PSII ASSEMBLY IN CHLOROPLASTS OF GREEN ALGAE AND PLANTS

The data presented in this work addresses the steps and mechanisms of PSII assembly in the cyanobacterium Synechocystis 6803. Chloroplasts of green algae and plants are generally accepted to be the evolutionary result of the engulfment of an ancient relative of nowadays cyanobacteria by an eukaryotic cell, known as the endosymbiotic theory (Gould et al., 2008). Hence, the main features and properties of PSII structure and assembly are conserved from cyanobacteria to higher plants (Figure 12). However, during evolution of nowadays chloroplasts, the majority (> 90%) of genes encoded by the endosymbiont has been transferred to the nuclear genome. As a consequence, the respective proteins are synthesized in the cytosol and have to be re-imported into the chloroplast post-translationally. About 50-200 proteins (dependent on the organism) are still encoded for in the chloroplast, resulting in formation of organellar protein complexes with subunits derived from different cell compartments (Barkan, 2011). In the case of PSII, the core subunits D1, D2, cyt b559, CP43 and CP47 as well as several low molecular weight subunits are still encoded by the chloroplast genome, whereas other low molecular weight subunits have to be imported from

Figure 12: PSII subunit composition in cyanobacteria and higher plant chloroplasts. The core of the complex is in both cases built by the D1 and D2 subunits surrounded by the inner antenna proteins CP47 and CP43. Besides the distinct peripheral antennae – soluble phycobilisomes in cyanobacteria (left; in blue) and membrane-intrinsic LHC complexes in plant chloroplasts (right, in dark green) – also differences in the composition of extrinsic proteins (pink) and low molecular mass subunits (grey) can be observed. Proteins encoded by the nuclear genome in plants are indicated by blue lettering. TM, thylakoid membrane. Adapted from Nickelsen and Rengstl, submitted (see appendix).

the cytosol (Figure 12; Allen et al., 2011). Hence, during PSII assembly, tight coordination of chloroplast and nuclear gene expression is necessary to ensure sufficient and reasonable availability of all subunits (Barkan, 2011).

Several PSII assembly factors are conserved throughout the green lineage, for example Alb3/Slr1471 (Moore et al., 2000; Ossenbühl et al., 2004; Göhre et al., 2006; Ossenbühl et al., 2006), HCF136/YCF48 (Meurer et al., 1998; Plücken et al., 2002; Komenda et al., 2008), PAM68/Sll0933 (see section 3.1), Psb28 (Dobáková et al., 2009; Shi et al., 2012) and Psb29/Thf1 (Wang et al., 2004; Keren et al., 2005b; Huang et al., 2006). However, also several exceptions have been described: some PSII assembly factors, e.g. LPA1 and LPA3, are present in chloroplasts of plants and green algae, but have no homologs in cyanobacteria, whereas others including LPA2 and HCF243 – a protein involved in stabilization of D1 in A. thaliana – are even restricted to higher plants (see Table 1 in Nickelsen and Rengstl, submitted, see appendix; Peng et al., 2006; Ma et al., 2007; Cai et al., 2010; Zhang et al., 2011). The presence of plant chloroplast-specific facilitating factors might reflect the different evolutionary development of chloroplasts from vascular plants compared to the prokaryotic cyanobacteria, as they had to adapt to other external influences and living conditions: During evolution, plants have developed a predominantly land-based life and thus have to cope with different environmental situations, which could be a reason for development of minor evolutionary changes in PSII composition and regulation of its assembly compared to cyanobacteria.

Interestingly, the existence of specialized PSII assembly proteins is not restricted to chloroplasts, as several factors are exclusively present in cyanobacteria, indicating that they have either been lost in chloroplasts of algae and plants during evolution or that they have been developed in cyanobacteria after the lineages spread (see Table 1 in Nickelsen and Rengstl, submitted, see appendix). The best studied example is the PratA protein, which does not possess any sequence homologs in e.g. C. reinhardtii or A. thaliana. Due to its important function in cyanobacteria in membrane organization required for PSII assembly, and as Mn2+ binding and transporting protein, it is likely that its role has been taken over by other proteins in chloroplasts. In this regard, a good candidate is represented by LPA1, which belongs, like PratA, to the family of TPR proteins and was shown to be able to directly bind to D1 as well (Peng et al., 2006). Initial analyses indeed revealed Mn2+ binding activity for LPA1 – at least in vitro (see section 3.3). However, substantial further analyses are required to clarify the mechanism of Mn4Ca cluster assembly and membrane organization in general and especially the role of LPA1 in A. thaliana during these processes.

Whereas the spatial organization of PSII de novo assembly initiation seems to be generally conserved in cyanobacteria and green algae (see section 4.4; Uniacke and Zerges, 2007), it is still unclear whether such an organization also exists in chloroplasts of higher plants. One striking difference is represented by the multicellularity of plants: whereas in unicellular photosynthetic organisms, PSII de novo assembly and repair occur (i) in the same cell and (ii) at the same time, these processes are – at least to a great extent – temporarily separated in plants. During plant development, their chloroplasts originate from undifferentiated proplastids at the shoot apex, which do not contain TMs and, thus, represent de novo TM/PSII biogenesis (Charuvi et al., 2012). Once the chloroplast TM system is fully developed, membrane and complex organization is largely sustained and – in case of PSII – complex dynamics are predominantly determined by operation of the repair cycle (Mulo et al., 2012). Hence, due to this temporal separation of both processes, a spatial differentiation does not necessarily have to be realized to the same extent in plants as in cyanobacteria, but more work is required to verify or disprove this hypothesis.

The exact site of PSII assembly in chloroplasts of higher plants and the existence of PDM-like biogenesis centers remain elusive; however, there is evidence that their TM system depicts a heterogeneous distribution. In these organisms, TMs appear either in non-appressed lamellar sheets or as appressed grana stacks (Arvidsson and Sundby, 1999). Photosynthetic protein complexes are not distributed evenly throughout this complex TM system, creating a lateral heterogeneity of TMs: Whereas PSII and LHCII (the light-harvesting complex of chloroplast PSII) are found in grana thylakoids, PSI, LHCI (the light-harvesting complex of chloroplast PSI) and ATP synthase are – likely due to steric reasons – located in stroma lamellae. Cyt b6f complexes are, on the other hand, present in both membrane types (Dekker and Boekema, 2005). Formation of grana stacks is thought to be mainly caused by electrostatic interactions between LHCII, although minor LHCs and PSII reaction centers may also play a role (Standfuss et al., 2005; Daum et al., 2010). Furthermore, analysis of distribution of PSII complexes, including the assembly intermediates RC and RC47, revealed that 80% of PSII can be found in the grana, especially the grana core fraction, whereas PSII supercomplexes were exclusively detected in grana TMs. The less the membranes are stacked, the lower is the proportion of PSII [2] and, concomitantly, the higher is the percentage of PSII [1], RC47 and RC complexes (Danielsson et al., 2006). Hence, this is a hint that PSII complexes might be assembled in non-appressed TM regions. This assumption of synthesis and assembly of membrane proteins taking place at the stromal lamellae of TMs is supported by the finding that ribosomes bind to the non-stacked membrane regions and proteins are inserted into the

membrane co-translationally (Yamamoto et al., 1981). Whether PSII assembly in higher plants starts at specialized domains within the stroma thylakoids comparable to the situation of biogenesis centers and T-zones in Synechocystis 6803 and C. reinhardtii, respectively, remains an issue for future work.

Further indications for a spatial organization of pigment/protein complex assembly is given by results on sublocalization of pigment synthesis enzymes in chloroplasts. Whereas early steps of chlorophyll synthesis take place in the chloroplast stroma, subsequent reactions converting protoporphyrinogen IX to chlorophyll a were reported to occur at the chloroplast envelope and TMs. The chlorophyll synthase enzyme using phytyl pyrophosphate as substrate for esterification of chlorophyllide a to chlorophyll a has even been exclusively detected in TMs (Figure 7; Soll et al., 1983; Eckhardt et al., 2004; Czarnecki and Grimm, 2012). Thus, chlorophyll synthesis in plants seems to underlie a strict compartmentalization. On the other hand, carotenoid synthesis in chloroplasts was reported to be almost restricted to envelope membranes, thereby raising the question how transport to the TM system is mediated (Joyard et al., 2009). Nonetheless, whether the organization of pigment synthesis in higher plant chloroplasts is directly linked to spatial sublocalization of PSII assembly, as it seems to be the case in biogenesis centers of Synechocystis 6803 (see section 4.1), remains unknown.

Besides synthesis and assembly of proteins and pigments, biogenesis of TMs additionally requires high rates of lipid synthesis, especially for newly synthesized TMs in developing chloroplasts. Interestingly, final steps of biosynthesis of TM lipids have been reported to occur in envelope membranes, thus necessitating either a transport system or a direct connection between the different membranes (Kelly and Dormann, 2004). In C. reinhardtii, it was suggested that TMs develop by local expansion and invagination of the inner envelope membrane, although it remains to be clarified whether the membrane systems are directly linked or whether the lipids are transferred to the TMs as vesicles (Hoober et al., 1991). Similar observations have been made in developing proplastids of higher plants, where the inner envelope and the TMs indeed seem to be connected (Mühlethaler and Frey-Wyssing, 1959). In mature chloroplasts, however, such connections are only observed rarely (Shimoni et al., 2005) and lipid transfer from the envelope to TMs rather may occur by a vesicle trafficking system (Garcia et al., 2010; Vothknecht et al., 2012).