4.6 Article

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

Journal

PLANT METHODS
Volume 18, Issue 1, Pages -

Publisher

BMC
DOI: 10.1186/s13007-022-00858-2

Keywords

Bundle sheath; Blue; Clear Native PAGE; Chloroplast NADH dehydrogenase-like complex; Lincomycin; Maize; Mesophyll; Photosystem I; Populus

Funding

  1. Eotvos Lorand University
  2. National Research, Development and Innovation Office [OTKA FK124748]
  3. Lendulet (Momentum) Program of the Hungarian Academy of Sciences
  4. Ion Mobility Mass Spectrometry Research Group of the Hungarian Academy of Sciences
  5. Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences
  6. New National Excellence Program of the Ministry of Technology and Innovation, Hungary [UNKP-20-3-I-ELTE-862]
  7. Synthesis + ELTE Thematic Excellence Programme - Hungarian Ministry for Innovation and Technology
  8. Bulgarian National Science Fund, Ministry of Education and Science [KPi-06-H21/8]
  9. Bulgarian Academy of Sciences [NKM 2018-107]
  10. Hungarian Academy of Sciences [NKM 2018-107]

Ask authors/readers for more resources

In this study, the Blue/Clear-Native polyacrylamide gel electrophoresis (BN PAGE) method was improved by using a detergent mixture of 1% (w/V) n-dodecyl-beta-d-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3-8% gel gradients for separation. Several large photosystem (PS) I containing bands were detected, and it was found that these bands were PSI-NADH dehydrogenase-like megacomplexes in maize bundle sheath thylakoids and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) in mesophyll thylakoids of lincomycin treated maize. Proposed methods for quantitative determination and comparison of the complexes in different samples were also provided.
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-beta-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.

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