Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on DpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.

Heterocyst-forming filamentous cyanobacteria are true multicellular prokaryotes, in which heterocysts and vegetative cells have complementary metabolism and are mutually dependent. The mechanism for metabolite exchange between cells has remained unclear. To gain insight into the mechanism and kinetics of metabolite exchange, we introduced calcein, a 623-Da fluorophore, into the Anabaena cytoplasm. We used fluorescence recovery after photobleaching to quantify rapid diffusion of this molecule between the cytoplasms of all the cells in the filament. This indicates nonspecific intercellular channels allowing the movement of molecules from cytoplasm to cytoplasm. We quantify rates of molecular exchange as filaments adapt to diazotrophic growth. Exchange among vegetative cells becomes faster as filaments differentiate, becoming considerably faster than exchange with heterocysts. Slower exchange is probably a price paid to maintain a microaerobic environment in the heterocyst. We show that the slower exchange is partly due to the presence of cyanophycin polar nodules in heterocysts. The phenotype of a null mutant identifies FraG (SepJ), a membrane protein localised at the cell-cell interface, as a strong candidate for the channel-forming protein.

Using a var2-2 mutant of Arabidopsis thaliana, which lacks a homologue of the zinc-metalloprotease, FtsH, we demonstrate that this protease is required for the efficient turnover of the D1 polypeptide of photosystem II and protection against photoinhibition in vivo. We show that var2-2 leaves are much more susceptible to lightinduced photosystem II photoinhibition than wild-type leaves. Furthermore, the rate of photosystem II photoinhibition in untreated var2-2 leaves is equivalent to that of var2-2 and wild-type leaves, which have been treated with lincomycin, an inhibitor of the photosystem II repair cycle at the level of D1 synthesis. This is in contrast to untreated wild-type leaves, which show a much slower rate of photosystem II photoinhibition due to an efficient photosystem II repair cycle. The recovery of var2-2 leaves from photosystem II photoinhibition is also impaired relative to wild-type. Using Western blot analysis in the presence of lincomycin we show that the D1 polypeptide remains stable in leaves of the var2-2 mutant under photoinhibitory conditions that lead to D1 degradation in wild-type leaves and that the abundance of DegP2 is not affected by the var2-2 mutation. We conclude, therefore, that the Var2 FtsH homologue is required for the cleavage of the D1 polypeptide in vivo. In addition, we identify a conserved lumenal domain in Var2 that is unique to FtsH homologues from oxygenic phototrophs. The Photosystem II (PSII)1 complex is a large protein-pigment assembly that catalyzes the light-dependent oxidation of water to molecular oxygen in chloroplasts and cyanobacteria. At the core of PSII lies the D1/D2 heterodimer, which binds the pigments and co-factors necessary for primary photochemistry (1). The D1 polypeptide is also important because of its high rate of turnover (2). This high turnover rate is related to the vulnerability of PSII to light, with D1 being the main target for photoinactivation and subsequent damage. An efficient repair cycle for D1 is therefore of paramount importance in oxygenic phototrophs. When the rate of photoinactivation and damage of D1 exceeds the capacity for repair, photoinhibition occurs, resulting in a decrease in the maximum efficiency of PSII photochemistry.A key feature of the D1 repair cycle is the degradation of the damaged polypeptide. It is generally accepted that damaged D1 is initially cleaved at a site on the stromal loop between transmembrane helices D and E yielding a 23-kDa N-terminal fragment (3) and a 10-kDa C-terminal fragment (4). This cleavage step is believed to be initiated by structural changes within the D1 polypeptide (5), although the precise nature of the cleavage event remains unclear. One proposal is that the action of active oxygen species acts to cleave the D1 polypeptide during strong illumination (6). However, the temperature dependence of the process (7) and its sensitivity to protease inhibitors (8) indicates the involvement of enzymatic proteolysis by an unidentified protease. Following cleavage, the breakdown fragments of ...

Surprisingly little is known about the physical environment inside a prokaryotic cell. Knowledge of the rates at which proteins and other cell components can diffuse is crucial for the understanding of a cell as a physical system. There have been numerous measurements of diffusion coefficients in eukaryotic cells by using fluorescence recovery after photobleaching (FRAP) and related techniques. Much less information is available about diffusion coefficients in prokaryotic cells, which differ from eukaryotic cells in a number of significant respects. We have used FRAP to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin. GFP was expressed in the cytoplasm, exported into the periplasm using the twin-arginine translocation (Tat) system, or fused to an integral plasma membrane protein (TatA). We show that TatA-GFP diffuses in the plasma membrane with a diffusion coefficient comparable to that of a typical eukaryotic membrane protein. A previous report showed a very low rate of protein diffusion in the E. coli periplasm. However, we measured a GFP diffusion coefficient only slightly smaller in the periplasm than that in the cytoplasm, showing that both cell compartments are relatively fluid environments.

No abstract

When plants, algae, and cyanobacteria are exposed to excessive light, especially in combination with other environmental stress conditions such as extreme temperatures, their photosynthetic performance declines. A major cause of this photoinhibition is the light-induced irreversible photodamage to the photosystem II (PSII) complex responsible for photosynthetic oxygen evolution. A repair cycle operates to selectively replace a damaged D1 subunit within PSII with a newly synthesized copy followed by the light-driven reactivation of the complex. Net loss of PSII activity occurs (photoinhibition) when the rate of damage exceeds the rate of repair. The identities of the chaperones and proteases involved in the replacement of D1 in vivo remain uncertain. Here, we show that one of the four members of the FtsH family of proteases (cyanobase designation slr0228) found in the cyanobacterium Synechocystis sp PCC 6803 is important for the repair of PSII and is vital for preventing chronic photoinhibition. Therefore, the ftsH gene family is not functionally redundant with respect to the repair of PSII in this organism. Our data also indicate that FtsH binds directly to PSII, is involved in the early steps of D1 degradation, and is not restricted to the removal of D1 fragments. These results, together with the recent analysis of ftsH mutants of Arabidopsis, highlight the critical role played by FtsH proteases in the removal of damaged D1 from the membrane and the maintenance of PSII activity in vivo.

Many filamentous cyanobacteria produce specialized nitrogen-fixing cells called heterocysts, which are located at semiregular intervals along the filament with about 10 to 20 photosynthetic vegetative cells in between. Nitrogen fixation in these complex multicellular bacteria depends on metabolite exchange between the two cell types, with the heterocysts supplying combined-nitrogen compounds but dependent on the vegetative cells for photosynthetically produced carbon compounds. Here, we used a fluorescent tracer to probe intercellular metabolite exchange in the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. We show that esculin, a fluorescent sucrose analog, is incorporated by a sucrose import system into the cytoplasm of Anabaena cells. The cytoplasmic esculin is rapidly and reversibly exchanged across vegetative-vegetative and vegetative-heterocyst cell junctions. Our measurements reveal the kinetics of esculin exchange and also show that intercellular metabolic communication is lost in a significant fraction of older heterocysts. SepJ, FraC, and FraD are proteins located at the intercellular septa and are suggested to form structures analogous to gap junctions. We show that a ΔsepJ ΔfraC ΔfraD triple mutant shows an altered septum structure with thinner septa but a denser peptidoglycan layer. Intercellular diffusion of esculin and fluorescein derivatives is impaired in this mutant, which also shows a greatly reduced frequency of nanopores in the intercellular septal cross walls. These findings suggest that FraC, FraD, and SepJ are important for the formation of junctional structures that constitute the major pathway for feeding heterocysts with sucrose.

Time-resolved femtosecond transient absorption measurements have been carried out at room temperature on light-harvesting chlorophyll a/b protein complex of photosystem II (LHC II) trimers prepared from spinach. Exciting in the chlorophyll (Chl) b region at 650 nm with very low intensity, virtually annihilation-free two-color transient absorption measurement of the kinetics over 100 ps, between 645 and 690 nm, yield global lifetimes of 175 fs, 625 fs, and 5 ps and a long component (≥790 ps) where the three fastest lifetimes reflect Chl b to Chl a energy transfer. Using a camera detection system, kinetics over 400 ps at still low annihilation levels and with much higher spectral resolution have been obtained. Short lifetime components of 180 fs, 480 fs, and 6 ps are comparable with the two-color data, but in addition, 34 and 85 ps components with small amplitudes are resolved and a long component (3.6 ns) is fixed at the longest lifetime value determined by fluorescence. Annihilation statistics have been calculated to compare these and earlier results. On the basis of these results and recent electron diffraction structural data, a preliminary three-pool Chl a, three-pool Chl b kinetic model is proposed. The possible influence of variable xanthophyll composition on quenching in LHC II preparations isolated from light- and dark-adapted leaves has been investigated using time-resolved picosecond fluorescence at room temperature. Global lifetimes of 5 ps, 170 ps and 3.6 ns, the lifetimes of the terminal LHC II excited state, were obtained. No discernable quenching effect due to the presence of zeaxanthin was observed.