Additional families of orange carotenoid proteins in the photoprotective system of cyanobacteria Han Bao 1 , Matthew R. Melnicki 1,2 , Emily G. Pawlowski 1 , Markus Sutter 1,2 , Marco Agostoni 1 , Sigal Lechno-Yossef 1 , Fei Cai 2 , Beronda L. Montgomery 1,3,4 and Cheryl A. Kerfeld 1,2,3,5 * The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Using phylogenomic analysis, we have revealed two new paralogous OCP families, each distributed among taxonomically diverse cyanobacterial genomes. Based on bioinformatic properties and phylogenetic relationships, we named the new families OCP2 and OCPx to distinguish them from the canonical OCP that has been well characterized in Synechocystis, denoted hereafter as OCP1. We report the first characterization of a carotenoprotein photoprotective system in the chromatically acclimating cyanobacterium Tolypothrix sp. PCC 7601, which encodes both OCP1 and OCP2 as well as the regulatory fluorescence recovery protein (FRP). OCP2 expression could only be detected in cultures grown under high irradiance, surpassing expression levels of OCP1, which appears to be constitutive; under low irradiance, OCP2 expression was only detectable in a Tolypothrix mutant lacking the RcaE photoreceptor required for complementary chromatic acclimation. In vitro studies show that Tolypothrix OCP1 is functionally equivalent to Synechocystis OCP1, including its regulation by Tolypothrix FRP, which we show is structurally similar to the dimeric form of Synechocystis FRP. In contrast, Tolypothrix OCP2 shows both faster photoconversion and faster back-conversion, lack of regulation by the FRP, a different oligomeric state (monomer compared to dimer for OCP1) and lower fluorescence quenching of the phycobilisome. Collectively, these findings support our hypothesis that the OCP2 is relatively primitive. The OCP2 is transcriptionally regulated and may have evolved to respond to distinct photoprotective needs under particular environmental conditions such as high irradiance of a particular light quality, whereas the OCP1 is constitutively expressed and is regulated at the post-translational level by FRP and/or oligomerization. T he orange carotenoid protein (OCP) is a 35 kDa water-soluble photoactive protein found in nearly all cyanobacteria 1 . The OCP is responsible for a photoprotective mechanism that enables cells to avoid photodamage and growth inhibition caused by high light or nutrient stresses 2 . On photoactivation with strong blue-green light, the OCP converts from an orange, stable form (OCP O ) to a red, metastable form (OCP R ) that facilitates thermal dissipation of excitation energy from the light-harvesting antenna through interaction with the phycobilisome (PBS) 2–5 . This process can be observed in the laboratory as a decrease in the emission of PBS fluorescence, and is referred to as ‘non-photochemical quench- ing’ (NPQ) 2,5,6 . As the quantum yield of photoconversion is very low, and the metastable OCP R form thermally converts back into the OCP O form without sufficient light 3 , OCP activity is gated by an intrinsic light sensor function that is specialized for high irradiance. The OCP-mediated energy quenching mechanism is further regulated by the fluorescence recovery protein (FRP), which accelerates the dark reversion of OCP R to the quenching- inactive OCP O (ref. 7). The OCP is structurally and functionally modular, consisting of a sensor and an effector domain, proposed to be the result of an ancient domain fusion event in a primordial cyanobacterium 1,8 . The OCP is composed of an α-helical amino (N)-terminal domain (NTD) and a mixed α/β carboxy (C)-terminal domain (CTD) connected by a flexible linker. The fold of the CTD is a member of the widespread nuclear transport factor 2-like (NTF2-like) superfamily, but the structure of the NTD is unique to cyanobacteria. The OCP also contains a non-covalently bound ketocarotenoid molecule which is embedded inside a conserved tunnel-like binding pocket that spans both domains 9,10 . A variety of carotenoids can be bound by the OCP, but it appears that keto- carotenoids such as canthaxanthin (CAN) or 3′-hydroxyechinenone (hECN) are required for photoconversion, as well as for energy quenching of excited PBS 11 . The OCP also performs a second photoprotective function by quenching singlet oxygen ( 1 O 2 ), which can accumulate under photoinhibitory conditions and is toxic to cells 10,12,13 . Almost all previous studies on the structure and function of the OCP have focused on Synechocystis sp. PCC 6803 (hereafter Synechocystis). For this well characterized OCP, crystal structures and X-ray footprinting analysis have shown that on photoactivation and domain dissociation, the NTD completely envelops the caroten- oid after a 12 Å translocation of the pigment 9,14 . It is also known that the NTD, when produced by expression of a truncated ocp gene or by proteolysis of the OCP, retains the carotenoid and appears red. It is constitutively active, no longer requiring photoactivation to induce PBS quenching 15 . As such, the NTD constitutes the effector module of the OCP. The CTD plays a role in stabilizing the carotenoid in the ‘orange’ inactive position, conferring photo- chemical activity to the OCP, and provides the site of interaction with FRP 7 . Accordingly, this domain regulates the quenching function of the NTD. In the past three years the number of available cyanobacterial genomes sequences has more than tripled, with a particular 1 MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, USA. 2 Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. 3 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA. 4 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, USA. 5 Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA. *e-mail: ckerfeld@lbl.gov ARTICLES PUBLISHED: 10 JULY 2017 | VOLUME: 3 | ARTICLE NUMBER: 17089 NATURE PLANTS 3, 17089 (2017) | DOI: 10.1038/nplants.2017.89 | www.nature.com/natureplants 1 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.