Requisitos previos al análisis por conglomerados

Analysis of oxygen evolution in ∆sll0408showed that PSII functions were impaired when cells were adapted to high light. However, Western analysis showed that

type and mutant. The activity and functionality of PSII depends on the amount of functional complex which is present in thylakoid membranes. It was investigated then whether wild-type and mutant, despite a similar amount of PSII subunits at high light, contained different amounts of functional PSII. The biogenesis of PSII complex can be operationally divided into several steps (Fig. 44):

Assembly

Photodamage, Repair and/or Degradation Functional PSII

Transcription Translation

Assembly

Photodamage, Repair and/or Degradation Functional PSII

Fig. 44. Scheme of PSII biogenesis and maintenance

Since D1 protein has the higher turnover rate than any other thylakoid proteins in light (Mattoo et al., 1984; Ohad et al., 1985), a lot of studies on PSII focused on D1 synthesis, assembly and turnover, both in cyanobacteria and in chloroplasts (Herrin and Michaels, 1985; Kim et al., 1991; Golden, 1994; Tyystjärvi et al., 2001). Following the same idea, the major steps of PSII biogenesis in cTLP40- deficient strain were monitored via the D1 protein.

4.3.1 Analysis of transcription, translation and degradation rate in wild-

type and

∆

It was shown that the amount of psbA mRNA was relatively constant when chloroplasts were adapted to different external conditions (Danon and Mayfield, 1991; Staub and Maliga, 1993; Hirose and Sogiura, 1996). On the other side, the

amount of psbA transcript in Synechocystis was highly dependent on environmental conditions (Mohamed and Jansson, 1989; Tyystjärvi et al., 1996, 2001) and increased several times when cells were acclimated to high light (Tyystjärvi et al., 2001). The reason for this difference in psbA mRNA expression between chloroplasts and Synechocystis resides in the fact that in chloroplasts D1 protein amounts are regulated at the level of transcription initiation, while in

Synechocystis the regulation is at the level of translational initiation that means that the increase of D1 protein amount requires an increase of the correspondent mRNA transcript (reviewed in Baena-Gonzalez and Aro, 2001). First, we checked whether the amount of psbA transcript in mutant ∆sll0408 could explain the lower amount of PSII subunits. In Synechocystis, there are three psbA genes (Williams, 1988), but only psbA2 and psbA3, that encode identical gene products are transcribed (Mohamed and Jansson, 1989; Mohamed et al., 1993; Komenda et al., 2000). The amount of D1 transcript in wild-type and mutant at standard growth conditions and after adaptation to high light were compared with real time PCR (Fig.23). When the cultures were adapted to high light, the amount of D1 transcript increased both in wild-type and mutant as it was previously reported (Tyystjärvi et al., 2001). Relative to the conditions used, psbA transcript increased about seven times in wild-type and three times in the ∆sll0408 mutant. However, the amount of

psbA mRNA was always higher in the mutant under both light conditions. Since the regulation of D1 transcript it is at the level of trascription (reviewed in Baena- Gonzalez and Aro, 2001), a higher amount of transcript suggested a higher requirement of D1 protein in the mutant. This hypothesis was also supported by the analysis of D1 synthesis in the wild-type and mutant strain at standard growth conditions and after short adaptation to high light. Under both light conditions, the synthesis of D1 protein was higher in the mutant than in wild-type according to a higher amount of psbA transcript in the mutant (Fig. 24). Pulse-chase experiments showed that newly synthesised D1 protein is inserted into PSII only when it is required to substitute a damaged one into the complex (Adir et al., 1990; Tyystjäri

et al., 2001). The higher amount of D1 transcript as well as a faster synthesis rate in the mutant under the growth conditions analysed supported the idea of a higher damage rate of PSII in the mutant. This conclusion was supported by measurements of oxygen evolution at high light (Section 4.3.4). In addition,

exchanged in the mutant than in the wild-type. When cells were grown at standard conditions, about 30% of D1 in the wild-type and 50% in the mutant was exchanged within three hours (Fig. 25). If it is considered that the amount of all subunits of PSII is strictly coordinated and regulated, a higher degradation rate of D1 protein in the mutant explained the Western results which showed then, despite a general higher synthesis rate, PSII subunits were reduced in the mutant. When cells were adapted to high light, the degradation rate increased in both strains according to a higher damage rate of D1. During high light adaptation the degradation of D1 was again faster in the mutant even if the difference with the degradation rate in the wild-type was not so pronounced as for standard conditions (55% in wild-type and 65% in the mutant after 3 h of high light adaptation). In this case is not possible to compare this result with that of the Western analysis since the adaptation time to high light was different between the two experiments.

4.3.2 Photosystem II assembly in wild-type and mutant

∆

The distribution of the major PSII subunits in thylakoid membranes was analysed by blue-native and subsequent SDS-PAGE. By this separation, PSII proteins can be found both, in free or assembled forms (Komenda et al., 2004; Herranen et al., 2004). In particular, by short pulse labelling of Synechocystis proteins followed by blue-native/SDS-PAGE separation of thylakoid membrane proteins, it was possible to identify several PSII intermediate in addition to the dimeric form (Komenda et al., 2004). These intermediates are thought to represent steps of PSII assembly or repair and were combined in a model for PSII turnover (Fig. 46) (Komenda et al., 2004). According to this model, the first step of PSII biogenesis comprises the association of the cytochrome b559 subunits, psbE and psbF, with D2 protein. Then

D1 (as pD1, D1 precursor) associates to the complex and after a first D1 processing, CP47 can associate as well. The formation of the D1/D2/CP47 complex (RC47) and the association of CP43 protein, leads to the PSII monomer and then to the dimer (Komenda et al., 2004; Ossenbühl et al., 2004). Before addition of CP43 to the D1/D2/CP47 complex, a second processing step of D1 takes place (Zhang et al., 2001).

The repair cycle of the PSII complex includes the removal of damaged D1 from the D1/D2/CP47 (RC47) complex, and the insertion of a newly synthesised one. It is important to note that according to this model, D1/D2/CP47 complex is an intermediate of both newly assembled and repaired PSII (Komenda et al., 2004).

Assembly D1/ D2 complex D1/D2/CP47 Complex Assembly PSII monomer D1 degradation Dimer

Fig. 45. Model for PSII assembly in Synechocystis. The scheme is adapted

from Komenda et al., (2004). Names of intermediate PSII complexes were modified according to this work. For explanations see the text.

The composition of PSII complexes in the wild-type and mutant strain was first analysed by Western analysis which shows a steady-state amount of PSII proteins in each complex. When cells were cultivated at normal conditions, it was possible to detect proteins in their free or assembled form. The D1/D2 complex was not detected in both strains, indicating that this complex is rapidly converted into the D1/D2/CP47 complex. At high light conditions PSII dimer form was not detectable in the mutant indicating that the amount present was below detection of ECL- based procedure. In addition, the relation between D1/D2/CP47 complex and PSII monomer was higher in the mutant than in the wild-type. Since the D1/D2/CP47 complex represents an assembly and a repair intermediate of PSII, these data

mutant. After acclimation to high light, increased amounts of free PSII proteins were found in the mutant, indicating an elevated requirement of PSII proteins high light. This was already concluded from psbA mRNA analysis and D1 synthesis rate of in the mutant. Radioactive labelling of Synechocystis cells provided new information on the dynamic of PSII assembly. During 25 min labelling under standard growth conditions most of radioactivity was incorporated in two PSII intermediates, D1/D2/CP47 and PSII monomer, in both strains. After high light adaptation, the highest amount of radioactivity was incorporated in PSII monomer in the wild-type, while in the mutant the D1/D2/CP47 complex and PSII monomer were equally synthesised. This finding suggested that while the two strains can assemble PSII until the monomer form with a comparable rate when grown under standard conditions, there is retardation in the assembly between D1/D2/CP47 intermediate and PSII monomer in the mutant after adaptation to high light. These results were confirmed by chase-labelling analysis where, after removal of radioactive methionine, the amount of D1/D2/CP47 complex relative to PSII monomer was higher in the mutant than in the wild-type. A lower amount of PSII dimer in the mutant at high light can depend on a retardation of PSII assembly although instability of PSII dimer (which would increase the amount of D1/D2/CP47 complex because of faster photoinhibition) could not be excluded.

4.4 Different mechanisms of regulation of PSII biogenesis in