A icle h ps://doi.o g/10.1038/s41467-025-57809-3
Pho o opin connec s blue ligh pe cep ion
o s a ch me abolism in g een algae
Yizhong Yuan
1,11
, An hony A. Ianne a
2
, Minjae Kim
3
,Pa icW.Sadecki
2
,
Ma ius A end
4,5,6
,AngelikiTsichla
1,12
,M.ÁguilaRuiz-Sola
1,13
,
Geo gios Kepesidis
1,14
, Denis Falcone
1
, Emmanuel The enon
1
,
Ma ianne Ta di
7
,SabineB ugiè e
7
, Yohann Cou é
7
,
Jean Philippe Kleman
8
,I inaSizo a
9
, Ma ion Schilling
1
, Julie e Jouhe
1
,
Pe e Hegemann
9
, Yonghua Li-Beisson
3
, Zo an Nikoloski
4,5,6
,
Oli ie Bas ien
1
,LeslieM.Hicks
2
&Dimi isPe ou sos
1,10
In pho osyn he ic o ganisms, ligh ac s as an en i onmen al signal o con ol
hei de elopmen and physiology, as well as ene gy sou ce o d i e he con-
e sion o CO
2
in o ca bohyd a es used o g ow h o s o age. The main s o-
age ca bohyd a e in g een algae is s a ch, which accumula es du ing he day
and is b oken down a nigh o mee cellula ene gy demands. The signaling
ole o ligh quali y in he egula ion o s a ch accumula ion emains unex-
plo ed. He e, we iden i y PHOTOTROPIN-MEDIATED SIGNALING KINASE 1
(PMSK1) as a key egula o o s a ch me abolism in Chlamydomonas ein-
ha d ii. In i s phospho yla ed o m (PMSK1-P), i ac i a es GLYCERALDEHYDE-
3-PHOSPHATE DEHYDROGENASE (GAP1), p omo ing s a ch biosyn hesis. We
show ha blue ligh , pe cei ed by PHOTOTROPIN, induces PMSK1 depho-
spho yla ion ha in u n ep esses GAP1 mRNA le els and educes s a ch
accumula ion. These findings e eal a p e iously uncha ac e ized blue ligh -
media ed signaling pa hway ha ad ances ou unde s anding o
pho o ecep o -con olled ca bon me abolism in mic oalgae.
Pho osyn he ic mic oalgae con e ligh in o chemical ene gy in he
o m o ATP and NADPH, which uel CO
2
fixa ion in he Cal in–Benson
cycle. This p ocess o CO
2
fixa ion is ini ia ed by he ac i i y o he CO
2
-
assimila ing enzyme ibulose 1,5-bisphospha e ca boxylase/oxygenase
(Rubisco)1. In euka yo ic algae, such as he model pho osyn he ic
g een mic oalga Chlamydomonas einha d ii (he ea e Chlamydomo-
nas), concen a ed CO
2
is deli e ed o Rubisco wi hin a specialized
mic ocompa men in he chlo oplas called he py enoid2.
Ligh is also a spa io empo al signal; ed ligh is de ec ed by bilin-
con aining phy och omes, while blue ligh is pe cei ed by fla in-
Recei ed: 5 Ap il 2024
Accep ed: 21 Feb ua y 2025
Check o upda es
1
Uni e si é G enoble Alpes, CNRS, CEA, INRAE, IRIG-LPCV, G enoble, F ance.
2
Depa men o Chemis y, Uni e si y o No h Ca olina a Chapel Hill, Chapel
Hill, NC, USA.
3
Ins i u e de Biosciences e Bio echnologies Aix-Ma seille, Aix Ma seille Uni e si y, CEA, CNRS, BIAM, Sain Paul-Lez-Du ance, F ance.
4
Bioin o ma ics Depa men , Ins i u e o Biochemis y and Biology, Uni e si y o Po sdam, Po sdam, Ge many.
5
Sys ems Biology and Ma hema ical Modeling
G oup, Max Planck Ins i u e o Molecula Plan Physiology, Po sdam, Ge many.
6
Bioin o ma ics and Ma hema ical Modeling Depa men , Cen e o Plan
Sys ems Biology and Bio echnology, Plo di , Bulga ia.
7
Uni e si é G enoble Alpes, INSERM, CEA, UA13 BGE, CNRS, CEA, FR2048, G enoble, F ance.
8
Uni-
e si é G enoble Alpes, CNRS, CEA, IBS, G enoble, F ance.
9
Ins i u e o Biology, Expe imen al Biophysics, Humbold -Uni e si ä zu Be lin, Be lin, Ge many.
10
Depa men o O ganismal Biology, Uppsala Uni e si y, Uppsala, Sweden.
11
P esen add ess: Depa men o Plan Biology, Ca negie Ins i u ion o Science,
S an o d, CA, USA.
12
P esen add ess: Depa men o O ganismal Biology, Uppsala Uni e si y, Uppsala, Sweden.
13
P esen add ess: Ins i u o de Bioquímica
Vege al y Fo osín esis, Consejo Supe io de In es igaciones Cien íficas-Uni e sidad de Se illa, Se illa, Spain.
14
P esen add ess: Sandia Na ional Labo a o ies,
Li e mo e, CA, USA. e-mail: di[email p o ec ed]
Na u e Communica ions | (2025) 16:2545 1
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1234567890():,;
con aining c yp och omes and/o PHOTs, he la e cha ac e ized by
wo pho osenso y ligh , oxygen, o ol age (LOV) domains3.Chlamy-
domonas no ably lacks phy och omes in i s genome4, ne e heless,
his mic oalga has e ol ed a ich epe oi e o pho o ecep o s,
including a single-copy PHOT, ou c yp och omes, eigh hodopsin-
like p o eins, and he UV-B pho o ecep o UVR85. Th ough his ne -
wo k o specialized pho o ecep o s, Chlamydomonas egula es
impo an cellula unc ions, including: gene exp ession, sexual li e
cycle, pho o axis, and pho op o ec ion6–13.
Fixed CO
2
in combina ion wi h ni ogen is used o syn hesize
amino acids— he building blocks o p o eins ha d i e biochemical
eac ions. I is also employed in he syn hesis o cellula ese es ha
ensu es ca bon and ene gy supply du ing he waning pe iod. The mos
abundan ca bon ese ein hemodelpho osyn he icg eenmic oalga
Chlamydomonas is s a ch; i s syn hesis occu s du ing he day and i s
deg ada ion is igge ed a nigh o sus ain ene gy-demanding cellula
unc ions14. S a ch also se es ano he c i ical ole as s a ch shea hs
encasing he py enoid ac as a ba ie , educing CO
2
leakage om his
s uc u e15. While ac i ely di iding, Chlamydomonas cells accumula e
s a ch mos ly a ound he py enoid. In con as , when subjec ed o a
s ess such as nu ien limi a ion, s a ch g anules a e massi ely accu-
mula ing in he chlo oplas s oma16. Li le is known abou he mole-
cula mechanisms unde lying he con ol exe ed by ligh on ca bon
s o age in mic oalgae, and cu en knowledge is limi ed o ac o s
impac ing s a ch accumula ion unde ad e se en i onmen al condi-
ions, such as ni ogen17 and phospho us18 limi a ion.
A link be ween ligh pe cep ion and s a chaccumula ion has been
sugges ed in ascula plan s. Fo ins ance, A abidopsis mu an s de oid
o he ed/ a - ed pho o ecep o phy och ome B ha e impai ed ca -
bon pa i ioning and al hough hey ha e educed CO
2
up ake, hey
o e -accumula e day ime suc ose and s a ch a he expense o
g ow h19. Fu he , he blue ligh ecep o PHOTOTROPIN (PHOT) has
been ound o media e s a ch deg ada ion in gua d cells in he ligh ,
hus ene gizing s oma al opening in A abidopsis20. Despi e ea ly find-
ings epo ing ha ligh quali y impac s ca bohyd a e accumula ion
and me abolism in g een algae21,22, he unde lying molecula mechan-
ism connec ing ligh pe cep ion o s a ch accumula ion emains
unexplo ed. He e we add ess his gap by combining gene ics, p o-
eomics, and phosphomime ics o un eil a signaling cascade linking
blue ligh pe cep ion by PHOT wi h s a ch accumula ion in Chlamy-
domonas. Pho -dependen de-phospho yla ion a a single se ine esi-
due o pho o opin-media ed signalling kinase 1 (PMSK1) ep esses
GAP1 (also known as GAPDH; glyce aldehyde-3-phospha e dehy-
d ogenase), which we ound o ac as an enhance o s a ch
me abolism.
Resul s
The pho mu an is a s a ch hype accumula o
We compa ed s a ch le els in wild- ype CC125 cells (WT) exposed o
whi e and ed ligh and ound ha ed ligh a o ed s a ch accumula-
ion, in ag eemen wi h p io wo k21. In e es ingly, when low fluence
blue ligh was supe imposed on ed ligh , he beneficial e ec o ed
ligh was los and cells accumula ed s a ch le els simila o hose o cells
exposed o whi e ligh (Supplemen a y Fig. 1). The e o e, we easoned
ha blue ligh ac s as a ep esso o s a ch accumula ion likely ia a blue
ligh ecep o . To es his hypo hesis, we measu ed s a ch con en in
WT and mu an cells lacking di e en blue-ligh ecep o s, including:
ac y, lacking he ANIMAL-TYPE CRYPTOCHROME6, gene a ed in his
s udy, pc y lacking he PLANT-TYPE CRYPTOCHROME23, gene a ed in
his s udy, he double ac ypc y mu an , gene a ed in his s udy, and he
pho mu an , de oid o PHOTOTROPIN (gene a ed in e . 24). When
g own asynch onously unde con inuous ligh , he s a ch con en o he
c yp och ome mu an s was indis inguishable om ha o he
WT (Fig. 1a) and he same was ue when cul u es we e synch onized
unde 12 h ligh /12 h da k cycles (Supplemen a y Fig. 2), a o ing
c yp och ome accumula ion in Chlamydomonas6,23.Incon as o he
c yp och ome mu an s, he pho mu an accumula ed app oxima ely
h ee imes mo e s a ch han he WT unde con inuous illumina ion
(Fig. 1a). This pheno ype was escued by ec opic exp ession o he WT
PHOT gene; i was ully escued in s ain pho -C125 (Fig. 1b), which
accumula es PHOT p o ein a WT le els (Supplemen a y Fig. 3a), and
pa ially escued in s ain pho -C2 (Fig. 1b), which accumula es PHOT
p o ein bu o a lesse ex en han he WT (Supplemen a y Fig. 3a).
Comple e escue o he pheno ype was also achie ed by com-
plemen a ion o a pe manen ly ac i e PHOT mu an lacking he LOV
senso y domains (pho -kin s ain; Fig. 1b and Supplemen a y Fig. 3a, b);
apho -kin s ain in a di e en gene ic backg ound has p e iously been
shown o exhibi PHOT kinase ac i i y in a ligh -quali y-independen
manne 13. The accumula ion o high le els o s a ch in he pho mu an
was also confi med by ansmission elec on mic oscopy (TEM),
e ealing s iking changes in s a ch deposi ion pa e ns compa ed o
he wild ype. As indica ed by as he a ows in he TEM images in Fig. 1c,
he chlo oplas s o he pho mu an a e filled wi h s a ch g anules and
i s py enoid, is su ounded by abno mally hick s a ch shea hs. In
con as , in he chlo oplas s o he WT and pho -C1 s ains s a ch is
p edominan ly localized as hin shea hs a ound he py enoid (Fig. 1c
and Supplemen a y Fig. 4).
We u he explo ed he link be ween PHOT and s a ch accumu-
la ion in synch onized cul u es unde whi e, blue o ed ligh . S a ch
accumula ed du ing he ligh phase and deg aded du ing he da k
phase, in acco d wi h14,26, unde all ligh quali ies and in all di e en
s ains es ed (Fig. 1d). We ound ha s a ch le els in WT a e highe
unde ed ligh , whe e PHOT is inac i e, han unde whi e o blue,
whe ePHOTis ac i e(Fig. 1d). In he pho mu an , s a ch le els a e high
in all h ee ligh quali ies, while he ully complemen ed pho -C1
beha es like WT and he pa ially complemen ed pho -C2 is in e -
media e be ween WT and pho .Incon as o hepho mu an , s a ch
le els in s ain pho -kin,inwhichPHOTisac i e,a elowanduna -
ec ed by he ligh quali y (Fig. 1d). These da a (Fig. 1) indica e ha
PHOT ac s as supp esso o s a ch accumula ion in Chlamydomonas.
We p o ided u he e idence o his claim by using ano he se o
pho mu an s in a di e en gene ic backg ound, he cw15-3028,13.In
acco dance wi h he da a in Fig. 1, we ound ha he cw15-302 pho
mu an accumula ed mo e s a ch han cw15-302 (Supplemen a y
Fig. 3c). This pheno ype was only pa ially escued in he com-
plemen ed ppho s ain, which accumula es lowe le els o PHOT
p o ein han he cw15-302 WT s ain13, and was ully escued in he
cw15-302 pho mu an exp essing he PHOT kinase domain (pkin
s ain; Supplemen a y Fig. 3c). Con e sely, complemen a ion o cw15-
302 pho cells wi h unca ed gene ca ying only he pho osenso y
domains LOV1 and 2 (plo s ain) did no escue s a ch accumula ion.
Impo an ly,we obse ed a simila pheno ype when he cw15-302 pho
mu an was complemen ed wi h he dead kinase PHOT (pkin-D s ain;
Supplemen a y Fig. 3c), demons a ing ha he supp ession o s a ch
accumula ion by PHOT equi es i s kinase ac i i y. G ow h (Supple-
men a y Fig. 5a) and pho osyn hesis (Supplemen a y Fig. 5b) we e no
a ec ed by he o e accumula ion o s a ch in pho (Fig. 1c). Finally,
pho showed no di e ence o WT in e ms o o al p o ein (Supple-
men a y Fig. 6), lipid con en (Supplemen a y Fig. 7) and composi ion
(Supplemen a y Fig. 8).
GAP1 is a key egula o o s a ch me abolism and is con olled
by PHOT
To gain mo e insigh in o he molecula mechanism unde lying he
PHOT-media ed ligh pe cep ion and s a ch accumula ion, we
applied mass spec ome y (MS)-based quan i a i e p o eomics o
compa e WT and pho cells g own in asynch onous pho o-
au o ophic condi ions unde con inuous whi e ligh . Gene on ology
en ichmen analyses e ealed ha ca bohyd a e me abolic p o-
cesses a e up egula ed in he pho mu an (Fig. 2a). We no ably ound
A icle h ps://doi.o g/10.1038/s41467-025-57809-3
Na u e Communica ions | (2025) 16:2545 2
ha GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAP1)
was exp essed 27.5- old highe in pho compa ed o WT (Fig. 2a,
Supplemen a y Da a 2). GAP1 is a chlo oplas iso o m o GAPDH in
Chlamydomonas, a Cal in-Benson cycle enzyme p edominan ly
localized in he s omal egion su ounding he py enoid27,28. The
significan accumula ion o GAP1 in pho was in acco d wi h he high
accumula ion o GAP1 mRNA (Fig. 2b). While mRNA accumula ion o
all es ed s a ch- ela ed genes was high in he pho mu an in he
middle o he ligh phase (Supplemen a y Fig. 9), GAP1 was he mos
highly exp essed, p omp ing u he in es iga ion in o he link
be ween PHOT and GAP1. We ound ha GAP1 mRNA o e -
accumula ion pheno ype o pho was comple ely escued in pho -C1
and pho -kin and pa ially escued in pho -C2 (Fig. 2b), in acco d wi h
he PHOT exp ession le els in he di e en complemen ed lines
(Supplemen a y Fig. 3a, b), sugges ing ha PHOT ac s as supp esso
o GAP1 upon illumina ion. Simila o he PHOT-media ed supp es-
sion o s a ch (Supplemen a y Fig. 3c), he PHOT-dependen sup-
p ession o GAP1 equi es he kinase ac i i y o PHOT
(Supplemen a y Fig. 10). The pkin-D s ain, a pho mu an exp essing
a dead kinase PHOT wi h poin mu a ions in he ATP binding si e8and
he plo s ain exp essing only he senso y domains LOV, bo h
accumula e GAP1 mRNA a he le el o pho . Complemen a ion o he
pho mu an wi h he ull-leng h PHOT (pho s ain) o i s kinase
domain (pkin s ain) escues GAP1 mRNA o WT le els (Supplemen-
a y Fig. 10).
We also measu ed GAP1 mRNA in cells synch onized o a 12/12
ligh -da kcycle. GAP1 mRNA in WT was ound o be s ongly influenced
by he diu nal cycle, s a ing a e y low le els a e he ini ial onse o
whi e ligh , ising o maximal le els a he end o he ligh phase, and
hen p og essi ely and sligh ly declining du ing he nigh phase
(Fig. 2c); his is in acco dance wi h da a om p e ious s udies29,30 on
he impac o diel cycle on he genome-wide gene exp ession
( eplo ed in Supplemen a y Fig. 11). GAP1 mRNA accumula ion p ofile
was iden ical in whi e and blue ligh condi ions. Howe e , unde ed
ligh , exp ession le els a he beginning o he day we e significan ly
bigge and eached app oxima ely 45 imes highe le els a he mid-
poin o he ligh phase (Fig. 2c), sugges ing ha he low le els o GAP1
mRNA a e he ini ial onse o ligh a e a esul o supp ession by blue
ligh . Indeed, in all ligh quali ies es ed, GAP1 mRNA le els in he
synch onized pho mu an (Fig.2d)we eiden ical o hoseo heWTin
ed ligh (Fig. 2c). The ully complemen ed pho -C1 beha ed like WT
(Fig. 2e) while he pa ially complemen ed pho -C2 only pa ially
WT pho -C1pho
ab
WT
ac y
pc y
ac ypc y
pho
20
WT
pho
pho -C1
pho -C2
pho -kin
S a ch con en
(μg/million cells)
WT pho pho -C1 pho -C2
pho -kin
0122436
0
5
10
15
20
25
0122436
d
c
01224
36
S a ch con en
(μg/million cells)
S a ch con en
(μg/million cells)
0
5
10
15
20
✱✱✱✱ ✱✱✱✱
✱
✱✱✱✱
0
5
10
15
✱✱✱✱
ns
ns
ns
Time (h)
Fig. 1 | PHOT inhibi s s a ch accumula ion in Chlamydomonas einha d ii.
aS a ch con en o WT, ac y, pc y, ac ypc y lines unde con inuous ligh . bS a ch
con en o WT and a iously pho -complemen ed lines unde con inuous whi e
ligh . cT ansmission elec on mic oscopy pic u es o WT, pho and pho -C syn-
ch onized o a12/12 da k/ligh cycle. Samples we e collec ed a he end o he ligh
phase. Red a ows indica e s a ch g anules. Rep esen a i e images om h ee
eplica es we e shown. dS a ch con en o WT and pho -complemen ed lines
synch onized o 12/12 da k/ligh cycle. Red iangles indica e sample collec ion
ime. The da k phase is indica ed by blackba s abo e he g aphs; he ligh phase by
whi e, blue o ed ba s, depending on heligh quali y used.In some cases, he e o
ba s a e smalle han he da a poin symbols. Da a a e ep esen ed as mean±SD
(n = 3 biologically independen samples). The s a is ical significance was de e -
mined using one-way ANOVA wi h Dunne ’s mul iple compa isons es s (a,b)and
wo-way ANOVA wi h Dunne ’s mul iple compa isons es s (d). As e isks indica ed
he p- alues (*p < 0.05; ****p < 0.0001; ns, no significan ). In some cases, he e o
ba s a e smalle han he da a poin symbols. De ailed s a is ical analyses a e p e-
sen ed in he Sou ce Da a File.
A icle h ps://doi.o g/10.1038/s41467-025-57809-3
Na u e Communica ions | (2025) 16:2545 3
escued he obse ed pheno ype (Fig. 2 ). No ably, he pho -kin s ain
in which PHOT is de oid o he pho osenso y LOV domains and is
always ac i e ega dless o ligh quali y13, accumula ed e y low le els
o GAP1 mRNA (Fig. 2g; no e he log scale). Howe e , in pho -kin cells
g own unde ed ligh , GAP1 mRNA le els a e highe a 6h compa ed
o cells g own in whi e o blue ligh (Fig. 2g). Despi e, he inc eased
GAP1 mRNA in ed ligh his was no su ficien o o e accumula ion o
s a ch (Fig. 1d). This indica es ha addi ional egula o s, beyond
PHOT, may be in ol ed in ep essing GAP1 in a blue ligh -dependen
manne .
Ou esul s so a show a s ong associa ion be ween s a ch and
GAP1 mRNA accumula ion in he di e en pho mu an s
(Figs. 1band2b). WT, pho -C1 and pho -kin accumula e low amoun s o
s a ch (Fig. 1b) and low mRNA GAP1 (Fig. 2b), pho accumula es high
Rel. GAP1 mRNA abundance
10
-1
10
0
10
1
10
2
10
3
WT
pho
pho -C1
pho -C2
pho -kin
ab
de
Ca bohyd a e
me abolism
Pho osyn hesis
Gene a ion o
p ecu so me boli es
Pho osyn hesis
ligh eac ion
Pho osyn hesis
ligh ha es ing
✱✱✱✱
✱✱✱✱
✱
✱✱✱✱
WT
gap1 oe-1
gap1 oe-2
gap1 oe-3
pho
pho gap1i-1
pho gap1i-2
pho gap1i-3
0
5
10
15
20
S a ch con en
(μg/million cells)
✱✱
✱
ns
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
WT
gap1 oe-1
gap1 oe-2
gap1 oe-3
pho
pho gap1i-1
pho gap1i-2
pho gap1i-3
10
-1
10
0
10
1
10
2
10
3
Rel. GAP1 mRNA abundance
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱
✱✱✱✱
✱✱
ns
WT pho pho -C1
pho -C2 pho -kin
c
10
-2
10
-1
10
0
10
1
10
2
10
3
Rel. GAP1 mRNA abundance
10
-2
10
-1
10
0
10
1
10
2
10
3
Rel. GAP1 mRNA abundance
061218
24
061218
24
Time (h)
g
hi
A icle h ps://doi.o g/10.1038/s41467-025-57809-3
Na u e Communica ions | (2025) 16:2545 4
le elso s a ch(Fig.1b) andhighGAP1 mRNAle els(Fig.2b), and finally
he pa ially complemen ed pho -C2 (Supplemen a y Fig. 3a) accu-
mula es in e media e le els o s a ch and GAP1 mRNA (Fig. 1band
Fig. 2b). To u he confi m he posi i e connec ion be ween GAP1
exp ession le el and s a ch amoun , we gene a ed WT s ains o e -
exp essing GAP1 (Supplemen a y Fig. 12). S ains gap1-oe1 and gap1-
oe2 accumula ed app oxima ely 15- old mo e GAP1 mRNA han WT
(Fig. 2h), esul ing in 1.5- old highe s a ch con en (Fig. 2i). The s ain
gap1-oe3, wi h a mo e modes o e exp ession o GAP1 mRNA (2.5- old
compa ed o WT; Fig. 2h), had s a ch con en a WT le els (Fig. 2i). Fo
compa ison, we included he pho mu an in hese analyses, which
exp essed 80- old mo e GAP1 and accumula ed 3- old mo e s a ch
han he WT. We also down egula ed GAP1 in he pho mu an and
gene a ed s ains pho -gap1-i1,2and 3 ha accumula ed 1.6-, 3.1- and
5.5- old less GAP1 mRNA (Fig. 2h) and 1.3-, 1.5- and 2- old less s a ch,
espec i ely, compa ed o pho (Fig. 2i). In conclusion, o e exp ession
o GAP1 in WT led o o e accumula ion o s a ch whe eas down-
egula ion o GAP1 in pho dec eased s a ch accumula ion (Fig. 2h, i).
These esul s (Fig. 2) s ongly sugges ha GAP1 plays a key ole in
s a ch me abolism in Chlamydomonas, ac ing unde he con ol
o PHOT.
PHOT al e s phospho yla ion s a e o PHOTOTROPIN-
MEDIATED SIGNALLING KINASE 1
To iden i y missing componen s in he pho o opin-media ed signal-
ing pa hway ha supp esss a ch accumula ion in Chlamydomonas,we
compa ed he phosphop o eome o WT and pho cells a e an o e -
nigh da k acclima ion and a 5-min exposu e o blue ligh . We iden i-
fied 1119 phosphopep ides, belonging o 747 phosphop o eins, and
applied unsupe ised hie a chical clus e ing o z- ans o med ela i e
abundance o phosphopep ides o ob ain an o e iew o he
condi ion-specific and pho o opin-dependen phospho yla ion
changes (Supplemen a y Fig. 13 and Supplemen a y Da a 3). Phos-
phopep ides alling wi hin clus e “E”we e highly phospho yla ed in
he da k and became de-phospho yla ed a e blue ligh illumina ion,
bu only in he case o WT; in he pho mu an hey emained highly
phospho yla ed a e blue ligh illumina ion. One such pep ide was he
ADGVSpSPHELTR, phospho yla ed a se ine120 (S120), which belongs
o he gene p oduc o C e16.g659400 encoding a Se /Th p o ein
kinase (Fig. 3b), localized in he cy osol, plasma memb ane and flagella
(Supplemen a y Fig. 14). We named his kinase pho o opin-media ed
signalling kinase 1 (PMSK1).
To alida e he phosphop o eomic findings, we gene a ed WT
and pho lines exp essing PMSK1 used o a FLAG epi ope. These lines
(WT/PMSK1-FLAG and pho /PMSK1-FLAG)we eda kacclima edp io
exposu e o ed o blue ligh and samples we e aken a 5minin e als
o a pe iod o 20 min o assess he PMSK1-FLAG phospho yla ion
s a us using Phos- ag SDS-PAGE, ollowed by immunode ec ion
agains FLAG (Supplemen a y Fig. 15). In acco dance wi h he ole o
PHOT in he ligh -dependen dephospho yla ion o PMSK1 de ec ed in
ou phosphop o eomic analyses (Fig. 3a), he Phos- ag da a e ealed a
apid dephospho yla ion o PMSK1-FLAG (Fig. 3c) upon exposu e o
ligh . In he WT backg ound, dephospho yla ion occu ed only unde
blue ligh (Fig. 3c). In con as , in he pho backg ound, depho-
spho yla ion was ba ely obse ed, e en unde blue ligh (Fig. 3c).
Fu he mo e, no dephospho yla ion was obse ed unde ed ligh in
ei he s ain (Fig. 3c). These da a (Fig. 3a–c) suppo ou hypo hesis
ha dephospho yla ion o PMSK1 equi es blue ligh -ac i a ed PHOT.
In e es ingly, he obse ed blue ligh dependen mobili y shi o
PMSK1-FLAG (Fig. 3c) was abolished when S120 was subs i u ed by an
alanine (A) o an aspa ic acid (D) esidue (Fig. 3d). These da a indica e
ha phospho yla iona esidueS120isessen ial o heshi .Howe e ,
his shi may no be solely a ibu ed o S120 phospho yla ion, bu
a he o addi ional phospho yla ion e en s ha depend on he ini ial
phospho yla ion o S120.
Phospho yla ion s a e o se ine 120 o PMSK1 con ols s a ch
accumula ion
We nex se ou o in es iga e he unc ional significance o he
phospho yla ion s a us o S120 in i o, aking ad an age o he
phosphomime ic mu a ion S120D and he non-phospho yla able
mu a ion S120A in PMSK1-FLAG, exp essed in WT and in pho (Sup-
plemen a y Fig. 15). We measu ed GAP1 mRNA and s a ch accumula-
ion in WT, pho and all abo e-men ioned gene a ed mu an s,
synch onized unde whi e, blue o ed ligh in a 12 h ligh /12 h da k
egime. O e exp ession o PMSK1S120D-FLAG in WT, esul ed in an
enhanced GAP1 exp ession le el and inc eased s a ch con en unde
all h ee ligh quali ies, exceeding hose eco ded in he pho mu an
(Fig. 4a, b and Supplemen a y Figs. 16 and 17). O e exp ession in pho
o PMSK1S120D-FLAG esul ed in an e en u he highe GAP1 exp es-
sionle el and inc eased s a ch con en as compa ed o pho (Fig.4a,b
and Supplemen a y Fig. 17). Con e sely, o e exp ession inpho o WT
o PMSK1S120A-FLAG, esul ed in low, WT-le el o e en lowe , GAP1
mRNA and s a ch le els, ac oss all h ee ligh quali y es s (Fig. 4a, b
and Supplemen a y Figs. 16 and 17). We also analyzed he impac o
o e exp ession o he unmodified PMSK1-FLAG in bo h WT and pho
mu an (Fig. 4a, b and Supplemen a y Figs. 15 and 17); WT and WT/
PMSK1-FLAG beha ed e y simila ly in all ligh colo s wi h espec o
GAP1 mRNA and s a ch accumula ion (Fig. 4a, b and Supplemen a y
Fig. 17). Howe e , pho /PMSK1-FLAG showed inc eased GAP1 mRNA
and accumula ed mo e s a ch (Fig. 4a, b and Supplemen a y Fig. 17).
This inc ease is likely due o he absence o PHOT, causing bo h
endogenous and ansgenic PMSK1-FLAG in he cells o emain
phospho yla ed (Fig. 3c).
Taken oge he , ou da a (Fig. 4) e eal ha he phospho yla ion
s a e o S120 o PMSK1 con ols s a ch me abolism h ough egula ion
o GAP1 mRNA le els.To in es iga e i PMSK1’s ole is dependen on i s
kinase ac i i y, we used a kinase-inac i e e sion o PMSK1, whe e Asp-
442 was subs i u ed wi h Asn (D442N) o dis up he ATP-binding si e,
alongside he phosphomime ic S120D o he non-phospho yla able
S120A mu a ions. Nei he mu a ion (S120D o S120A) a ec ed s a ch
con en (Supplemen a y Fig. 18), indica ing ha PMSK1 ulfills he
obse ed egula o y ole on s a ch me abolism h ough i s kinase
ac i i y.
Fig. 2 | GAP1 is inhibi ed by blue ligh ia PHOT and plays key ole in PHOT-
dependen s a ch me abolism in Chlamydomonas. aGene on ology en ichmen
analyses o di e en ially abundan p o eins in pho compa ed o WT based on
whole cell p o eomics da a. LFC, log2 old change (en iched GO e ms we e only
ound o p o eins wi h inc eased abundance in pho in compa ison o WT); colo
code indica es adjus ed p- alue o GO se en ichmen (null hypo hesis: numbe o
di e en ially abundan p o eins in GO se is hype geome ic andom dis ibu ed.
P- alue we e adjus ed acco ding o Benjamini-Hochbe g p ocedu e bGAP1 ela i e
mRNA abundance o WT and pho -complemen ed lines unde con inuous whi e
ligh . c–g ela i e mRNA abundance o GAP1 in WT and pho -complemen ed lines
synch onized o a 12/12 da k/ligh cycle. Phases a e indica ed by whi e and g ay
shading. Line colo s indica ed ligh quali ies. Black, whi e ligh ; Red, ed ligh ; Blue,
blue ligh . The GAP1 ansc ip ion le el (h) and s a ch con en (i)o WT,GAP1
o e exp ession, and pho gap1 knockdown lines unde con inuous ligh . Da a a e
p esen ed as mean± SD (n = 3 biologically independen samples). The s a is ical
significance was de e mined using one-way ANOVA wi h Dunne ’s mul iple com-
pa isons es s(b,h) and wo-way ANOVA wi h Dunne ’smul iple compa isons es s
(c–g) o log10 ans o med mRNA da a as indica ed in he g aphs. The s a is ical
significance was de e mined using one-way ANOVA wi h Dunne ’s mul iple com-
pa isons es s (i). As e isks indica ed he p- alues (*p < 0.05; **p< 0.01;
****p< 0.0001; ns, no significan ). In some cases, he e o ba s a e smalle han he
da a poin symbols. De ailed s a is ical analyses a e p esen ed in he Sou ce
Da a File.
A icle h ps://doi.o g/10.1038/s41467-025-57809-3
Na u e Communica ions | (2025) 16:2545 5
PMSK1 ac s downs eam o PHOT o egula e s a ch me abolism
in esponse o blue ligh
We applied CRISPR-CAS9 o dis up PMSK1 in he WT and in pho , hus
gene a ing he single pmsk1 and he double pho pmsk1 mu an s
(Supplemen a y Fig. 19). In compa ison o WT, he pmsk1 mu an
accumula ed 3-, 4- and 10- old lowe GAP1 mRNA le els in whi e, blue
and ed ligh , espec i ely (Fig. 5a; when compa ing GAP1 mRNA le els
a he middle o he day). Ne e heless, GAP1 exp ession in pmsk1
emained dependen on ligh -quali y used, eaching highe exp ession
le els unde illumina ion wi h ed ligh (Fig. 5a). Howe e , his o e -
accumula ion o GAP1 mRNA in ed ligh was no su ficien o o e -
accumula ion o s a ch (Fig. 5b and Supplemen a y Fig. 20), as in he
case o he gap1 oe-3 line, which sligh ly o e accumula ed GAP1 mRNA
bu s a ch le els emained compa able o WT le els (Fig. 2handi).Asa
esul , we concluded ha s a ch accumula ion in pmsk1 was ligh -
quali y-independen . The double mu an pho pmsk1 accumula ed
3- old less GAP1 mRNA and 2- old less s a ch as compa ed o pho
(Fig. 5a, b and Supplemen a y Fig. 20).
Taken oge he , ou esul s show ha PMSK1 plays a c i ical ole in
ansducing he PHOT-media ed blue ligh signal o egula e s a ch
me abolism in Chlamydomonas.Indeed, hepmsk1 mu an has been
ins umen al in ge ing a clea pic u e o his dis inc ligh -signalling
pa hway, summa ized g aphically in Fig. 6. Based on ou da a, we p o-
pose ha PMSK1 is an ac i a o o GAP1 mRNA accumula ion, and i s
ac i i y is egula ed by he phospho yla ion s a us o S120 in esponse
o ligh quali y. When PMSK1 is phospho yla ed, i becomes ac i e and
p omo es GAP1 mRNA accumula ion. Con e sely, when i is no phos-
pho yla ed, PMSK1 is inac i e and does no u he p omo e GAP1
mRNA accumula ion. In addi ion o his egula ion, GAP1 exp ession is
influenced by o he signals, possibly ci cadian, pho osyn he ic, o
diu nal hy hms, esponsible o he exp ession pa e n obse ed o e
he cou se o he day. Red ligh d i es he phospho yla ed o m o
PMSK1 (PMSK1-P; Fig. 3c), esul ing in high GAP1 mRNA accumula ion
(Fig. 4a). Howe e , in whi e and blue ligh , dephospho yla ion o ac i e
PMSK1-P esul s in an accumula ion o un-phospho yla ed PMSK1
(PMSK1-U; Fig. 3c), educing he ela i e abundance o PMSK1-P wi hin
he o al PMSK1 pool. This shi owa d PMSK1-U dec eases he p o-
po ion o PMSK1-P, leading o educed GAP1 mRNA le els (Fig. 4a). In
he pmsk1 mu an , GAP1 mRNA le els (Fig. 5a) a e simila o hose in WT/
PMSK1-S120A (unphospho yla ed o m; Fig. 4a), showing lowe
exp ession a 12 h. Howe e , he e is s ill egula ion in ed ligh , which is
abolished in he pho pmsk1 double mu an (Fig. 5a), sugges ing he
exis ence o o he egula o s o GAP1 ha a e con olled by blue ligh
independen ly o PMSK1, al hough PMSK1 appea s o play a majo ole.
Ou model (Fig. 6), which sugges s ha PMSK1-P ac s as an ac i-
a o o GAP1 mRNA accumula ion, explains why he PMSK1 mu a ion
has he g ea es impac on GAP1 mRNA le els in ed ligh (Fig. 5a). In
ed ligh , PMSK1-P is mos abundan in WT, making he mu a ion’s
PMSK1
585 aa
(ca. 63.9 kDa)
3670 bp
S120
ADGVSpSPHELTR
Kinase domain
500bp
P
U
ATPB
WT 0’ -P con ol
WT pho
0’ 5’ 10’ 20’ 0’ 5’ 10’ 20’
P
U
pho 0’ -P con ol
PMSK1-
FLAG
ATPB
Phospho-le el (log2)
50
75
50
75
kDa
a
b
d
c
PMSK1-
FLAG
WT D
WT BL
pho D
pho BL
7
8
9
10
11 ✱✱✱ ns
-P con ol
0’ 20’
PMSK1
0’ 20’
S120A
0’ 20’
S120D
PMSK1-
FLAG
ATPB
P
U
50
75
kDa
Fig. 3 | Iden ifica ion o PMSK1 as a key p o ein on PHOT-dependen s a ch
me abolism in Chlamydomonas einha d ii. aChanges in he phospho yla ion
le el o S120 o PMSK1 in esponse o BL in WT and pho ,quan ified by phospho-
p o eomics. Samples we e collec ed a e 24h o acclima ion o da kness and 5 min
a e he s a o blue ligh . D da k, BL blue ligh . Da a a e p esen ed as log2-
ans o med phospho-le els (n = 5 biologically independen samples). E o ba s
indica e mean±SEM. Box plo s ep esen he median (cen e line), in e qua ile
ange (bounds o he box), minima, and maxima (whiske s) wi h indi idual da a
poin s o e laid. The s a is ical significance was de e mined using one-way ANOVA
wi h Dunne ’s mul iple compa isons es s. As e isks indica ed he p- alues
(****p < 0.0001; ns no significan ). bUppe : Genomic s uc u es o PMSK1. Black
boxes and lines indica e exons and in ons, espec i ely. Lowe : Schema ic s uc-
u es o PMSK1. Red lines indica e he phospho yla ed esidue in PMSK1. The
o ange box indica es he kinase domain. cPhospho yla ion le el o PMSK1-FLAG as
a unc iono ime inWT/PMSK1-FLAG and pho /PMSK1-FLAG, exposed oblueo ed
ligh a e 24 h acclima ion o da kness (indica ed as = 0 in he g aph). De ec ion
was pe o med by Phos- ag SDS-PAGE; ATPB was used as a loading con ol.
Phospha ase- ea ed WT/PMSK1-FLAG and pho /PMSK1-FLAG samples we e also
loaded on he gels. A ep esen a i e expe imen om a leas h ee independen
expe imen s is shown. dChanges in he phospho yla ion le el o PMSK1-FLAG in
WT/PMSK1-FLAG, WT/PMSK1S120A-FLAG and WT/PMSK1S120D-FLAG lines. Samples we e
collec ed a e 24 h o acclima ion oda kness ( = 0′)and20′a e exposu e oblue
ligh (100 μmol pho ons m−2s−1). De ec ion was pe o med by Phos- ag SDS-PAGE;
ATPB was used as a loading con ol. Phospha ase- ea ed WT/PMSK1-FLAG sample
was also loaded on he gels. “U”and “P”indica e he unphospho yla ed and
phospho yla ed PMSK1-FLAG espec i ely. The blue o ed ba o he le o he
immunoblo s indica es he ligh quali y used. A ep esen a i e expe imen om a
leas h ee independen expe imen s is shown.
A icle h ps://doi.o g/10.1038/s41467-025-57809-3
Na u e Communica ions | (2025) 16:2545 6
e ec mo e p onounced. In con as , in whi e o blue ligh , PHOT
p omo es PMSK1 dephospho yla ion, educing PMSK1-P le els and
diminishing he mu a ion’s impac in hese condi ions.
Discussion
In his wo k we epo he disco e y o an un ecognized ligh -
signalling pa hway linking blue-ligh pe cep ion by PHOT wi h s a ch
accumula ion in Chlamydomonas. Ou da a sol e a ou -decades-long
ques ion abou he easons o s a ch accumula ion in g een algae
p edominan ly unde ed ligh 31. We showed ha PHOT egula es he
phospho yla ion o a specificse ine esidue(S120)onaye uncha -
ac e ized kinase, PMSK1. In i s phospho yla ed s a e, PMSK1 ans-
duces a signal ha con ols he accumula ion o GAP1 mRNA (Fig. 6).
Since GAP1 is in ol ed in he gene a ion o phospho yla ed suga s, as
p ecu so s o s a ch syn hesis, ou findings demons a e how his
ligh -signalling pa hway leads o imp o emen o s a ch me abolism.
Ou wo k es ablishes PMSK1 as a kinase in ol ed in PHOT-
dependen signaling in algae, highligh ing i s dis inc ole in his pa h-
way. Ye , PHOT may no be he only blue-ligh esponsi e p o ein
supp essing s a ch accumula ion in Chlamydomonas; as ou da a show,
applica ion o ed ligh in he pho mu an esul s in highe accumula-
ion o s a ch compa ed o whi e o blue illumina ed cells (Fig. 1d),
sugges ing he p esence o addi ional blue-ligh esponsi e p o ein(s)
ep essing s a ch accumula ion, as illus a ed in ou model (Fig. 6). How
PHOT, a kinase, is in ol ed in he dephospho yla ion o PMSK1 is an
in iguing ques ion ha needs u he in es iga ion. One o he possi-
bili ies is ha a p o ein phospha ase (indica ed PPase in Fig. 6)isa
missing componen in ou p oposed model; PHOT may media e he
ac i i y o his PPase o dephospho yla e S120 o PMSK1. In A abidopsis,
which encodes wo pho o opins, Pho 1 and Pho 2, blue ligh induces
he phospho yla ion o NPH3 (NON-PHOTOTROPIC HYPOCOTYL3) a
se ine 744 (S744) in a Pho 1-dependen manne . This phospho yla ion
c ea es a 14-3-3 binding si e enabling NPH3 o associa e wi h 14-3-3
p o eins. Subsequen ly NPH3 ge s dephospho yla ed32,33.Whe he
Chlamydomonas PHOT ope a es ia a simila mechanism—in e ac ing
wi h and phospho yla ing PMSK1 p io o i s dephospho yla ion a
S120— emains o be de e mined.
PMSK1 is ound o belong o a amily o Se ine/ h eonine-p o ein
kinases (named PMSK-like amily, see ma e ial and me hods) which is
conse ed in g een algae and ascula plan s. When sea ching o
PMSK1-like sequences in he A abidopsis genome in he NCBI
da abase34,HIGHLEAFTEMPERATURE1(HT1)
35 and CONVERGENCE
OF BLUE LIGHT AND CO2 1/2 (CBC1/2)36 a e ound in addi ion o he
PMSK-like A abidopsis membe s (Supplemen a y Figs. 21–24 and
Supplemen a y Tex ), p o eins ha ha e been shown o media e
esponses o CO
2
and blue ligh in A abidopsis. Ye , while A abidopsis
CBC1/2/HT1 ac s o s imula e s oma al opening by inhibi ing S- ype
anion channels36,ou findings demons a e ha he egula o y unc-
ion o i s Chlamydomonas coun e pa PMSK1 con ols s a ch me a-
bolism h ough he ansc ip ional egula ion o GAP1.
WT
pho
WT/PMSK1 -FLAG
S120A
WT/PMSK1 -FLAG
S120D
pho /PMSK1 -FLAG
S120A
pho /PMSK1 -FLAG
S120D
WT/PMSK1-FLAG
pho /PMSK1-FLAG
ns
✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
ns ns
ns ns
ns
✱✱✱✱
✱✱✱✱ ✱✱✱✱
✱✱✱✱
✱✱✱✱ ✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱
10
-2
10
-1
10
0
10
1
10
2
10
3
Rel. GAP1 mRNA abundance
10
-2
10
-1
10
0
10
1
10
2
10
3
Rel. GAP1 mRNA abundance
WT
pho
WT/PMSK1-FLAG
0
10
20
30
S a ch con en
(μg/million cells)
0 6 12 18
24
0 6 12 18
24
0 6 12 18
24
0 6 12 18
24
WT/PMSK1 -FLAG
S120A
WT/PMSK1 -FLAG
S120D
pho /PMSK1-FLAG pho /PMSK1 -FLAG
S120A
pho /PMSK1 -FLAG
S120D
a
b
Time (h)
Fig. 4 | PMSK1 phospho yla ion s a us con ols s a ch me abolism in Chla-
mydomonas einha d ii. GAP1 ansc ip ion le el (a) and s a ch con en (b)o
a ious PMSK1 o e exp ession lines synch onized o a 12/12 ligh da k cycle unde
di e en ligh quali ies. Da a a e p esen ed as mean ± SD (n = 3 biologically inde-
penden samples).Phases a e indica ed bywhi e and g ay shadingin(a). Line colo s
indica ed ligh quali ies. Black, whi e ligh ; Red, ed ligh ; Blue, blue ligh . In b ed
iangles indica e sample collec ion ime. The da k phase is indica ed by black ba s
abo e he g aphs; he ligh phase by whi e, blue, o ed ba s, depending on he ligh
quali y used. The s a is ical significance was de e mined using wo-way ANOVA
wi h Dunne ’s mul iple compa isons es s (a) o log10 ans o med mRNA da a as
indica ed in he g aphs. The s a is ical significance was de e mined using one-way
ANOVA wi h Dunne ’s mul iple compa isons es s (b). As e isks indica ed he
p- alues compa e o WT (*p< 0.05; ***p < 0.001; ****p< 0.0001; ns, no significan ).
In some cases, he e o ba s a e smalle han he da a poin symbols. De ailed
s a is ical analyses a e p esen ed in he Sou ce Da a File.
A icle h ps://doi.o g/10.1038/s41467-025-57809-3
Na u e Communica ions | (2025) 16:2545 7
In A abidopsis, pho o opins (Pho 1 and Pho 2) play a c ucial ole
in egula ing s a chdeg ada ioningua dcellsin esponse oblue ligh ,
a p ocess essen ial o s oma al opening, whichmanages gas exchange
and wa e balance in plan s. Upon exposu e o blue ligh , pho o opin
signaling igge s a pa hway ha leads o he apid b eakdown o
s a ch in gua d cells. This in ol es he coo dina ed ac ion o he
enzymes β-amylase 1 (BAM1) and α-amylase 3 (AMY3). The s a ch
deg ada ion is linked o he ac i a ion o he plasma memb ane H
+-ATPase, which p omo es s oma al opening and con ibu es o
o e all plan g ow h20.Ou findings e eal ye ano he ascina ing
di e encebe ween ascula plan s andChlamydomonas pho o opins;
in con as o he A abidopsis Pho s, Chlamydomonas PHOT ac s on
he mRNA le el o a key me abolic enzyme GAP1 o modula e cellula
accumula ion o s a ch.
In he g een mic oalgae Chlo ella, a possible explana ion o why
blue ligh ep esses s a ch accumula ion22 may lie in ea ly s udies
showing ha unde blue ligh ca bohyd a e ca abolism is
enhanced37,38.While hespecific oleo GAP1inChlo ella emains o be
in es iga ed, we ecen ly demons a ed ha unde ed ligh , Chlo ella
accumula ed significan s a ch le els e en a e a p olonged 7-day
exposu e39, in acco dance o ea lie sho - e m expe imen s22.In
Chlamydomonas, ou esul s showed ha bo h s a ch syn hesis- and
ca abolism- ela ed genes we e up egula ed in he pho mu an (Sup-
plemen a y Fig. 9). Al hough p o eomics does no necessa ily cap u e
S a ch con en
(μg/million cells)
a
b
WT
pmsk1
pho
pho pmsk1
WT
pmsk1
pho
pho pmsk1
WT
pmsk1
pho
pho pmsk1
0
10
20
30
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱
✱✱
✱✱
✱
ns
10
-2
10
-1
10
0
10
1
10
2
10
3
Rel. GAP1 mRNA abundanceRel. GAP1 mRNA abundance
WT pmsk1
10
-2
10
-1
10
0
10
1
10
2
10
3
pho
0 6 12 18
24
0 6 12 18
24
pho pmsk1
Time (h)
Fig. 5 | PHOT egula es s a ch me abolism ia PMSK1 and GAP1 in Chlamydo-
monas einha d ii. aGAP1 ansc ip ion le el and bs a ch con en in single and
double pho and pmsk1 mu an s synch onized o a 12/12 ligh da k cycle unde
di e en ligh quali ies. Da a a e ep esen ed as mean± SD (n = 3 biologically
independen samples). Line colo s indica ed ligh quali ies. Black, whi e ligh ; Red,
ed ligh ; Blue, blue ligh . Red iangles indica e sample collec ion ime. Da k phase
is indica ed by black ba s abo e he g aphs; he ligh phase by whi e, blue, o ed
ba s, depending on he ligh quali y used. Red iangles indica ed samples
collec ion ime. The s a is ical significance was de e mined using wo-way ANOVA
wi h Dunne ’s mul iple compa isons es s (a) o log10 ans o med mRNA da a as
indica ed in he g aphs. The s a is ical significance was de e mined using one-way
ANOVA wi h Dunne ’s mul iple compa isons es s (b). As e isks indica ed he
p- alues compa e o WT (*p < 0.05; **p <0.01; ***p< 0.001; ****p < 0.0001; ns, no
significan ). In some cases, he e o ba s a e smalle han he da a poin symbols.
De ailed s a is ical analyses a e p esen ed in he Sou ce Da a File.
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enzyme ac i i y, i is wo h men ioning ha ou whole-cell p o eomics
da a (Supplemen a y Da a 2) did no e eal any s a is ically significan
changes in he le els o alpha-amylase, he enzyme esponsible o he
majo hyd oly ic ac i i y in ol ed in s a ch deg ada ion in
Chlamydomonas40.
In summa y, ou findings no only demons a e ha he PHOT-
PMSK1 pa hway egula es s a ch me abolism in Chlamydomonas,bu
hey also shed ligh on he impo ance o his mechanism wi hin he
con ex o he o ganism’s diu nal hy hm. The egula o y e ec o
PHOT-PMSK1 plays a c ucial ole in modula ing GAP1 mRNA le els,
pa icula ly a he onse o illumina ion ollowing he da k phase. By
ac ing h ough blue ligh , PHOT-PMSK1 fine- unes he induc ion o
GAP1, p e en ing an immedia e spike in i s exp ession. This con olled
esponse ensu es ha s a ch syn hesis does no o e whelm he cell’s
ene gy ese es, allowing o a balanced dis ibu ion o esou ces
be ween s a ch p oduc ion and o he essen ial cellula unc ions. This
egula ion likely p e en s excessi e s a ch accumula ion, which could
o he wise comp omise cellula heal h, highligh ing he c i ical na u e
o his finely uned-mechanism.
None heless, he highe s a ch accumula ion in he pho mu an
unde he low ligh in ensi y condi ions used in ou expe imen s, does
no a ec g ow h and pho osyn hesis (Supplemen a y Fig. 5). This
finding indica es ha his PHOT-media ed ligh -signaling pa hway
o e comes a key esou ce alloca ion ade-o p esen in Chlamydo-
monas, whe eby ca bon, fixed by pho osyn hesis, is alloca ed o
ene gy ese es (e.g. s a ch) a he cos o g ow h14.Ou findings pa e
he way o he applica ion o p ecise kinase enginee ing o PMSK1 as a
sus ainable way o p oduce s a ch om g een mic oalgae in bio-
echnological applica ions.
Me hods
S a is ics
S a is ical me hods we e no used o p ede e mine he sample size.
The expe imen s we e no andomized, and he in es iga o s we e no
blinded o alloca ion du ing expe imen al p ocedu es and da a
assessmen .
Algal Ma e ial
The s ains used in his s udy included Chlamydomonas pho (de ec-
i e in PHOT; gene ID: C e03.g199000) and pho -C1 (pho s ain
complemen ed wi h WT PHOT gene), as well as hei backg ound
s ain CC-125, which ha e been p e iously desc ibed25. Addi ionally,
Chlamydomonas ac y24 (de ec i e in animal- ype c yp och ome, aka
aCRY;geneID:C e06.g278251),pc y (de ec i e in plan - ype c yp o-
ch ome, aka pCRY; gene ID: C e06.g295200), and ac ypc y (de ec i e
in bo h animal- ype and plan - ype c yp och ome) we e gene a ed
h ough CRISPR-CAS9 p o ided by ollowing he p o ocol desc ibed
in e . 24.Thepmsk1 (de ec i e in pho o opin-media ed signaling
kinase 1, aka PMSK1; gene ID: C e16.g659400) and pho pmsk1
(de ec i e in bo h PHOT and PMSK1) mu an s we e gene a ed using
inse ional CRISPR-Cas9 RNP me hod desc ibed by Kim e al.41 wi h a
ew modifica ions. The a ge sgRNA sequence o PMSK1 was
Fig. 6 | P oposed model depic ing he mechanisms o blue-ligh dependen
egula ion o s a ch me abolism in Chlamydomonas einha d ii. Phospho y-
la ed PMSK1 a S120 (PMSK1-P) ac i a es GAP1, leading o inc eased s a ch bio-
syn hesis. In he p esence o blue ligh , PHOTOTROPIN de ec s he signal and
ini ia es he dephospho yla ion o PMSK1, esul ing in dec easedGAP1 mRNA le els
and educed s a ch accumula ion. PPase is a hypo he ical phospha ase ha could
dephospho yla e PMSK1-P in a PHOTOTROPIN-dependen manne . The model also
includes a PHOTOTROPIN-dependen bu PMSK1-independen ep ession o GAP1,
as GAP1 in pmsk1 mu an s emains egula ed by ed ligh —a egula ion los in he
pho pmsk1 double mu an (Fig. 5a). Addi ionally, o he blue-ligh - esponsi e p o-
eins a e p oposed o independen lyinhibi s a chaccumula iono GAP1 ou side o
he PHOT and GAP1 pa hways. This is based on obse a ions ha : (i) pho mu an s
accumula e mo e s a ch unde ed ligh (Fig. 1d), despi e consis en ly high GAP1
mRNA le els ac oss ligh condi ions (Fig. 2d), and (ii) ed ligh enhances GAP1
mRNA in he pho -kin s ain (Fig. 2g).
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