DIFFERENTIAL MOBILITY OF PIGMENT-PROTEIN COMPLEXES IN GRANAL AND AGRANAL THYLAKOID MEMBRANES OF C 3 AND C 4 PLANTS

Photosynthetic performance of plants is crucially dependent on the mobility of the molecular complexes that catalyze conversion of sunlight to metabolic energy equivalents in the thylakoid membrane network inside chloroplasts. The role of the extensive folding of thylakoid membranes leading to structural differentiation into stacked grana regions and unstacked stroma lamellae for diffusion-based processes of the photosynthetic machinery is poorly understood. This study examines, for the first time, the mobility of photosynthetic pigment-protein complexes in unstacked thylakoid regions in the C 3 plant Arabidopsis thaliana and agranal bundle sheath chloroplasts of the C 4 plants Sorghum bicolor and Zea mays by the fluorescence recovery after photobleaching technique. In unstacked thylakoid membranes, more than 50% of the protein complexes are mobile whereas this number drops to about 20% in stacked grana regions. The higher molecular mobility in unstacked thylakoid regions is explained by a lower protein packing density compared to stacked grana regions. It is postulated that thylakoid membrane stacking to form grana leads to protein crowding that impedes lateral diffusion processes, but is required for efficient light-harvesting of the modular organized photosystem II and its light harvesting antenna system. In contrast, the arrangement of photosystem I – light-harvesting complex I in separate units in unstacked thylakoid membranes does not require dense protein packing which is advantageous for protein diffusion. constituents.


Introduction
In higher plants, the photosynthetic apparatus is compartmentalized in the specialized chloroplast organelle. The molecular machinery for the primary photosynthetic processes, the sunlight-driven generation of metabolic energy equivalents, is harbored in an intricate thylakoid membrane system within the chloroplasts. Recent improvements in electron tomography have led to 3D models of the complex architecture of thylakoids membranes (Kouril et al. 2011, Daum  An essential feature of the thylakoid membrane system is its high flexibility, which is required for adaptability and maintenance of the photosynthetic machinery in plants. Highly responsive to environmental conditions, both the overall thylakoid architecture (e.g. number of grana discs) and the molecular membrane composition can change remarkably to optimize, protect, and maintain the photosynthetic apparatus (Chuartzman et al. 2008 highlights the evolutionary pressure to preserve this complex structural feature. Recently, the importance of grana formation was highlighted in Arabidopsis mutants, which lack the GDC1 (grana-deficient-chloroplast 1) gene; they grow much slower than wildtype and exhibit seed lethargy due to missing grana formation (Cui et al. 2011). The functional advantages of grana formation have been discussed extensively (e.g. Anderson et al. 2008, Mullineaux 2005, Trissl andWilhelm 1993). It was hypothesized that grana could (i) increase the thylakoid membrane area, and the pigment concentration, in chloroplasts, (ii) avoid energy spillover from PSII to PSI, (iii) regulate the balance of energy distribution between PSII and PSI by state transition, and (iv) enable transversal exciton energy transfer between adjacent grana discs. Although there are good arguments that these possibilities are important for photosynthetic energy conversion, the basis for the evolutionary development of grana has not been determined (Mullineaux 2005, Anderson et al. 2008. A less considered aspect of grana formation is that it leads to a concentration of protein complexes (Murphy 1986, Kirchhoff 2008). The membrane area fraction that belongs to integral photosynthetic protein complexes is about 70%, making grana discs one of the most crowded biomembranes (Kirchhoff 2008). Light-harvesting by photosystem II benefits from a high protein packing density for two reasons. First, a concentration of PSII and LHCII in grana ensures a high concentration of light-absorbing pigments that increase the probability of capturing sunlight, which is a "dilute" energy source on the molecular scale (Blankenhship 2002). Second, it has been demonstrated that a high protein packing density in grana thylakoids is required for efficient intermolecular exciton energy transfer between LHCII and PSII (Haferkamp et al. 2010).
Macromolecular crowding ensures that weakly interacting LHCII and PSII complexes come in close contact, allowing efficient Förster type energy transfer.
Besides these advantages, lateral protein traffic is challenged by macromolecular crowding (Mullineaux 2005, Kirchhoff 2008). The molecular mobility of proteins in grana thylakoids is reduced by numerous collisions of the diffusing object in the 2D reaction space of the membrane with obstacles, integral membrane proteins, that increase computer simulations (Kirchhoff et al. 2004, Tremmel et al. 2003) and by diffusion measurement on isolated grana membranes (Kirchhoff et al. 2008) and intact chloroplasts (Goral et al. 2010 Recently, evidence has accumulated that photoprotective high-energy quenching also requires large-scale diffusion-based structural reorganization within grana thylakoids (Betterle et al. 2009, Johnson et al. 2011).
In contrast to our current understanding of diffusion-based processes in thylakoid membranes, knowledge about the factors that determine the mobility of photosynthetic protein complexes in different thylakoid domains is still fragmentary (Mullineaux 2008).
The protein packing density is very likely a main element that determines protein mobility (Kirchhoff et al. 2008). However, other factors like electrostatic interactions between proteins by membrane surfaces charges (Tremmel et al. 2005) or the size and molecular shape of protein complexes (Tremmel et al. 2003) can contribute significantly.
However, data only exists about protein mobility for isolated grana thylakoids (Kirchhoff et al. 2008) and for chloroplasts from the grana-containing C 3 plant Arabidopsis (Goral et al. 2010). The diffusion characteristics of the latter are almost completely determined by granal proteins. Limiting information on protein diffusion exists for stroma lamellae of C 3 plants (Consoli et al. , Vladimirou et al. 2009) and no data is available for agranal thylakoids, which occur in BS cells of some C 4 species.
The current study fills this gap in the knowledge base by studying lateral protein diffusion in unstacked thylakoid membranes in BS chloroplasts of two NADP-ME type C 4 species, maize and sorghum, in comparison to the grana containing mesophyll chloroplasts. The analysis was also complemented by studies on isolated thylakoid subfragments (grana core, grana and stroma lamellae) from Arabidopsis. The protein mobility was measured by FRAP (Mullineaux and Kirchhoff 2007) which has been shown to be a straightforward method to analyze protein diffusion in photosynthetic membranes by utilizing natural chlorophyll fluorescence (Kirchhoff et al. 2008, Goral et al. 2010). The comparison to diffusion characteristics in unstacked versus stacked membrane areas highlights the significance of grana formation on the lateral mobility of photosynthetic pigment-protein complexes.

Microscopic, spectroscopic and biochemical characterization of chloroplasts in isolated bundle sheath strands and mesophyll protoplasts of maize and sorghum
In NADP-ME type C 4  The lipid composition was measured from total M and BS cells that include extrachloroplast lipids. Information about chloroplast lipids can be extracted from these data by quantification of the three lipid classes; monogalactosyldiacylglycerol (MGDG), digalactosyldiglyceride (DGDG), and sulfoquinovosyl diacylglycerol (SQDG), that are exclusively found in chloroplasts (Benning 2009). Since more than 80% of the chloroplast lipids are found in thylakoid membranes and less than 20% in envelope membranes (Kirchhoff et al. 2002), the quantification of MGDG, DGDG, and SQDG allows estimation of the lipid content in thylakoid membranes. We also quantified the fourth thylakoid lipid class, phosphatidylglycerol (PG) ( Table 1) that is also found in other cell membranes. Since the ratio of chloroplast to extrachloroplast lipids is high, the contamination with non-chloroplast PG is low. Both the relative abundance of the four lipid classes and the lipid/chlorophyll ratio for Arabidopsis (Table 1) are in good agreement with numbers determined for isolated thylakoid membranes from spinach (Kirchhoff et al. 2002). This indicates the validity of the approach to quantify the lipid content for thylakoid membranes from whole cell lipid extracts.
Since virtually all chlorophylls are bound to membrane-integral photosynthetic protein complexes, the chlorophyll content is a good measure of the protein content in thylakoid membranes. Consequently, the thylakoid-lipid/chlorophyll ratio is a good estimate for the protein packing density in thylakoids (Haferkamp and Kirchhoff 2008).
From the lipid/chlorophyll ratios in Table 1, it follows that the protein packing density in agranal sorghum and maize BS strands is significantly lower (higher lipid/chlorophyll ratio) compared to grana containing M cells. The impact of these differences in protein packing on the mobility of photosynthetic pigment-protein complexes in thylakoid membranes is reported in the next sections.

Arabidopsis
For further characterization of a differential protein mobility in stacked and unstacked thylakoid regions, FRAP measurements were performed on isolated stroma lamellae, grana (including grana margins), and grana core subfragments from Arabidopsis plants.
In particular, the comparison of the mobile fractions in isolated stroma lamellae with agranal BS cells is interesting, since the protein organization in these unstacked membranes is different (see Discussion). www The identities of the three membrane domains were verified by their chl a/b ratios, and by their protein composition as analyzed by denaturative gel electrophoresis (SDS-PAGE) (Fig. 5). The chl a/b ratios of 4.92 for stroma lamellae, 2.56 for grana, and 2.34 for grana core are in accordance with literature values for these three thylakoid subdomains (Albertsson 2000). The identity of the three subdomains is further supported by the abundance of α-subunit (~54 kDa, atpA gene product) and β-subunit (~50 kDa, atpB gene product) of the CF 1 part of the ATP synthase complex in stroma lamellae and its depletion in grana and grana core as seen in SDS gels (Fig. 5D) since the ATPase complex is excluded from stacked thylakoid regions by steric hindrance (Albertsson 2001). In addition, densiometric analysis of the AtpA and AtpB bands reveals that the grana core membranes (16% AtpA and 14% AtpB abundance relative to thylakoids) are more depleted in these subunits than in grana preparations (24% and 27%). This indicates that the grana, compared to the grana core, contains a higher amount of ATPasecontaining grana margins (Albertsson 2001), which is in line with the higher chl a/b ratio      protein packing density can be lower. As shown in this study, this is an advantage for lateral protein traffic. Since BS thylakoids in NADP-ME type C 4 plants are optimized to produce mainly ATP by cyclic electron transport, and are depleted in PSII but enriched in PSI, their thylakoid membranes have a protein packing density similar to stroma lamellae, which consequently allows for higher protein mobility.

CONCLUSION
Grana formation leads to a macromolecular crowding that is required for efficient light harvesting by the modularly organized PSII/LHCII system. Optimizing this function seems to be a higher evolutionary priority than allowing a high mobility of grana-hosted protein complexes. It is important to note that although 70% to 80% of the protein complexes in grana are virtually immobile, the remaining protein complexes are very mobile (Kirchhoff et al. 2008). It is likely that the mobile fraction increases under environmental conditions that require brisk lateral protein transport through the crowded grana. In unstacked thylakoid membranes, the need to pack protein complexes tightly is not required because of the non-modular organization of the PSI/LHCI system. Thus, the physicochemical forces that govern tight protein packing in grana stacks are not operative in unstacked regions, leading to a lower protein packing density and higher protein mobility. Obviously, high protein packing densities are only formed if required, e.g. for efficient light-harvesting, but high packing densities do not occur if not needed due to interference in the mobility of membrane constituents.  Bundle sheath (BS) strands were isolated mechanically and M protoplasts enzymatically from maize and sorghum, following a process similar to that described in Sheen (1995)

Membrane preparations
Arabidopsis thaliana plants were grown in soil under short-day conditions (9 h light/15 h dark) cycles with 100 PPFD at a constant temperature of 20°C in a growth chamber.
Leaves of 4-to 6-week-old plants were harvested at the end of the dark period and isolation procedures were carried out in darkness under dim green light in a cold room. were isolated by mechanical fractionation of isolated thylakoids followed by aqueous two-phase partition according to Svensson and Albertsson (1989).

Lipid analysis
BS strands or protoplasts from maize and sorghum or Arabidopsis leaves were ground in liquid nitrogen. The organic components were extracted with chloroform/methanol (2:1, v/v) and separated from aqueous components by a two-phase system established by addition of a 1 M KCl solution. The green organic phase was harvested, dried completely with nitrogen gas, and dissolved in chloroform (lipid extract). Lipids were separated by two-dimensional thin layer chromatography (TLC) and stained by bathing the TLC plates in 10% copper sulphate and 7.5% phosphoric acid followed by heating at 170° C as

Protein Analysis
BS strands or mesophyll protoplasts from maize, sorghum, or Arabidopsis leaves were homogenized in a protoplast buffer (without enzymes) with a Brinkman homogenizer then ground in liquid nitrogen. The extract was pottered for 12 plunges then filtered through an 80 μm followed by a 20 μm mesh. The eluate was centrifuged at max speed with Eppendorf 5417R table centrifuge (25000 x g). The pellet was then resuspended in dH20 and subjected to a membrane pretreatment (exposure to 5M NaSCN to bind extrinsic proteins) according to (Fiedler et al 1994). Protein concentration is then determined using a standard Lowry assay via. Folin−Ciocalteu Reagent. We determined that the lipid/protein ratio increases with increasing amounts of membranes used for protein determination. Therefore, all protein determinations were performed at chlorophyll concentrations between 100 to 150 μg/mL. In this concentration, range the lipid/chlorophyll ratio levels off.

FRAP measurements
Mesophyll protoplast or bundle sheath samples were directly placed on a glass slide and covered with a cover slip. For measurements with isolated grana core, grana and stroma lamellae of Arabidopsis the glass slide was covered with a bilayer of phosphatidylcholine (PC) to avoid artificial glass-membrane interactions (Kirchhoff et al. 2008). For isolated stroma lamellae, the mobile fraction with and without the PC-support system was 55% and 33.5%, respectively. This indicates that the glass membrane interactions impede protein mobility and it highlights the importance of using a fluid support system to www               μ g of chlorophyll per lane was loaded into each well. Apparent molecular weights were estimated by co-electrophoresis of a low molecular weight protein standard (MW) (Invitrogen). Bands for the ATPase subunits (AtpA and AtpB protein) as an indicator of unstacked stroma lamellae are depicted. In addition, the LHCII band is boxed as an indicator of stacked grana. MW is given in kDa.

Figure 6
Dependency of the mobile fraction determined from FRAP measurements on the lipid to chlorophyll ratio (A) or the lipid to protein ratio (B). Data represent the mean with standard deviation. Further statistical information for the mobile fraction are given in Fig. 4 and for the lipid/chlorophyll data in Table 1. The lipid/protein data were collected from two to four independent measurements. The regression line for A is y = -17.3 + 20.8•x (r 2 = 0.973). For B the regression equation is is y = -6.9 + 663.1•x (r 2 = 0.973).
A B