Procesamiento e interpretación de los resultados de las entrevistas a los señores,

The steady state level of proteins in wild-type and ∆sppA2 mutant after adaptation to different light intensities was examined. The cells grown under LL were transferred to HL for 72 h. Thylakoid membrane proteins were separated on denaturing 10 - 17.5 % SDS-PAGE and stained by silver. The protein stained analysis showed higher content of phycobiliproteins and other proteins in molecular weight range of 36 and 45 kDa in the ∆sppA2 samples in comparison to the wild-type (Fig. 51).

Figure 51. Protein profile of wild-type and ∆sppA2 mutant acclimated to LL and HL. Wild-type and ∆sppA2 strains were adapted under LL and HL for 36 h. Thylakoid proteins were separated by 10 - 17.5% SDS-PAGE. Proteins were revealed by silver staining. The position of main proteins of ATP synthase (ß subunit), photosystem I (PsaA/B), photosystem II (D1, D2 and CP47) and phycobiliproteins were detected by Western analysis and shown at the right of figure. Molecular weight in kDa is indicated at the left.

To check the content of the major photosynthetic complexes in the ∆sppA2 mutant in comparison to Synechocystis wild-type, thylakoid proteins extracted from cells adapted to LL and HL were tested with antisera against various photosynthetic proteins. In wild-type cyanobacterial cells general response to HL acclimation exhibited the reduction of PBS and degradation of PSII and PSI complexes. No differences in protein amounts from all photosynthetic complexes were observed in the ∆sppA2 mutant compare to the wild-type (Fig. 52). However, ∆sppA2 mutant contained slightly more of phycobiliproteins under HL.

Figure 52. Western analysis of wild-type and

∆sppA2 mutant adapted to LL and HL.

Thylakoid proteins from wild-type and ∆sppA2

mutant were separated on the 12% SDS-PAGE, transferred onto nitrocellulose membrane and incubated with antisera against proteins of ATP synthase (ß subunit), photosystem I (PsaA/B), photosystem II (D1, D2 and CP47) and phycobiliproteins

3.2.2.10 Characterization of wild-type and ∆sppA2 strains after recovery from HL to LL

Physiological analysis revealed that the ∆sppA2 strain exhibited bleaching of cells and a slower growth rate even under standard light conditions. The detection of SppA2 under LL but not under HL proved that this protease is important for LL and recovery from HL to LL. These data suggest that SppA2 can be involved in turnover of proteins essential under HL but not required under LL conditions.

Photoprotection mechanism of cyanobacteria evolves different ways including the synthesis and recruitment of enzymes essential under stress conditions with followed degradation of that after recovery to the standard conditions. One group of proteins that accumulate upon exposure to HL (as well as under other stress conditions, e.g. cold stress, sulfur and nitrogen deprivation) is the HLIP (high light inducible proteins; He et al., 2001). These low molecular weight polypeptides are requires for survival and acclimation of cells to the absorption of excess of light energy, and perhaps could bind and store free chlorophyll specifically when cells are absorbing excess excitation (Havaux et al., 2003). Accumulation of HLIP occurred within 1 h of transfer to HL with maximum peak in abundance over 24 h of light stress and accompanied by decreasing of photosynthetic activities of cells. All HLIP proteins are rapidly degraded during first the hours of recovery of cells from excess excitation (He et al., 2001). To check whether SppA2 could be involved in the recovery step, wild-type and ∆sppA2

strains were characterized after exposure to LL and HL, and after recovery of HL treated cells to LL.

3.2.2.10.1 Photosynthetic activity of cells under different light regimes

The photosynthetic activity of the cells was estimated by measuring the O2 evolution under

different light regimes with the Clark electrode. Cells of wild-type and ∆sppA2 strains grown at LL were exposed to HL for 24 h and then after transferred back to LL for further 24 h. The samples for O2 measurements were taken and diluted to a chlorophyll a concentration of 2

µg/ml. As shown in Fig. 53, photosynthetic O2 evolution in mutant cells was lower than that

measured in the wild-type already under LL. However, the strains adapted to HL showed roughly identical activity of O2 evolution. Measuring the O2 evolution after recovery from HL

to LL showed that both strains could restore photosynthetic activities to LL level, however, in mutant cells this process occurred slower as in wild-type.

Figure 53. Effect of different light intensities on photosynthetic O2

evolution in wild-type and ∆sppA2 strains. For the measurements cells of both strains from different light regimes were concentrated till ncentration

of 2 µg/ml. Net O2

evolut

chlorophyll co

ion of cells was measured with white light of photon flux density 50 µmol m-2 s-1.

3.2.10.2 Biochemical analysis of ∆sppA2 thylakoid proteins under LL and HL regimes

Figure 54 shows comparison of thylakoid membrane proteins from wild-type and ∆sppA2

strains grown under LL, HL and recovery from HL to LL regimes. The separation of proteins on denaturing SDS-PAGE and following silver staining revealed accumulation of unknown low molecular weight polypeptides in the ∆sppA2 as well as in the wild-type under HL. However, in contrast to the wild-type these polypeptides remained stable in the mutant strain after recovery from HL to the LL. The accumulation of polypeptides with molecular masses between 4 and 6 kDa was observed only under HL in the wild-type cells that assumed the light-inducible expression of these proteins.

Figure 54. Separation of thylakoid membrane polypeptides from wild- type and ∆sppA2 strains grown at different light regimes. Cells of both strains were grown at LL, transferred to the HL and then cells were recovered back to LL for 24 h (Rec). Proteins were revealed by silver staining. Arrows indicate the positions of two unknown polypeptides, which are absent in wild- type cells after recovery from HL to LL.