Christiane Funk

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of δ-aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL−/scpE− strain, whereas PChlide accumulated in the PS I-less/chlL−/scpB− strain. In the PS I-less/chlL− control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.

Novel approach reveals localisation and assembly pathway of the PsbS and PsbW proteins into the photosystem II dimer

A blue‐native gel electrophoresis system was combined with an in organello import assay to specifically analyse the location and assembly of two nuclear‐encoded photosystem II (PSII) subunits. With this method we were able to show that initially the low molecular mass PsbW protein is not associated with the monomeric form of PSII. Instead a proportion of newly imported PsbW is directly assembled in dimeric PSII supercomplexes with very fast kinetics; its negatively charged N‐terminal domain is essential for this process. The chlorophyll‐binding PsbS protein, which is involved in energy dissipation, is first detected in the monomeric PSII subcomplexes, and only at later time points in the dimeric form of PSII. It seems to be bound tighter to the PSII core complex than to light harvesting complex II. These data point to radically different assembly pathways for different PSII subunits.

Expression of ELIPs and PS II-S protein in spinach during acclimative reduction of the Photosystem II antenna in response to increased light intensities

The PS II-S protein and the so-called early light-inducible proteins (ELIPs) are homologous to the chlorophyll a/b-binding (Cab) gene products functioning in light-harvesting. The functional significance of these two CAB homologues is not known although they have been considered to bind pigments and in the case of the PS II–S protein this has been experimentally supported. The role of these two proteins does not appear to be light-harvesting but instead they are suggested to play a role as quenchers of free chlorophyll molecules during biogenesis and/or degradation of pigment-binding proteins. Such a role would be essential to eliminate the toxic and damaging effects that can be induced by free chlorophyll in the light. To this end the expression and characteristics of the ELIPs and the PS II–S protein were investigated in spinach leaves acclimating from low to high light intensities. Under these conditions there is a reduction in the antenna size of Photosystem II due to proteolytic digestion of its major chlorophyll a/b-binding protein (LHC II). During this acclimative proteolysis, up to one third of LHC II can be degraded and consequently substantial amounts of chlorophyll molecules will lose their binding sites. Our results reveal that there is a close correlation between ELIP accumulation and the onset of the LHC II degradation as low light-grown spinach leaves are subjected to increased light intensities. In contrast, there was no change in the relative level of the PS II–S protein during the acclimation process. It is concluded that the role for the ELIPs may be related to binding of liberated chlorophyll molecules and quenching of the toxic effects during LHC II degradation. In addition it was shown that in spinach four different ELIP species can be expressed and that they show different accumulation patterns in response to increased light intensities.

Photoactive protochlorophyllide regeneration in cotyledons and leaves from higher plants.

Abstract Chlorophyll accumulation during greening implies the continuous transformation of photoactive protochlorophyllide (Pchlide) to chlorophyllide. Since this reaction is a light-dependent step, the study of regeneration of photoactive Pchlide under a continuous illumination is difficult. Therefore this process is best studied on etiolated plants during a period of darkness following the initial photoreduction of photoactive Pchlide. In this study, the regeneration process has been studied using spinach cotyledons, as well as barley and bean leaves, illuminated by a single saturating flash. The regeneration was characterized using 77 K fluorescence emission and excitation spectra and high-performance liquid chromatography. The fluorescence data indicated that the same spectral forms of photoactive Pchlide are regenerated by different pathways: (1) photoactive Pchlide regeneration starts immediately after the photoreduction through the formation of a nonphotoactive Pchlide form, emitting fluorescence at approximately 651 nm. This form is similar to the large aggregate of photoactive Pchlide present before the illumination, but it contains oxidized form of nicotinamide adenine dinucleotide phosphate, instead of the reduced form (NADPH), in the ternary complexes; and (2) after the dislocation of the large aggregates of chlorophyllide–light-dependent NADPH:Pchlide a photooxidoreductase–NADPH ternary complexes, the regeneration occurs at the expense of the several nonphotoactive Pchlide spectral forms present before the illumination.

The PsbS Protein: A Cab-protein with a Function of Its Own

Chlorophyll a/b binding proteins (Cab proteins) are the most abundant membrane proteins on earth. The intrinsic PsbS protein of Photosystem II is very peculiar among the family of the Cab proteins. It differs from the conventional light harvesting proteins by an additional putative fourth transmembrane helix. PsbS is able to bind chlorophyll a and b, but unlike other chlorophyll-binding proteins it does not take part in the process of light harvesting. It is present in etiolated plants and seems to be stable also in the absence of pigments. There fore, it was suggested to have a function in transient pigment binding and act as a chlorophyll carrier protein, a role that is also postulated for its relatives, the early light induced proteins (ELIPs). Recently the PsbS protein received broad attention when it was shown, that an Arabidopsis thaliana mutant, which is not able to perform non-photochemical quenching, is deficient in the psbS gene. This chapter provides an overview of the data obtained for the PsbS protein so far, emphasizing its similarities and differences to the Cab-antenna proteins and ELIPs and discusses its possible function.

Engineering of N-terminal threonines in the D1 protein impairs photosystem II energy transfer in Synechocystis 6803

Mutants of the cyanobacterium Synechocystis sp. PCC 6803 with N‐terminal changes in the photosystem (PSII) II D1 protein were analysed by flash‐induced oxygen evolution, chlorophyll a fluorescence decay kinetics and 77 K fluorescence emission spectra. The data presented here show that mutations of the Thr‐2, Thr‐3 and Thr‐4 in D1 do not influence the oxygen evolution. A perturbation on the acceptor side was observed and the importance of the N‐terminal threonines for an efficient energy transfer between the phycobilisome and PSII and for stability of the PSII complex was demonstrated.

D1′ centers are less efficient than normal photosystem II centers

One prominent difference between the photosystem II (PSII) reaction center protein D1′ in Synechocystis 6803 and normal D1 is the replacement of Phe‐186 in D1 with leucine in D1′. Mutants of Synechocystis 6803 producing only D1′, or containing engineered D1 proteins with Phe‐186 substitutions, were analyzed by 77 K fluorescence emission spectra, chlorophyll a fluorescence induction yield and decay kinetics, and flash‐induced oxygen evolution. Compared to D1‐containing PSII centers, D1′ centers exhibited a 50% reduction in variable chlorophyll a fluorescence yield, while the flash‐induced O2 evolution pattern was unaffected. In the F186 mutants, both the P680+/QA − recombination and O2 oscillation pattern were noticeably perturbed.