LHCSR1-dependent fluorescence quenching is mediatedby excitation energy transfer from LHCII tophotosystem I in Chlamydomonas reinhardtiiKotaro 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 LifeScience, Graduate University for Advanced Studies, 444-8585 Okazaki, Japan; cCore Research for Evolutional Science and Technology, Japan Science andTechnology Agency, 332-0012 Saitama, Japan; dGraduate School of Science, Kobe University, 657-8501 Kobe, Japan; and eInstitute of Low TemperatureScience, 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 betransferred from excited chlorophyll in the triplet state (3Chl*) tomolecular O2, which leads to the production of harmful reactiveoxygen species. To avoid this photooxidative stress, photosyn-thetic organisms must respond to excess light. In the green algaChlamydomonas reinhardtii, the fastest response to high light isnonphotochemical quenching, a process that allows safe dissipa-tion of the excess energy as heat. The two proteins, UV-inducibleLHCSR1 and blue light-inducible LHCSR3, appear to be responsiblefor this function. While the LHCSR3 protein has been intensivelystudied, the role of LHCSR1 has been only partially elucidated. Toinvestigate the molecular functions of LHCSR1 in C. reinhardtii, weperformed biochemical and spectroscopic experiments and foundthat 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 excitationtransfer allows remarkable fluorescence quenching under high light.Our findings suggest that there is a PSI-dependent photoprotectionmechanism 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 photodamageto the photosystems (1). To minimize the harmful effects of excesslight-dependent reactions, photosynthetic organisms have estab-lished protection mechanisms referred to as nonphotochemicalquenching (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 energyinto thermal energy (3). Utilizing this mechanism, photosyntheticorganisms, including land plants and aquatic algae, can survive innatural light environments.Certain key molecules, PSBS and LHCSRs, are responsible for

NPQ (2). These molecules represent a family of light-harvestingcomplexes (LHCs) that are used in photosynthesis, while otherLHCs (LHCI and LHCII) function as antennae for the photo-systems (PSI and PSII). PSBS and LHCSRs have been identifiedin land plants (4, 5), mosses (6), and eukaryotic algae (7). Mu-tants deficient in these proteins are highly stressed under highlight. In contrast to land plants, which constitutively expressPSBS (4), the model green alga Chlamydomonas reinhardtiiinducibly expresses LHCSRs (LHCSR3 and LHCSR1) whenexposed to specific colors of light (8). Interestingly, althoughLHCSR3 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 inC. reinhardtii have been extensively studied during the past de-cade. Bonente et al. (11) suggested that LHCSR3 is itself aquencher and does not require interaction with other photo-synthetic protein complexes. Reconstituted LHCSR3 isolatedfrom Escherichia coli is capable of quenching light energy underconditions similar to high-light conditions (low-pH buffer). Thisprevious 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 energyquencher. We previously reported that LHCSR3 can associatewith PSII−LHCII supercomplexes (12, 13), likely mediated bythe PSII subunit PSBR (14), thereby contributing to low-pH-inducible energy quenching in PSII. LHCSR3 is also known tobe protonated due to high light-dependent thylakoid luminalacidification as well as other light-harvesting proteins (11, 12).Ballottari et al. (15) reported that mutants with modified aminoacid residues in LHCSR3 were incapable of efficient NPQ andshowed 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 beensufficiently investigated to date. In addition to characterizingrecombinant LHCSR3, Bonente et al. (11) investigated theLHCSR1 protein expressed in E. coli; however, the obtained

Significance

Unlike another effector protein for algal nonphotochemicalquenching (NPQ)—LIGHT HARVESTING COMPLEX II STRESSRELATED PROTEIN 3 (LHCSR3)—the role of LHCSR1 in NPQ hasbeen very limited. In this report, we studied the fluorescencequenching event occurring in the presence and the absence ofLHCSR1 and demonstrated that there is a significant excitationenergy transfer from Light-harvesting complex II (LHCII) toPhotosystem I (PSI), and not only to Photosystem II, upon ac-tivation of LHCSR1 by low pH. The results suggest another layerof photoprotection mechanism based on this UV-inducibleprotein 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.

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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, Croceand coworkers (18) used a vitamin repressor system to com-pletely eliminate photosystem core components (PSII and PSI)while maintaining the LHCs (and LHCSR1), allowing functionalanalysis of LHCSR1 in vivo. When the authors measured chlo-rophyll fluorescence, they found that the cells containing bothLHCs and LHCSR1 without the photosystems exhibited low-pH-inducible NPQ. Thus, they concluded that the excitation energyquenching 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 isequally related to LHCSR1 and LHCSR3 in green algae (19).Recently, Kondo et al. (20) reported a reconstituted P. patensLHCSR1 protein, as revealed via single-molecule spectroscopy.They showed that the protein exhibits both pH-dependent andcarotenoid-dependent energy dissipative states and thereforeconcluded that the protein itself is capable of controlling quenchingdynamics during photoprotective energy dissipation. These findingssuggested molecular functions of the LHCSR1 found in moss, butthe moss protein is clearly distinct from LHCSR1 in C. reinhardtii.Therefore, the molecular functions of LHCSR1 in the green algaremain unclear.The molecular mechanism of LHCSR1-dependent NPQ in-

duction in C. reinhardtii is still poorly understood. To elucidatehow LHCSR1 activates NPQ, we characterized excitation energydynamics in thylakoid membranes isolated from C. reinhardtii.Our results show that, at low pH, there is energy transfer fromLHCII to PSI, mediated by LHCSR1. Time-resolved chlorophyllfluorescence analysis of mutants lacking the photosystems revealedremarkable activation of LHCSR1-dependent fluorescencequenching by PSI. We propose that LHCSR1 in C. reinhardtiiactivates PSI-dependent fluorescence quenching in addition todissipating excitation energy in LHCIIs to avoid photooxidativestress under excess light.

ResultsTo evaluate the amplitude of LHCSR1-dependent fluorescencequenching in vitro, we first attempted to isolate the thylakoidmembranes from LHCSR1-expressing C. reinhardtii strains. UVtreatment is one of the most effective methods for inducingLHCSR1 (9). Therefore, we applied UV treatment before thy-lakoid membrane isolation from four different strains: WT,

lhcsr1 (LHCSR1-lacking mutant), npq4 (LHCSR3-lacking mu-tant), and npq4/lhcsr1. Consistent with a previous report (9), theWT and lhcsr1 strains showed clear accumulation of the LHCSR3protein, as the UV treatment was also effective for inducingLHCSR3 expression (Fig. S1). In contrast, neither the npq4 northe npq4/lhcsr1 strain, which lack the LHCSR3 gene (7),showed an LHCSR3 signal (Fig. 1A and Fig. S1). Moreover, theaccumulations of PSBS and the other LHCIIs between npq4and npq4/lhcsr1 strains were comparable (Fig. 1 A and B),suggesting that the difference in NPQ (Fig. S2) between these twostrains is largely based on the presence or absence of LHCSR1protein expression. To avoid the contribution of LHCSR3 in furtheranalyses, we focused on the npq4 and npq4/lhcsr1 strains.To investigate pH-inducible fluorescence quenching in the

isolated thylakoid membranes from the npq4 and npq4/lhcsr1mutants, we performed room-temperature fluorescence decayanalysis. The isolated thylakoids from npq4 showed a drasticdecrease in the fluorescence lifetime when treated with acidicbuffers (pH 5.5 in Fig. 1C). The npq4/lhcsr1 thylakoids, on theother hand, showed only a small decrease in fluorescence decayfrom pH 7.5 to pH 5.5 (Fig. 1D). These data suggest that thefunction of the LHCSR1 protein (i.e., low-pH-dependent energyquenching) can be observed not only in vivo (Fig. S2) but also inisolated thylakoid membranes.Since isolated npq4 thylakoids exhibited LHCSR1-dependent

quenching at low pH, we next attempted to characterize thequenching mechanism via both steady-state and time-resolvedfluorescence spectra analyses at low temperature (77 K). Con-sistent with the changes in room-temperature fluorescence decayobserved using buffers with different pH levels (shown in Fig.1C), the measurement of 77 K steady-state fluorescence spectrarevealed that npq4 thylakoids showed a lower fluorescence in-tensity when exposed to low-pH buffer (Fig. 2, solid red line),whereas the npq4/lhcsr1 thylakoids did not show lower fluores-cence (Fig. 2, dashed red line). Although we observed LHCSR1-mediated fluorescence quenching in thylakoid membranes, asshown above, the details of the excitation energy transfer dynamics inthylakoid membranes still need to be characterized. To characterizeexcitation energy dynamics, fluorescence decay kinetics at wave-lengths of 660 nm to 760 nm were measured upon excitation at459 nm, which predominantly excited LHCII, and then subjected to aglobal fitting analysis to identify the decay components [fluorescencedecay associated spectra (FDAS) analysis shown in Fig. 3, Fig. S4,and Table S1; see Materials and Methods]. When we treated thethylakoid membranes with acidic buffer, a remarkable expansion ofthe negative peak around 710 nm to 720 nm was observed in the first

Fig. 1. Time-resolved fluorescence analysis of the isolated thylakoid membranes. (A) Immunoblotting analysis of purified thylakoid membranes from UV-treated cells using antibodies against either ATPB or LHCSRs, or PSBS. (B) Thylakoid membrane samples were analyzed by SDS/PAGE stained by CoomassieBrilliant Blue G-250. The LHCII bands were indicated as CP26 (Lhcb5), CP29 (Lhcb4), LHCII type I (LhcbM3, LhcbM 4, LhcbM 6, LhcbM 8, LhcbM 9); LHCII type III(LhcbM2, -7), or LHCII Type IV (LhcbM1). (C and D) The time-correlated single-photon counting of fluorescence for the thylakoids of (C) npq4 and (D) npq4/lhcsr1 were recorded at 682 nm (slit = 8 nm) at pH 5.5 (red) and 7.5 (blue). Instrumental response function (IRF) is shown as gray line in C. The samples,normalized to 1 μg Chl/mL, were excited at 463 nm.

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FDAS (τ = ∼30 ps) of npq4 (Fig. 3, blue line), indicating greaterenergy transfer to PSI and/or energy dissipation in LHCIIs underacidic conditions. In addition, the relative amplitudes of PSIIfluorescence in the third FDAS decreased, indicating fasterexcitation energy quenching around PSII in npq4 under lowpH. Although the fluorescence lifetime components showedsimilar lifetimes (Table S1), the shortening of the average lifetimefor npq4 at pH 5.5 (Fig. S4, solid red line) was expressed as acombination of increases in amplitudes of the 85-ps componentand decreases in those of the 500-ps component (second andthird FDAS in Fig. 3, blue line). Indeed, in the fourth FDAS(∼2.2 ns), which is assigned to fluorescence from the final energytraps in PSII (685 nm to 700 nm) and PSI, the PSII amplitudewas reduced relative to that of PSI in npq4 (Fig. 3, blue line).This finding implies that both excitation energy dissipation atLHCII and excitation energy transfer from LHCII to PSI becomemore active under lower pH in the presence of the LHCSR1 protein(npq4) but not in the absence of the LHCSR1 protein (npq4/lhcsr1).The latter excitation dynamics may reflect the increase in excitationenergy transfer from LHCII to PSI, rather than to PSII. These ob-servations led us to hypothesize that LHCSR1-dependent fluores-cence quenching is specifically correlated with excitation energytransfer from LHCII to PSI.The marked excitation energy transfer from LHCII to PSI ob-

served under low pH implies a contribution of PSI to LHCSR1-dependent fluorescence quenching. To obtain direct evidence thatPSI contributes to quenching, we used photosystem mutants andconducted further spectroscopic measurements. Because visiblelight (photosynthetically active radiation) is not required forLHCSR1 expression (9), UV treatment of these strains suc-cessfully induced LHCSR1 protein expression, even though thestrains are incapable of photosynthesis (Fig. 4). On the otherhand, the photosystem-lacking mutants cannot form ΔpH, atrigger for energy quenching, due to a deficiency of the light-driven proton flux. We therefore applied the previously reportedpH adjustment method (18) as follows. After UV treatment forLHCSR1 protein expression, the pH of all strains was adjusted to

5.5 or 7.5, using acetic acid or sodium hydroxide, respectively. Allstrains showed low-pH (acetic acid)-inducible energy quenching,as demonstrated by rapid fluorescence decay (Fig. 4 A–C). Themutants lacking PSI (ΔPSI and ΔPSI/II) exhibited relativelysmall changes in the average fluorescence lifetime (τave) betweendifferent pH levels compared with the difference observed inΔPSII (Table 1). The amplitudes of these τave changes in theΔPSI and ΔPSI/II strains were also calculated as pH-induciblequenching, and ∼46% and ∼51% of chlorophyll fluorescence wasshown to be quenched at low-pH in the ΔPSI and ΔPSI/II strains,respectively. These quenching amplitudes are comparable to theamplitude observed for the LHCII+LHCSR1 cells in a previousstudy (∼50% in ref. 18), implying that energy dissipation occursat LHCII and is mediated by LHCSR1 in both theΔPSI andΔPSI/IIstrains. In contrast, the ΔPSII strain showed a remarkable amplitude

Fig. 2. Low-temperature absolute fluorescence spectra of isolated thyla-koid membranes. Fluorescence spectra of the isolated thylakoid membranesfrom npq4 (solid line) and npq4/lhcsr1 (dashed line). The membranes weretreated with either pH 7.5 (black) or pH 5.5 (red) buffers. The fluorescencespectra were recorded with an integration sphere to obtain the absolutefluorescence photon counts for the samples. Samples normalized to 8 μg Chl/mLwere excited at 480 nm.

Fig. 3. Time-resolved fluorescence decay-associated spectrum analysis ofisolated thylakoid membranes at 77 K. FDAS were derived from the time-resolved fluorescence profiles of thylakoid membranes obtained via excita-tion at 459 nm. The colored lines represent npq4 at pH 7.5 (green) andpH 5.5 (blue) and npq4/lhcsr1 at pH 7.5 (yellow) and pH 5.5 (red). The spectrawere normalized to the maximum intensity of the slowest component(∼2.2 ns).

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of low-pH-inducible quenching (∼74%, Table 1), although theprotein expression levels of both LHCSR1 and LHCIIs in the mu-tant were almost identical to the levels found in other mutants (Fig.4D and Fig. S6). In addition, the ΔPSII strain showed relatively lessexpression of LHCSR3 among the mutants (Fig. S5), strongly im-plying that the large fluorescence quenching observed in the mutant(ΔPSII in Fig. 4 and Table 1) is not significantly contributed by theLHCSR3 protein. FDAS of the ΔPSII strain also indicates that thePSI-related peak at around 710 nm in the first component (20 ps to30 ps) became larger in the ΔPSII at low pH, while the other strains(ΔPSI and ΔPSI/II) showed little change in this region (Fig. S3,ΔPSII). Following the fast component, positive fluorescence peaks at710 nm in the second (120 ps) and the third (500 ps) componentsincreased only in the ΔPSII. These results support an efficient ex-citation energy transfer to PSI from LHCII at low pH. Taking intoaccount that only the ΔPSII strain harbored PSI (Fig. 4D, PsaA/Bsignals), the large quenching observed in this strain was most likelydependent on the PSI machinery. We also estimated the NPQ ca-pability of the strains, which was calculated with τave in both pHenvironments [Table 1, NPQcalc = τave (pH 7.5)/τave (pH 5.5) − 1].These values showed that the ΔPSII strain exhibited a large degreeof quenching (NPQcalc = ∼2.8) compared with the other strains(NPQcalc = ∼0.85 in ΔPSI and ∼1.0 in ΔPSI/II). Based on theseresults, we conclude that LHCSR1-mediated fluorescence quench-ing under acidic conditions is stimulated by excitation energy dissi-pation among LHCIIs and efficient excitation energy transfer fromLHCII to PSI in C. reinhardtii.

DiscussionNPQ is the mechanism of feedback regulation for excess pho-tosystem excitation, which functions by dissipating absorbed lightenergy as heat (21). This mechanism is based on the contributionsof two stress-related LHC proteins, LHCSR1 and LHCSR3, inC. reinhardtii (7, 9, 16). Although LHCSR3 has been well studied,information about the molecular functions of LHCSR1 in this alga

is limited. In addition to the energy dissipation at LHCII asreported previously (18), we provide evidence that LHCSR1-dependent fluorescence quenching is mediated by excitation en-ergy transfer from LHCII to PSI.Acidic pH conditions in the thylakoid lumen triggered fluo-

rescence quenching in the double mutant lacking both photo-systems (see ΔPSI/II in Fig. 4 and Table 1). To estimate thepotential amplitude of NPQ in the mutants, we calculatedNPQcalc using the average fluorescence lifetime obtained atpH 5.5 and 7.5 (Table 1, NPQcalc). The ΔPSI/II mutant showed asimilar quenching ability (NPQcalc near to 1.0) to that observedby Dinc et al. (18) in the LHCII+LHCSR1 cells. These resultsstrongly suggest that LHCSR1 activates quenching at LHCIIs,even in the absence of photosystems, which is consistent with aprevious report proposing that LHCSR1-mediated quenchingoccurs at free LHCII (18).In addition to the quenching of free LHCIIs described above,

the results of time-resolved FDAS measurements implied that C.reinhardtii controls the transfer of excitation energy from LHCIIto PSI via UV-inducible LHCSR1 at low pH (Fig. 2 and Fig. S3).Although this excitation energy transfer from LHCII to PSIcontributes to additional layer of fluorescence quenching, itsunderlying mechanism is not energy dissipation but PSI chargeseparation (Fig. 3, Fig. S4, and Table S1).In our study, we observed even a larger quenching when both

PSI and LHCSR1 present in the cells (NPQcalc = 2.8 in ΔPSII,Table 1), suggesting PSI-dependent fluorescence quenching in C.reinhardtii. FDAS of the npq4 and ΔPSII strains at low pH showsa clear increase of the PSI fluorescence (700 nm to 710 nm) afterexcitation energy transfer from LHCII to PSI (see the secondand third component of FDAS in Fig. 3 and Fig. S3). The ob-served lifetimes are similar to those reported previously, repre-senting charge separation at PSI (22, 23). It is well known that,compared with PSII, PSI exhibits very low chlorophyll fluores-cence emission at room temperature, due to its efficient energy

Fig. 4. In vivo characterization of photosystem mutants. (A−C) The time-correlated single-photon counting of the fluorescence of (A) ΔPSI, (B) ΔPSII, and(C) ΔPSI/II cells after 6 h of UV treatment were recorded at 682 nm (slit = 8 nm) at pH 5.5 (red) and 7.5 (blue). The samples, normalized to 2 μg Chl/mL, wereexcited at 480 nm. (D) UV-treated cells (2 μg Chl) were subjected to immunoblotting analysis with antibodies specific to ATPB, PsaA/B (PSI), PsbA (D1), andLHCSR1.

Table 1. Estimated pH-inducible quenching in vivo

Average fluorescence lifetime(τave), ns

pH-inducible quenching, % NPQcalcStrain name At pH 7.5 At pH 5.5

ΔPSI 2.21 ± 0.22 1.19 ± 0.04 45.8 ± 0.05 0.85 ± 0.18ΔPSII 2.29 ± 0.15 0.60 ± 0.08 73.9 ± 0.02 2.84 ± 0.27ΔPSI/II 2.39 ± 0.13 1.17 ± 0.05 50.8 ± 0.03 1.04 ± 0.12

The efficiency of pH-inducible energy quenching was calculated as 1 − τave (pH 5.5)/τave (pH 7.5) (%). NPQcalc = τave(pH 7.5)/τave (pH 5.5) − 1; n = 3 biological replicates, mean ± SE.

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excitation−relaxation turnover (24, 25). This phenomenon indi-cates that PSI exhibits shorter lifetime of excited singlet chlo-rophyll (1Chl*) and lower frequencies of conversion to a tripletchlorophyll (3Chl*) state, which leads to harmful singlet oxygen(1O2*) formation (26). In other words, it is reasonable to use PSIas a quencher when excess light energy accumulates around PSII.Based on our findings, we propose a tentative model of

LHCSR1-mediated NPQ in C. reinhardtii (Fig. 5). When thethylakoid lumen becomes acidified under a high light intensity,LHCSR1 and LHCSR3 may sense the change in pH (15, 18).LHCSR1 plays two distinct roles in transferring excitation energyto PSI−LHCI supercomplexes (Fig. 5 process A and this study)or free LHCII (Fig. 5B process B, ref. 18, and this study). As aresult, excess light energy harvested by LHCIIs is safely trappedby PSI and/or dissipated at free LHCIIs, if any. Although wepresent a fluorescence quenching mechanism mediated by LHCSR1,it is still unclear whether the LHCSR1 protein associates withphotosynthetic pigments such as chlorophylls and/or caroten-oids. It is also not clear where it localizes within the thylakoidmembranes, and it is unknown whether the protein itself ex-hibits quenching ability. To answer these questions, more spe-cific biochemical techniques using both native and recombinantLHCSR1 protein complexes will be required, as in Bonenteet al. (11).Recently, an NPQ effector zeaxanthin was modeled at an

atomic resolution in PSI of land plants (27, 28). Although therehas been debate about the contribution of zeaxanthin to PSIquenching, the detailed molecular mechanisms of the fluores-cence quenching in land plants have been reported (29, 30).Direct excitation energy quenching by LHCSRs surrounding PSIhas also been observed in moss (31), implying that the quenchingaround PSI could be conserved in the green lineage. Our findingsalso show fluorescence quenching via excitation energy trans-ferred from LHCII to PSI (Fig. 3 and Fig. S3), and the LHCSR1-

mediated mechanisms thus can reduce the excitation of PSII atthe cost of increasing PSI excitation. Taken together with the PSIquenching established in land plants and mosses, it is plausiblethat LHCSR1-mediated fluorescence quenching by PSI in greenalgae is the primitive photoprotection mechanism of greenphotosynthetic eukaryotes.

Materials and MethodsCulture Conditions. The C. reinhardtii strain 137c (mt+) was obtained fromthe Chlamydomonas Center (https://www.chlamycollection.org/) and wasused as the WT strain. The mutant strains npq4 and npq4/lhcsr1 were iso-lated in previous reports (7, 9, 15, 16) and were then backcrossed with theWT strain at least three times. The ΔPSI (ΔPsaA) and ΔPSII (Fud7 as ΔPsbA)strains were obtained as described previously (32). The ΔPSI/II mutant wasgenerated in a previous study (33). All strains were grown in Tris-acetate-phosphate medium (34) under dim light (

12. Tokutsu R, Minagawa J (2013) Energy-dissipative supercomplex of photosystem IIassociated with LHCSR3 in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 110:10016–10021.

13. Kim E, Akimoto S, Tokutsu R, Yokono M, Minagawa J (2017) Fluorescence lifetimeanalyses reveal how the high light-responsive protein LHCSR3 transforms PSII light-harvesting complexes into an energy-dissipative state. J Biol Chem 292:18951–18960.

14. Xue H, et al. (2015) PHOTOSYSTEM II SUBUNIT R is required for efficient binding ofLIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 to photosystem II-light-harvesting supercomplexes in Chlamydomonas reinhardtii. Plant Physiol 167:1566–1578.

15. Ballottari M, et al. (2016) Identification of pH-sensing sites in the light harvestingcomplex stress-related 3 protein essential for triggering non-photochemicalquenching in Chlamydomonas reinhardtii. J Biol Chem 291:7334–7346.

16. Truong TB (2011) Investigating the role(s) of LHCSRs in Chlamydomonas reinhardtii.Doctoral dissertation (Univ California, Berkeley, CA).

17. Berteotti S, Ballottari M, Bassi R (2016) Increased biomass productivity in green algaeby tuning non-photochemical quenching. Sci Rep 6:21339.

18. Dinc E, et al. (2016) LHCSR1 induces a fast and reversible pH-dependent fluorescencequenching in LHCII in Chlamydomonas reinhardtii cells. Proc Natl Acad Sci USA 113:7673–7678.

19. Alboresi A, Caffarri S, Nogue F, Bassi R, Morosinotto T (2008) In silico and biochemicalanalysis of Physcomitrella patens photosynthetic antenna: Identification of subunitswhich evolved upon land adaptation. PLoS One 3:e2033.

20. Kondo T, et al. (2017) Single-molecule spectroscopy of LHCSR1 protein dynamicsidentifies two distinct states responsible for multi-timescale photosynthetic photo-protection. Nat Chem 9:772–778.

21. Horton P, Ruban A (2005) Molecular design of the photosystem II light-harvestingantenna: Photosynthesis and photoprotection. J Exp Bot 56:365–373.

22. Gobets B, van Grondelle R (2001) Energy transfer and trapping in photosystem I.Biochim Biophys Acta 1507:80–99.

23. van Amerongen H, van Grondelle R, Valkunas L (2000) Excitation energy transfer andtrapping: Experiments. Photosynthetic Excitons (World Sci, Singapore), pp 449–478.

24. Savikhin A (2006) Ultrafast optical spectroscopy of photosystem I. Photosystem I: TheLight-Driven Plastocyanin. Ferredoxin Oxidoreductase (Springer, Dordrecht, TheNetherlands), Vol 24, pp 155–175.

25. Croce R, van Amerongen H (2013) Light-harvesting in photosystem I. Photosynth Res116:153–166.

26. Krieger-Liszkay A (2005) Singlet oxygen production in photosynthesis. J Exp Bot 56:337–346.

27. Mazor Y, Borovikova A, Nelson N (2015) The structure of plant photosystem I super-complex at 2.8 Å resolution. eLife 4:e07433.

28. Qin X, Suga M, Kuang T, Shen JR (2015) Photosynthesis. Structural basis for energytransfer pathways in the plant PSI-LHCI supercomplex. Science 348:989–995.

29. Ballottari M, et al. (2014) Regulation of photosystem I light harvesting by zeaxanthin.Proc Natl Acad Sci USA 111:E2431–E2438.

30. Tian L, Xu P, Chukhutsina VU, Holzwarth AR, Croce R (2017) Zeaxanthin-dependentnonphotochemical quenching does not occur in photosystem I in the higher plantArabidopsis thaliana. Proc Natl Acad Sci USA 114:4828–4832.

31. Pinnola A, et al. (2015) Light-harvesting complex stress-related proteins catalyze ex-cess energy dissipation in both photosystems of Physcomitrella patens. Plant Cell 27:3213–3227.

32. Tokutsu R, Kato N, Bui KH, Ishikawa T, Minagawa J (2012) Revisiting the supramo-lecular organization of photosystem II in Chlamydomonas reinhardtii. J Biol Chem287:31574–31581.

33. Iwai M, Yokono M, Inada N, Minagawa J (2010) Live-cell imaging of photosystem IIantenna dissociation during state transitions. Proc Natl Acad Sci USA 107:2337–2342.

34. Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: Their sequence in thephotosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl AcadSci USA 54:1665–1669.

35. Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonasreinhardtii. Proc Natl Acad Sci USA 46:83–91.

36. Ueno Y, Aikawa S, Kondo A, Akimoto S (2015) Light adaptation of the unicellular redalga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.Photosynth Res 125:211–218.

37. Correa-Galvis V, et al. (2016) Photosystem II subunit PsbS is involved in the inductionof LHCSR protein-dependent energy dissipation in Chlamydomonas reinhardtii. J BiolChem 291:17478–17487.

38. Akimoto S, et al. (2012) Adaptation of light-harvesting systems of Arthrospira platensis tolight conditions, probed by time-resolved fluorescence spectroscopy. Biochim BiophysActa 1817:1483–1489.

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