-
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:
[email protected].
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
Dow
nloa
ded
by g
uest
on
Mar
ch 3
1, 2
021
http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.1720574115&domain=pdfhttp://www.pnas.org/site/aboutpnas/licenses.xhtmlmailto:[email protected]://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalwww.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,
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.
Kosuge et al. PNAS | April 3, 2018 | vol. 115 | no. 14 |
3723
PLANTBIOLO
GY
Dow
nloa
ded
by g
uest
on
Mar
ch 3
1, 2
021
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplemental
-
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).
3724 | www.pnas.org/cgi/doi/10.1073/pnas.1720574115 Kosuge et
al.
Dow
nloa
ded
by g
uest
on
Mar
ch 3
1, 2
021
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalwww.pnas.org/cgi/doi/10.1073/pnas.1720574115
-
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.
Kosuge et al. PNAS | April 3, 2018 | vol. 115 | no. 14 |
3725
PLANTBIOLO
GY
Dow
nloa
ded
by g
uest
on
Mar
ch 3
1, 2
021
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720574115/-/DCSupplemental
-
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.
Kosuge et al. PNAS | April 3, 2018 | vol. 115 | no. 14 |
3727
PLANTBIOLO
GY
Dow
nloa
ded
by g
uest
on
Mar
ch 3
1, 2
021