Interactions between the Photosystem II Subunit PsbS and Xanthophylls Studied in Vivo and in Vitro

Giulia Bonente,Barry D Howes,Stefano Caffarri,Giulietta Smulevich,Roberto Bassi

doi:10.1074/jbc.m708291200

Giulia Bonente, Barry D Howes + Show 3 more

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https://doi.org/10.1074/jbc.m708291200

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Abstract

The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.

Highlights

Lakoid lumen acidification as an essential factor for Nonphotochemical quenching (NPQ) [1]
Screening of the F2 progeny for xanthophyll composition, in dark conditions and after high light exposure, and for the PsbS protein enabled four double mutants, E122Q/npq1, E122Q/lut2, E226Q/npq1, and E226Q/ lut2, to be obtained
Four-week-old plants of control genotypes npq1, lut2, npq1/lut2, PsbS E122Q, PsbS E226Q, the double mutant PsbS E122Q/E226Q, npq4, and the four newly obtained double mutants were assayed for NPQ (Figs. 1–3)

Summary

Introduction

Lakoid lumen acidification as an essential factor for NPQ [1]. Excess light increases proton pumping into the thylakoid lumen, which elicits chlorophyll fluorescence quenching dependent on protein protonation events. The need for two xanthophyll species and two activated DCCD binding domains, for full expression of qE, has been rationalized in the “two xanthophyll-binding sites” model [12, 13], which proposes that protonation of each glutamate residue (Glu-122 and Glu-226) activates a corresponding binding site for de-epoxidized xanthophylls This model was supported by the following: (i) the finding that a 535 nm spectral feature appearing during establishment of qE was PsbS-dependent and exhibited the Raman spectrum of zeaxanthin [14], and (ii) the. An alternative model that reconciles these observations can be proposed in which the phenotypes related to lumen acidification are due to PsbS activation, whereas the phenotypes of xanthophyll biosynthesis mutations are mediated by xanthophyll binding to Lhc proteins (19 –22). The results are discussed in terms of their consistency with the above models

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