In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants

Abstract

Dissipation of excess light energy in plant photosynthetic membranes plays an important role in the response of plants to the environment, providing short-term balancing between the intensity of sunlight and photosynthetic capacity. The carotenoid zeaxanthin and the photosystem II subunit PsbS play vital roles in this process, but the mechanism of their action is largely unexplained. Here we report that the isolated photosystem II subunit PsbS was able to bind exogenous zeaxanthin, the binding resulting in a strong red shift in the absorption spectrum, and the appearance of characteristic features in the resonance Raman spectrum and a distinct circular dichroism spectrum, indicating pigment-protein, as well as specific pigment-pigment, interaction. A strong shift in the absorption spectrum of PsbS phenylalanine residues after zeaxanthin binding was observed. It is concluded that zeaxanthin binding to PsbS is the origin of the well known energy dissipation-related 535-nm absorption change that we showed in vivo to arise from activation of 1-2 molecules of this pigment. The altered properties of zeaxanthin and PsbS that result from this interaction provide the first direct indication about how they regulate energy dissipation.

Fig 1.

Purification of PsbS. Silver-stained SDS polyacrylamide gel showing spinach thylakoids (lane 1), PSII membrane fragments (lane 2), pellet after extraction with DM (lane 3), supernatant after extraction with cholate (lane 4), and after passing down a Sephadex G-25 column (lane 5). A Western blot of lane 5 with anti-PsbS antibody is shown in lane 6. Molecular weight markers (See Blue, Invitrogen) are shown in the far-left lane.

Fig 2.

Absorption (A) and CD (B) spectra of the preparation of purified PsbS. The absorption spectrum was deconvoluted, revealing the bands at 257 and 274 nm arising from phenylalanine and tyrosine, respectively.

Fig 3.

(A) Absorption spectra of zeaxanthin in ethanol (1) and in detergent buffer used in reconstitution experiments (2). (B) 1, Absorption spectrum of PsbS reconstituted with zeaxanthin; 2, second derivative of spectrum 1; 3, CD spectrum of PsbS reconstituted with zeaxanthin; 4, simulated CD spectrum of two excitonically coupled zeaxanthin molecules with the higher and lower exciton components at 507 and 536 nm, respectively.

Fig 4.

UV absorption spectrum of PsbS reconstituted with zeaxanthin (1) and a (PsbS+zeaxanthin) − (PsbS only) absorption difference spectrum (2).

Fig 5.

RR spectra of zeaxanthin in ν₄. 1, qE-activated zeaxanthin absorbing at 535 nm (modified from ref. 14). 2, zeaxanthin bound to PsbS obtained as described for Fig. 3; and 3, zeaxanthin in detergent-lipid micelles purified on a sucrose gradient (modified from ref. 14). The arrows indicate the five main transitions associated with zeaxanthin activation.