes separated by the BN-PAGE were denatured in gel by incubation in Laemmli buffer and transferred to a nitrocellulose membrane via western blot. Phosphorylation state of thylakoid membrane proteins was detected by incubation with anti-phospho-threonine antibodies. The amido-black stained membrane at the height of LHCII trimers is shown as loading control. Bands were labeled as described (Järvi et al., 2011). The experiment was repeated three times with results showing only marginal variations, thus one representative result is given. Note that the phosphorylation states of the free LHCII trimers correspond well to those shown in Figure 2. (B–D) Thylakoid protein samples corresponding to 30 μg total chlorophyll were separated by BN-PAGE. Material separated on the same gel always was isolated from the same growth batch of plants that were all grown under LD conditions for 2 weeks prior to the different short-term illumination treatments indicated on top of each well. Dark samples were harvested at the end of the night period and served as control. All material was harvested directly under the respective light source. (B) Dark - PSII 50: 50 min PSII-light after dark; Dark - PSII 50 - PSI 30: 50 min PSII- light after dark followed by 30 min PSI-light; Dark: control at end of dark period; Dark - WL 50: 50 min WL after dark (same in panels (B) and (C)). (C) Dark - WL 50 - dark 15: 50 min WL after dark followed by a 15 min shift into dark. (D) Dark - WL 2 h: 2 h WL after dark; Dark - WL 2 h - dark 15: 2 h WL after dark followed by a 15 min shift into dark. Panels (B–D) are from individual gels each and display representative results. Each experiment was done at least three times. Bands were labeled (right margin) as described (Dietzel et al., 2011; Järvi et al., 2011). As common control Dark - WL 50 was included in all gels.