Lipid Polymorphism of the Subchloroplast—Granum and Stroma Thylakoid Membrane—Particles. I. 31P-NMR Spectroscopy

Build-up of the energized state of thylakoid membranes and the synthesis of ATP are warranted by organizing their bulk lipids into a bilayer. However, the major lipid species of these membranes, monogalactosyldiacylglycerol, is a non-bilayer lipid. It has also been documented that fully functional thylakoid membranes, in addition to the bilayer, contain an inverted hexagonal (HII) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31P-NMR spectroscopy experiments on sub-chloroplast particles of spinach: stacked, granum and unstacked, stroma thylakoid membranes. These membranes exhibited similar lipid polymorphism as the whole thylakoids. Saturation transfer experiments, applying saturating pulses at characteristic frequencies at 5 °C, provided evidence for distinct lipid phases—with component spectra very similar to those derived from mathematical deconvolution of the 31P-NMR spectra. Wheat-germ lipase treatment of samples selectively eliminated the phases exhibiting sharp isotropic peaks, suggesting easier accessibility of these lipids compared to the bilayer and the HII phases. Gradually increasing lipid exchanges were observed between the bilayer and the two isotropic phases upon gradually elevating the temperature from 5 to 35 °C, suggesting close connections between these lipid phases. Data concerning the identity and structural and functional roles of different lipid phases will be presented in the accompanying paper.


Introduction
In plants, the light reactions of photosynthesis occur in chloroplast thylakoid membranes (TMs), flattened lipid vesicles which separate the inner luminal and the outer, stroma-side aqueous phases. These membranes embed the two photosystems (PSs) PSII and PSI, containing the photochemical reaction center core complexes and their lightharvesting antenna proteins (LHCII and LHCI, respectively), the cytochrome b 6 f complex and some additional components of the electron transport system, and the ATP-synthase.
The coexistence of the bilayer phase with an isotropic phase in fully functional isolated plant thylakoid membranes has been first demonstrated by Krumova and coworkers [24] by employing 31 P-NMR spectroscopy. The use of this technique for fingerprinting the phase behavior of phospholipids in native and artificial membranes has been thoroughly documented [25][26][27]. In TMs, PG is the only phospholipid, about 60% of which is present in the bulk phase [28]; it has been shown to be a sensitive marker of the lipid phases [29,30]. It is important to stress that there is no lateral heterogeneity of the distribution of PG and all other lipids in the bulk phase of thylakoid membranes [31]. Further, data from molecular dynamics simulations on thylakoid lipids also revealed "a well-mixed system in both the lamellar and inverted hexagonal state" [10].
Our more recent 31 P-NMR spectroscopy experiments have revealed the presence of two isotropic phases and an H II phase, in addition to the bilayer phase [32]. The heterogeneity of the packing of lipids has also been confirmed using time-resolved fluorescence spectroscopy using the lipophylic dye Merocyanine 540 [32,33]. The lipid phases of TMs have been shown to undergo different, largely reversible reorganizations, induced by varying the temperature, the pH, and the ionic and osmotic strengths of the medium. These variations in the polymorphic phase behavior of lipids were associated with characteristic changes in the macro-organization of proteins, were correlated with the fine-tuning of the permeability of membranes, and appeared to regulate the photoprotective activity of the water-soluble lipocalin-like enzyme, the violaxanthin de-epoxidase (VDE) [34][35][36]. These observations are in harmony with the basic predictions of the DEM of TMs and with the tentative assignments of the non-lamellar lipid phases. The H II phase was proposed to be formed by lipids expelled from the bilayer, the two isotropic phases were hypothesized to indicate the presence of VDE-lipid (and possibly other lipocalin-lipid) assemblies, and to arise from the interwoven network of granum and stroma TMs, containing regions where membranes fuse together [37] or divide by forking [38,39]. However, the origin of the different lipid phases, in terms of structural entities of the TM system, has remained elusive.
Here, using 31 P-NMR spectroscopy, we investigated the lipid polymorphism of the two main membrane constituents of plant TM systems, the granum and stroma TMs. These sub-chloroplast membrane particles possess strikingly different protein compositions [40], which, as inferred from the LPM and the FSM, might require different lipid polymorphisms for their optimal functioning. However, we show that both of these membrane fractions exhibit four distinct lipid phases, resembling the polymorphism of TMs; lipase and heat treatments of granum and stroma TMs induced marked and specific effects on the lipid phases. In the accompanying paper, to obtain information on the origin and significance of TM lipid polymorphism, we examined the effects of the same treatments on the molecular organization and functional activity of the photosynthetic machineries and analyzed the ultrastructural features of the granum and stroma sub-chloroplast membrane particles.

Isolation of Granum and Stroma Thylakoid Membranes
The granum and stroma sub-chloroplast thylakoid membrane particles were isolated by digitonin fragmentation of spinach TMs, followed by differential centrifugation, using modified protocols of [41,42], respectively. Spinach leaves, purchased from the local market, were washed in chilled deionized water and kept for overnight at 4 • C in darkness before use. About 250 g leaves were homogenized in 200 mL medium containing 50 mM Tricine-KOH buffer (pH 7.8), 300 mM sorbitol, 25 mM NaCl, 25 mM KCl, and 5 mM MgCl 2 . The crude extract was filtered through four layers of perlon net, followed by a 1.5 min centrifugation at 3000× g. The pellet was suspended for 1.5 min in 30 mL of 10 mM MgCl 2 , after which 30 mL 40 mM Tes-KOH buffer (pH 7.8), containing 500 mM sorbitol, 50 mM KCl, and 50 mM NaCl, was added. After centrifugation at 3500× g for 10 min, the pellet was resuspended in 20-30 mL of 20 mM Tes-KOH buffer (pH 7.8), containing 250 mM sorbitol, 25 mM KCl, 25 mM NaCl, and 5 mM MgCl 2 . Digitonin (Merck, Darmstadt, Germany; twice recrystallized from ethanol) was applied in this medium at a final concentration of 0.2% Cells 2021, 10, 2354 4 of 16 (w/v) and a digitonin/Chl (w/w) ratio of 1 (Chl, chlorophyll). The mixture was stirred for 30 min in the dark at 4 • C, followed by a 3-fold dilution with the same medium. After centrifugations for 30 min at 5000× g, the supernatant was further centrifuged for 30 min at 10,000× g. The pellet (D10, granum TMs) was resuspended in a medium containing 5 mM Tes-KOH (pH 7.8), 20 mM Tricine-KOH (pH 7.8), 20 mM NaCl, 20 mM KCl, and 5 mM MgCl 2 . The remaining supernatant was further centrifuged for 30 min at 50,000× g and the pellet was resuspended in the same medium. The final centrifugation of the supernatant was carried out for 60 min at 130,000× g to obtain D130, the stroma TMs, which were resuspended in small volumes of the same medium as used for D10. The Chl content of the isolated particles, determined according to [43], were always higher than 5 mg Chl (a + b) mL −1 . The Chl a/b ratios of D10 and D130 were 3.05 ± 0.23 (n = 4) and 9.68 ± 2.7 (n = 4), respectively. All isolation procedures were performed at 4 • C in dim green laboratory light and the samples were stored at −80 • C until use.

Lipase Treatments
In the experiments on lipase-treated granum and stroma TMs, a substrate non-specific [44], general tri-, di-, and monoglyceride hydrolase-lipase from wheat germ (L3001, Sigma-Aldrich, Burlington, MA, USA) was used. A stock solution of 0.5 U µL −1 activity was prepared in MilliQ water, and volumes containing the desired activity of the lipase were added into the sample and thoroughly mixed prior to the start of the measurement.

31 P-NMR Measurements
31 P-NMR measurements were performed as described earlier [35]. Spectra were recorded using Avance Neo 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) equipped with a BBFO probe tuned at the resonance frequency of the 31 P nucleus. Circa 1 mL of sample was placed into a 5 mm outer diameter NMR tube. Our earlier experiments have shown that-due to the very high density of the TMs-no magnetic orientation of the sample occurs [24]. (N.B.: intact TMs possess considerably higher diamagnetic anisotropy values than the sub-chloroplast particles, and thus TMs are easier to align in an external magnetic field than their fragments.) Spectra were recorded using a 40 • rf pulse, an inter-pulse time of 0.5 s and no 1 H-decoupling. The temperature was controlled to within ±0.1 • C. Chemical shifts were referenced externally to 85% solution of H 3 PO 4 in water.
For saturation experiments, a low power RF pulse was applied at the selected chemical shift for 0.3 s, followed by a 40 • pulse and 0.2 s of acquisition, for a repetition time of 0.5 s. The power of the pre-saturation pulse was adjusted according to the peaks of interest-the RF pulse field strength was 80 Hz for pre-saturation of lamellar and H II phase and 40 Hz for the isotropic phases.
During averaging of the 31 P-NMR spectra, weighting factors were applied to correct for the different chlorophyll contents of the samples, and were normalized to 10 mg Chl (a + b) mL −1 in all cases. Weighting factors were also applied if the number of scans of the spectra used for averaging were not the same.

31 P-NMR Fingerprints of Lipid Phases in Isolated Granum and Stroma Thylakoid Membranes
The 31 P-NMR spectra of isolated granum and stroma thylakoid membranes recorded at 5 • C revealed that the signals originated from several different chemical environments of the phosphorous nucleus ( Figure 1). As already pointed out in the case of isolated intact thylakoid membranes [24], the signals arise predominantly from PG, the only phospholipid molecules of TMs. Although the spectra of the two sub-chloroplast TM particles were not identical, and in both cases the spectral distributions varied from batch to batch, the basic features remained very similar to one another and resembled the spectra of isolated intact TMs [35,36].

31 P-NMR Fingerprints of Lipid Phases in Isolated Granum and Stroma Thylakoid Membranes
The 31 P-NMR spectra of isolated granum and stroma thylakoid membranes recorded at 5 °C revealed that the signals originated from several different chemical environments of the phosphorous nucleus ( Figure 1). As already pointed out in the case of isolated intact thylakoid membranes [24], the signals arise predominantly from PG, the only phospholipid molecules of TMs. Although the spectra of the two sub-chloroplast TM particles were not identical, and in both cases the spectral distributions varied from batch to batch, the basic features remained very similar to one another and resembled the spectra of isolated intact TMs [35,36]. Figure 1. 31 P-NMR spectra (a,c) and relative intensities (b,d) of isolated spinach granum (a,b) and stroma (c,d) thylakoid membranes at 5 °C. Average of (a) five spectra from three batches and (c) six spectra from five batches. Integrated areas of the component spectra (b,d) associated with the different lipid phases, relative to the overall integrated area; mean values ± SD.
As shown in Figure 1, the spectra could be deconvoluted to four component spectra. Similar to intact TMs, both membrane particles exhibited a well-defined lamellar phase, peaking around −10 ppm and displaying an asymmetric shape extending to the low-field side. A spectral shape with reversed symmetry, characteristic of the HII phase, was also observed with a peak position at around 20 ppm. This peak appeared to be somewhat shifted compared to TMs, where it was typically found at ~25-30 ppm [36]. The spectra also contained two sharp, symmetric bands (I1 and I2) in the region between about 2.5 and 4.5 ppm; these resonances are of isotropic origin. The relative contributions of the four different phases were also very similar in the granum and stroma TMs ( Figure 1, Panels b and d). The lamellar phases displayed large integrated areas; somewhat surprisingly, equally large or somewhat (albeit statistically insignificantly) larger average integral values were obtained for the HII phases. Although the I2 peak was usually weaker than I1, and in some cases could only be discerned as a shoulder (as in Figure 1a), it was clearly present in all samples. The peak positions of I1 and I2 were essentially the same in the Figure 1. 31 P-NMR spectra (a,c) and relative intensities (b,d) of isolated spinach granum (a,b) and stroma (c,d) thylakoid membranes at 5 • C. Average of (a) five spectra from three batches and (c) six spectra from five batches. Integrated areas of the component spectra (b,d) associated with the different lipid phases, relative to the overall integrated area; mean values ± SD.
As shown in Figure 1, the spectra could be deconvoluted to four component spectra. Similar to intact TMs, both membrane particles exhibited a well-defined lamellar phase, peaking around −10 ppm and displaying an asymmetric shape extending to the low-field side. A spectral shape with reversed symmetry, characteristic of the H II phase, was also observed with a peak position at around 20 ppm. This peak appeared to be somewhat shifted compared to TMs, where it was typically found at~25-30 ppm [36]. The spectra also contained two sharp, symmetric bands (I 1 and I 2 ) in the region between about 2.5 and 4.5 ppm; these resonances are of isotropic origin. The relative contributions of the four different phases were also very similar in the granum and stroma TMs (Figure 1, Panels b and d). The lamellar phases displayed large integrated areas; somewhat surprisingly, equally large or somewhat (albeit statistically insignificantly) larger average integral values were obtained for the H II phases. Although the I 2 peak was usually weaker than I 1 , and in some cases could only be discerned as a shoulder (as in Figure 1a), it was clearly present in all samples. The peak positions of I 1 and I 2 were essentially the same in the granum and stroma TMs, 2.66 ± 0.07 and 2.68 ± 0.16, and 3.98 ± 0.40 and 4.19 ± 0.13, respectively; and only the half-bandwidths appeared to be somewhat narrower in the stroma TMs (Table 1).

Saturation-Transfer Experiments
To probe the spectral shapes of different phases, and to test the validity of the above mathematical deconvolution of the 31 P-NMR spectra, we performed saturation-transfer experiments, i.e., applied saturating pulses, to suppress contributions from different phases, at selected frequencies at or near the peak positions of the spectral components tested [24,46]. This approach was practicable due to the relatively high stability of the sub-chloroplast particles, as shown by the data in Figure S1. It can be seen that the spectra recorded in the first and second 30 min of the acquisition time were essentially identical; in addition, the lamellar phases were retained with very little decrease during several hours, in contrast to intact TMs under similar conditions [34]; further, the isotropic phases increased only moderately during the time of up to several-hours long measuring periods.
As shown in Figure 2a, a saturating pulse applied at −12 ppm eliminated the lamellarphase signal of granum TMs, while it exerted virtually no effect on the isotropic region. At the same time, in harmony with the sizeable overlap of the H II -phase spectral component with the lamellar phase, the intense pulse at −12 ppm also caused a moderate decrease in the H II signal. Vice versa, when the (nearly) saturating pulse was applied at 20 ppm (i.e., at the peak position of the H II -phase), a decrease in the intensity of the lamellar-phase component was observed (Figure 2b). With the same pulses, the isotropic phases did not decrease; in fact, here, the I 1 intensity increased, which evidently could be accounted for by the time lapse between recording the control and the saturation-transfer spectra. The two isotropic peaks, I 1 and I 2 could also be suppressed by selective pulses at characteristic frequencies, near the peak positions (Figure 2 Panels c and d). The selectivity of suppressions was high, if taking into account the small frequency difference between the two peaks. Saturation-transfer experiments on stroma TMs yielded very similar results as obtained for granum TMs. In particular, the 31 P-NMR signals of the lamellar and H II phases as well as those associated with the intense I 1 phase and the somewhat weaker I 2 phase could be suppressed with reasonable degree of selectivity (Figure 3 Panels a-d).
In general, these data, in line with the above deconvolution analyses, show that the sub-chloroplast TMs at 5 • C contain four lipid phases, each of which originate from distinct structural entities. Further, it can also be seen that the spectral shapes of the different phases obtained by using the mathematical deconvolution, and those which can be inferred from the saturation transfer experiments are in good agreement with one another.

Temperature Dependences
The lamellar phase of granum TMs was gradually destabilized upon increasing the temperature stepwise from 5 to 15; from 15 to 25 and then to 35 • C; shifts and broadenings were also observed in the isotropic region ( Figure 4). Deconvolution of the spectra (Panels be of Figure 4), and an inspection of the temperature-dependent variations of the integrated areas of the component spectra ( Figure S2) revealed a substantial increase in the intensity of the I 1 phase at the expense of the lamellar and H II phases. At the same time, the sharp peak of I 1 , at~2.6 ppm gradually shifted to about 1 ppm and broadened considerably. For I 2 , no such shift was observed, and the broadening appeared only at 35 • C (Figure 4e). Very similar trends were observed in stroma TMs (Figure 5a): a gradual loss of the lamellar phase ( Figure 5b); some diminishment of the H II phase-albeit its overall low intensity in

Effect of Wheat Germ Lipase
To test the potentially distinct lipase sensitivity of the different lipid phases of granum and stroma TMs, we performed 31 P-NMR spectroscopy measurements in the absence and presence of different concentrations of wheat-germ lipase. As shown in Figure 6a, whereas the H II phase of granum TMs was essentially insensitive to the lipase and the lamellar phase was only marginally affected by 5 U lipase treatment, the sharp I 1 isotropic phase was essentially eliminated and replaced by a broad band-indicating the appearance of a large, immobilized molecular assembly replacing a highly mobile lipid-containing domain. Again, as for the thermal treatment, the I 2 phase was less sensitive to the lipase treatment. These effects depended on the concentration of the lipase and progressed with increasing the enzyme concentration-as also reflected in the deconvoluted component spectra (Figure 6b-e). Because the broad isotropic band largely overlapped the bands of the lamellar and H II phases, we applied saturation pulses, and confirmed the existence of these latter phases even after the lipase treatments ( Figure S3). These experiments also confirmed the appearance of the broad isotropic phase, which could be suppressed, with reasonable selectivity, with a pulse at 2.8 ppm. It is clear that already after treating the membranes with the lipase at 5 U, the isotropic signals were replaced with a broad featureless peak, which was broadened into the baseline upon further addition of the lipase. The observed broadening of the signal is indicative of a reduction of T2* relaxation time, which we interpret as the result of a reduction of molecular mobility due to the redistribution of lipids from the isotropic phase to a larger phospholipid formation [47]. Very similar data were obtained on stroma TMs, which appeared to be more susceptible to the same lipase treatment: 2.5 U exerted similar effect on these membranes as 5 U on the grana (Figure 7a). Here, too, the lamellar phase (Figure 7b) and the H II phase (Figure 7c) were only marginally sensitive. In contrast, the two sharp isotropic peaks were essentially eliminated (Figure 7d,e) and the broad resonance attributed to the emergence of a larger formation composed of released or partially cleaved lipid molecules resulting from the action of the lipase was observed. In general, these data, in line with the above deconvolution analyses, show that sub-chloroplast TMs at 5 °C contain four lipid phases, each of which originate from d tinct structural entities. Further, it can also be seen that the spectral shapes of the differ the increase of the temperature; most remarkably, elevating the temperature desta the lamellar phases both in the granum and the stroma TMs; different responses o I2 should also be noticed.  In general, these data show that the different lipid phases do not react uniformly to the increase of the temperature; most remarkably, elevating the temperature destabilized the lamellar phases both in the granum and the stroma TMs; different responses of I 1 and I 2 should also be noticed.

Effect of Wheat Germ Lipase
To test the potentially distinct lipase sensitivity of the different lipid ph granum and stroma TMs, we performed 31 P-NMR spectroscopy measurements in sence and presence of different concentrations of wheat-germ lipase. As shown in These data, in good agreement with the data from the saturation transfer experiments (Section 3.2), indicate that the lamellar, H II , the two isotropic phases originate from distinct structural entities both in the granum and the stroma TMs, and that the I 1 isotropic phases in the two membrane particles share very similar susceptibilities to wheat-germ lipase. x 6. Effects of wheat-germ lipase treatments on the 31 P-NMR spectra (a) of granum thylakoid membranes a mponent spectra: L, the lamellar phase (b), HII, the inverted hexagonal phase (c), and the two isotropic ph I2 (e). The spectra represent averages of two measurements from two batches exhibiting similar spectra; rec  These data, in good agreement with the data from the saturation transfer experime (Section 3.2), indicate that the lamellar, HII, the two isotropic phases originate from distin structural entities both in the granum and the stroma TMs, and that the I1 isotropic pha in the two membrane particles share very similar susceptibilities to wheat-germ lipase

Discussion
In this paper, we investigated the origin of different non-bilayer lipid phases in t context of the lateral heterogeneity of TMs. In chloroplasts of higher plants, the photosy

Discussion
In this paper, we investigated the origin of different non-bilayer lipid phases in the context of the lateral heterogeneity of TMs. In chloroplasts of higher plants, the photosynthetic membranes are differentiated into granum and stroma TMs: the cylindrical stacks of grana are interconnected by unstacked stroma membranes, which are helically wound around the granum [38]. Nevertheless, the two types of membranes form one single continuum membrane, enclosing a contiguous inner aqueous (luminal) phase [48]. Whereas the TM lipids are evenly distributed within the plane of the membrane [31], the granum and stroma TMs display strikingly different protein compositions. Indeed, the differentiation of TMs into granum and stroma membranes appears to be governed by sorting of their proteins, via the self-assembly of LHCII-PSII macrodomains, stabilized by stacking [49]. As a consequence, grana are enriched in LHCII-PSII supercomplexes; in contrast, LHCI-PSI and the ATP synthase are found predominantly in the stroma TM and the end-membranes of grana [40].
The marked protein-composition differences between the two types of TMs could, in principle, exert effects on the lipid phase behavior of membranes, especially when considering the predictions of the LPM [16,17] and the FSM [19] on the protein functions. In other terms, the functional differences in the two regions could, in principle, be associated with differences in the lipid phase behavior. However, no such difference was observed ( Figure 1). This can be explained by the fact that both the granum and the stroma TMs contain large, compact supercomplexes, robustly organized LHCII-PSII and LHCI-PSI, respectively. These supercomplexes are composed of dozens of tightly packed protein subunits and highly organized pigment systems, containing more than 200 pigment molecules (chlorophylls and carotenoids) [50,51]. They also bind tens of different lipid moleculeswith no apparent distinction between bilayer and non-bilayer lipids, i.e., between PG, SQDG and DGDG, and MGDG, respectively. These non-annular or structural lipids appear to mediate interactions between protein subunits and might fill the grooves in the membrane-embedded protein structures. The structural lipids, and the slowly exchanging annular or shell (boundary) lipids around the supercomplexes do not contribute to the 31 P-NMR signal of TMs (cf. [22] and references therein).
Regarding the role of the non-bilayer lipid MGDG in the bulk, its presence lends non-bilayer propensity to the lipid mixture, which, in turn, keeps the TMs in a frustrated state-as proposed in all models taking into account the presence of non-bilayer lipids in bilayer membranes: the LPM [16], the FSM [19], and the DEM [22]. This, in general, might be of high physiological significance but, per se, does not explain the observed lipid polymorphism of intact TMs and their constituent fractions. The coexistence of bilayer and non-bilayer lipid phases is one of the basic postulations of the DEM. Thus, our data and our earlier observations concerning the polymorphism of lipid phases in functional thylakoid membranes [24,32] are consistent with this latter model. In this context, it is also worth mentioning that the coexistence of bilayer and non-bilayer lipid phases has also been documented in the other main energy-converting membranes, in functionally active inner mitochondrial membranes [52].
The mathematical deconvolution of the spectra of granum and the stroma TMs (Figure 1), similarly to intact TMs [36], confirmed the presence of two isotropic phases and an H II phase, in addition to the bilayer. A more direct, experimental support on the validity of this spectral deconvolution was obtained from saturation transfer experiments. In particular, by applying saturating pulses at different, characteristic frequencies, we have shown that the signals of the different distinct lipid phases, exhibiting characteristic spectral distributions, can be selectively suppressed by irradiating the samples at or near their peak frequencies (Figures 2 and 3). At the same time, at higher temperatures, we also observed exchanges between different lipid phases (Figures 4 and 5)-in agreement with our earlier works [33,36] and in harmony with the DEM of TMs.
As to the origin of these distinct, yet apparently interconnectable lipid phases, we tested their accessibilities to wheat-germ lipase (Figures 6 and 7). These experiments, while provided no clue concerning the structural entities associated with the non-bilayer lipid phases, have clearly shown that the isotropic phases originate from structural units which are more easily digestible with this lipase than the lipids of the bilayer membrane or of the H II phase. Wheat-germ lipase preferentially destroyed the I 1 phase and transformed it to a largely immobile molecular assembly.
The elucidation of the questions concerning the identity and physiological roles of the observed non-bilayer phases requires further structural and functional data. This type of experiments, combined with measurements testing the effects of temperature and lipase treatments on the molecular organization and functioning of the photosynthetic machineries in the granum and stroma TMs, have been carried out-and will be reported in the accompanying paper.

Conclusions
In this work we have shown that the granum and stroma TMs exhibit marked lipidphase polymorphisms, which are very similar to each other and to the intact TMs: in addition to the bilayer, they exhibit an H II phase and two isotropic phases. These data clearly show that the protein composition of the membranes does not exert a significant effect on the phase behavior of TM lipids. Saturation transfer experiments and lipase treatments at 5 • C have revealed that the different lipid phases are associated with distinct structural entities, which are nevertheless capable of exchanging lipids with one another at higher temperatures. In general, these data are in harmony with the DEM of TMs. In the accompanying paper, by investigating the ultrastructure, spectroscopy, and functional parameters of these preparations, and analyzing the effects of different treatments, we provide insight into the structural identity and origin of different lipid phases and their role in the self-assembly of TM systems; we will also discuss the possible physiological significance of non-bilayer lipid phases in thylakoid membranes and in energy-converting membranes in general.
Supplementary Materials: The following materials are available online at https://www.mdpi.com/ article/10.3390/cells10092354/s1, Figure S1: Short-term and medium-term lipid-phase stability of granum and stroma thylakoid membranes at 5 • C-as reflected by the temporal variations of the 31 P-NMR spectra recorded at different intervals after suspending the sample; Figure S2: integrated areas of 31 P-NMR spectral components, which are assigned to the L, I 1 , I 2 and H II lipid phases of granum TMs, plotting also the total integrated area of the corresponding spectra; Figure S3: 31 P-NMR spectra of wheat-germ treated granum thylakoid membranes in the absence and presence of saturation pulses applied at different frequencies, corresponding to or close to the peak position of different phases.
Author Contributions: G.G. and V.Š. conceived the study. The sub-chloroplast particles were isolated by O.Z. in Szeged and were shipped to Ljubljana for 31 P-NMR measurements, which were performed by U.J., with the help of P.Š. and supervised by J.P.; averaging and data analyses were carried out by O.D., with the help of U.J. and V.K., supervised by G.G. Spectral characterization of the membranes were performed by O.Z. and O.D. and V.K., supervised by V.Š. The paper was written by G.G., O.D. and U.J., with all authors contributing to the writing. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The original data were recorded at the Slovenian NMR Center. Processed and derived data are available from the corresponding author G.G. on request.