Phycobilisome diffusion is required for light-state transitions in cyanobacteria

Abstract

Phycobilisomes are the major accessory light-harvesting complexes of cyanobacteria and red algae. Studies using fluorescence recovery after photobleaching on cyanobacteria in vivo have shown that the phycobilisomes are mobile complexes that rapidly diffuse on the thylakoid membrane surface. By contrast, the PSII core complexes are completely immobile. This indicates that the association of phycobilisomes with reaction centers must be transient and unstable. Here, we show that when cells of the cyanobacterium Synechococcus sp. PCC7942 are immersed in buffers of high osmotic strength, the diffusion coefficient for the phycobilisomes is greatly decreased. This suggests that the interaction between phycobilisomes and reaction centers becomes much less transient under these conditions. We discuss the possible reasons for this. State transitions are a rapid physiological adaptation mechanism that regulates the way in which absorbed light energy is distributed between PSI and PSII. Immersing cells in high osmotic strength buffers inhibits state transitions by locking cells into whichever state they were in prior to addition of the buffer. The effect on state transitions is induced at the same buffer concentrations as the effect on phycobilisome diffusion. This implies that phycobilisome diffusion is required for state transitions. The main physiological role for phycobilisome mobility may be to allow such flexibility in light harvesting.

Figure 1.

The 77 K fluorescence emission spectra for cells of Synechococcus 7942 adapted to State 1 or to State 2. Cells were adapted to State 1 (black line) by incubation in red light or to State 2 (gray line) by incubation in the dark before freezing in liquid nitrogen. Fluorescence spectra were recorded with excitation at 600 nm and normalized to the phycocyanin fluorescence peak (654 nm).

Figure 2.

The 77 K fluorescence emission spectra for cells of Synechococcus 7942 in 0.5 m phosphate buffer. Fluorescence spectra recorded with excitation at 600 nm and normalized to the phycocyanin fluorescence peak (654 nm). Cells were adapted to red light (black line) or to dark (gray line) before addition of phosphate buffer. A, Cells readapted to red light after addition of phosphate buffer. B, Cells readapted to dark after addition of phosphate buffer.

Figure 3.

Fixation of cells in State 1 and State 2 as a function of phosphate concentration. Cells were adapted to State 1 (black) or to State 2 (gray) before addition of phosphate buffer. Cells were then adapted to the opposite light regime before being frozen for 77 K fluorescence spectroscopy. Fixation of light state is calculated from F₆₈₅/F₆₅₄ from 77 K spectra. Fixation in State 1 is defined as (LD−DD)/(LL−DD) and fixation in State 2 is defined as (DL−DD)/(LL−DD), where LD is F₆₈₅/F₆₅₄ for cells adapted to red light and then readapted to dark after addition of phosphate buffer, DL is F₆₈₅/F₆₅₄ for cells adapted to dark and then readapted to red light after addition of buffer, and so on. Mean data from five samples is presented. sds are shown by the error bars.

Figure 4.

The 77 K fluorescence emission spectra for cells of Synechococcus 7942 in 0.2 m phosphate buffer. Fluorescence spectra recorded with excitation at 600 nm and normalized to the phycocyanin fluorescence peak (654 nm). Cells were adapted to red light (black line) or to dark (gray line) before addition of phosphate buffer. A, Cells readapted to red light after addition of phosphate buffer. B, Cells readapted to dark after addition of phosphate buffer.

Figure 5.

Effect of phosphate buffers on the kinetics of state transitions. Cells were dark adapted (State 2) prior to addition of phosphate buffer. Cells were then illuminated in the presence of DCMU as described in “Materials and Methods.” The faster phase of the fluorescence rise (appearing immediate on this timescale) has been subtracted. Fluorescence is expressed relative to this initial fluorescence for cells with no added phosphate.

Figure 6.

FRAP image sequence for a cell of Synechococcus 7942 in growth medium. Confocal fluorescence images taken from a typical FRAP sequence. Scale bar = 5 μm. Fluorescence from the phycobilisomes is imaged. After recording the prebleach image, a line was bleached across the cell by increasing the laser power and scanning the laser for 2 s in the X-direction. The laser power was then decreased again and a series of images were recorded. For each image, the time after recording the first post-bleach image is shown.

Figure 7.

FRAP image sequence for a cell of Synechococcus 7942 in 0.5 m phosphate buffer Confocal fluorescence images taken from a typical FRAP sequence. Scale bar = 5 μm. Fluorescence from the phycobilisomes is imaged. After recording the prebleach image, a line was bleached across the cell by increasing the laser power and scanning the laser for 2 s in the X-direction. The laser power was then decreased again and a series of images were recorded. For each image, the time after recording the first post-bleach image is shown.

Figure 8.

Phycobilisome diffusion coefficients in Synechococcus 7942 cells in different concentrations of phosphate buffer. Diffusion coefficients are means from measurements on six cells. sds are shown by the error bars.