- Methodology
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An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants
Plant Methods volumeĀ 20, ArticleĀ number:Ā 38 (2024)
Abstract
Background
The extraction of thylakoids is an essential step in studying the structure of photosynthetic complexes and several other aspects of the photosynthetic process in plants. Conventional protocols have been developed for selected land plants grown in controlled conditions. Plants accumulate defensive chemical compounds such as polyphenols to cope with environmental stresses. When the polyphenol levels are high, their oxidation and cross-linking properties prevent thylakoid extraction.
Results
In this study, we developed a method to counteract the hindering effects of polyphenols by modifying the grinding buffer with the addition of both vitamin C (VitC) and polyethylene glycol (PEG4000). This protocol was first applied to the marine plant Posidonia oceanica and then extended to other plants synthesizing substantial amounts of polyphenols, such as Quercus pubescens (oak) and Vitis vinifera (grapevine). Native gel analysis showed that photosynthetic complexes (PSII, PSI, and LHCII) can be extracted from purified membranes and fractionated comparably to those extracted from the model plant Arabidopsis thaliana. Moreover, total protein extraction from frozen P. oceanica leaves was also efficiently carried out using a denaturing buffer containing PEG and VitC.
Conclusions
Our work shows that the use of PEG and VitC significantly improves the isolation of native thylakoids, native photosynthetic complexes, and total proteins from plants containing high amounts of polyphenols and thus enables studies on photosynthesis in various plant species grown in natural conditions.
Background
The ecological crisis is accelerating the need to study photosynthesis in free-growing wild plants to complement data obtained on plants grown under controlled laboratory conditions. Indeed, the latter conditions do not consider the ecological and evolutionary relevance of the underlying photosynthetic mechanisms. For example, studies of marine plants rarely address the response mechanisms to a light environment that strongly differ depending on the depth, at which the plants live (1ā40Ā m). One of the most important molecular approaches for studying photosynthesis is the biochemical characterization of the photosystems II and I (PSII and PSI). Extraction of thylakoid membranes and photosynthetic protein complexes is crucial to performing such studies. However, thylakoid extraction protocols have been optimized for model plants grown in laboratory conditions, such as Arabidopsis thaliana or Pisum sativum [1,2,3], and proved very inefficient in extracting membranes from wild plants and crops like oak and grapevine. Protocols that extract proteins by denaturing them directly from leaves are available for some refractory species. However, those protocols require protein precipitation in organic buffers such as acetone, acetone/trichloroacetic acid, or phenol solvents followed by several washing and drying steps [4,5,6,7]. They have the additional inconvenience of being time-consuming due to the need for multiple steps.
Posidonia oceanica, an endemic Mediterranean seagrass is the engineer of an ecosystem of major importance that provides goods and services to the growing human populations in coastal areas. Initial attempts to extract thylakoids and proteins from leaves of P. oceanica with the conventional protocol used for model plants failed. Thylakoid preparations turned brown and resisted solubilization by non-ionic detergents, likely due to the presence of polyphenols [8,9,10]. Plants in natural conditions are subjected to continuous changes in environmental factors such as light quantity and quality, temperature, UV radiation, and water availability. Many plant species synthesize specialized secondary metabolic compounds like polyphenols [11]. These organic compounds are characterized by the presence of several hydroxyl groups linked to an aromatic ring, which confer protective redox and reactive properties upon abiotic and biotic stress [12,13,14]. The hydroxyl groups connected through a CāC delocalized double bond promote the oxidation of oxygen atoms within the molecule by molecular oxygen or reactive oxygen species. In this reaction, polyphenols turn into quinones, which display highly oxidizing properties in vitro [8, 10, 15]. Those molecules also possess in vitro cross-linking and reticulating properties mediated by hydrogen bonds between their hydroxyl and carbonyl groups [14, 16,17,18].
In plants, polyphenols are compartmentalized within the cell vacuole, but also in specialized polyphenol-rich cells [19,20,21]. Grinding leaves during the thylakoid membrane extraction releases polyphenols and allow them to interact with oxygen leading to harmful effects on macromolecules.
In this study, we developed a method to isolate thylakoid membranes and total proteins from the leaves of P. oceanica, a polyphenol-rich plant. We showed that, by neutralizing the reactivity of polyphenols using both VitC and PEG, we can easily and efficiently extract thylakoid proteins from P. oceanica. The addition of PEG and VitC in the denaturing buffer enhances the total protein extraction from leaves. These compounds were previously used to extract thylakoids from Picea abies [22], but no explanation was given for their use in the extraction buffer and no relationship was established with the plant's polyphenol content. Our method extended to other plant species enriched in phenols or in polyphenols: Quercus pubescens (oak), Vitis vinifera (grapevine), and Ocimum basilicum (basil) [23, 24]. In Q. pubescens and V. vinifera, using the new optimized protocol allows the efficient isolation of thylakoids compared to conventional protocols.
Results
Extraction of thylakoids from P. oceanica leaves using a conventional procedure
When P. oceanica (from 2-m depth) leaves were ground according to the conventional protocol (i.e. without Asc and PEG,), the resulting mixture appeared brownish (Fig.Ā 1A left). In addition, the pellet obtained at the last centrifugation step (cf Method section for details) displayed membranes that appeared entrapped in a mucilaginous mass (Fig.Ā 1A middle). The brownish color of the supernatant indicated the possible formation of quinones as a result of polyphenol oxidation during grinding [8, 9, 15] and the mucilaginous mass was likely a consequence of both the presence of cross-linked quinones and polyphenols. The final pellet described above was then resuspended in a solubilization buffer for Native-PAGE analysis. Solubilization of thylakoids by dodecyl maltoside detergent (DDM) was ineffective as seen by the clear color of the supernatant after solubilization (Fig.Ā 1A top right). The separation of the solubilized sample in the BN-PAGE revealed only free LHCII trimers.
Extraction of total proteins from P. oceanica leaves
Extracting total proteins from P. oceanica leaves was tested to prepare soluble and membrane protein fractions for SDS-PAGE and immunoblot analyses. Similar amounts of fresh Posidonia leaves were used for protein extraction, (ca 1Ā g) in 2Ā mL of buffer. To evaluate the relative quantity and quality of the extracted proteins, we performed SDS-PAGE gels. First, a typical Tris extraction buffer containing, sodium dodecyl sulfate (SDS), a reducing agent (Ī²-mercaptoethanol), and a protease inhibitor (PMSF) was used. That protocol is efficient for preparing protein extracts from glycophytes such as Arabidopsis thaliana [25], Solanum tuberosum [26], and halophytes such as Thellungiella halophila [27] or Atriplex halimus [28]. As shown in Fig.Ā 1B, after separating proteins by SDS-PAGE and Coomassie-blue staining, little protein was recovered from samples collected at 2Ā m depth. A similar profile was observed in the sample harvested at 15Ā m depth. Intriguingly, more proteins were recovered from samples at 26Ā m depth although the bands appeared quite smeary, likely due to a distinct composition of P. oceanica leaves in this environment. These data indicate that the solubilization problem persists even under denaturing conditions and that a strong ionic detergent such as SDS is not enough to inhibit the probable deleterious effects of polyphenols. To test whether the presence of polyphenols was involved in the poor protein yield, we assayed the polyphenol content of P. oceanica dried leaves at the three depths (Fig.Ā 1C). The samples from 2 and 15Ā m depths displayed a polyphenol concentration in the same range (6.75āĀ±ā0.46 and 7.83āĀ±ā0.18Ā mg Gallic Acid Equivalent (GAE)/mg chlorophyll, respectively) whilst the 26-m depth samples displayed a threefold lower polyphenol concentration (2.63āĀ±ā0.07 GAE/mg chlorophyll). This result suggests that the polyphenol concentration could be a reason for the inefficient extraction of thylakoids or proteins from P. oceanica.
Microscopy analysis of P. oceanica leaf tissues
In land plants, phenolic compounds are generally stored in vacuoles and cell walls. However, the polyphenols in P. oceanica are also confined in mesophyll-specialized structures [20, 21, 29], in contrast to those found in Australian Posidonia, which are deposited in the epidermis [30, 31]. Since optical microscopy did not provide a good visualization of the mesophyll structures (Fig.Ā 2A), P. oceanica leaves (from 15Ā m depth) were observed using electronic microscopy (Fig.Ā 2B). Observations revealed the presence of both low-contrasted aerenchyma cells (filled with gas) and cells characterized by a stronger grey coloration assigned to polyphenol deposit (Fig.Ā 2B) [21]. The close-up view of a polyphenol-containing cell (PC) highlighted large vacuoles filled with polyphenols and numerous mitochondria suggesting high metabolic activity in polyphenol-containing cells (Fig.Ā 2C). These PCs could contribute to the formation of the cross-linking and gelation, observed during extraction of thylakoids.
Effect of PEG and VitC on thylakoid membranes extraction from P. oceanica
In an attempt to neutralize the effects of polyphenols, we extracted thylakoids with an extraction buffer supplemented with 5% VitC (concentration arbitrarily chosen for this assay) to prevent polyphenol oxidation and thus quinone formation. The ground leaf mixture appeared much clearer and greener (Fig.Ā 3A left) than in the absence of VitC (Fig.Ā 1A left). However, as shown in Fig.Ā 3A (middle), the thylakoids were still trapped in mucilage and poorly solubilized (Fig.Ā 3A right). To prevent reticulation of polyphenols and mucilage formation, we tested several conditions for extracting thylakoids such as NaCl instead of sorbitol, adding 5% milk powder or a reducing agent such as DTT,Ā and acidic (pH 5) or alkaline (pH 8.5) buffers. None of these approaches were successful (data not shown). Furthermore, even the physical separation of thylakoids from mucilage using Percoll gradient centrifugation did not provide satisfactory results (data not shown). Therefore, we continued our assays focusing on the action of PEG together with VitC by adding 5% PEG4000 and 5% VitC in the grinding buffer (concentrations of chemicals were arbitrarily chosen for this assay). Using the PEG and VitC Protocol (PVC protocol), the pellet after centrifugation appeared differentiated into three layers, the dark green upper one, being the thylakoid membranes (Fig.Ā 3B middle). After DDM treatment, the sample was successfully solubilized (Fig.Ā 3B right), and photosynthetic complexes could be separated using BN-PAGE (compare with Figs. 1A and 3A right). We noted a similar yield across all species with the implementation of the PVC protocol (Additional file 2: Fig. S1).
Effect of vitamin C and PEG on the extraction of total proteins from P. oceanica leaves
Total protein isolation from P. oceanica leaves was performed as shown in Fig.Ā 1B but with the addition of VitC and/or PEG in the extraction buffer. Adding 5% VitC greatly improved the extraction procedure since proteins were successfully obtained from the samples harvested at different depths and bands could be observed in the gel (Fig.Ā 4A). When extraction was performed in the presence of both 5% VitC and 5% PEG (Fig.Ā 4B), very similar protein patterns in terms of content and band intensity were shown for the three samples. Furthermore, the resolution of the bands was slightly increased. Similarly, to that observed for thylakoid extraction (Fig.Ā 3B), these results highlighted the beneficial effect of the presence of both VitC and PEG for preparing P. oceanica total protein extracts and their action on polyphenols irrespective of the extraction conditions (native or denaturing). We then aimed to validate this procedure by performing immunoblot analyses of protein extracts using various antibodies raised against photosynthetic proteins from land plant species involved in either the electron transfer chain or the Calvin-Benson cycle (Fig.Ā 4C). Cross-reactions were observed for several of these sera. Proteins from PSII, PSI, cytochrome b6f, and ATP synthase complexes were probed in P. oceanica extracts. Similar abundances of these thylakoid proteins were noticed in the three types of samples. Similar amounts of RubisCO large subunit and phosphoribulokinase (PRK) were also revealed in these extracts. Altogether, these analyses validate the use of VitC and PEG for preparing P. oceanica protein extracts suitable for immunological experiments.
Optimization of PEG and VitC concentrations and selection of the native gel system
While several photosynthetic complexes were extracted with the new protocol, the PSII-LHCII supercomplexes were weakly distinguishable in the BN-PAGE profile (Fig.Ā 3B). To solve this problem, we tried to optimize the working concentration of PEG and VitC. For this purpose, P. oceanica thylakoid membranes were extracted using a gradually increasing concentration of VitC in the presence of 5% PEG. Better separation of solubilized complexes by CN-PAGE was found to correlate with increasing VitC concentration (Fig.Ā 5A). In the absence of VitC, a smeared migration profile was noticed. When VitC concentration was raised to 0.1%, supercomplexes still appeared smeary but were better resolved than without VitC. The best band resolution was observed at 5% and 10% of VitC. While PSII-LHCII bands were still weak and smeary (suggesting poor solubilization of PSII), the well-separated bands highlighted the presence of PSI-LHCI, free LHCII trimeric antennae, and two PSI-(LHCI-)LHCII supercomplexes as determined by 2D Urea-PAGE analysis (Additional file 2: Fig. S2). Adding 0.1% PEG, in the presence of 5% VitC, solubilizes the thylakoids with supercomplexes being visible in CN-native gels (Fig.Ā 5B). The best band resolution was obtained by raising concentration from 1% up to 5%. Finally, the concentrations used at the outset of the trials (5% of each compound) were used for the rest of the study. As proper concentrations of PEG and VitC were determined, the best native gel system was determined to separate and visualize the photosystem supercomplexes. When using 2% Ī±-DDM, separating photosystem supercomplexes using a Blue-Native PAGE system allowed a better solubility, separation, and visibility of PSII-LHCII (Fig.Ā 5C) in line with data from [1].
Oxygen production in thylakoids extracted with the PVC protocol
We assessed the integrity of the OEC (Oxygen Evolving Complex) of PSII by measuring the oxygen production rate of thylakoids from P. oceanica (2Ā m), extracted with or without PEG and VitC. The results revealed that thylakoids extracted without PEG and VitC, or with VitC alone, exhibited a null oxygen production. However, employing the PVC protocol allowed us to measure a maximum oxygen production rate (OPR) of nearly 22.6āĀ±ā5.7Ā ĀµmolĀ O2/mL/h/mg chlorophyll in P. oceanica, which is lower than the rate measured in A. thaliana. In the last species, a maximal rate of 81.9āĀ±ā4.2Ā ĀµmolĀ O2/mL/h/mg chlorophyll was recorded in thylakoids extracted with the PVC protocol (TableĀ 1, Additional file 2: Fig. S3). These findings indicate that the PVC protocol preserves OEC activity in P. oceanica and A. thaliana.
Extraction of thylakoids from various plant species using the PVC protocol
Since the addition of PEG and VitC enabled the proper extraction of thylakoids in P. oceanica, the procedure was extended to other plant species known for their agronomic, economic, or ecological value, and which are different in terms of growth environment, morphology, and polyphenol content: Quercus pubescens (oak), Vitis vinifera (grapevine) and Ocimum basilicum (basil). The model plant Arabidopsis thaliana was also used as a control.
BN-PAGE analysis of photosystem supercomplexes from various plant species using the PVC protocol
For each species, thylakoids were extracted using either the conventional or the PVC protocol.
As observed with P. oceanica (Fig.Ā 1), V. vinifera and Q. pubescens supercomplexes were not or poorly solubilized when thylakoids were extracted without PEG and VitC even with a strong DDM concentration (3%) (Fig.Ā 6A, B left). When using the PVC protocol, thylakoids were partially solubilized in the presence of 1% DDM as shown by smeared bands in the gels. By increasing the concentration of DDM to 2%, the complexes were visible as well-resolved distinct bands (Fig.Ā 6A, B right). At first glance, the migration pattern was somewhat different between V. vinifera and Q. pubescens, but 2D-PAGE analysis (Additional file 2: Fig. S4) confirmed the presence of free LHCII, PSI-LHCI, PSII-LHCII supercomplexes and of a band assigned to the PSI-LHCI-LHCII complex in V. vinifera. While free LHCII was observed in the gel using the conventional protocol with V. vinifera extracts, no complex was observed in the Q. pubescens migration profile when using the same protocol. On the other hand, the PVC protocol allowed the solubilization of all photosynthetic complexes in the thylakoids, demonstrating its efficiency. In A. thaliana, the migration profile was similar when using conventional or PVC protocol, but differed in band resolution (Fig.Ā 6D). Indeed, while the PSII supercomplex bands appeared to spread out using 3% DDM with the conventional protocol, these bands were already resolved from 2% DDM when using the PVC protocol. In O. basilicum, few differences were observable between the two procedures despite that the PSI-LHCI bands and free LHCII antennae appeared greener with the conventional protocol (Fig.Ā 6C) whereas, with the PVC protocol, the highest PSII-LHCII bands appeared more intense (Additional file 2: Fig. S4). This might suggest better grana solubilization when using the PVC method or better stability of supercomplexes during solubilization. However, the slight differences observed, between protocols in the resolution of PSII-LHCII bands for O. basilicum and A. thaliana might be related to our working conditions, i.e. the use of frozen leaves. Many reports show nice native gels from fresh tissue thylakoids are available for A. thaliana [1], but using the PVC protocol is not necessary for this species.
Soluble polyphenol content in leaves from various plant species
We hypothesized that polyphenols would have inhibitory effects on thylakoid and protein extraction independent of their localization in parenchyma cells (P. oceanica) or epidermis cells (land plants). To test this hypothesis, the soluble polyphenol content of dried leaves from P. oceanica (15Ā m depth) Q. pubescens, V. vinifera, O. basilicum and A. thaliana was determined (Fig.Ā 7A). As these plant species do not share the same leaf structure (e.g., leaf thickness, the structure of vascular tissues, epidermis, and parenchyma organization), the polyphenol content is likely to differ (Fig.Ā 2A and Additional file 2: Fig. S5). The soluble polyphenol content was therefore related to the leaf chlorophyll content (Fig.Ā 7 and Additional file 2: Fig. S6). Of the five species tested, V. vinifera had the highest polyphenol content (19.6āĀ±ā2Ā mg GAE/mg Chl), followed by Q. pubescens (12.1āĀ±ā0.7Ā mg GAE/mg Chl) and P. oceanica (7.0āĀ±ā0.3Ā mg GAE/mg Chl). In contrast, O. basilicum (2.1āĀ±ā0.1Ā mg GAE/mg Chl) and A. thaliana (0.9āĀ±ā0.1Ā mg GAE/mg Chl) displayed the lowest contents. Q. pubescens and V. vinifera, which exhibit the highest polyphenol contents, are the species, for which the extraction of thylakoids required the PVC protocol like P. oceanica. Given that the inhibitory effects of polyphenols may be associated with specific compounds, the polyphenol composition of the five plants was determined. FigureĀ 7B shows that O. basilicum leaves contain a wide array of polyphenolic compounds compared to the other species (16 of the 19 tested molecules). However, the PVC protocol was not required for thylakoid extraction from this plant. Moreover, all polyphenols identified in P. oceanica (9 polyphenols), V. vinifera (5), and Q. pubescence (10) were detected in O. basilicum. This result suggests that the concentration of polyphenols, rather than their specific chemical composition, is a critical factor in preventing thylakoid membrane extraction.
Discussion
In this study, we developed a protocol for the extraction of proteins and thylakoids from polyphenol-rich plants, for which conventional procedures are not suitable. Initially, as we suspected cross-reaction of cell wall components with thylakoids, we performed pretreatments of leaves using enzymes acting directly against the cell wall polymers (e.g., lignin and cellulose, data not shown). These treatments did not improve the extraction of thylakoids and the quality of the final protein preparation. Several reports showed that polyphenols are characterized by gelling properties (reticulation and cross-linking) and high sensitivity to oxidation [14, 16,17,18]. To neutralize the oxidation of polyphenols leading to the formation of highly oxidizing quinones, the conventional extraction protocols for thylakoids and leaf proteins were first modified by adding VitC (5%), which is a soluble and inexpensive antioxidant. The optimal VitC concentration probably depends on other factors such as the polyphenol content and properties, which then determine the ability of oxidized polyphenols and VitC to establish a redox equilibrium. For example, in other marine plant species tested, the VitC concentration needs to be raised to 10%, (data not shown). The redox equilibrium reached through the optimal VitC concentration may favor the redox reaction of quinones with VitC molecules instead of proteins and other leaf cell components [32,33,34]. However, VitC alone was not sufficient to obtain proper thylakoid preparations as we also encountered issues attributed to the reticulation of polyphenols. Indeed, the cross-linking reaction of polyphenols leads to the formation of a mucilaginous mass trapping the thylakoids (Fig.Ā 1). We were able to inhibit this deleterious process by adding 0.1 up to 5% PEG to the grinding buffer (5% being the concentration we use for our work in Posidonia oceanica). PEG molecules own repeated ether groups all along the molecules, which bind to the hydroxyl groups of phenolic compounds via hydrogen bonds, thereby blocking their very high reactivity [35, 36].
Moreover, in the plants studied in the present work, we have also shown that the concentration of polyphenols in leaves is an essential parameter for the successful preparation of the thylakoid membranes since we excluded the implication of specific polyphenol species (Fig.Ā 7). This hypothesis is corroborated by the fact that polyphenol concentrations in the range of 1 to 2 GAE/mg chlorophyll were measured in A. thaliana and O. basilicum, species for which thylakoid extraction does not require the PVC protocol, while much higher levels were determined in Posidonia, oak, and grapevine. Interestingly, denaturing extraction of proteins in Posidonia in the absence of VitC and PEG led to very poor yield in samples collected at 2 and 15Ā m depth, which are characterized by polyphenol contents of ~ā7 GAE/mg chlorophyll (Fig.Ā 1B). Extraction was much more efficient in the samples collected at 26Ā m depth, which display a lower polyphenol content of 2.6 GAE/mg chlorophyll. Those data suggest a threshold value in the range of this concentration, above which extraction of native or denatured proteins becomes difficult in Posidonia.
Other biological and metabolic barriers could also prevent the successful extraction of thylakoids in plant species characterized by very thick and leathery leaves such as conifers, palm trees, or olive trees (Olea europaea) [37], which exhibit an elevated wax content in the cuticle. Nevertheless, the optimized protocol works well on the various plant species tested in this study, particularly Posidonia oceanica, but also Vitis vinifera and Quercus pubescens, which are very rich in polyphenols. Moreover, the PVC protocol did not seem to decrease the extraction yield of thylakoids since we obtained a similar amount of chlorophyll per fresh weight using the two protocols in O. basilicum and A. thaliana (Additional file 2: Fig. S1).
Although we did not assess the efficacy of Vitamin C (Vit C) alone in the two additional species necessitating the PVC protocol (Vitis vinifera and Quercus pubescens), its application is unlikely to facilitate thylakoid extraction. VitC, being a small molecule unlike PEG, a substantial molecular weight polymer, prevents polyphenol cross-linking by capturing them through hydrogen bonds and van der Waals forces [16,17,18]. As demonstrated in Posidonia leaves (Fig.Ā 3A), VitC alone fails to impede polyphenol cross-linking, rendering it ineffective for thylakoid isolation in Posidonia. Moreover, polyphenol levels are significantly higher in Vitis vinifera and Quercus pubescens compared to P. oceanica (Fig.Ā 7), suggesting that the use of VitC would not prevent polyphenol reticulation due to the elevated polyphenol concentrations in Vitis vinifera and Quercus pubescens.
Conclusion
This protocol can be applied to a large number of species that stand out by a high polyphenol content since most plants synthesize polyphenols, especially under environmental constraints. Thus, such a protocol will allow improving the knowledge not only in the photosynthesis field but also in other areas of plant physiology and metabolism that could not be easily studied up to now in plants grown in natural conditions.
Methods
Samples collection
Posidonia oceanica individuals (sheat-bundles and leaves) were harvested in the south of Frioul Island, Marseille, France (Coordinate 43Ā°Ā 16ā²Ā 11ā³Ā N 5Ā°Ā 17ā²Ā 32ā³Ā E). The collections were performed in January 2019 and January 2021 between 9 and 11 a.m. at 2, 15, and 26-m depths. The samplings were carried out within the framework of a protected species exemption with a prefectural decree authorizing them.
Immediately after harvesting, plants were kept in the dark in cold seawater. Leaves were separated from bundles at 4Ā Ā°C under green light, and epiphyte organisms were removed by washing with seawater. A. thaliana leaves were harvested from 4-week-old plants grown under 120Ā Ī¼mol photons/m2/s white light; O. basilicum stalks were purchased in a garden center store. Q. pubescens and V. vinifera leaves were harvested from a set of four different trees located in Luminy campus park (Marseille, France) and a vineyard (Aix-en-Provence, France, respectively. Young and adult-developing leaves were selected. Due to logistic constraints in sample collection, leaves from all species were frozen in liquid nitrogen following collection and stored at āā80Ā Ā°C for further studies.
Thylakoid membrane extraction
The thylakoid membrane extraction protocols further called āthe conventional protocolā and āPVC protocolā (the PEGā+āvitamin C) were inspired by [38] with some modifications in the buffer composition. For optimization experiments using Posidonia oceanica, we used leaves from different growing depths. Detailed instructions about the complete extraction procedure are shown in Additional file 3: Method S1. The composition of the buffers and the main step of the procedure are described below:
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Thylakoid membranes were extracted by grinding leaves in two distinct buffers (GB). For the conventional protocol, the buffer was composed of 20Ā mM Tricine pH 7.8, 0.3Ā M sorbitol, 10Ā mM EDTA, 10Ā mM NaHCO3, 0.15% bovine serum albumin (BSA), 10Ā mM of phosphatase inhibitor NaF, 5Ā mM benzamidine, 5Ā mM caproic acid. For the PVC protocol, the grinding buffer described above was supplemented with 5% Polyethylene glycol 4000 (PEG4000) and 5% ascorbic acid (vitamin C, Sigma).
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Chloroplasts were then burst using a hypotonic buffer (25Ā mM HepesāKOH pH 7.5, 25Ā mM sorbitol, 5Ā mM NaCl, 5Ā mM MgCl2, 5Ā mM KCl, 10Ā mM NaF, and 5Ā mM Benzamidine, 5Ā mM caproic acid, cOmpleteā¢ Protease Inhibitor Cocktail).
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The extracted thylakoid membranes were stored in the storage buffer (50Ā mM Hepes pH 7.5, 0.3Ā M sorbitol, 10Ā mM NaCl, 5Ā mM MgCl2, 10Ā mM NaF, cOmpleteā¢ Protease Inhibitor Cocktail).
Isolation and separation of photosystem supercomplexes
Large pore native-PAGE preparation
Gels were prepared using a 40T and 3C acrylamide/bis-acrylamide (w/v) mixture. This represents 38.8Ā g of acrylamide added to 1.2Ā g of bis-acrylamide diluted in 100Ā mL distilled water. This T/C ratio was chosen according to [1]. Gels containing 3ā12% (w/v) T, and 3% C were cast according to [1] with minor modification as no stacking gel was cast above the resolving gel (3ā12%).
Thylakoid membrane solubilization
Thylakoid membranes were washed twice in a solubilization buffer (50Ā mM BisāTris pH 7, 20% glycerol, 20Ā mM EDTA, 10Ā mM NaF). The membranes were then diluted to 1Ā mg/mL of chlorophyll. Then, an equal volume of 1ā6% (w/v) n-dodecyl-É-d-maltopyranoside (DDM, Anatrace, Ref: D310HA), or detergents specified otherwise, prepared in the solubilization buffer was added. Membranes and detergents were then incubated at 4Ā Ā°C for 20Ā min under gentle agitation. After solubilization, samples were centrifuged for 30Ā min at 18,000Ćg to remove insoluble materials. The supernatant was then used for Clear-Native and Blue Native-PAGE sample preparation.
Native PAGE sample preparation
Large pore Clear Native-PAGE (referred to as CN-PAGE) and Large pore Blue Native-PAGE (referred to as BN-PAGE) were carried out similarly in [1] and the solubilizate was supplemented with 1/10 volume of Serva Blue loading buffer for BN-PAGE (100Ā mM BisāTris methane pH 7.0, 0.5Ā M amino-n-caproic acid, 30% (w/v) sucrose and 50Ā mg/mL Serva Blue G) or 1/10 DOC (Deoxycholate) loading buffer for CN-PAGE (100Ā mM BisāTris methane pH 7.0, 0.5Ā M amino-n-caproic acid, 30% (w/v) sucrose and 5% DOC).
Separation of photosystem supercomplexes by lp-Clear and Blue native-PAGE
An anode buffer (25Ā mM BisTris, 25Ā mM Tricine, pH 7.0) was used for electrophoresis for both lp-CN and lp-BN gels. For lp-CN, the cathode buffer was composed of 50Ā mM BisTris-HCl, 50Ā mM Tricine, pH 7.0, and supplemented with 0.015% DDM and 0.05% DOC (Deoxycholate). For lp-BN, the cathode buffer was composed of 50Ā mM BisTris-HCl, 50Ā mM Tricine, pH 7.0, and supplemented with 0.015% DDM and 0.01% Serva Blue G 250. About 5ā10Ā Ī¼g of chlorophyll were loaded onto the gel wells. Electrophoresis was performed at 4Ā Ā°C for 7Ā h. The current intensity was fixed and adjusted so that at the start of the run, the voltage was around 70Ā V.
Second dimension urea-PAGE separation of supercomplexes isolated by BN-PAGE
The lanes of interest were cut out from 1st Dimension CN or BN-PAGE gels and incubated for 10Ā min in Laemmli denaturing sample buffer (125Ā mM TrisāHCl, pH 6.8, 20% glycerol, 2% SDS, 100Ā mM DTT, 0.01% bromophenol blue). The lanes were further loaded on the top of a 13% acrylamide/bis-acrylamide (37.5/1) resolving gel and fixed with agarose (0.125Ā M TrisāHCl, pH 6.8, 2% agarose, 0.001% bromophenol blue). For the run, the anode and cathode chambers were filled up with Laemmli running buffers (25Ā mM TrisāHCl, pH 8.3, 0.192Ā M glycine, 0.1% SDS). The migration was performed for approximately 2Ā h at 90Ā V.
Leaf total protein extraction
Posidonia oceanica leaves were ground in liquid nitrogen and the powder (ca. 1Ā g) was resuspended in 2Ā ml of a buffer containing 50Ā mM TrisāHCl pH 8, 1% SDS, 1Ā mM PMSF, 50Ā mM Ī²-mercaptoethanol and supplemented or not with 5% (w/w) vitamin C (Sigma) and/or 5% PEG-4000 (Sigma) to prepare extracts containing soluble and membrane proteins. After homogenization for a few minutes and vigorous shaking for at least 3Ā h at room temperature, extracts were centrifuged at room temperature for 30Ā min at 18,000Ćg. Four volumes of acetone were added to the supernatant to precipitate proteins at āā20Ā Ā°C overnight. After centrifugation, the protein pellet was then let to dry and resuspended in the Laemmli denaturing buffer.
SDS-PAGE protein electrophoresis and Western blot analysis
Proteins were separated in 13% acrylamide SDS-PAGE gel either for staining using Coomassie Blue (Imperialā¢ Protein Stain, Thermo Scientific) or for electroblotting onto 0.45Ā Āµm nitrocellulose (Pall Corporation) to perform immunoblot analysis. To evaluate protein yield in equal extract volumes, SDS-PAGE gels were carried out to quantify band intensity, following staining with Imperial Coomassie Blue (ThermoFisher), using the āOdyssey Infrared Imagerā scanning system at 680Ā nm (Licor, Lincoln, NE, USA). Membranes were stained using Ponceau Red (Sigma) to ensure homogenous transfer and loading of lanes (Additional file 4). Antibodies against ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and ATP synthase coupling factors (kindly provided by Dr. GH Schmid, University of Bielefeld, Germany) were diluted 1:10,000, and those raised against cytochrome f, PsaD and PRK (kindly provided by Dr. X. Johnson, BIAM, Aix-Marseille UniversitĆ©, France) were used diluted 1:5000, 1:5000 and 1:2000, respectively. The sera against PsbO and PsbQ (PSII subunits) were purchased from Agrisera (VƤnnƤs, Sweden) and used diluted 1:5000. Bound antibodies were detected using either an anti-rabbit immunoglobulin G coupled to alkaline phosphatase (Sigma) or a goat anti-rabbit secondary antibody coupled to a fluorescent molecule at a dilution of 1:10,000 (Alexa Fluor 680, Invitrogen) using the āOdyssey Infrared Imagerā at 680Ā nm (Licor, Lincoln, NE, USA).
Leaf chlorophyll extraction and assay
Leaves were ground in liquid nitrogen using a mortar and pestle, and the powder was then freeze-dried. The powder was further pulverized using a Vibro-crusher. The chlorophyll was then extracted in 80% acetone (buffered with 5Ā mM Hepes pH 7.0) containing 0.5% ascorbic acid (VitC) to avoid polyphenol oxidation. The acetone (80%)ā+āVitC buffer was prepared freshly to avoid VitC oxidation. The chlorophyll concentration of the purified thylakoids and the leaf extracts were measured as described in [39, 40].
Extraction and content determination of leaf hydrosoluble polyphenols
Polyphenols were extracted from the same powder used for chlorophyll extraction as described in [41]. The powder (50 or 100Ā mg) was mixed in a 15Ā mL tube containing 2.4Ā mL of 50% ethanol in water. The 15Ā mL tube was filled with water and placed in a microwave. The sample was then heated at 100Ā W power for 5Ā min. Every 30Ā s, the sample was homogenized and put back into the microwave for another 30-s run. Quantification of the extracted polyphenols was performed as described in [42]. The standard curve was performed using gallic acid. The quantity of extracted polyphenols was expressed in terms of Gallic Acid Equivalent (GAE) per dry leaf weight or normalized to the total chlorophyll content per dry weight.
Polyphenol profiling
Extraction and UHPLC-ESI-HRMS profiling analysis was carried out according to [46]. The major phenolic compounds in each species were identified with available standards or annotated with bibliographic data [45,46,47,48]. Common logarithm of the peak area means (>ā19,000Ā area/mL) for each condition (five technical replicates) and for each metabolite expressed per one microliter injected of the extract solution (one mg of the starting plant dry material per mL). Heatmaps were performed with the R software (version 3.6.3, company Foundation for Statistical Computing, Vienna, Austria) using the heatmap package (version 1.0.12). Heatmap data was clustered using Wardās method.
Electron microscopy
Samples were prepared as described in [43]. Grids were observed under the Tecnai G2 electron microscope.
Clark-type O2 measurement on isolated thylakoids
Light-induced O2 evolution was measured using a Clark-type electrode (Hansatech Instruments, England; Oxytraceā+āsoftware) under 1500Ā ĀµmolĀ photonsĀ mā2Ā sā1 of white LED illumination. Calibration followed Oxytraceā+ārecommendations: initially, the maximal oxygen concentration in an O2-saturated solution (storage buffer) was determined, followed by degassing with N2 bubbling until a minimal electrode potential indicated zero O2 concentration. Thylakoids, diluted to 10Ā Ī¼g/ml chlorophyll in storage buffer, were introduced into the measurement chamber in a volume of 2Ā ml. For P. oceanica thylakoids, 50Ā mM HCO3ā was added. Electron acceptors DCBQ (Dichlorobenzoquinone) and FeCy (Potassium ferricyanide) were incorporated at final concentrations of 250Ā ĀµM and 500Ā ĀµM, respectively, with NH4Cl at 4Ā mM to ensure membrane H+ proton permeability. The reaction medium was thoroughly degassed with nitrogen until the oxygen concentration reached zero, followed by hermetic sealing of the chamber. Degassing completion preceded the start of recording and illumination activation.
Data Availability
Data generated or analyzed during this study are included in this published article or are available upon request.
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Acknowledgements
We are grateful to Sandrine Ruitton, Pascal Mirleau, Deny Malengros, Fabrice Garcia, Michel Lafont, Sandrine Chenesseau, FrĆ©dĆ©rique Legendre and Bruno Belloni from the OSU Institut Pytheas (Aix Marseille University, CNRS, IRD, IRSTEA, OSU Institut PythĆ©as, Marseille, France) for the Posidonia oceanica samples collection. The electron microscopy experiments were performed on the PiCSL-FBI core facility (Nicolas BROUILLY and/or Fabrice RICHARD and/or AĆÆcha AOUANE, IBDM, AMU-Marseille), member of the France-BioImaging national research infrastructure (ANR-10-INBS-04). A special thanks to AĆÆcha Aouane for her expertise and help in the plant samples preparation for electron microscopy. Thanks to David Mathiron (Plateforme-Analytique, Institut de Chimie de Picardie, FR CNRS 3085, UniversitĆ© de Picardie Jules Verne, F-80039 Amiens, France), Jean Xavier Fontaine and Romain Roulard (UMR INRAE 1158 TransfrontaliĆØre BioEcoAgro, BIOlogie des Plantes et Innovation, BIOPI, UPJV, Amiens, France) for the help on the polyphenols analysis. We are grateful to Marina Siponen for the review of the manuscript.
Funding
This research was partly funded by the Swedish Foundation for Strategic Research SSF (Grant Number ARC19-0051).
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CJ, and QC planned and designed the research. QC, PR, FD, HL, RM, MI, and CR conducted experiments, collected data, and performed analysis. SP designed the statistical analysis of numeric data. QC, PR, and RM prepared figures and QC made the additional files. DG and colleagues performed P. oceanica sampling. CJ, QC, and PR wrote the manuscript. PR, SC, MI, and CR authors reviewed the manuscript. All authors read and approved the manuscript.
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Supplementary Information
Ā Original and uncropped gels, blots and pictures.
Additional file 1.
Ā Data set 1: Leaf chlorophyll and polyphenolsĀ content determination. Summary table data set 1:Ā Summary of computedĀ and normalized data from data set 1. Data Set 2 :Ā Determination of the yield of extractedĀ thylakoids chlorophyll.
Additional file 2: Figure S1.
Chlorophyll extraction yield using conventional and PVC protocols from selected plant species. Figure S2. 2D Urea-PAGE for the identification of P. oceanica supercomplexes isolated by CN-PAGE. Figure S3. Oxygen production rate from A. thaliana thylakoidsĀ (PVC protocol) and P. oceanica (2Ā m) thylakoids (conventional protocol, conventional protocolā+ā5% VitC and PVC protocol). Figure S4. 2D Urea-PAGE for the identification of peptides of supercomplexes isolated by BN-PAGE from various plant species. Figure S5. Organization and ultrastructure of leaf tissues from A. thaliana and Q. pubescens. Figure S6. Leaf chlorophyll content from various plant species.
Additional file 3: Method S1.
Detailed protocol for the extraction of the thylakoid membranes from P. oceanica and various plant species.
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Charras, Q., Rey, P., Guillemain, D. et al. An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants. Plant Methods 20, 38 (2024). https://doi.org/10.1186/s13007-024-01166-7
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DOI: https://doi.org/10.1186/s13007-024-01166-7