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  • LHCSR1-dependent fluorescence quenching is mediated by excitation energy transfer from LHCII to photosystem I in Chlamydomonas reinhardtii Kotaro Kosugea,b, Ryutaro Tokutsua,b,c, Eunchul Kima, Seiji Akimotod, Makio Yokonoe,1, Yoshifumi Uenod, and Jun Minagawaa,b,c,2

    aDivision of Environmental Photobiology, National Institute for Basic Biology, 444-8585 Okazaki, Japan; bDepartment of Basic Biology, School of Life Science, Graduate University for Advanced Studies, 444-8585 Okazaki, Japan; cCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, 332-0012 Saitama, Japan; dGraduate School of Science, Kobe University, 657-8501 Kobe, Japan; and eInstitute of Low Temperature Science, Hokkaido University, 060-0819 Sapporo, Japan

    Edited by Elisabeth Gantt, University of Maryland, College Park, MD, and approved March 1, 2018 (received for review November 27, 2017)

    Photosynthetic organisms are frequently exposed to light intensi- ties that surpass the photosynthetic electron transport capacity. Under these conditions, the excess absorbed energy can be transferred from excited chlorophyll in the triplet state (3Chl*) to molecular O2, which leads to the production of harmful reactive oxygen species. To avoid this photooxidative stress, photosyn- thetic organisms must respond to excess light. In the green alga Chlamydomonas reinhardtii, the fastest response to high light is nonphotochemical quenching, a process that allows safe dissipa- tion of the excess energy as heat. The two proteins, UV-inducible LHCSR1 and blue light-inducible LHCSR3, appear to be responsible for this function. While the LHCSR3 protein has been intensively studied, the role of LHCSR1 has been only partially elucidated. To investigate the molecular functions of LHCSR1 in C. reinhardtii, we performed biochemical and spectroscopic experiments and found that the protein mediates excitation energy transfer from light- harvesting complexes for Photosystem II (LHCII) to Photosystem I (PSI), rather than Photosystem II, at a low pH. This altered excitation transfer allows remarkable fluorescence quenching under high light. Our findings suggest that there is a PSI-dependent photoprotection mechanism that is facilitated by LHCSR1.

    photosynthesis | algae | stress | light | fluorescence

    Solar energy is essential for photosynthetic organisms, but theamount of light frequently exceeds the capacity of photo- chemical reactions, leading to potentially serious photodamage to the photosystems (1). To minimize the harmful effects of excess light-dependent reactions, photosynthetic organisms have estab- lished protection mechanisms referred to as nonphotochemical quenching (NPQ) (2). One of the NPQ mechanisms, energy- dependent quenching, can be activated rapidly (within a minute) under high-intensity light conditions to safely convert light energy into thermal energy (3). Utilizing this mechanism, photosynthetic organisms, including land plants and aquatic algae, can survive in natural light environments. Certain key molecules, PSBS and LHCSRs, are responsible for

    NPQ (2). These molecules represent a family of light-harvesting complexes (LHCs) that are used in photosynthesis, while other LHCs (LHCI and LHCII) function as antennae for the photo- systems (PSI and PSII). PSBS and LHCSRs have been identified in land plants (4, 5), mosses (6), and eukaryotic algae (7). Mu- tants deficient in these proteins are highly stressed under high light. In contrast to land plants, which constitutively express PSBS (4), the model green alga Chlamydomonas reinhardtii inducibly expresses LHCSRs (LHCSR3 and LHCSR1) when exposed to specific colors of light (8). Interestingly, although LHCSR3 and LHCSR1 are induced by different colors of light, they behave similarly to NPQ effectors under high-light condi- tions in C. reinhardtii (9, 10).

    The molecular functions and physiological role of LHCSR3 in C. reinhardtii have been extensively studied during the past de- cade. Bonente et al. (11) suggested that LHCSR3 is itself a quencher and does not require interaction with other photo- synthetic protein complexes. Reconstituted LHCSR3 isolated from Escherichia coli is capable of quenching light energy under conditions similar to high-light conditions (low-pH buffer). This previous report also showed that there are photosynthetic pig- ments (chlorophylls, xanthophylls) associated with the protein, strongly suggesting that the protein itself can be a direct energy quencher. We previously reported that LHCSR3 can associate with PSII−LHCII supercomplexes (12, 13), likely mediated by the PSII subunit PSBR (14), thereby contributing to low-pH- inducible energy quenching in PSII. LHCSR3 is also known to be protonated due to high light-dependent thylakoid luminal acidification as well as other light-harvesting proteins (11, 12). Ballottari et al. (15) reported that mutants with modified amino acid residues in LHCSR3 were incapable of efficient NPQ and showed that protonation of three residues exposed to the thy- lakoid lumen side are essential for quenching. Although LHCSR1, a paralog of LHCSR3, significantly con-

    tributes to the NPQ process (16, 17), this protein has not been sufficiently investigated to date. In addition to characterizing recombinant LHCSR3, Bonente et al. (11) investigated the LHCSR1 protein expressed in E. coli; however, the obtained

    Significance

    Unlike another effector protein for algal nonphotochemical quenching (NPQ)—LIGHT HARVESTING COMPLEX II STRESS RELATED PROTEIN 3 (LHCSR3)—the role of LHCSR1 in NPQ has been very limited. In this report, we studied the fluorescence quenching event occurring in the presence and the absence of LHCSR1 and demonstrated that there is a significant excitation energy transfer from Light-harvesting complex II (LHCII) to Photosystem I (PSI), and not only to Photosystem II, upon ac- tivation of LHCSR1 by low pH. The results suggest another layer of photoprotection mechanism based on this UV-inducible protein LHCSR1.

    Author contributions: R.T. and J.M. designed research; K.K., R.T., E.K., S.A., and Y.U. performed research; R.T., E.K., S.A., and M.Y. analyzed data; and K.K., R.T., E.K., S.A., and J.M. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission.

    Published under the PNAS license. 1Present address: Innovation Center, Nippon Flour Mills Co., Ltd., 243-0041 Atsugi, Japan. 2To whom correspondence should be addressed. Email: minagawa@nibb.ac.jp.

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1720574115/-/DCSupplemental.

    Published online March 19, 2018.

    3722–3727 | PNAS | April 3, 2018 | vol. 115 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1720574115

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    http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.1720574115&domain=pdf http://www.pnas.org/site/aboutpnas/licenses.xhtml mailto:minagawa@nibb.ac.jp http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplemental http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplemental www.pnas.org/cgi/doi/10.1073/pnas.1720574115

  • yields were insufficient for further characterization. The in- duction conditions for this gene were discovered very recently (9), and there is a report suggesting that LHCSR1 triggers pH- dependent quenching in vivo (18). In this reported study, Croce and coworkers (18) used a vitamin repressor system to com- pletely eliminate photosystem core components (PSII and PSI) while maintaining the LHCs (and LHCSR1), allowing functional analysis of LHCSR1 in vivo. When the authors measured chlo- rophyll fluorescence, they found that the cells containing both LHCs and LHCSR1 without the photosystems exhibited low-pH- inducible NPQ. Thus, they concluded that the excitation energy quenching triggered by LHCSR1 occurs in free (non-photosystem- associated) LHCIIs. The LHCSR1 sequence of the moss Physcomitrella patens does

    not directly correspond to LHCSR1 in C. reinhardtii but is equally related to LHCSR1 and LHCSR3 in green algae (19). Recently, Kondo et al. (20) reported a reconstituted P. patens LHCSR1 protein, as revealed via single-molecule spectroscopy. They showed that the protein exhibits both pH-dependent and carotenoid-dependent energy dissipative states and therefore concluded that the protein itself is capable of controlling quenching dynamics during photoprotective energy dissipation. These findings suggested molecular functions of the LHCSR1 found in moss, but the moss protein is clearly distinct from LHCSR1 in C. reinhardtii. Therefore, the molecular functions of LHCSR1 in the green alga remain unclear. The molecular mechanism of LHCSR1-dependent NPQ in-

    duction in C. reinhardtii is still poorly understood. To elucidate how LHCSR1 activates NPQ, we characterized excitation energy dynamics in thylakoid membranes isolated from C. reinhardtii. Our results show that, at low pH, there is energy transfer from LHCII to PSI, mediated by LHCSR1. Time-resolved chlorophyll fluorescence analysis of mutants lacking the photosystems revealed remarkable activation of LHCSR1-dependent fluorescence quenching by PSI. We propose that LHCSR1 in C. reinhardtii activates PSI-dependent fluorescence quenching in addition to dissipating excitation energy in LHCIIs to avoid photooxidative stress under excess light.

    Results To evaluate the amplitude of LHCSR1-dependent fluorescence quenching in vitro, we first attempted to isolate the thylakoid membranes from LHCSR1-expressing C. reinhardtii strains. UV treatment is one of the most effective methods for inducing LHCSR1 (9). Therefore, we applied UV trea