Qualitative and quantitative evaluation of thylakoid complexes separated by Blue Native PAGE

Abstract

Background Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging. Results Using a detergent mixture of 1% (w/V) n-dodecyl-β-d-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3–8% gel gradients for separation as methodological improvements in BN PAGE, several large photosystem (PS) I containing bands were detected. According to BN(/BN)/SDS PAGE and mass spectrometry analyses, these PSI bands proved to be PSI-NADH dehydrogenase-like megacomplexes more discernible in maize bundle sheath thylakoids, and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) more abundant in mesophyll thylakoids of lincomycin treated maize. For quantitative determination of the complexes and their comparison across taxa and physiological conditions, sample volumes applicable to the gel, correct baseline determination of the densitograms, evaluation methods to resolve complexes running together, calculation of their absolute/relative amounts and distribution among their different forms are proposed. Conclusions Here we report our experience in Blue/Clear-Native polyacrylamide gel electrophoretic separation of thylakoid complexes, their identification, quantitative determination and comparison in different samples. The applied conditions represent a powerful methodology for the analysis of thylakoid mega- and supercomplexes.

Etioplasts are more susceptible to salinity stress than chloroplasts and photosynthetically active etio‐chloroplasts of wheat (Triticum aestivum L.)

“…The unchanged thylakoid structure under osmotic stress of wheat proteome agrees with the results of Chen et al (2017). Though the increase in PSI‐LHCII, the state transition specific complex (Pesaresi et al, 2009), may be artefactual because LHCII association is very sensitive to n‐dodecyl‐ β ‐D‐maltoside (Järvi et al, 2011; Sárvári et al, 2022), the higher amount of this complex (Figure 4) is in agreement with the results showing enhanced cyclic electron transport under osmotic or drought stress (Dulai et al, 2014; Lehtimäki et al, 2010). Taken together, our data indicate that the observed changes – first of all, the swelling of the PT lumen of etioplasts – are not associated with the osmotic stress induced by the high ion concentration of the applied solutions but must be connected with the direct and specific effects of the ions.…”

“…After filtration, chloroplasts or etio‐choroplasts were immediately centrifuged in a swing‐out rotor (1000/3000 × g , 5 min, 4°C), and washed in isolating buffer without BSA and Na‐ascorbate. Isolation and washing of thylakoid membranes (osmotic shock and washing out part of the coupling factor 1) was carried out as in Sárvári et al (2022). Light‐grown, 16 h greened and 4 h greened thylakoids (500, 500, and 20 μg Chl ml −1 ) were solubilized with 1%, 1% and 2% (w/v) n‐dodecyl‐ β ‐D‐maltoside ( β ‐DM, SIGMA), respectively, plus 1% digitonin (SERVA) on ice for 30 min.…”

“…After filtration, chloroplasts or etiochoroplasts were immediately centrifuged in a swing-out rotor (1000/3000 Â g, 5 min, 4 C), and washed in isolating buffer without BSA and Na-ascorbate. Isolation and washing of thylakoid membranes (osmotic shock and washing out part of the coupling factor 1) was carried out as in Sárvári et al (2022). Light-grown, 16 h greened and 4 h F I G U R E 1 Outline of the used plant material, applied light regimes, and treatments as described in the manuscript.…”

“…The supernatant obtained after centrifugation (18,000 Â g for 15 min at 4 C) was supplemented with 1/5 volume of 5% (w/v) Serva Blue G dissolved in 500 mM aminocaproic acid, and 10-15 μL of the solubilized material was applied per lanes. For the separation of thylakoid complexes, Blue Native polyacrylamide gel electrophoresis (BN PAGE), subsequently second dimension SDS-PAGE and densitometric analyses of the gels were performed as described in Sárvári et al (2022). BN patterns of 4 h greened samples were stained using the Blue-Silver method (Candiano et al, 2004).…”

Protein Changes in Shade and Sun Haberlea rhodopensis Leaves during Dehydration at Optimal and Low Temperatures

“…SOD isoenzymes were identified based on the selective inhibition (KCN sensitivity of Cu/ZnSODs and H 2 O 2 sensitivity of both Cu/ZnSODs and FeSOD) results of Yahubyan et al [ 32 ]. Total protein content of the crude extracts was determined by separating the proteins using SDS-PAGE and comparing the cumulative density of the Coomassie-stained bands with reference [ 100 ].…”