Plant psbS proteins are essential for regulated thermal dissipation of excess light referred to as non-photochemical quenching of chlorophyll fluorescence yield (NPQ). Amino acid sequences derived from 65 psbS genes from 44 species were aligned to reveal extensive conservation consistent with of motifs that underlie intrinsic aspects of the NPQ mechanism. Site-directed mutagenesis was employed to block presumptive zeaxanthin or chlorophyll-binding sites in Arabidopsis psbS by disrupting ion-bonding between two pairs of non-adjacent glutamate and arginine residues. Transgenic Arabidopsis lines synthesizing only the altered psbS forms exhibited severely impaired NPQ capacity. In addition, the phylogenetic depth of the psbS database permitted identification of cryptic sites of adaptive evolution. Instances of localized positive selection were rare and largely limited to the family Poaceae (grasses). Specifically, adaptive evolution was detected in a hydrophilic stroma-exposed region and was correlated with the presence of the C4 pathway of carbon fixation. (c) 2007 Elsevier Inc. All rights reserved.