Effect of Light Acclimation on the Organization of Photosystem II Super- and Sub-Complexes in Arabidopsis thaliana

Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1-2 d.

Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plant's capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

We have reviewed the current state of multidisciplinary knowledge of the photoprotective mechanism in the photosystem II antenna underlying non-photochemical chlorophyll fluorescence quenching (NPQ). The physiological need for photoprotection of photosystem II and the concept of feed-back control of excess light energy are described. The outline of the major component of nonphotochemical quenching, qE, is suggested to comprise four key elements: trigger (ΔpH), site (antenna), mechanics (antenna dynamics) and quencher(s). The current understanding of the identity and role of these qE components is presented. Existing opinions on the involvement of protons, different LHCII antenna complexes, the PsbS protein and different xanthophylls are reviewed. The evidence for LHCII aggregation and macrostructural reorganization of photosystem II and their role in qE are also discussed. The models describing the qE locus in LHCII complexes, the pigments involved and the evidence for structural dynamics within single monomeric antenna complexes are reviewed. We suggest how PsbS and xanthophylls may exert control over qE by controlling the affinity of LHCII complexes for protons with reference to the concepts of hydrophobicity, allostery and hysteresis. Finally, the physics of the proposed chlorophyll-chlorophyll and chlorophyll-xanthophyll mechanisms of energy quenching is explained and discussed. This article is part of a Special Issue entitled: Photosystem II. © 2011 Elsevier B.V. All rights reserved.