Dynamic control of protein diffusion within the granal thylakoid lumen

Abstract

The machinery that conducts the light-driven reactions of oxygenic photosynthesis is hosted within specialized paired membranes called thylakoids. In higher plants, the thylakoids are segregated into two morphological and functional domains called grana and stroma lamellae. A large fraction of the luminal volume of the granal thylakoids is occupied by the oxygen-evolving complex of photosystem II. Electron microscopy data we obtained on dark- and light-adapted Arabidopsis thylakoids indicate that the granal thylakoid lumen significantly expands in the light. Models generated for the organization of the oxygen-evolving complex within the granal lumen predict that the light-induced expansion greatly alleviates restrictions imposed on protein diffusion in this compartment in the dark. Experiments monitoring the redox kinetics of the luminal electron carrier plastocyanin support this prediction. The impact of the increase in protein mobility within the granal luminal compartment in the light on photosynthetic electron transport rates and processes associated with the repair of photodamaged photosystem II complexes is discussed.

Fig. 1.

Ultrastructural analysis of thylakoid membranes in Arabidopsis chloroplasts. (A, B, D, and E) Low- (A and D) and high- (B and E) magnification cryo-TEM images of vitrified dark- (A and B) and light- (D and E) adapted leaf samples. The stacking repeat unit, which includes the thylakoid lumen, the two encasing membrane bilayers, and the width of one partition gap (white arrow), is marked by a brace. (A, Inset and D, Upper Inset). Defocus images of the grana in which the membrane bilayers (white arrowheads) are clearly discerned. The luminal space is marked by a black arrow. (D, Lower Inset). Scheme depicting the relation between the stacking repeat distance (R), membrane bilayers (M), lumen width (L), and partition gap (P); R = 0.5P + M + L + M + 0.5P. (C and F) Power spectra of grana from dark- (C) and light- (F) adapted leaves. [Scale bars, 200 nm (A and D); 50 nm (B and E, and Insets).]

Fig. 2.

Models of dark- (Left) and light-adapted (Right) granal thylakoids. The structures of PSII and plastocyanin (PC, small globule inside the lumen) are drawn to scale. In the dark, the measured luminal thickness does not allow a face-to-face arrangement of OECs residing on opposite membranes (Lower), indicating that they assume a staggered arrangement (Upper Left). Although expansion of the lumen in the light (Upper Right) may allow for OECs to arrange face to face, adopting such an arrangement upon illumination is unlikely (see text).

Fig. 3.

Models of OEC packing and available diffusion space in the lumen of dark- and light-adapted thylakoids. (A–C) Structure of the OEC (15) sliced near the plane or ∼2 or 3 nm away from the membrane bilayer surface (black and white) and corresponding projections (colored). The yellow and orange contours are mirror images of each other and represent OEC complexes that reside on opposite bilayers of one granum disk. (Scale bars, 10 nm.) (D–F, Upper) Predicted 2D density maps of OECs at the middle of the lumen of dark-adapted (D) or light-adapted (E) thylakoids, or ∼3 nm from one of the membranes in light-adapted thylakoids (F). The distributions were generated using the contours shown in A, B, and C, respectively. (D–F, Lower) Maps depicting the available diffusion area (black) for PC at the middle of the lumen of dark- (D) or light-adapted (E) thylakoids, or near one of the encasing membrane bilayers (F), as illustrated in J–L. Expansion of the lumen in the light allows PC to travel in the gap between an OEC and the opposing membrane surface, further (and substantially) increasing its available diffusion space (L). (G–I) Histograms of areas available for PC diffusion, as derived from the maps shown in D–F, Lower. For dark-adapted thylakoids (G), PC diffusion is restricted to small domains. The space available for PC diffusion increases considerably in light-adapted thylakoids (H) and becomes almost continuous once diffusion near the membrane surfaces is taken into account (L). [Scale bars, 250 nm (D–F); 20 nm (J–L).]

Fig. 4.

Redox kinetics and equilibration between cytochrome f and P700 measured in dark-adapted (A and B) and light-adapted (C and D) leaves. (A) Equilibration plot of cytochrome f and P700 in dark-adapted leaves, generated from relaxation kinetic measurements following a 100-ms light pulse. The dashed line shows the theoretical equilibration curve (K_thermo), calculated using +352 and +472 mV for the redox midpoint potentials of cytochrome f and P700, respectively. The gray delineation of the line represents the range of values reported in the literature for the potentials (22). Error bars represent SE (n = 8). (B) Oxidation kinetics of cytochrome f and P700 induced by a saturating light pulse in dark-adapted leaves. The oxidation kinetics of P700 is monophasic, whereas that of cytochrome f is biphasic, exhibiting clearly distinguished slow and fast components. (C) Equilibration plot of cytochrome f and P700 in light-adapted leaves (black circles). Gray squares show the steady state oxidation levels in leaves exposed to different light intensities, in the absence of methylviologen. Sat, saturated light intensity. (D) Oxidation kinetics of cytochrome f and P700 in light-adapted leaves. Note that the slow cytochrome f component seen in dark-adapted leaves is no longer apparent. The absolute (nonnormalized) cytochrome f amplitudes are virtually identical for light- and dark-adapted samples.