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Dec 2020

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Solubilization Method for Isolation of Photosynthetic Mega- and Super-complexes from Conifer Thylakoids
从针叶树类囊体中分离光合巨复合体和超复合体的增溶方法    

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Abstract

Photosynthesis is the main process by which sunlight is harvested and converted into chemical energy and has been a focal point of fundamental research in plant biology for decades. In higher plants, the process takes place in the thylakoid membranes where the two photosystems (PSI and PSII) are located. In the past few decades, the evolution of biophysical and biochemical techniques allowed detailed studies of the thylakoid organization and the interaction between protein complexes and cofactors. These studies have mainly focused on model plants, such as Arabidopsis, pea, spinach, and tobacco, which are grown in climate chambers even though significant differences between indoor and outdoor growth conditions are present. In this manuscript, we present a new mild-solubilization procedure for use with “fragile” samples such as thylakoids from conifers growing outdoors. Here, the solubilization protocol is optimized with two detergents in two species, namely Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). We have optimized the isolation and characterization of PSI and PSII multimeric mega- and super-complexes in a close-to-native condition by Blue-Native gel electrophoresis. Eventually, our protocol will not only help in the characterization of photosynthetic complexes from conifers but also in understanding winter adaptation.

Keywords: Blue-Native gel electrophoresis (凝胶电泳), Norway spruce (挪威云杉), Scots pine (苏格兰松), Picea abies (云杉), Pinus sylvestris (樟子松), Photosystem I (光系统 I), Photosystem II (光系统II), 2nd Dimension SDS-PAGE (二维 SDS-PAGE), Thylakoids (类囊体)

Background

Being the basis of life on Earth, photosynthesis has become a major research focus from the mid-19th century (Hill, 1937 and 1939; Benson and Calvin, 1950; Benson et al., 1950; Porter, 1950; Huzisige and Ke, 1993). Among several biochemical characterization methods, separation in polyacrylamide gel-based matrix of photosynthetic multi-subunit complexes, such as Photosystem I and II (PSI and PSII), was optimized from previous methods (Schägger and von Jagow, 1991; Kügler et al., 1997; Järvi et al., 2011; Haniewicz et al., 2015; Farci et al., 2017; Rantala et al., 2017). Furthermore, the solubilization approach to extract those complexes evolved over the last decades, moving from “harsh” toward “gentle” procedures by using detergents with lower critical micelle concentration (CMC) and at lower concentrations. The same is true for all procedures aimed at extracting membrane protein complexes for structural and functional analyses. Yet, sample preparation and biochemical characterization remain challenging.


In almost all biochemical studies, the isolation of protein complexes is of fundamental importance for the success of any project. Protocols need to be robust and reproducible to minimize variations between extractions. A crucial factor for successful isolation and characterization is the employment of the best-suited detergent depending on the material used, considering the CMC, incubation time, and the temperature during solubilization but also the mixing procedure. It is an even more delicate task to obtain protocols that keep the isolated membrane protein complexes intact, in a close-to-native state. A protein complex in solution does not necessarily have a native and stable structure; additionally, a detergent useful for extraction can be unsuitable for purification procedures and subsequent studies. Thylakoid membranes are solubilized with different detergents, such as n-Dodecyl-β-D-Maltopyranoside (β-DDM), n-Dodecyl-α-D-Maltopyranoside (α-DDM), and digitonin, at an efficiency depending on the thylakoid region (grana or stoma lamellae) and species (Barera et al., 2012; Haniewicz et al., 2013 and 2015; Albanese et al., 2016; Rantala et al., 2017). Isolated photosynthetic complexes have been fully characterized in Arabidopsis thaliana, Pisum sativum L., and Spinacia oleracea L. (Albanese et al., 2016; Rantala et al., 2017; Wood et al., 2018), and partially characterized in other species, such as Nicotiana tabacum L., Physcomitrella patens, Picea abies L. (Norway spruce), Selaginella martensii, Oryza sativa L., and Zea mays L. (D’Amici et al., 2008; Romanowska et al., 2008; Haniewicz et al., 2013 and 2015; Ferroni et al., 2014; Shen et al., 2017; Iwai et al., 2018; Grebe et al., 2019; Kouřil et al., 2020). Different detergent concentrations, mixtures of two detergents, and multi-step strategies (e.g., differential solubilization) are also used to optimize solubilization (Wientjes et al., 2013; Haniewicz et al., 2015). These approaches gained detailed knowledge on oligomeric states, subunit composition, and dynamics in PSII. To achieve reproducibility, most of these studies were performed on chamber-grown plants, but they do not necessarily yield answers to how photosynthetic regulation takes place under natural conditions. Conversely, studies on tree species (e.g., P. abies) growing in a challenging condition (like a boreal winter) provided a more complex picture of photosynthetic acclimation to environmental stress (Bag et al., 2020; Grebe et al., 2020; Chang et al., 2021). For these species, several differences in composition and abundance in photosynthetic complexes were reported: the presence of triple phosphorylation of the novel LHCB1 variant, needed to achieve winter-adapted chloroplast structures (Bag et al., 2020; Grebe et al., 2020); higher-order mega complexes (Kouřil et al., 2020), which are still a matter of debate in higher plants; and the absence of the PSI-NDH complex, mega-complexes, and the so-called M-LHCII complex (Bassi and Dainese, 1992; Nystedt et al., 2013; Kouřil et al., 2016). The latter is most likely an LHCII assembly containing both Lhcb3 and Lhcb6 that are missing in the Pinaceae family of conifers (Kouřil et al., 2016; Grebe et al., 2019) but present in A. thaliana.


Consequently, optimized biochemical procedures for the isolation and characterization of membrane complexes are needed to pursue such challenging studies, compare different species, and gain meaningful insights on structural/functional differences. In this manuscript, we use digitonin to apply and improve a mild-solubilization procedure for isolating thylakoid complexes from Norway spruce (P. abies) and Scots pine (P. sylvestris). The complexes are characterized by Blue-Native gel electrophoresis (BN-PAGE) and denaturing second dimension gel electrophoresis (2D SDS-PAGE). This newly optimized protocol will enable further characterization of PSI and PSII complexes by improving the extraction procedure in a close-to-native state, which, in these species, cannot be achieved by classic α-/β-DDM solubilization. Finally, the availability of this method opens paths for obtaining an in-depth understanding of the winter adaptation processes in evergreen pine trees.

Materials and Reagents

  1. Consumables

    1. Conical bottom microcentrifuge tubes, 1.5 ml

    2. Conical bottom centrifuge tubes, 50 ml and 15 ml

    3. Pipet tips (P1000, P200, and P10)


  2. Plant material

    P. abies and P. sylvestris L. plant materials collected from naturally grown trees in the Umeå University campus (63.8202° N, 20.3054° E)


  3. Reagents

    1. Invitrogen NativePAGE Bis-Tris (3-12% w/v gradient and 1.0 mm thickness, catalog number: BN1001BOX)

    2. Precision Plus Protein Dual Color Standards (SDS-PAGE molecular marker; Bio-Rad, catalog number: 1610374)

    3. SYPRO-ruby staining (ThermoFisher, catalog number: S12001)

    4. APS (Ammonium persulfate, Sigma, catalog number: A3678)

    5. Trizma (Sigma-Aldrich, catalog number: 93362)

    6. Tricine (Sigma-Aldrich, catalog number: T0377)

    7. Sodium Dodecyl Sulphate (SDS) (Sigma-Aldrich, catalog number: 436143)

    8. Serva Coomassie Blue G (SERVA Electrophoresis, catalog number: 17524.02)

    9. Bis-Tris (Sigma-Aldrich, catalog number: 14879)

    10. 6-aminocaproic acid (Sigma-Aldrich, catalog number: A7824)

    11. Glycerol (Sigma-Aldrich, catalog number: G5516)

    12. miracloth (Merk Millipore)

    13. Grinding buffer (B1) (see Recipes)

    14. Shock buffer (B2) (see Recipes)

    15. Storage buffer (B3) (see Recipes)

    16. Aqueous acetone solution (80%) (see Recipes)

    17. Washing buffer 1 (WB1) (see Recipes)

    18. Washing buffer 2 (WB2) (see Recipes)

    19. Washing buffer 3 (WB3) (see Recipes)

    20. Solubilizing BTH buffer (SB) (see Recipes)

    21. n-Dodecyl-α-D-Maltopyranoside (α-DDM) (see Recipes)

    22. Digitonin (see Recipes)

    23. 100 mM α-DDM working solution (see Recipes)

    24. 100 mM digitonin working solution (see Recipes)

    25. Loading buffer (LB) (see Recipes)

    26. Running buffer (RB) (see Recipes)

    27. Soft-staining solution (see Recipes)

    28. Acrylamide solution A (ThermoFischer) (see Recipes)

    29. Acrylamide solution B (ThermoFischer) (see Recipes)

    30. Cathode SDS buffer (CB) (see Recipes)

    31. Anode SDS buffer (AB) (see Recipes)

Equipment

  1. Blender (Multiblender Coline, Clas Ohlson)

  2. Automatic pipettes (P1000, P200, and P10)

  3. Heating block (95°C)

  4. Cooled microcentrifuge (Eppendorf, model: 4530)

  5. Microcentrifuge tube shaker (OrbitTM Digital Microtube and Microplate Shaker)

  6. Hoefer electrophoretic chamber (model: SE260 Mighty Small II)

  7. Bio-Rad electrophoretic chamber (mini)

  8. Bio-Rad mini-PAGE casting apparatus

  9. Power Supply (Bio-Rad Power pack ultra)

  10. pH meter (Bio-Rad)

  11. UV-VIS spectrophotometer (Lumina, scanning range 150-800 nm)

Software

  1. Chemidoc imaging system (Bio-Rad Image Lab version 6)

Procedure

  1. Sample harvesting

    1. Mature needles were cut from branches with a pair of scissors, collected into a glass beaker, and kept on ice (T ≤ 0°C) in the dark.

    2. Needles were harvested on sunny days during June and July from south-facing branches of five different P. sylvestris and P. abies trees growing in the Umeå University campus (63.8202° N, 20.3054° E).

    3. Upon cutting, the samples were collected into a glass beaker (a separate icebox for each biological replicate was used) and placed in an icebox (35-40 cm deep; ¾ filled with ice and ¼ headspace).

    4. After collection of approximately 3-4 small branches (10-15 g of needles), the whole icebox was covered with a double layer of thick-black cloth to ensure darkness in the sunny summer days during the 2-5 min transportation time to the laboratory.

    5. In the laboratory, samples were immediately subjected to isolation in the cold room (T ≤ 4°C) under dim green light. At no point from collecting branches until completion of isolation were samples moved from either icebox/cold room or darkness (see Figure S1 for sampling collection process and working setup).


  2. Thylakoid isolation

    Note: This step must be performed in a cold room.

    (Buffers required - B1, B2, and B3; water and buffers were kept in the cold room overnight)

    1. Samples were prepared according to Bag et al. (2020) with slight modifications to maximize protein complex isolation. Briefly, approximately 10-15 g (fresh weight) of needles were washed in ice-cold distilled water; then, 100-150 ml of ice-cold Grinding buffer (B1) was added (1:10 ratio of needles to buffer), and the mixture blended with a high-speed blender and pre-cooled at 4°C for 15 s in five repetitions, with a 30-s gap between each blending step [a high number of blending steps (5-6) at low blending time (15-20 s) and time lapse between each blending steps (25-40 s) are important for isolating intact higher-order complexes].

    2. The blended needles were filtered through two layers of miracloth (Merk Millipore) and centrifuged (4,500 × g for 8 min, at 4°C). The obtained pellet was resuspended in 30-35 ml Shock buffer (B2) and centrifuged (4,500 × g for 8 min, at 4°C).

    3. A subsequent resuspension, this time on Storage buffer (B3), was followed by a two-step centrifugation, first at low speed (200 × g for 2 min, at 4°C, discard pellet), then at higher speed (4,500 × g for 8 min, at 4°C).

    4. The final obtained pellet was resuspended on 800 µl of B3 buffer, aliquoted in small fractions (50-100 µl), and either used immediately or frozen in liquid nitrogen and stored at -80°C for further use. In all steps, the pellet was resuspended with thin hair paintbrushes to prevent excessive mechanical shearing, which might damage the higher-order protein complexes.


  3. Chlorophyll concentration determination

    Considering the stoichiometric relationship between Chlorophyll and Chl-binding proteins, the sample concentration is expressed as milligrams of Chls in a milliliter of sample.

    1. Chlorophyll concentration was calculated from 10 µl of samples extracted in 0.99 ml of 80% ice-cold acetone (1 ml final volume) by rigorous vortexing until no clumps could be seen (approximately 2 min; more time may be needed if the sample is very thick).

    2. After vortexing, tubes were centrifuged at 8,000 × g for 5 min and measured according to Porra et al. (1989).


  4. Sample preparation, electrophoresis, and isolation of protein complexes in native conditions

    Note: This step must be performed in a cold room.

    Major optimization methods for conifer samples start from this part. From here onwards, steps are denoted chronologically using the same numbers as they appear on the graphical illustration (Figure 1). The volumes and concentrations of the samples, buffers, and detergents only focus on the optimal detergent concentrations that allow maximum separation. Samples, buffers, and detergents required for different tested detergent concentrations are provided in Tables 1 and 2.



    Figure 1. Graphical illustration of the solubilization, gel running, and band identification


    Table 1. Solubilization with increasing concentrations of α-DDM. The table shows the volumes of solubilizing buffer (SB) and volumes/concentrations of α-DDM used for obtaining the increasing detergent to chlorophyll ratios presented in Figure 2.

    *The volumes are referred to the 100 mM α-DDM working solution (see Recipes).


    Table 2. Solubilization with increasing concentrations of digitonin. The table shows the volumes of SB and volumes/concentrations of digitonin used for obtaining the increasing detergent to chlorophyll ratios presented in Figure 3. Here, a two-steps solubilization was applied: first, 5 mM α-DDM, denoted as a constant +1 µl in the detergent volume column, were added 5 min prior to digitonin, which is added according to the desired concentration (changing volumes in the detergent volume column).


    *The volumes are referred to the 100 mM digitonin working stocking solution (see Recipes).


    1. Washing thylakoid samples

      (Buffers required - WB1, WB2, and WB3)

      Considering the high tannin and polyphenolic contents in conifer needles, separation of native complexes needs several washing steps before solubilization; otherwise, the detergent activity becomes limited. Here, we used three different washing buffers before solubilization. First, 250 µl of WB1 was aliquoted in Eppendorf tubes and a certain volume of thylakoids corresponding to 10 µg of chlorophylls was suspended by mixing by swirling the pipette in the tube (vortexing or pipetting up and down is strictly prohibited since this can destroy protein complexes). After that, the sample was centrifuged (3,000 × g for 2 min, at 4°C) and the supernatant discarded. Then, the same process was repeated twice, once with WB2 and once with WB3.

    2. Thylakoid solubilization

      (Buffers required - SB)

      For thylakoid solubilization, two different approaches are followed based on the detergent used.

      1. First, for α-DDM solubilization, we solubilized the washed thylakoid pellet in 8-9 µl of SB (depending on pellet size, the bigger the pellet, the smaller the volume of SB that should be added) to obtain 10 µl final volume of the resuspended washed thylakoids. In this way, the chlorophyll concentration of the sample becomes 1 mg/ml. After solubilization in SB, 100 mM of n-dodecyl-α-D- maltoside (α-DDM) is added at equal volume (obtaining a final chlorophyll concentration equal to 0.5 mg/ml and a detergent to chlorophyll ratio of 50:1) and solubilized on ice for 30 min at 4°C.

      2. The fractional solubilization process was applied as digitonin solubilization was reported to be unsuccessful in Arabidopsis protocols (Järvi et al., 2011; Rantala et al., 2017). First, thylakoids were solubilized in SB as before to a chlorophyll concentration of 1 mg/ml, then 1 µl of 100 mM n-dodecyl-α-D- maltoside (α-DDM) was added and the sample kept on ice for 5 min. Then, 6 µl of 100 mM digitonin was added and the final volume filled to 20 µl with SB (the final chlorophyll concentration was 0.5 mg/ml and the detergent to chlorophyll ratio 30:1). Gentle shaking was applied to tubes (max 200 rpm in OrbitTM Digital Microtube and Microplate Shake) and solubilization performed for 25 min at 4°C.

      3. Finally, both solubilized samples were centrifuged (14,000 × g for 25 min, at 4°C) and the obtained supernatant was used as for all analyses presented in this work.

    3. BN-PAGE process

      1. Blue-Native Polyacrylamide Gel Electrophoresis

        (Buffer required - LB and RB)

        BN-PAGE was carried out using 3-12% w/v gradient and 1.0 mm thickness pre-casted 10 well gels. Before loading, samples were prepared at a ratio of 1:10 in loading buffer. A sample volume of 16 µl was loaded, which corresponds to a final concentration of 8 µg/ml Chls in each electrophoretic lane. Before loading the samples on the gel, the precast gels need to be installed on the electrophoretic chamber and the tank needs to be filled with running buffer and cooled down to 4°C. For our samples, we used a sub-zero water bath to keep the temperature of the electrophoretic system continuously below 2°C throughout the running period.

      2. Native electrophoretic conditions

        The run was performed on a Hoefer electrophoretic chamber at 65 V for 14 h (or overnight) at 4°C in the dark. The running buffer had the same composition for both anode and cathode, but before use, the cathode buffer was supplemented with loading buffer at a ratio of 1:1,000.

    4. Post BN-PAGE processes

      1. Soft staining and gel image acquisition

        After the run, the gels were unmounted from the electrophoretic chambers and casting plates and washed in distilled water, fixed, and stained. BN-PAGEs were stained with the soft-staining solution (Figure 2) according to the procedure described by Farci et al. (2017). Gels were imaged on a white-light LED trans-illuminator using a digital camera (Canon EOS RP digital).



        Figure 2. Solubilization of pine (A, B) and spruce (C, D) thylakoids with different concentrations of α-DDM. A and C are RGB images of soft-stained gels taken with a digital camera. B and D are Coomassie-stained images of the same gels.


      2. 2D Denaturing Polyacrylamide Gel Electrophoresis

        For 2D separation, denaturing Sodium-Dodecyl-Sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Haniewicz et al. (2013). Briefly, the strips from the BN-PAGE were excised and denatured with Rotiload (Roth) at room temperature for 20 min. After denaturation, the strips were placed on top of a 10% denaturing SDS-PAGE (1.5 mm thickness) and sealed with 0.5% (w/v) agarose in cathode buffer. The molecular weight of the complexes isolated by 2D SDS-PAGE was estimated by plotting the retardation factor values (Rf, length of the band migration/length of the dye front) versus the log of the molecular weight of the molecular marker using a polynomial curve fit (second-order polynomial best-fit), according to the manufacturer's instructions. The obtained curve was used for calculating the apparent masses at the equivalent Rf of the 2D SDS-PAGE spots (data not shown). The masses were combined with western-blotting data for the identification of PSI and PSII subunits (see Step 5b).

      3. Denaturing electrophoretic conditions

        The run was performed on a Bio-Rad electrophoretic chamber and was carried out at 75 V for 3 h, at 4°C, in the dark. Freshly made cathode and anode running buffers were used.

    5. Post 2D SDS-PAGE processes

      1. Staining and gel image acquisition

        After the run, the gels were unmounted from the electrophoretic chambers and casting plates and washed in distilled water, fixed, and stained. 2D SDS-PAGEs were stained with SYPRO-ruby staining (Figures 4 and 5) according to the manufacturer’s protocol. Gels were imaged on a white-light LED trans-illuminator using a digital camera (Chemidoc imaging system).

      2. Western blotting analysis

        After 2D SDS-PAGE, proteins were transferred on PVDF membranes and further used for western blotting analysis and identification of PSI and PSII antibodies, as described in Bag et al. (2020).

Data analysis

  1. Optimizing concentrations of detergents

    Isolated native thylakoid membranes were solubilized as per the methods in two different detergents n-dodecyl-alpha-D-maltoside (α-DDM) and digitonin. The separation of thylakoid membrane complexes greatly depends on the optimal solubilization of samples (Haniewicz et al., 2015), and the detergent to chlorophyll ratio may vary from species to species (Kouřil et al., 2016 and 2020). For both detergents, we used a series of concentrations, as shown in Figure 2. For α-DDM, we started with the lowest concentration of detergent (0.5% w/vol =10 mM = 10:1 D:Chls) known to release complexes (to our knowledge) in other plant species (Shao et al., 2011), and concentrations were increased up to 70 mM in 10 mM increments (Figure 2). With increasing detergent concentrations, the release of complexes increases and reaches the maximum at 50 mM. It is worth noting that previously it was reported that the use of 40 mM β-DDM releases C2S2M2 as the largest complex, but here, 50 mM α-DDM released even larger complexes from the membrane (Figures 2 and 3), much larger than C2S2M2 complexes found in Arabidopsis. Most likely, these bands could be similar to higher-order super-complexes previously identified in conifers (Kouřil et al., 2020).

      For digitonin solubilization, similar increasing detergent concentrations from 4 mM (0.4% w/vol = 4:1 D:Chls) have been used, but protocols like those used for other species (Järvi et al., 2011) failed to release any complexes at all (Figure S2). We then supplemented digitonin with a very low concentration of α-DDM (5 mM) as this solubilization method is known to result in a better release of higher-order complexes (Wientjes et al., 2013). Moreover, we obtained clearer results with a two-step solubilization (Figure 3), as explained in the methods and previously reported for other species where it was named “differential solubilization” (Haniewicz et al., 2015). From different detergent concentrations, we chose 32 mM of digitonin to achieve maximum release of complexes when used in combination with 0.25 mM α-DDM in a two-steps solubilization process. By this, a total of six higher-order complexes (higher molecular weights than PSII dimer and PSI monomer) could be separated. The bands were named from 1-6 starting from the higher molecular weight (Figure 3E and 3F).



    Figure 3. Solubilization of pine (A, B) and spruce (C, D) thylakoids with different concentrations of digitonin supplemented with 5 mM α-DDM. A and C are RGB images of soft-stained gels taken with a digital camera. B and D are Coomassie-stained images of the same gels. The red boxes marked in B and D are enlarged and depicted at high contrast in the insets E and F, respectively. These show six higher-order complexes (higher molecular weights than PSII dimer and PSI monomer) here named from B1 to B6 starting from the higher molecular weight.


  2. Identification of complexes from 2D SDS-PAGE of 1D BN-PAGE strips

    Identification of thylakoid membrane protein complexes separated on native gels have been done in Arabidopsis and Tobacco (Järvi et al., 2011; Haniewicz et al., 2015; Farci et al., 2017). Recently, Grebe et al. (2019) identified proteins from native P. abies complexes released by β-DDM solubilization. We used the information from these maps to identify the protein composition of the complexes from pine (Figure 4C-ii, D-ii) and spruce (Figure 5C-ii, D-ii) and compared these with known Arabidopsis maps by running Arabidopsis thylakoids in parallel (Figure 4 and Figure 5C-i, D-i).



    Figure 4. Identification of possible presence of thylakoid proteins by 2D SDS-PAGE of 1D BN-PAGE strips. Comparison of α-DDM (A) and digitonin solubilization (B) of pine (Psl) and Arabidopsis thaliana (Ath) thylakoid samples. 2D SDS-PAGE of 1D strips (C) cut from α-DDM solubilized Arabidopsis (C-i) and pine (C-ii); 2D SDS-PAGE of 1D strips (D) cut from digitonin solubilized Arabidopsis (D-i) and pine (D-ii).



    Figure 5. Identification of possible presence of thylakoid proteins by 2D SDS-PAGE of 1D BN-PAGE strips. Comparison of α-DDM (A) and digitonin solubilization (B) of spruce (Pab) and Arabidopsis thaliana (Ath) thylakoid samples. 2D SDS-PAGE of 1D strips (C) cut from α-DDM solubilized Arabidopsis (C-i) and spruce (C-ii); 2D SDS-PAGE of 1D strips (D) cut from digitonin solubilized Arabidopsis (D-i) and spruce (D-ii).


      The 2D SDS-PAGE of α-DDM solubilized BN strips (Figure 4C-ii and Figure 5C-ii) shows that both first and second bands contain mostly PSII proteins, i.e., CP47, CP43, D1, D2, and LHCII (in comparison with band patterns from Grebe et al., 2020). No spots or bands could be assigned to PSI proteins; thus, we conclude that these bands most likely comprised higher-order PSII complexes (either super- or mega- or oligomers of PSII).

      Conversely, in 2D SDS-PAGE of digitonin solubilized BN strips, the first band (Figure 4D-ii and Figure 5D-ii) contains both PSI and PSII proteins, suggesting the presence of any of the three complexes, PSI-LHCI-PSII-LHCII (Rantala et al., 2017), PSII-LHCII, and PSI-LHCI oligomer (Kouřil et al., 2020). The second and third bands were mostly enriched in PSII subunits, while the fourth and fifth bands contain both PSI and PSII subunits. Finally, the last band was mainly represented by PSI core subunits, LHCI, and LHCII, but contained no PSII core subunits, suggesting this band to be either PSI-LHCI or PSI-LHCI-LHCII. This possibility of multiple complexes arises from the fact that, in conifers, there are unique higher-order complexes that are not present in so-called model plants such as Arabidopsis.


  3. Future perspectives

    The establishment of this protocol for thylakoid solubilization with non-ionic detergents facilitates further studies of higher-order complexes from conifers, for example, by mass spectroscopy or cryo-EM. This will enable a better understanding of the basis of photosynthetic adaptations in conifers, for example, during the boreal winter.

Recipes

Note: Stock/working solutions and buffers are prepared in Milli-Q water.

  1. Grinding buffer (B1)

    50 mM HEPES-KOH pH 7.5

    330 mM sorbitol

    5 mM MgCl2

    10% (w/v) PEG 6000

    10 mM NaF

    1 mM BSA

    0.65 mM Na-ascorbate

  2. Shock buffer (B2)

    50 mM HEPES-KOH pH 7.5

    5 mM MgCl2

    10 mM NaF

  3. Storage buffer (B3)

    50 mM HEPES-KOH pH 7.5

    5 mM MgCl2

    100 mM sorbitol

    10 mM NaF

  4. Aqueous acetone solution

    80% acetone

    20% water

  5. Washing buffer 1 (WB1)

    50 mM Bis-Tris HCl (pH 7.0)

    4% sorbitol

    10 mM NaF

    2.5% Pefabloc

  6. Washing buffer 2 (WB2)

    40 mM Bis-Tris HCl (pH 7.0)

    5% sorbitol

    10 mM NaF

    2.5% Pefabloc

  7. Washing buffer 3 (WB3)

    35 mM Bis-Tris HCl (pH 7.0)

    6% sorbitol

    10 mM NaF

    2.5% Pefabloc

  8. Solubilizing BTH buffer (SB)

    25 mM Bis-Tris HCl (pH 7.0)

    20% glycerol

    10 mM NaF

    2.5% Pefabloc

  9. α-DDM

    200 mM stock solution in distilled water

  10. Digitonin (200 mM stock solution in distilled water)

    Dissolving digitonin requires heating at 92-95°C for 10-12 min.

    If stock solution is frozen for further use, it needs similar heat treatment before it can be used again.

  11. 100 mM α-DDM working solution

    25 mM Bis-Tris HCl (pH 7.0)

    20% glycerol

    10 mM NaF

    2.5% Pefabloc

    200 mM α-DDM

    This solution needs to be freshly made every time.

  12. 100 mM digitonin working solution

    25 mM Bis-Tris HCl (pH 7.0)

    20% glycerol

    10 mM NaF

    2.5% Pefabloc

    200 mM digitonin

    This solution needs to be freshly made every time.

  13. Loading buffer (LB)

    5% serva Coomassie Blue G

    750 mM aminocaproic acid

    50 mM Bis-Tris HCl (pH 7.0)

    35% sucrose

  14. Running buffer (RB)

    50 mM BisTris, pH 6.8

    50 mM Tricine

  15. Soft-staining solution

    7% acetic acid

    10% ethanol

    83% distilled water

  16. Acrylamide 40% (w/v) solution A (ready-to-use) (ThermoFischer)

  17. Acrylamide 2% (w/v) solution B (ready-to-use) (ThermoFischer)

  18. Cathode SDS buffer (CB)

    0.1 M Tris

    0.1 M Tricine

    0.1% (w/v) SDS

  19. Anode SDS buffer (AB)

    0.2 M Tris, pH 8.9

Acknowledgments

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 675006 to P.B. and S.J. (Umeå University), The Swedish Research council (VR) and the Kempe Foundation to S.J. (Umeå University), and The Carl Tryggers Foundation for Scientific Research CTS 19:324) to D.F and W.P.S. (Umeå University). We would also like to thank Prof. Eva-Mari Aro and Prof. Roman Kouřil for helpful discussions.

Competing interests

The authors declare no conflict or competing interests.

References

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简介

[摘要]光合作用是收获阳光并将其转化为化学能的主要过程,几十年来一直是植物生物学基础研究的重点。在高等植物中,该过程发生在两个光系统(PSI 和 PSII)所在的类囊体膜中。在过去的几十年里,生物物理和生化技术的发展允许对类囊体组织以及蛋白质复合物和辅因子之间的相互作用进行详细研究。这些研究主要集中在模式植物,如拟南芥,p EA,小号pinach和牛逼obacco,这是 尽管室内和室外生长条件存在显着差异,但在气候室中生长。在这个手稿中,我们提出了一种新的温和溶过程的使用与“脆弱”的样品,如从不断增长的针叶树类囊体室外。在这里,增溶方案用两种物种的两种洗涤剂进行了优化,即挪威云杉 ( Picea abies ) 和苏格兰松 ( Pinus sylvestris) 。我们通过 Blue-Native 凝胶电泳优化了 PSI 和 PSII 多聚体巨型和超复合物在接近天然条件下的分离和表征。最后,我们的协议不仅会从针叶树光合复合物的特征也有助于在了解冬季适应性。


[背景]作为地球上生命的基础,光合作用已成为从中期的19大研究热点个世纪(山,1937年和1939年,本森和卡尔文,1950年,本森等人,1950年;波特,1950年; Huzisige和柯, 1993)。在几种生化表征方法中,在聚丙烯酰胺凝胶基质中分离光合多亚基复合物,如光系统 I 和 II(PSI 和 PSII),是从以前的方法中优化的(Schägger 和 von Jagow,1991;Kügler等人,1997;Järvi等人,2011 年;Haniewicz等人,2015 年;Farci等人,2017 年;Rantala等人,2017 年)。此外,增溶的方法,以提取演变在过去几十年的那些复合物,从“苛刻”朝向“温柔”程序移动小号通过使用清洁剂具有较低的临界胶束浓度(CMC)和在较低浓度。这同样适用于旨在为结构性和功能性analys提取膜蛋白复合物的所有程序真正ê秒。然而,样品制备和生化特性保持challeng ING 。

在几乎所有的生化研究,蛋白复合物的分离是一个成功的根本重要性NY项目。协议需要稳健且可重复,以最大限度地减少提取之间的差异。对于成功的分离和表征的一个关键因素是所述雇用的精神疾病根据使用的材料最适合的洗涤剂,考虑荷兰国际集团的CMC,孵育时间,和溶解过程中的温度,而且在混合过程。获得使分离的膜蛋白复合物保持完整、接近天然状态的协议是一项更加精细的任务。溶液中的蛋白质复合物不一定具有天然和稳定的结构;此外,用于提取的去污剂可能不适用于纯化程序和后续研究。类囊体膜中溶解有不同的洗涤剂,如正十二烷基β-d-D-吡喃麦芽糖苷(β-DDM),正-十二烷基α-d-D-吡喃麦芽糖苷(α-DDM),和毛地黄皂苷,在效率取决于类囊体区域(谷物或气孔薄片)和物种(Barera等人,2012 年;Haniewicz等人,2013 年和 2015 年;Albanese等人,2016 年;Rantala等人,2017 年)。分离的光合复合物已在拟南芥、豌豆和菠菜中得到充分表征(Albanese等,2016;Rantala等,2017;Wood等,2018),并在其他物种中部分表征,例如烟草L.、Physcomitrella patens 、Picea abies L. (Norway spruce)、Selaginella martensii 、Oryza sativa L.和Zea mays L. (D'Amici et al. , 2008; Romanowska et al. , 2008; Haniewicz等人,2013 年和 2015 年;Ferroni等人,2014 年;Shen等人,2017 年;Iwai等人,2018 年;Grebe等人,2019 年;Kouřil等人,2020 年)。不同的去污剂浓度、两种去污剂的混合物和多步骤策略(例如差异溶解)也用于优化溶解(Wientjes等人,2013 年;Haniewicz等人,2015 年)。这些方法获得了关于 PSII 中的寡聚状态、亚基组成和动力学的详细知识。为了实现可重复性,这些研究中的大多数是在室内种植的植物上进行的,但它们不一定能得出在自然条件下光合作用调节如何发生的答案。相反,在树种(研究例如,P.冷杉)是一个具有挑战性的生长条件(如北半球冬季)提供光合适应的更复杂的图像对环境胁迫(袋等人,2020;鷉等人,2020; Chang等人,2021 年)。对于这些种类,在组成和数量在光合络合物几个不同的报道:三重磷酸化的存在的新颖LHCB1变体,实现冬季需要-适于叶绿体结构(包。等人,2020;鷉。等人,2020) ; 高阶巨型复合物(Kouřil et al. , 2020),这在高等植物中仍然是一个有争议的问题;和不存在的PSI-NDH复杂,大型复合物,和所谓的M-LHCII复合物(巴斯和傣族,1992; Nystedt等人,2013;Kouřil 。等人,2016)。所述后者是最有可能的组件含有Lhcb3和Lhcb6其缺少在LHCII的松科针叶树家族的(Kouřil等人,2016;鷉等人,2019),但存在于拟南芥。

对于因此,优化的生物化学程序的分离和表征的膜复合物,需要寻求这样的挑战螺柱IES ,比较不同的物种,并获得有意义的见解对结构/功能上的差别。在这份手稿中,我们使用毛地黄皂苷应用并改进了一种温和的增溶程序,用于从挪威云杉 ( P. abies ) 和苏格兰松 ( P. sylvestris ) 中分离类囊体复合物。该复合物是特征分析d由蓝天然凝胶电泳(BN-PAGE)和变性第二维凝胶电泳(2D SDS-PAGE)。这种新优化的协议将通过改进接近天然状态的提取程序来进一步表征 PSI 和 PSII 复合物,在这些物种中,经典的 α-/β-DDM 增溶无法实现。˚F inally ,这种可用性方法开小号在常绿松树获得冬季适应过程的深入了解路径。

关键字:凝胶电泳, 挪威云杉, 苏格兰松, 云杉, 樟子松, 光系统 I, 光系统II, 二维 SDS-PAGE, 类囊体


材料和试剂

 
耗材
锥形底微量离心管,1.5 ml
锥形底离心管,50 ml 和 15 ml
移液器吸头(P1000、P200 和 P10)
 
植物材料
P 。冷杉和P 。西尔维斯特 L . 从于默奥大学校园 (63.8202° N, 20.3054° E) 自然生长的树木中收集的植物材料
 
试剂
Invitrogen NativePAGE Bis-Tris(3 - 12% w/v 梯度和 1.0 mm 厚度,目录号:BN1001BOX )
Precision Plus 蛋白质双色标准品(SDS-PAGE 分子标记;Bio - Rad ,目录号:1610374 )
SYPRO-红宝石染色(ThermoFisher ,目录号:S12001 )
APS(过硫酸铵,Sigma ,目录号:A3678 )
Trizma(Sigma-Aldrich ,目录号:93362 )
Tricine(Sigma-Aldrich ,目录号:T0377 )
十二烷基磺酸钠小号ulphate(SDS)(Sigma-Aldrich公司,目录号:436143 )
Serva Coomassie Blue G(SERVA Electrophoresis ,目录号:17524.02 )
Bis-Tris(Sigma-Aldrich ,目录号:14879 )
6-氨基己酸(Sigma-Aldrich ,目录号:A7824 )
甘油(Sigma-Aldrich ,目录号:G5516 )
奇迹布(默克密理博)
研磨缓冲液 (B1) (见配方)
冲击缓冲器(B2)(见配方)
存储缓冲区(B3)(见配方)
丙酮水溶液(80%)(见配方)
洗涤缓冲液 1(WB1)(见配方)
洗涤缓冲液 2(WB2)(见配方)
洗涤缓冲液 3(WB3)(见配方)
溶解 BTH 缓冲液(SB)(见配方)
正十二烷基-α-D-吡喃麦芽糖苷(α-DDM)(见食谱)
洋地黄皂苷(见食谱)
100 mM α-DDM 工作溶液(见配方)
100 mM 毛地黄皂苷工作溶液(见配方)
加载缓冲液(LB)(见配方)
运行缓冲液 (RB) (见配方)
软染色溶液(见配方)
丙烯酰胺溶液 A(ThermoFischer)(见配方)
丙烯酰胺溶液 B(ThermoFischer)(见配方)
阴极 SDS 缓冲液(CB)(见配方)
阳极SDS缓冲液(AB)(见配方)
 
设备
 
Blender (Multiblender Coline, Clas Ohlson)
自动移液器(P1000,P200 ,和P10)
加热块 (95°C)
冷却微量离心机(Eppendorf ,型号:4530)
微量离心管摇床(Orbit TM Digital Microtube and Microplate Shaker)
Hoefer 电泳室(型号:SE260 Mighty Small II)
Bio - R ad 电泳槽 (迷你)
Bio - Rad mini-PAGE 浇铸装置
电源(Bio - Rad Power pack ultra)
pH计(Bio - Rad )
UV-VIS 分光光度计 (Lum ina, 扫描范围 150-800 nm)
 
软件
 
Chemidoc 成像系统(Bio - Rad Image Lab 版本 6)
 
程序
 
样品采集
成熟针从树枝剪有一对剪刀,在收集到玻璃烧杯中,并保持在冰上(T≤0℃)中的暗。
在六月和七月的阳光明媚的日子里,从五个不同P的朝南枝条收获针叶。sylvestris和P . 于默奥大学校园内生长的冷杉(63.8202° N, 20.3054° E)。
切割后,将样品收集到玻璃烧杯中(每个生物重复使用一个单独的冰箱)并放置在冰箱中(35-40 厘米深;3/4 充满冰和 1/4 顶空)。
收集大约 3-4 根小树枝(10-15 克针)后,整个冰箱用双层厚黑布覆盖,以确保在2-5分钟的运输时间内在阳光明媚的夏日里保持黑暗。实验室。
在实验室中,样品立即在冷室(T ≤ 4°C)中在昏暗的绿光下进行隔离。在没有从收集分支点未直到隔离完成了从移动样品或者冰箱/冷室或黑暗(参见图小号1用于采样收集过程和工作设置)。
 
类囊体分离
注:这一步必须执行主编在寒冷的房间。
(需要缓冲液 - B1、B2 和 B3;水和缓冲液在冷藏室中保存过夜)
根据 Bag等人的方法制备样品。(2020)的方法稍加修改š最大化蛋白复合物隔离。简而言之,在冰冷的蒸馏水中洗涤大约 10-15 克(鲜重)的针;然后,100-150毫升冰冷的研磨缓冲液(B1)中加入(1:10比率针的缓冲),并且将混合物混合有高速搅拌器和在4℃下在预冷却的15秒5次重复,具有一个30 - s各自共混步骤之间间隙[高数量的混合步骤(5-6)在低混合时间(15-20 S)和时间的流逝各混合步骤之间(25-40或多个)是重要用于分离完整的高阶复合物] 。
通过两个过滤的共混针MIRACLOTH的层(默克Millipore)中并离心(4 ,500 ×克8分钟,在4℃)。将得到的沉淀重新悬浮于30-35毫升休克缓冲液(B2)并离心(4 ,500 ×克8分钟,在4℃)。
随后在存储缓冲液 (B3) 上重新悬浮,然后进行两步离心,首先以低速(200 × g ,2 分钟,4°C,丢弃沉淀),然后以更高速度(4 ,500 × g 8 分钟,4°C)。
将最终获得的沉淀物重新悬浮在 800 µl B3 缓冲液中,分成小份(50-100 µl),立即使用或在液氮中冷冻并储存在-80°C 以备进一步使用。在所有步骤中,将沉淀物resuspen DED薄头发漆刷以防止过度的机械剪切,这可能会损坏高阶蛋白复合物。
 
叶绿素浓度测定
考虑叶绿素和叶绿素结合蛋白之间的化学计量关系,样品浓度表示为毫克的在一个的Chls毫升样品。
叶绿素浓度从10微升在0.99毫升80%的提取的样品的计算出的冰冷的丙酮(1毫升最终体积)通过严格的涡旋直至没有团块可以看出(约2分钟;更多的时间可能需要如果样品是非常厚的)。
涡旋后,将管以8 , 000 × g离心5 分钟,并根据 Porra等人的方法进行测量。(1989)。
 
天然条件下蛋白质复合物的样品制备、电泳和分离
注:这一步必须执行主编在寒冷的房间。
针叶树样品的主要优化方法从这部分开始。从这里开始,步骤按时间顺序使用与图形说明(图 1)中出现的相同数字表示。的体积和浓度的样品,缓冲剂,和洗涤剂只着眼于最佳去污剂浓度,允许最大的分离。表 1 和 2 中提供了不同测试去污剂浓度所需的样品、缓冲液和去污剂。
 
 
图 1. 溶解、凝胶运行和条带识别的图示
 
表 1.随着 α-DDM 浓度的增加而增溶。该表显示增溶缓冲液(SB)和体积的体积/用于获得增加洗涤剂到F中呈现叶绿素比值α-DDM的浓度IG URE 2。
 
*体积是指 100 mM α-DDM工作溶液(参见配方)。
 
表 2.增加毛地黄皂苷浓度的增溶作用。该表显示SB和体积的体积/毛地黄皂苷用于获得增加洗涤剂到F中呈现叶绿素比的浓度IG URE 3.这里,两步溶解涂布:第一,5mM的α-DDM,表示为常数1微升在洗涤剂体积柱,加入前5分钟,以洋地黄皂苷,其中加入根据所需的浓度(改变量在洗涤剂体积列)。
 
*体积指的是 100 mM毛地黄皂苷工作储存溶液(参见配方)。
 
洗涤类囊体样品
(需要缓冲液 - WB1、WB2 和 WB3)
考虑到针叶树针中单宁和多酚含量高,天然复合物的分离在溶解前需要几个洗涤步骤;否则,去污剂的活性就会受到限制。在这里,我们在增溶前使用了三种不同的洗涤缓冲液。首先,将250μl WB1的是在Eppendorf管等分和类囊体的一定体积小号对应于10微克的叶绿素被通过混合悬浮通过旋动移液管在所述管(涡旋或吹打上下严禁因为这可以破坏蛋白质复合体)。此后,样品进行离心(3 ,0 00 ×克2分钟,在4℃) ,弃去上清液。然后,同样的过程被重复了两次,一次与WB2和曾经与WB3。
类囊体增溶
(需要缓冲器 - SB)
对于类囊体增溶,根据所使用的去污剂可采用两种不同的方法。
首先,对α-DDM溶解,我们溶解的洗涤类囊体沉淀在8-9 SB的微升(取决于粒料尺寸,颗粒越大,将较小的SB的体积其应该被添加)以获得10微升最终体积重新悬浮的洗涤类囊体。以这种方式,所述叶绿素升样品的浓度为1毫克/毫升。在SB,100mM的溶解后Ñ十二烷基-α-D-麦芽糖苷(α-DDM)加入在等体积(获得最终浓度叶绿素等于0.5毫克/毫升和一个洗涤剂叶绿素升比的50:1 ) 并在 4°C 下在冰上溶解 30 分钟。
施加分数增溶方法如洋地黄皂苷增溶据报道,在不成功拟南芥协议(贾维等人,2011; Rantala等人,2017)。首先,在类囊体SB中溶解作为befor E要1:1的叶绿素浓度毫克/毫升,然后将1μl的100mM Ñ十二烷基-α-D-麦芽糖苷(α-DDM)中并将样品在冰上放置5分钟 然后,6微升100mM毛地黄皂苷的是加入最终体积填充至20微升用SB(最终叶绿素浓度为0.5毫克/毫升和所述洗涤剂叶绿素比30:1) 。ģ entle摇动被施加到管(最大200rpm下在轨道TM数字微型试管及微孔板摇),并溶解在4℃下25分钟进行。
最后,既溶解样品进行离心(14 ,000 ×克25分钟,在4℃),将得到的上清液被用作所有analys ê S IN这项工作中提出。
BN-PAGE工艺
Blue-Native 聚丙烯酰胺凝胶电泳
(需要缓冲区 - LB 和 RB)
BN-PAGE进行了使用3 - 12%w / v的梯度和1.0毫米的厚度预铸10个孔凝胶。装载前,制备样品在1:10在上样缓冲液的比。甲样品16微升的体积装填,其对应š至8微克/毫升的Chls的每个电泳泳道的最终浓度。在上样之前,预制凝胶需要安装在电泳室上,槽需要填充运行缓冲液并冷却至 4°C。对于我们的样品,我们使用低于零的水浴在整个运行期间将电泳系统的温度保持在 2°C 以下。
天然电泳条件
运行在 65 V 的 Hoefer 电泳室上在 4°C 下在黑暗中进行 14 小时(或过夜)。运行缓冲液为具有阳极和相同的组成的阴极,但在使用前,阴极缓冲液补充有加样缓冲液以1:1的比率,000。
后BN-PAGE过程
软染色和凝胶图像采集
电泳结束后,将凝胶从卸除的电泳室和铸造板和在蒸馏水中洗涤,固定并染色。BN-页面用染色的根据Farci描述的程序软染色溶液(图2)等。(2017)。使用数码相机(Canon EOS RP digital)在白光LED 透射照明器上对凝胶成像。
 
 
图 2. 松树 (A, B) 和云杉 (C, D) 类囊体与不同浓度的 α-DDM 的增溶作用。A和C是用数码相机拍摄的软染色凝胶的 RGB 图像。B和D是相同凝胶的考马斯染色图像。
 
二维变性聚丙烯酰胺凝胶电泳
对于 2D 分离,根据 Haniewicz等人的方法进行了变性十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE) 。(2013)。简而言之,将来自 BN-PAGE 的条带在室温下用 Rotiload (Roth) 切除并变性 20 分钟。变性后,将条带置于 10% 变性 SDS-PAGE(1.5毫米厚)的顶部,并用阴极缓冲液中的 0.5% (w/v) 琼脂糖密封。通过使用多项式曲线绘制延迟因子值(Rf,带迁移的长度/染料前沿的长度)与分子标记的分子量对数来估计由 2D SDS-PAGE 分离的复合物的分子量配合(二阶多项式的最佳拟合),根据该制造商的说明。获得的曲线用于计算 2D SDS-PAGE 点的等效 Rf 处的表观质量(数据未显示)。将质量与w酯印迹数据相结合,用于识别 PSI 和 PSII 亚基(参见步骤 5b)。
变性电泳条件
物上的执行的运行生物- Rad公司电泳室和在75伏进行了3小时,在4℃下,在黑暗中。使用新鲜制作的阴极和阳极运行缓冲液。
后 2D SDS-PAGE 工艺
染色和凝胶图像采集
电泳结束后,将凝胶从卸除的电泳室和铸造板和在蒸馏水中洗涤,固定并染色。根据制造商的方案,使用 SYPRO-ruby 染色(图 4 和 5)对 2D SDS-PAGE 进行染色。使用数码相机(Chemidoc成像系统)在白光 LED透射照明器上对凝胶成像。
蛋白质印迹分析
2D SDS-PAGE后,将蛋白质转移的PVDF膜上,并进一步用于瓦特西部时代印迹分析和PSI和PSII抗体的识别,如在描述的袋等。(2020)。
 
数据分析
 
优化洗涤剂的浓度
分离的天然类囊体膜按照方法在两种不同的去污剂正十二烷基-α-D-麦芽糖苷(α-DDM)和毛地黄皂苷中溶解。的类囊体膜的复合物的分离在很大程度上取决于样品的最佳增溶(Haniewicz等人,和洗涤剂以叶绿素比可以变化,2015)从物种的物种(Kouřil等人,2016;Kouřil 。等人,2020) . 对于这两种去污剂,我们使用了一系列浓度,如图 2 所示。对于 α-DDM,我们从已知的最低去污剂浓度 (0.5% w/vol =10 mM = 10:1 D:Chls) 开始释放复合物(就我们所知)在其他植物物种(邵等人,2011) ,和浓度为营养不良的人口增加编高达70毫米的10mM增量(图2)。随着增加去污剂浓度,配合物的增加,并且达到释放的最大在50毫米。值得注意的是,以前据报道,使用的40mM的β-DDM释放Ç 2 š 2中号2为最大的复合物,但在这里,释放的50mMα-DDM更大从膜复合物(图2和图3 ),远大于在拟南芥中发现的C 2 S 2 M 2复合物。最有可能的是,这些带可能类似于先前在针叶树中发现的高阶超级复合体(Kouřil等,2020)。
  对于毛地黄皂苷增溶,使用了类似的从 4 mM(0.4% w/vol = 4:1 D:Chls)增加洗涤剂浓度的方法,但与用于其他物种的协议(Järvi等人,2011 年)未能释放任何复合物完全(图S 2)。然后我们用非常低浓度的 α-DDM (5 mM)补充毛地黄皂苷,因为已知这种增溶方法可以更好地释放高阶复合物(Wientjes等人,2013 年)。此外,我们获得了清晰的ER与两步溶解(图3)的结果,在这些方法中所解释和先前报道的其他物种,其中它被命名为“差异溶解”(Haniewicz等人,2015)。从不同的洗涤剂浓度中,我们选择了 32 mM 的毛地黄皂苷,以在两步溶解过程中与 0.25 mM α-DDM 结合使用时实现复合物的最大释放。通过这种方式,总共六个高阶复合物(分子量较高的权重比PSII二聚体和PSI单体)可被分离。所述条带从1-6从较高分子量的起始命名(图URE 3E和3 F)。
 
 
图 3. 松树 (A, B) 和云杉 (C, D) 类囊体与不同浓度的毛地黄皂苷的增溶作用,辅以 5 mM α-DDM。A和C是用数码相机拍摄的软染色凝胶的 RGB 图像。B和D是相同凝胶的考马斯染色图像。B 和 D 中标记的红色框分别在插图 E 和 F中以高对比度放大和描绘。这些节目6高阶复合物(较高分子量的卫克HTS比PSII二聚体和PSI单体)ħ ERE从B1到B6从较高分子量的起始命名。
 
从 1D BN-PAGE 条带的 2D SDS-PAGE 鉴定复合物
已在拟南芥和烟草中鉴定在天然凝胶上分离的类囊体膜蛋白复合物(Järvi等人,2011 年;Haniewicz等人,2015 年;Farci等人,2017 年)。ř ecently ,鷉等。(2019) 从天然P 中鉴定出蛋白质。β-DDM 增溶释放的冷杉复合物。我们使用从这些图中的信息来识别从松复合物的蛋白质组合物(图4C- II ,D- II )和云杉(图5C- II ,D- II ),并比较这些已知拟南芥通过运行映射拟南芥类囊体并行(图 4 和图5C- i 、D- i )。
 
 
图 4. 通过 1D BN-PAGE 条带的 2D SDS-PAGE 鉴定可能存在的类囊体蛋白。松树 (Psl) 和拟南芥(Ath) 类囊体样品的 α-DDM ( A ) 和毛地黄皂苷增溶 ( B ) 的比较。从 α-DDM 溶解的拟南芥( Ci ) 和松树 ( C-ii )切下的 1D 条带 ( C ) 的2D SDS-PAGE ;从毛地黄皂苷溶解的拟南芥( Di ) 和松树 ( D-ii )切下的 1D 条 ( D ) 的2D SDS-PAGE 。
 
 
图 5. 通过 1D BN-PAGE 条带的 2D SDS-PAGE 鉴定可能存在的类囊体蛋白。云杉 (Pab) 和拟南芥(Ath) 类囊体样品的 α-DDM ( A ) 和毛地黄皂苷增溶 ( B ) 的比较。从 α-DDM 溶解的拟南芥( Ci ) 和云杉 ( C-ii )切下的 1D 条带 ( C ) 的2D SDS-PAGE ;从毛地黄皂苷溶解的拟南芥( Di ) 和云杉 ( D-ii )切下的 1D 条带 ( D ) 的2D SDS-PAGE 。
 
  α-DDM 溶解的 BN 条带的2D SDS-PAGE(图 4C- ii和图 5C- ii )显示第一条带和第二条带主要包含 PSII 蛋白,即CP47、CP43、D1、D2 和 LHCII(在compari儿子从鷉带图案等人,2020)。没有斑点或条带可以分配给 PSI 蛋白;因此,我们得出结论,这些条带很可能包含更高阶的 PSII 复合物(PSII 的超聚体或超聚体或寡聚体)。
相反,在毛地黄皂苷的2D SDS-PAGE溶解BN条,所述第一条带(图4D- II和图5D- II )同时含有PSI和PSII蛋白,提示的任何三种复合物的存在,PSI-LHCI-PSII- LHCII(Rantala等,2017)、PSII-LHCII 和 PSI-LHCI 低聚物(Kouřil等,2020)。第二和第三条带主要富含 PSII 亚基,而第四和第五条带同时包含 PSI 和 PSII 亚基。最后,最后一个波段主要由 PSI 核心亚基 LHCI 和 LHCII 代表,但不包含PSII 核心亚基,表明该波段是 PSI-LHCI 或 PSI-LHCI-LHCII。多种复合物的这种可能性源于这样一个事实,针叶树,还有独特的高层次复合物中不存在所谓的模型植物,如拟南芥。
 
未来展望
此协议用于类囊体增溶的建立与非离子型去污剂促进小号高阶复合物的进一步研究从针叶树,例如,通过质谱或冷冻电镜。这将有助于更好地了解针叶树光合适应的基础,例如,在北方冬季。
 
食谱
 
不è :库存/工作方案和缓冲区在Milli准备- Q水。
研磨缓冲液(B1)
50 mM H EPES -KOH pH 7.5
330 mM 山梨糖醇
5 毫米氯化镁2
10% (w/v) PEG 6000
10 毫米氟化钠
1 毫米牛血清白蛋白
0.65 mM 抗坏血酸钠
减震器(B2)
50 mM HEPES -KOH pH 7.5
5 毫米氯化镁2
10 毫米氟化钠
存储缓冲区(B3)
50 mM HEPES -KOH pH 7.5
5 毫米氯化镁2
100 mM 山梨糖醇
10 毫米氟化钠
丙酮水溶液
80% 丙酮
20% 水
洗涤缓冲液 1 (WB1)
的50mM的Bis-Tris HC升(pH为7 0.0)
4% 山梨糖醇
10 毫米氟化钠
2.5% 派法布洛克
洗涤缓冲液 2 (WB2)
的40mM的Bis-Tris HC升(pH为7 0.0)
5% 山梨糖醇
10 毫米氟化钠
2.5% 派法布洛克
洗涤缓冲液 3 (WB3)
的35mM的Bis-Tris HC升(pH为7 0.0)
6% 山梨糖醇
10 毫米氟化钠
2.5% 派法布洛克
增溶 BTH 缓冲液(SB)
的25mM的Bis-Tris HC升(pH为7 0.0)
20% 甘油
10 毫米氟化钠
2.5% 派法布洛克
α-DDM
200 mM 蒸馏水储备液
洋地黄皂苷(200 mM 蒸馏水储备液)
D溶解毛地黄皂苷需要在 92-95°C 下加热 10-12 分钟。
如果原液冷冻以备进一步使用,则需要类似的热处理才能再次使用。
100 mM α-DDM 工作溶液
的25mM的Bis-Tris HC升(pH为7 0.0)
20% 甘油
10 毫米氟化钠
2.5% 派法布洛克
200 mM α-DDM
每次都需要重新制作此解决方案。
100 mM 毛地黄皂苷工作溶液
的25mM的Bis-Tris HC升(pH为7 0.0)
20% 甘油
10 毫米氟化钠
2.5% 派法布洛克
200 mM 毛地黄皂苷
每次都需要重新制作此解决方案。
加载缓冲区(LB)
5% serva 考马斯蓝 G
750 mM 氨基己酸
的50mM的Bis-Tris HC升(pH为7 0.0)
35%蔗糖
运行缓冲液(RB)
50 mM BisTris,pH 6.8
50 mM Tricine
软染色溶液
7%醋酸
10%乙醇
83% 蒸馏水
丙烯酰胺 40% (w/v) 溶液 A(即用型)(ThermoFischer)
丙烯酰胺 2% (w/v) 溶液 B(即用型)(ThermoFischer)
阴极 SDS 缓冲液(CB)
0.1 M 三角
0.1 M 三辛胺
0.1% (w/v) SDS
阳极 SDS 缓冲液(AB)
0.2 M Tris,pH 8.9
 
致谢
 
该项目已根据 Marie Skłodowska-Curie 赠款协议 2020 获得欧盟地平线 2020 研究和创新计划的资助。675006 给 PB 和 SJ(于默奥大学),瑞典研究委员会 (VR) 和 Kempe 基金会给 SJ(于默奥大学),卡尔·特里格斯基金会科学研究 CTS 19:324)给 DF 和 WPS(于默奥大学)。我们还要感谢 Eva-Mari Aro 教授和 Roman Kou ř il教授的有益讨论。
 
利益争夺
 
作者声明没有冲突或竞争利益。
 
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引用:Bag, P., Schröder, W. P., Jansson, S. and Farci, D. (2021). Solubilization Method for Isolation of Photosynthetic Mega- and Super-complexes from Conifer Thylakoids. Bio-protocol 11(17): e4144. DOI: 10.21769/BioProtoc.4144.
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