Regulation of Photosynthetic Light Harvesting Involves Intrathylakoid Lumen pH Sensing by the PsbS Protein

Abstract

The biochemical, biophysical, and physiological properties of the PsbS protein were studied in relation to mutations of two symmetry-related, lumen-exposed glutamate residues, Glu-122 and Glu-226. These two glutamates are targets for protonation during lumen acidification in excess light. Mutation of PsbS did not affect xanthophyll cycle pigment conversion or pool size. In conditions of excess light, photosynthetic light harvesting is regulated by a feedback de-excitation mechanism termed energy-dependent quenching (qE), 1 which increases thermal dissipation of excess absorbed light energy in photosystem II (PSII). The qE mechanism is triggered by conditions that limit photosynthetic carbon fixation and result in increased acidification of the chloroplast thylakoid lumen (1-4). The thermal dissipation of excess excitation energy is most commonly measured and referred to as nonphotochemical quenching (NPQ) of PSII chlorophyll (Chl) a fluorescence. Although there are several components of NPQ, in higher plants qE can account for the major part of NPQ and is characterized by its relatively fast induction and relaxation kinetics, on a physiological time scale of seconds to minutes. The decrease in the intensity of Chl fluorescence is the result of the decrease in the electronic excited state lifetime of Chl caused by an increased thermal dissipation rate constant (5). The rapid response of the qE process is chemically associated with changes in the trans-thylakoid membrane pH gradient (⌬pH). The ⌬pH change has at least two functions in qE. First, it activates the violaxanthin de-epoxidase that converts violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) (6). A and/or Z are essential elements of qE (7-9). Second, the lower pH in the lumen results in protonation of PSII proteins, including the 22-kDa PSII subunit, PsbS, which plays a key role in qE (10). When both pH-induced changes occur together it is believed that Chls in PSII can transfer their excess energy to Z, which can return to the ground state via thermal decay (7,11,12). Plants containing PsbS mutations of both glutamatesThe pH-sensing mechanism of the PsbS protein is influenced by two pairs of symmetrically arranged glutamate residues, each located within or close to the two lumen-exposed loops of the protein (13). Dicyclohexylcarbodiimide (DCCD), a well known inhibitor of qE (14 -16) is a carboxylate-modifying agent (17) that binds to PsbS (18). Although it was suggested that the DCCD binding site is in the lumenal loops of PsbS, the exact binding site has not been determined. Importantly, site-directed mutagenesis experiments indicated that two of the PsbS glutamates, Glu-122 and Glu-226, are necessary for the function of PsbS (13).In this article we used single and double mutations of PsbS (E122Q/E226Q) to make a detailed biochemical and biophysical analysis of the role of these two glutamates in pH sensing and DCCD binding. We probed the role of the Glu-122 and Glu-226 residues by monitoring the changes in the PSII Chl a fluores-

The xanthophyll cycle pool size controls the kinetics of non‐photochemical quenching in Arabidopsis thaliana

“…The control of the rate of the transition into and out of this state by the DES is consistent with the proposed role of the xanthophyll cycle carotenoids as allosteric regulators of qE [9,10]. The data could also be accommodated within a model in which the only role of zeaxanthin is as the direct quencher [8], either bound to PsbS [21], LHCII [22] or to a minor antenna complex [23,24]. However, important new features would need to be invoked: there must be competition between violaxanthin and zeaxanthin for the quenching site; and the rate of binding and release from this site must be rate limiting for qE formation and relaxation respectively.…”

Photosynthesis Control: An underrated short-term regulatory mechanism essential for plant viability

“…25,26 Similar results have been reported in pgr5 mutants of rice 37 and Chlamydomonas, 40 but not in mutants devoid of NPQ, such as in the Arabidopsis npq4 plants lacking PsbS protein, 43,45,49 supporting a key and specific role for the PGR5 protein in the Photosynthesis Control mechanism. Clearly, by ensuring the pH-dependent control of PQH 2 oxidation at the level of Cyt b 6 f, PGR5 prevents electrons from flowing freely from PSII to PSI, which would over-reduce the electron carriers on the acceptor side of PSI.…”

“…31,32,34 Thus, CET appears to be important for providing a sufficient proton motive force, which in turn attenuates PSII activity through the induction of the NPQ photoprotective mechanism. 9 That is obtained through the protonation of two glutamic acid residues in the lumenexposed loop of PsbS protein, [43][44][45] which is a component of PSII, and the activation of the Violaxanthin De-Epoxidase (VDE) that converts violaxanthin into zeaxanthin within the xanthophyll cycle. 13,14,46 Secondly, the pgr5 mutants of both Arabidopsis and rice have also been shown to exhibit an elevated proton conductance of the ATP-synthase, 37,47 as well as an increased amount of the ATP synthase b subunit.…”

Nonphotochemical Quenching of Chlorophyll Fluorescence inChlamydomonas reinhardtii

“…In contrast, the extensive nonphotochemical quenching measured in Arabidopsis was sensitive to addition of nigericin (not shown), indicating that this quenching reflects the occurrence of high-energy state quenching (1,8,9,37). The absence of high-energy state quenching in Chlamydomonas was confirmed by the lack of absorbance changes at 535 nm during illumination (data not shown), a linear indicator for the generation of this type of quenching in plants (38).…”