In order to find some basis of salinity resistance in the chloroplastic metabolism, a halophytic Thellungiella salsuginea was compared with glycophytic Arabidopsis thaliana. In control T.s. plants the increased ratios of chlorophyll a/b and of fluorescence emission at 77 K (F730/F685) were documented, in comparison to A.t.. This was accompanied by a higher YII and lower NPQ (non-photochemical quenching) values, and by a more active PSI (photosystem I). Another prominent feature of the photosynthetic electron transport (PET) in T.s. was the intensive production of H2O2 from PQ (plastoquinone) pool. Salinity treatment (0.15 and 0.30 M NaCl for A.t. and T.s., respectively) led to a decrease in ratios of chl a/b and F730/F685. In A.t., a salinity-driven enhancement of YII and NPQ was found, in association with the stimulation of H2O2 production from PQ pool. In contrast, in salinity-treated T.s., these variables were similar as in controls. The intensive H2O2 generation was accompanied by a high activity of PTOX (plastid terminal oxidase), whilst inhibition of this enzyme led to an increased H2O2 formation. It is hypothesized, that the intensive H2O2 generation from PQ pool might be an important element of stress preparedness in Thellungiella plants. In control T.s. plants, a higher activation state of carboxylase ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was also documented in concert with the attachment of Rubisco activase (RCA) to the thylakoid membranes. It is supposed, that a closer contact of RCA with PSI in T.s. enables a more efficient Rubisco activation than in A.t.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) can be divided into two branches: the "red-like type" of marine algae and the "green-like type" of cyanobacteria, green algae, and higher plants. We found that the "green-like type" rubisco from the thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher specificity factor compared with rubiscos of mesophilic cyanobacteria, reaching the values of higher plants, and simultaneously revealing an improvement in enzyme thermostability. The difference in the activation energies at the transition stages between the oxygenase and carboxylase reactions for Thermosynechococcus elongatus rubisco is very close to that of Galdieria partita and significantly higher than that of spinach. This is the first characterization of a "green-like type" rubisco from thermophilic organism.

In the cyanobacterial RuBisCO operon from Thermosynechococcus elongatus the rbcX gene is juxtaposed and cotranscribed with the rbcL and rbcS genes which encode large and small RuBisCO subunits, respectively. It has been suggested that the rbcX position is not random and that the RbcX protein could be a chaperone for RuBisCO. In this study, the RbcX protein from T. elongatus was overexpressed, purified and preliminary functional studies were conducted. The recombinant protein purified from Escherichia coli extracts was predominantly present in a soluble fraction in a dimeric form. Coexpression experiments have demonstrated that RbcX can mediate RbcL dimer (L(2)) formation, and that it is essential for the L(8) core complex assembly. This is the first characterization of the RbcX protein from a thermophilic organism.

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In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present.