The Plan Cell, 2025, 37, koa 033
h ps://doi.o g/10.1093/plcell/koa 033
Ad ance access publica ion 11 Feb ua y 2025
La ge-Scale Biology
Mul iomics in eg a ion un eils pho ope iodic plas ici y
in he molecula hy hms o ma ine phy oplank on
Ana B. Rome o-Losada,
1,2,†
Ch is ina A ani idou(Χριστίνα Αρβανιτίδου),
1,2,†
M. Elena Ga cía-Gómez,
1
Ma ía Mo ales-Pineda,
1
M. José Cas o-Pé ez,
3
Yen Peng Chew,
4
Ge ben an Ooijen,
4
Me cedes Ga cía-González,
1,
* F ancisco J. Rome o-Campe o
1,2,
*
1
Ins i u e o Plan Biochemis y and Pho osyn hesis, Uni e sidad de Se illa – Consejo Supe io de In es igaciones Cien í icas, A . Amé ico Vespucio 49,
Se ille 41092, Spain
2
Depa men o Compu e Science and A i icial In elligence, Uni e sidad de Se illa, A . Reina Me cedes s/n, Se ille 41012, Spain
3
Ins i u e o Biomedicine in Se ille, Uni e sidad de Se illa – Consejo Supe io de In es igaciones Cien í icas, A . Manuel Siu o s/n, Se ille 41012, Spain
4
School o Biological Sciences, Uni e si y o Edinbu gh, Max Bo n C escen , Edinbu gh EH9 3BF, UK
*Au ho o co espondence: [email p o ec ed] (F.J.R.-C.), [email p o ec ed] (M.G.-G.)
†
These au ho s con ibu ed equally and bo h should be conside ed as i s au ho s.
The au ho esponsible o dis ibu ion o ma e ials in eg al o he indings p esen ed in his a icle in acco dance wi h he policy desc ibed in he Ins uc ions o
Au ho s (h ps://academic.oup.com/plcell/pages/Gene al-Ins uc ions) is: F ancisco J. Rome o-Campe o ([email p o ec ed]).
Abs ac
Ea h’s il ed o a ion and ansla ion a ound he Sun p oduce pe asi e hy hms on ou plane , gi ing ise o pho ope iodic changes in
diel cycles. Al hough ma ine phy oplank on plays a key ole in ecosys ems, mul iomics analysis o i s esponses o hese pe iodic
en i onmen al signals emains la gely unexplo ed. The ma ine picoalga Os eococcus au i was chosen as a model o ganism due o i s
cellula and genomic simplici y. Os eococcus was subjec ed o di e en ligh egimes o in es iga e i s esponses o pe iodic
en i onmen al signals: long summe days, sho win e days, cons an ligh , and cons an da k condi ions. Al hough <5% o he
ansc ip ome main ained oscilla ions unde bo h cons an condi ions, 80% p esen ed diel hy hmici y. A d as ic educ ion in diel
hy hmici y was obse ed a he p o eome le el, wi h 39% o he de ec ed p o eins oscilla ing. Pho ope iod-speci ic hy hms we e
iden i ied o key physiological p ocesses such as he cell cycle, pho osyn hesis, ca o enoid biosyn hesis, s a ch accumula ion, and
ni a e assimila ion. In his s udy, a pho ope iodic plas ic global o ches a ion among ansc ip ome, p o eome, and physiological
dynamics was cha ac e ized o iden i y pho ope iod-speci ic empo al o se s be ween he iming o ansc ip s, p o eins, and
physiological esponses.
Recei ed June 07, 2024. Accep ed Feb ua y 3, 2025
© The Au ho (s) 2025. Published by Ox o d Uni e si y P ess on behal o Ame ican Socie y o Plan Biologis s.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h ps://c ea i ecommons.o g/licenses/by/4.0/), which pe -
mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
In oduc ion
Ma ine phy oplank on plays a pi o al ole in Ea h’s ecosys ems,
ac ing as p ima y p oduce s by con ibu ing o ∼45% o
global pho osyn he ic ne p ima y p oduc ion (Field e al. 1998).
Consequen ly, i no only sus ains he exis ence o mos oceanic
li e bu also suppo s li e ac oss he en i e plane . Ligh a ailabili y
has a p o ound impac on ma ine phy oplank on g ow h and phys-
iology (Edwa ds e al. 2015). Ea h’s o a ion p oduces he mos pe -
asi e hy hmic en i onmen al signal, gi ing ise o al e na ing
cycles o days (ligh pe iods o pho ope iods) and nigh s (da k pe i-
ods o sko ope iods), collec i ely known as diel cycles. Ea h’s il ed
o a ional axis and i s ansla ion a ound he Sun also lead o pho-
ope iodic a ia ions in diel cycles, esul ing in long days (LDs) in
summe and sho days (SDs) in win e in empe a e zones, he
egions be ween he opics and he pola ci cles. Seasonali y plays
a cen al egula o y ole in ma ine phy oplank on dynamics
(Bolaños e al. 2020; Chen e al. 2021; Mondal and Bane jee 2022).
Howe e , he di e en molecula hy hms unde pinning hese e-
sponses a e no ye cha ac e ized.
Ch onobiology is a mul idisciplina y ield ha ocuses on he
s udy o he iming and egula ion o biological hy hms and hei
synch oniza ion wi h en i onmen al cycles (Kuhlman e al. 2018).
Speci ically, he au onomous oscilla ing molecula sys ems ha
ha e e ol ed o an icipa e and espond o diel cycles a e e e ed
o as ci cadian clocks. These sys ems a e en ained o ex e nal
inpu s, diel cycles, and p oduce hy hmic ou pu o biological
p ocesses. Ci cadian hy hms a e sel -sus ained, main aining
hy hmici y o abou 24 h unde cons an condi ions. To dis in-
guish hese biological hy hms om diel hy hms ha espond
o he en i onmen , ch onobiology expe imen s a e ypically de-
signed as a sequence o se e al consecu i e days unde al e na -
ing ligh /da k cycles, ollowed by se e al consecu i e days o
cons an ligh (LL) o da k (DD), called ee- unning condi ions
(Kuhlman e al. 2018). Ex ensi e ch onobiological s udies on ci ca-
dian and diel hy hms ha e been conduc ed a he molecula and
physiological le els in many model o ganisms (Bläsing e al. 2005;
Mille e al. 2007; Pa ke e al. 2020; Ma e al. 2021; Pe e sen e al.
2022; Hä ke e al. 2023) unco e ing key molecula mechanisms
and egula o s con olling ci cadian hy hms. Howe e , only ini-
ial s eps ha e been aken o analyze such sys ems in ma ine phy-
oplank on using omics echnologies (Monnie e al. 2010;
Annunzia a e al. 2019; Kay e al. 2021), which a e beginning o
cha ac e ize molecula hy hms a he ansc ip omic and p o-
eomic le els and iden i y hei ci cadian clock egula o s. A e-
cen kingdom-wide compa a i e analysis o diel gene exp ession
p o iles ac oss A chaeplas ida has highligh ed e olu iona y
ends in hy hmic pa e ns ac oss he g een lineage (Fe a i
e al. 2019). Howe e , his s udy solely ocused on he ansc ip-
omic laye and did no explo e he e ec o di e en pho ope -
iods and ee- unning condi ions.
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To cha ac e ize he esponses o ma ine phy oplank on o pho o-
pe iodic a ia ions in diel cycles, he model phy oplank onic pi-
coeuka yo e Os eococcus au i (Os eococcus) was selec ed due o i s
cellula simplici y (Hende son e al. 2007) and ully sequenced
and anno a ed genome (De elle e al. 2006; Palenik e al. 2007;
Blanc-Ma hieu e al. 2014). Mo eo e , Os eococcus occupies a key
posi ion in he g een lineage (Vi idiplan ae) as a ep esen a i e o
he Class Mamiellophyceae, which di e ged ea ly om he g oup
ha would gi e ise o land plan s and, he e o e, could p o ide
insigh s in o he ances al ai s o g een plan s and hei e olu ion-
a y p ocesses ( an Ba en e al. 2016; de los Reyes e al. 2017; Bachy
e al. 2022). In ensi e and ex ensi e s udies on he physiology o
Os eococcus ha e p o ided a solid ounda ion o sys ems biology
and omics s udies. Ch onobiological s udies (Co ellou e al. 2009;
Monnie e al. 2010; O’-Neill e al. 2011; an Ooijen e al. 2011;
F eeney e al. 2016) ha e unco e ed he undamen al componen s
o he Os eococcus ci cadian clock e ealing key hy hmic p ocesses.
Cell-cycle analysis (Co ellou e al. 2005; Robbens e al. 2005;
Gil-Rod íguez e al. 2024) ha e p o ided insigh s in o he molecula
componen s egula ing cell-cycle p og ession in Os eococcus unde
di e en ligh egimes while pho obiology app oaches (Sands e al.
2023) ha e highligh ed he ole o ligh quali y in di e en biological
p ocesses. Resea ch in o sexual ep oduc ion (Beni es e al. 2021),
i al in ec ions dynamics (De elle e al. 2018; Cas illo e al. 2021),
and biomass composi ion (Ral e al. 2004; Deg ae e-Guilbaul
e al. 2017, 2020; Guyon e al. 2018) ha e expanded he knowledge
o Os eococcus’ po en ial s a egies o cope wi h bio ic and nu i ion-
al s esses. Finally, omics analysis (Monnie e al. 2010; Le Bihan
e al. 2011, 2015; Hen íquez-Cas illo e al. 2018; Bousque e al.
2020; Kay e al. 2021) ha e p o ided global cha ac e iza ions o
he ansc ip ome and p o eome sepa a ely. These in es iga ions
ha e un eiled ha he Os eococcus ci cadian clock is no ably sim-
ple han ha o o he pho osyn he ic o ganisms like A abidopsis
haliana. I has been shown o be cons i u ed only by a educed cen-
al loop comp ising po en ial o hologues o CIRCADIAN CLOCK
ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1).
In con as , A abidopsis has a mo e complex clock composed o
in e locking loops, including a mo ning loop cons i u ed among
o he s by PSEUDO RESPONSE REGULATORS 9, 7, and 5 (PRR9,
PRR7, and PRR5), he cen al loop consis ing in CCA1 and TOC1,
and an e ening loop in ol ing addi ional componen s such as
ZEITLUPE and GIGANTEA (Se ano-Bueno e al. 2017).
Despi e hese ad ances, Os eococcus esponses o pho ope iod-
ic a ia ions in diel cycles, as well as ee- unning condi ions, e-
main o be explo ed, pa icula ly h ough mul iomics in eg a i e
analysis. This s udy aimed o cha ac e ize he Os eococcus
ansc ip ome, p o eome, and physiological hy hmici y, unde
long and sho pho ope iods. Addi ionally, o de e mine ci cadian
hy hms, ou expe imen s we e ex ended o ee- unning
condi ions consis ing o LL o DD. In eg a ion o ansc ip omic
and p o eomic da a wi h physiological measu emen s un eiled
a pho ope iodic plas ic global o ches a ion be ween ansc ip-
ome, p o eome, and physiological dynamics, wi h pho ope iod-
speci ic empo al o se s be ween he phase o ansc ip , p o ein,
and physiological hy hms.
Resul s
T ansc ip hy hmici y unde diel cycles and
ee- unning condi ions
In his s udy, pho ochemos a s ope a ed in a con inuous egime
(Supplemen a y Fig. S1A) we e used simula ing LD condi ions
(16 h ligh :8 h da k) o SD condi ions (8 h ligh :16 h da k). The
illumina ing sys em simula ed he p og essi e ligh in ensi y in-
c ease and dec ease du ing sola dayligh cycles wi h a maximum
ligh i adiance o 1,500 μE m
−2
s
−1
. Tempe a u e and pH we e
main ained cons an a 20 °C and 8. Samples we e collec ed o 3
consecu i e days e e y 4 h s a ing a ZT0, Zei gebe ime 0, he
ime poin co esponding o he beginning o he pho ope iod simu-
la ing dawn (Supplemen a y Fig. S1B). Robus global hy hmici y
was de ec ed in he ansc ip omes unde LD and SD condi ions
by using hie a chical clus e ing (HC) and p incipal componen
analysis (PCA) (Supplemen a y Fig. S1, C o E). A cyclic ci cula o -
ganiza ion o he ansc ip omes was e ealed o e diel cycles
(Supplemen a y Fig. S1, D and E). Pho ope iodic a ia ions did no
a ec ansc ip ome hy hmici y, as he se s o hy hmic genes
unde LD and SD en ainmen we e nea -iden ical, comp ising
∼80% o he genome unde ei he condi ion (Fig. 1A). A hy hmic
genes we e ei he no exp essed o lowly exp essed. Indeed, hy h-
mic genes p esen ed signi ican maximum exp ession le els 3- old
g ea e han a hy hmic genes (Fig. 1B). These genes a e mainly in-
ol ed in s ess esponses such as i al in ec ion, and migh no be
exp essed unde hese labo a o y condi ions.
In o de o dis inguish be ween ci cadian- egula ed genes and
hose esponding o he hy hmic en i onmen , cul u es we e
ans e ed o ee- unning condi ions consis ing o LL o DD. No
samples we e collec ed du ing he i s day o cons an condi ions
o allow cul u e acclima ion, and samples we e collec ed he e-
a e o gene a e ansc ip omic da a o 2 consecu i e days s a -
ing a ci cadian ime 0 (CT0), he ime poin co esponding o
subjec i e dawn (Supplemen a y Fig. S1B). A clea educ ion in
hy hmici y was obse ed unde cons an condi ions: ∼21% o
he ansc ip ome was comple ely elian on diel cycles o main-
ain hy hmici y since hese genes los hei hy hms unde
bo h LL and DD (Fig. 1, C and E; Supplemen a y Table S1), inde-
penden ly om he en ainmen egime. In con as , he an-
sc ip ome p opo ion ha main ained oscilla ions exclusi ely
unde LL o DD, bu no bo h, was dependen on he p e ious en-
ainmen . Whe eas 15% kep cycling unde LL a e LD en ain-
men , only 6% we e hy hmic a e SD en ainmen . The
oscilla ing genes unde LL, bo h a e LD and SD en ainmen ,
we e ound o be signi ican ly in ol ed in DNA eplica ion and
ch omosome o ganiza ion (Fig. 1D; Supplemen a y Fig. S2). The
de imen al e ec o DD o e ansc ip ome hy hmici y was
smalle han ha o LL, wi h almos 22% main aining oscilla ions
a e LD en ainmen and 39% a e SD en ainmen . This obse -
a ion is ema kable, as p e ious s udies ha e ound a comple e
lack o ansc ip ion unde DD (O’-Neill e al. 2011). This can be a -
ibu ed o he g ow h condi ions in ou expe imen s, which allow
Os eococcus cells o accumula e subs an ial amoun s o s a ch o
be used as ene gy sou ce unde DD o main ain oscilla ions (see
sec ion on s a ch con en analysis). The biological p ocesses
RNA p ocessing and ibosome biogenesis we e ound o be signi i-
can ly en iched among he hy hmic genes unde DD, bo h
a e LD and SD en ainmen (Fig. 1F; Supplemen a y Fig. S3).
O hologues o he cen al componen s o he ci cadian clock,
os a06g02340 (CCA1) and os a13g01820 (TOC1), exhibi ed hy h-
mici y unde bo h LL and DD ee- unning condi ions. CCA1 p e-
sen ed highe le els o exp ession unde DD, in con as o he
hal in oscilla ions epo ed p e iously unde di e en g ow h
condi ions (O’-Neill e al. 2011), while TOC1 showed highe ex-
p ession le els unde LL (Supplemen a y Fig. S4). O hologues o
he ligh ecep o s, os a15g0100 (CRYPTOCHROME 1, CRY1), os -
a01g06470 (CRYPTOCHROME 3, CRY3), and os a03g05620 (UV
RESISTANCE 2, UVR2), we e hy hmic unde bo h LL and DD
2 | The Plan Cell, 2025, Vol. 37, No. 2
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ee- unning condi ions, wi h highe exp ession le els unde LL
han DD. Thei hy hmic exp ession p o iles we e a ec ed by pho-
ope iodic a ia ions excep CRY3, which p esen ed an iden ical
pa e n unde bo h LD and SD bu wi h an addi ional peak o
exp ession a he beginning o he pho ope iod unde LD. This
sugges s a cen al posi ion o hese genes a he egula o y co e
o he ci cadian clock esponding o pho ope iodic a ia ions in
Os eococcus (Supplemen a y Fig. S4).
Genes p esen ing hy hmici y unde bo h LD and SD as well as
main aining hei hy hmic exp ession p o iles unde bo h ee-
unning condi ions we e conside ed in his s udy as hose mo e
di ec ly egula ed by he ci cadian clock in Os eococcus. A clea de-
pendence on he p e ious en ainmen egime was obse ed, wi h
1,647 genes keeping hy hmici y unde bo h ee- unning condi-
ions a e LD en ainmen , e sus 1,034 ollowing SD en ain-
men . These 2 se s o e lapped pa ially, iden i ying 350 genes
Figu e 1. T ansc ip ome hy hmici y unde al e na ing ligh /da k cycles and iden i ica ion o ci cadian genes including ee- unning condi ions.
A) Venn diag am compa ing hy hmic genes unde LD (blue ci cle) and SD (ligh ed ci cle). B) Boxplo ep esen ing he maximum exp ession le el o
hy hmic and a hy hmic genes. Medians a e ep esen ed by cen al ho izon al lines, uppe and lowe qua iles by boxes, minimum and maximum
alues by whiske ends. Gene exp ession le els a e measu ed as FPKM. Signi icance was compu ed acco ding o Mann–Whi ney–Wilcoxon
nonpa ame ic es . C) Ba plo ep esen ing wi h blue colo s di e en hy hmic gene se s unde LD condi ions. D) Gene exp ession p o iles du ing 3
consecu i e days unde LD, 2 consecu i e days unde LL, and 2 consecu i e days unde DD o DNA polyme ase alpha subuni B (os a11g01400, POLA2).
Whi e ec angles ep esen pho ope iods (ligh pe iods o days), blue- illed ec angles co espond o sko ope iods (da k pe iods o nigh s) unde LD,
ligh blue ec angles ma k subjec i e nigh s o days unde LL and DD, espec i ely a e LD en ainmen . ZTN, Zei gebe ime N, ma ks he ime poin
N hou s a e dawn (ligh s on, ZT0). CTN, ci cadian ime N deno es he ime poin N hou s a e subjec i e dawn. A discon inui y is shown on he ime
axis o indica e ha samples we e collec ed a e 24 h acclima ion o he co esponding ee- unning condi ions. E) Ba plo ep esen ing wi h ed colo s
di e en hy hmic gene se s unde SD condi ions. F) Gene exp ession p o iles du ing 3 consecu i e days unde SD and 2 consecu i e days unde LL and
DD o Ribosomal p o ein L7Ae (os a02g01210, RPL7AE). Whi e ec angles ep esen pho ope iods, ed- illed ec angles co espond o sko ope iods unde
SD, ligh ed ec angles ma k subjec i e nigh s o days unde LL and DD, espec i ely, a e SD en ainmen . G) Venn diag am compa ing ci cadian
genes iden i ied a e LD en ainmen (blue ci cle) and a e SD en ainmen ( ed ci cle). H) T eemap summa izing he biological p ocesses signi ican ly
en iched o e hy hmic genes unde LD, SD, LL, and DD. Rec angle sizes ep esen signi icance le els. I) Gene exp ession p o iles du ing 3 consecu i e
days unde LD and SD; and 2 consecu i e days unde LL and DD o RuBisCO Ac i ase (os a04g02510) exempli ying hy hmic genes unde LD, SD, LL, and
DD in ol ed in pho osyn hesis ela ed p ocesses.
Pho ope iodic plas ici y in ma ine phy oplank on hy hms | 3
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co esponding o 4.6% o he Os eococcus ansc ip ome (Fig. 1G;
Supplemen a y Table S1). Func ional en ichmen analysis e-
ealed ha hese genes a e signi ican ly in ol ed in pho osyn he-
sis, chlo oplas o ganiza ion, and pigmen me abolic p ocesses
(Fig. 1, H and I). The speci ic ci cadian genes de ec ed a e LD
en ainmen a e signi ican ly in ol ed in ibosome biogenesis
whe eas hose ound a e SD en ainmen a e p ima ily associ-
a ed wi h cell cycle. This indica es a s ong in luence o he ci ca-
dian clock o e hese p ocesses. Howe e , genes iden i ied as
ci cadian a e LD en ainmen lose hy hmici y unde LL a e
SD en ainmen , and he e e se o he speci ic ci cadian genes
a e SD en ainmen ha lose hy hmici y unde DD a e LD en-
ainmen . This sugges s a s ong de imen al impac on hy h-
mici y o LL a e SD en ainmen wi h a sho pho ope iod, as
well as cons an da kness a e LD en ainmen wi h a sho
da k pe iod (Supplemen a y Fig. S5).
In LD-en ained cul u es, signi ican educ ions in ampli ude
we e de ec ed unde bo h LL and DD condi ions o he LD/LL
and LD/DD hy hmic genes, espec i ely (Fig. 2A). In SD-en ained
cul u es, ampli ude educ ion was only no ed when ans e ed
o LL o SD/LL hy hmic genes bu no subs an ial change was
ound when ans e ed o DD o SD/DD hy hmic genes
(Fig. 2D). F ee- unning condi ions also a ec ed he phase, o
ime poin o maximum exp ession le el, in hy hmici y pa e ns.
Independen ly om he p e ious en ainmen egime, phases
we e delayed when cul u es we e ans e ed o LL (Fig. 2, B and
C, E and F, le ). Whe eas ad anced phases (i.e. phases occu ing
ea lie ) we e ound when cul u es we e ans e ed o DD (Fig. 2,
B and C, E and F, igh ), wi h his e ec mo e p onounced o
LD-en ained cul u es and only sligh o SD-en ained cul u es.
The phase delays in LL we e mo e d as ic o SD han
LD-en ained cul u es al hough he ad ances in DD we e mo e
e iden o LD han SD-en ained cul u es.
E ec s o pho ope iodic a ia ions o e gene
exp ession p o iles
Di e ences we e iden i ied in phase and ampli ude o hy hmic
gene exp ession pa e ns be ween LD and SD (Fig. 3A) wi h only
31 genes exhibi ing he same exp ession p o ile unde bo h pho o-
pe iods. A signi ican educ ion in o e all ampli ude was obse ed
in SD compa ed o LD-en ained cul u es (Fig. 3B). Unde bo h LD
and SD, mos genes peaked a nigh (Fig. 3C), indica ing a “noc u -
nal ansc ip ome p o ile. Ad anced phases we e no ed unde SD
wi h espec o LD condi ions. Unde LD en ainmen , peak phases
occu uni o mly om he end o he day (ZT12) o he end o he
nigh (ZT20), while unde SD-en ainmen peak phases p edomi-
nan ly occu du ing he i s hal o he nigh (ZT8 o ZT16; Fig. 3C).
Gene clus e s we e de ined based on peak phases
(Supplemen a y Table S2). Dis inc diel imings o maximum
gene exp ession le els o speci ic biological p ocesses we e iden-
i ied by pe o ming unc ional en ichmen analysis o e each
gene clus e unde LD (Fig. 3D; Supplemen a y Fig. S6) o SD con-
di ions (Fig. 3E; Supplemen a y Fig. S7). Changes in he phase o bi-
ological p ocesses unde LD and SD condi ions we e de ec ed.
These di e ences in diel imings we e p oduced by he p e iously
men ioned ad anced gene phases unde SD wi h espec o LD.
Fo example, genes in ol ed in pho osyn hesis peak a ZT8 unde
LD and a ZT4 unde SD, midday coinciding wi h he momen o
maximal ligh i adiance in bo h condi ions. Simila ly, genes
in ol ed in DNA eplica ion eached maximum exp ession le el
a la e day (ZT12) unde LD, and a ea ly nigh (ZT8) unde SD.
Rea angemen s in he o de o peak phases o biological
p ocesses we e also ound. Fo ins ance, unde LD condi ions,
genes in ol ed in amino acid biosyn hesis, ibosome biogenesis,
and ansla ion eached hei highes le el a ZT20, ZT0, and
ZT4, espec i ely. Howe e , in SD condi ions, ibosome biogenesis
genes we e up egula ed a ZT12, while bo h amino acid biosyn-
hesis and ansla ion- ela ed genes eached maximum exp es-
sion le els a ZT16.
To iden i y po en ial egula o s o hese dis inc diel imings,
ansc ip ion ac o -binding si es (TFBS) en ichmen analysis
was pe o med o e he p omo e s o genes in each ime poin
clus e (Fig. 4). DNA mo i s ecognized by he amilies o plan
ansc ip ion ac o s myeloblas osis (MYB), DNA-binding wi h
one inge (DOF), basic leucine zippe (bZIP), cys eine- ich
polycomb-like p o ein (CPP), and Homeobox we e iden i ied, sug-
ges ing a key ole in egula ing diel hy hmic gene exp ession pa -
e ns. Gene clus e s co esponding o di e en ime poin s unde
LD and SD condi ions exhibi ed en ichmen o he same DNA mo-
i s, as hese clus e s sha e many genes in common due o he ad-
anced gene phases unde SD compa ed o LD. Fo ins ance, gene
clus e s co esponding o ZT12 unde LD and ZT8 unde SD
showed en ichmen o he DNA sequence ecognized by he
CPP ansc ip ion ac o amily. This amily has been implica ed
in cell p oli e a ion in A abidopsis (Wang e al. 2018). Consis en
wi h hese indings he genes os a20g00800 and os a09g04220,
encoding CPP ansc ip ion ac o s, peaked a ZT12 unde LD
and ZT8 unde SD condi ions. No ably, he e ening elemen (EE)
mo i , AAATATCT, was signi ican ly iden i ied in he p omo e s
o he genes peaking a ZT12 unde LD condi ions and ZT0 and
ZT4 unde SD condi ions. In A abidopsis, he EE is associa ed
wi h he binding si e o CCA1 (Ha me e al. 2000). This ansc ip-
ion ac o binds o i s a ge s in he mo ning o ep ess hei ex-
p ession, esul ing in maximum exp ession la e in he e ening
(Kamioka e al. 2016). A simila gene exp ession pa e n is ob-
se ed in ou ansc ip omic da a om Os eococcus, sugges ing a
s ong conse a ion a he cen al co e o he ci cadian clock be-
ween A abidopsis and Os eococcus. Based on he p esence o he
EE in he gene p omo e s discussed abo e, CCA1 Os eococcus o -
hologue, os a06g02340, would be expec ed o bind in he mo ning
o genes peaking a ZT12 unde LD o la e in he nigh o genes
peaking a ZT0 o ZT4 unde SD.
To cap u e he p e iously desc ibed changes in he phase and
ampli ude o hy hmic gene exp ession p o iles esul ing om
pho ope iodic a ia ions, a co-sinusoidal dynamical model was
de eloped. This model p edic s he phase and ampli ude o each
hy hmic gene o a speci ic day o he yea using linea in e pola-
ion be ween he phase and ampli ude iden i ied unde LD o SD
en ainmen (Video 1). P e iously published mic oa ay da a, gen-
e a ed unde neu al day condi ions (ND) (12 h ligh :12 h da k)
(Monnie e al. 2010) we e used o e alua e i s p edic i e powe .
The phases o app oxima ely wo- hi ds (63%) o he hy hmic
genes we e success ully p edic ed wi h ±4 h e o . The limi ed
p edic i e powe o ou model sugges s ha while wo- hi ds o
he hy hmic genes espond o changes in pho ope iod by g adu-
ally adjus ing hei phases, he emaining one- hi d exhibi a
mo e complex esponse o pho ope iod changes.
Ano he di e ence obse ed be ween LD and SD condi ions
was ela ed o he occu ence o gene exp ession p o iles wi h 2
daily peaks (Fig. 3F). Al hough gene p o iles wi h 2 peaks we e de-
ec ed in bo h LD and SD cul u es (Supplemen a y Table S3), a
d as ic inc ease in he numbe o genes wi h bimodal hy hmici y
was obse ed unde SD condi ion (1,855 e sus 376 genes; Fig. 3G).
Bimodal hy hmici y was no main ained unde ee- unning con-
di ions in which dis inc single-peak p o iles we e appa en unde
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LL and DD (Fig. 3H). Nonlinea squa es (Ba y e al. 2015) we e
applied o decompose he obse ed bimodal gene p o ile in
SD-en ained cul u es in o 2 di e en single-peak p o iles, one
peaking du ing he pho ope iod and he o he du ing he sko ope -
iod. Unde LL ee- unning condi ion, only he pho ope iod-peaking
p o ile was main ained, whe eas unde DD condi ion only he
sko ope iod-peaking p o ile was p esen . Fo genes wi h bimodal
hy hmici y, a dynamical model was de eloped combining 2 dis-
inc co-sinusoidal p o iles o cap u e changes in he phase and
ampli ude, esul ing om changes in pho ope iod leng h. Unde
LD condi ions, hese p o iles o e lap in ime p oducing a single-
peak p o ile, whe eas unde SD condi ion hey become ou o
phase, esul ing in a bimodal p o ile (Video 2). To es his model,
p edic ions we e un o assess hy hmici y p o iles unde ND
Figu e 2. F ee- unning condi ions e ec s o e gene exp ession p o iles. A) Boxplo ep esen ing hy hmic genes ampli ude eached unde LD
condi ions, when cul u es we e ans e ed o ee- unning condi ions consis ing o LL (ligh blue) and DD (da k blue) a e LD en ainmen . Medians
a e ep esen ed by cen al ho izon al lines, uppe and lowe qua iles by boxes, minimum and maximum alues by whiske ends. Gene exp ession
le els a e measu ed as FPKM. Signi icance was compu ed acco ding o Mann–Whi ney–Wilcoxon nonpa ame ic es . B) His og ams showing he
dis ibu ion o he numbe o genes exhibi ing ad anced and delayed phases when cul u es a e ans e ed om LD o LL (le ) and DD ( igh ). Ve ical
dashed lines ma k no shi . C) Gene exp ession p o iles du ing 3 consecu i e days unde LD and 2 consecu i e days unde LL and DD o
sedohep ulose-bisphospha ase (os a03g05500, SBPase). Ve ical black a ows ma k LD phases, e ical g ay a ows ma k LL and DD phases and ho izon al
black a ows ep esen delayed o ad anced phases. Whi e ec angles ep esen pho ope iods (ligh pe iods o days), blue- illed ec angles co espond
o sko ope iods (da k pe iods o nigh s) unde LD, ligh blue ec angles ma k subjec i e nigh s o days unde LL and DD, espec i ely a e LD
en ainmen . ZTN, Zei gebe ime N, ma ks he ime poin N hou s a e dawn (ligh s on, ZT0). CTN, ci cadian ime N, deno es he ime poin N hou s
a e subjec i e dawn. A discon inui y is shown on he ime axis o indica e ha samples we e collec ed a e 24 h acclima ion o he co esponding
ee- unning condi ions. D) Boxplo ep esen ing hy hmic genes ampli ude o maximum exp ession le el eached unde SD condi ions, when cul u es
we e ans e ed o LL and DD ee- unning condi ions a e SD en ainmen . Signi icance was compu ed acco ding o Mann–Whi ney–Wilcoxon
nonpa ame ic es . E) His og ams showing he dis ibu ion o he numbe o genes exhibi ing ad anced and delayed phases when cul u es a e
ans e ed om SD o ee- unning condi ions consis ing in LL (le ) and DD ( igh ). Ve ical dashed lines ma k no shi . F) Gene exp ession p o iles
du ing 3 consecu i e days unde LD and 2 consecu i e days unde LL and DD o SBPase. Ve ical black a ows ma k SD phases, e ical g ay a ows ma k
LL and DD phases, and ho izon al black a ows ep esen delayed phases.
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Figu e 3. Pho ope iodic e ec s o e gene exp ession p o iles. A) Gene exp ession p o iles du ing 3 consecu i e days unde LD (blue) and SD ( ed)
condi ions o CYCB (os a01g06150, op) and del a-9 acyl-lipid desa u ase 1 (os a01g00790, ADS1, bo om). Blue and ed e ical do ed a ows ma k LD and
SD phases. Ho izon al black a ow ep esen s ad anced phase unde SD when compa ed wi h LD. Blue and ed ho izon al dashed lines ma k LD and SD
ampli udes. Ve ical black a ows ep esen he educ ions in ampli ude unde SD wi h espec o LD. Ligh egimes a e ep esen ed as desc ibed in Fig. 1.
B) Boxplo ep esen ing hy hmic genes ampli ude unde LD in blue and SD in ed. Medians a e ep esen ed by cen al ho izon al lines, uppe and lowe
qua iles by boxes, minimum and maximum alues by whiske ends. Gene exp ession le els a e measu ed as FPKM. Signi icance was compu ed
acco ding o Mann–Whi ney–Wilcoxon nonpa ame ic es . C) His og ams showing he dis ibu ion o he numbe o genes wi h phase a speci ic ime
poin s du ing he day unde LD (blue, op) and SD condi ions ( ed, bo om). ZTN, Zei gebe ime N, ma ks he ime poin N hou s a e dawn (ligh s on). D)
Ci cula hea map ep esen ing he empo al o ganiza ion o gene exp ession p o iles unde LD condi ions. Da k blue s ands o low exp ession, whe eas
yellow ep esen s high exp ession. Genes a e clus e ed depending on hei phases. Genes wi h phase a ZT0 a e loca ed in he ou e ci cle, while genes
wi h phase a subsequen zei gebe (ZT) a e placed sequen ially in o inne ci cles. Biological p ocesses en iched in he gene se wi h phase a each speci ic
ime poin a e depic ed cap u ing he ansc ip ional p og am o e diel cycles unde LD condi ion. E) Simila ly, ci cula hea map ep esen ing he
empo al o ganiza ion o he ansc ip ional p og am unde SD condi ion. F) Gene exp ession p o iles du ing 3 consecu i e days unde LD (blue line) and
SD ( ed line) o Fe edoxin-NADP + educ ase (os a18g01250, FNR). G) Ba plo s ep esen ing in di e en g een colo s om op o bo om a e he numbe o
a hy hmic, single-peak hy hmic, and 2 peaks hy hmic genes unde LD and SD condi ions. H) Gene exp ession p o iles du ing 3 consecu i e days unde
SD and 2 consecu i e days unde ee- unning condi ions consis ing o LL o DD o FNR (os a18g01250). This gene exempli ies how 2 peaks exp ession
pa e ns unde SD condi ions could eme ge as he combina ion o 2 dis inc hy hmic p o iles. One depending on he pho ope iod (do ed line) wi h phase
ma ked wi h a g ay e ical a ow main aining i s hy hmici y only unde LL ( op). Ano he exp ession p o ile is appa en depending on he sko ope iod
(dashed line) wi h phase ma ked wi h a black e ical a ow main aining i s hy hmici y only unde DD (bo om).
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condi ions. The model accu a ely p edic ed he eme gence o
bimodal hy hmic p o iles, as iden i ied in mic oa ay da a om
ND condi ions, alida ing ou model (Supplemen a y Fig. S8).
P o eome hy hmici y unde pho ope iodic
a ia ions and in eg a ion wi h ansc ip omic
hy hmic pa e ns
As p o eins a e he p ima y ac o s o biological p ocesses, p o eo-
mic analyses p o ide a di ec assessmen o he unc ional conse-
quences o en i onmen al hy hms (Kay e al. 2021). In his s udy,
a o al o 3,672 p o eins we e success ully quan i ied unde LD o
SD condi ions (Supplemen a y Fig. S9A), accoun ing o ∼48% o
he p edic ed Os eococcus p o eome (Supplemen a y Table S4).
The p edic ed subcellula loca ion o hese p o eins indica es co -
e age o all subcellula loca ions (Supplemen a y Fig. S9B).
The es ima ed numbe o hy hmic p o eins was no ably much
lowe han ha o hy hmic genes. Speci ically, 928 and 1,442
hy hmic p o eins we e iden i ied unde LD and SD condi ions, e-
spec i ely (Fig. 5A; Supplemen a y Fig. S9, C and Dand Table S5).
Globally, empo al phase o se s we e de ec ed be ween hy h-
mic p o ein abundance p o iles and he co esponding ansc ip
p o iles wi h gene exp ession p eceding p o ein abundance se -
e al hou s (Fig. 5B). A di ec ela ionship be ween ansc ip and
p o ein abundance becomes clea upon calcula ing co ela ion
ollowing phase alignmen o ansc ip and p o ein p o iles
Figu e 4. TFBS en iched in each ime poin gene clus e unde LD and SD condi ions.
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(Fig. 5, C and D). This sugges s ha p o ein and ansc ip abun-
dance ela e linea ly bu wi h a clea empo al sepa a ion. In LD
condi ions, he dis ibu ion o ansc ip /p o ein phase empo al
o se s cen e ed a ound 5 o 6 h, consis en wi h p o ein abun-
dance ollowing ansc ip abundance. Howe e , unde SD, o -
se s ollowed a mo e uni o m dis ibu ion and we e signi ican ly
longe han unde LD (Fig. 5E), indica ing ha many p o eins do
no ollow on p edic ably om ansc ip s. Unde bo h condi ions,
a “diu nal cha ac e was obse ed o he Os eococcus p o eome
wi h mos p o eins eaching maximum abundance du ing he
day (Fig. 5F). No ably, his is in con as wi h he “noc u nal cha -
ac e obse ed o he ansc ip ome (Fig. 3B). T ansc ip /p o ein
phase o se s esul in di e en diel imings o biological p ocesses
based on he ime poin when he co esponding p o eins each
hei maximum abundances (Fig. 5G) compa ed o he diel imings
o maximum gene exp ession le els (Fig. 3, D and E).
T ansc ip /p o ein phase o se s did no co ela e o any bio-
chemical p ope ies compu ed om p o ein sequences, such as ami-
no acid composi ion, cha ge, o hyd ophobici y (Supplemen a y Fig.
S10). Simila ly, ansc ip /p o ein phase o se did no co ela e
wi h ansc ip phase unde LD condi ion. Howe e , unde SD
condi ion ansc ip /p o ein o se s we e signi ican ly longe o
ansc ip s peaking du ing he sko ope iod, ZT8, ZT12, ZT16,
and ZT20, when compa ed o hose genes wi h ansc ip phases
du ing he pho ope iod, ZT0 and ZT4 (Fig. 6A). This may be due o
ansla ion occu ing p e e en ially du ing he ligh pe iod as
sugges ed in ou analysis (Fig. 5G) and in p e ious s udies o
A abidopsis (Sea on e al. 2018). No ably, dis inc sho o long an-
sc ip /p o ein phase o se s we e obse ed be ween genes in ol ed
in di e en biological p ocesses, as iden i ied by gene on ology
(GO) analyses (Fig. 6B). Speci ically, biological p ocesses associa ed
wi h sho o se s we e DNA eplica ion and pho osyn hesis.
T ansla ion on he o he hand was one o he mos ep esen a i e
biological p ocesses exhibi ing long ansc ip /p o ein o se s
(Fig. 6, C and D).
P o eome and ansc ip ome hy hmici y combine
o o ches a e physiological hy hms
Rhy hmic pa e ns ha e been desc ibed in mic oalgae, including
Os eococcus, o physiological p ocesses such as cell di ision, pho-
osyn hesis, and me abolism (Mi ag 2001; Moulage e al. 2007;
So okina e al. 2011; Shi e al. 2022). In his s udy, ansc ip omic
and p o eomic da a ha e been in eg a ed wi h measu emen s o
cell-cycle p og ession and me abolism o elucida e he empo al
o ches a ion ha unde lies dynamic physiology.
Cell-cycle p og ession
Flow cy ome y was used o assess he cell cycle based on o al
DNA con en in Os eococcus obse ing hy hmici y in all cell-cycle
phases unde bo h LD and SD condi ions (Fig. 7A; Supplemen a y
Table S6). A educ ion o ∼24% was obse ed in he numbe o cells
en e ing S phase unde SD compa ed o LD. In SD condi ions, cells
emained in ei he G1 o G2 phase. Fu he mo e, signi ican delays
o ∼4 h we e obse ed in he cell-cycle phases unde LD compa ed
o SD (Fig. 7A). These obse a ions a e consis en wi h he shi s in
he ime poin s o maximum ansc ip and p o ein abundance
ound unde sho e sus long pho ope iods. Fo key genes associ-
a ed wi h di e en cell-cycle phases (Robbens e al. 2005), he co -
esponding phase o ansc ip and p o ein abundance unde LD
and SD condi ions we e assessed (Supplemen a y Table S6).
T ansc ip and p o ein abundance p o iles we e compa ed o he
cell-cycle phases using iolin plo s. Clea empo al o se s we e
obse ed wi h gene exp ession p eceding p o ein abundance le els
and sho e empo al o se s be ween p o ein abundance and
he cell pe cen age in each cell-cycle phase (Fig. 7B). These gene
exp ession and p o ein abundance p o iles e e o he genes
associa ed wi h di e en cell-cycle phases in Supplemen a y
Table S6.
Cyclins and cyclin-dependen kinases (CDKs) a e essen ial com-
ponen s o he molecula machine y go e ning cell-cycle p og es-
sion (Co ellou e al. 2005). Thei gene exp ession and p o ein
abundance p o iles we e examined and in eg a ed wi h he empo-
al cell-cycle phase p og ession unde LD and SD (Fig. 7, C o E).
CyclinD (CYCD, os a18g01570) exp ession inc eased om he
Video 1. Changes in he phase and ampli ude o hy hmic gene
exp ession p o iles as a consequence o seasonal pho ope iod
leng hening/sho ening. Anima ion illus a ing how seasonal
pho ope iod leng hening may esul in a g adually delayed phase
coupled o a g adual inc ease in ampli ude. Acco dingly, seasonal
pho ope iod sho ening may esul in a g adually ad anced phase
coupled o a g adual dec ease in ampli ude. Whi e ec angles ep esen
pho ope iods (ligh pe iods o days) and colo - illed ec angles
co espond o sko ope iods (da k pe iods o nigh s). T ansi ions om
ed o blue colo s and ice e sa ep esen seasonal changes in
pho ope iods and sko ope iods.
Video 2. Eme gence o 2 peaks gene exp ession pa e ns as a
consequence o seasonal pho ope iod o day sho ening (sko ope iod o
nigh leng hening). Anima ion illus a ing how seasonal day o
pho ope iod sho ening (nigh o sko ope iod leng hening) may esul in
he eme gence o 2 peaks gene exp ession pa e ns as a consequence o
he 2 cons i uen gene exp ession p o iles becoming ou o phase. The
obse ed gene exp ession pa e n ep esen ed by a con inuous hick
line can esul om he combina ion o 2 dis inc exp ession p o iles
ep esen ed by hin do ed and dashed lines. One o hese exp ession
p o iles (do ed line) may no espond o seasonal changes in
pho ope iod/sko ope iod, whe eas he o he one (dashed line) may
expe ience phase shi s and ampli ude changes. Unde LD condi ions
(al e na ing 16 h ligh /8 h da k) he phases o maximum
exp ession-le el ime poin o bo h exp ession p o iles may coincide
esul ing in a single-peak exp ession pa e n. Whe eas unde SD
condi ions (al e na ing 8 h ligh /16 h da k) he phases may be eached
a di e en ime poin s p oducing a 2 peaks exp ession pa e n.
T ansi ions om ed o blue colo s and ice e sa ep esen seasonal
changes in pho ope iods and sko ope iods esul ing in g adual
ansi ions om 2 peaks o a single-peak exp ession pa e n.
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Figu e 5. P o eome hy hmici y unde al e na ing ligh /da k cycles and empo al o se s wi h espec o he co esponding ansc ip ome. A) Ba plo s
ep esen ing he numbe o iden i ied p o eins unde LD ( op) and unde SD (bo om) condi ions. The numbe o hy hmic p o eins unde LD condi ion
is ep esen ed in blue and unde SD condi ion in ed. A hy hmic p o eins a e ep esen ed in whi e. B) Scaled ansc ip (ligh line) and p o ein (da k
line) abundance p o iles du ing 3 consecu i e days unde LD ( op, blue) and SD (bo om, ed) condi ions o Minich omosome Main enance 2
(os a11g00910, MCM2). Whi e ec angles ep esen pho ope iods (ligh pe iods o days), blue- and ed- illed ec angles co espond o sko ope iods (da k
pe iods o nigh s) unde LD and SD, espec i ely. ZTN, Zei gebe ime N, ma ks he ime poin N hou s a e dawn (ligh s on, ZT0). C) Boxplo s
ep esen ing he global dis ibu ion o he co ela ions be ween ansc ip and p o ein abundance p o iles (whi e box) and shi ed aligned p o iles wi h
coinciden phases (g ay). Medians a e ep esen ed by cen al ho izon al lines, uppe and lowe qua iles by boxes, minimum and maximum alues by
whiske ends. D) Top, p o ein abundance (con inuous blue line) and gene exp ession (dashed ligh blue line) p o iles du ing 3 consecu i e days unde LD
condi ion o MA3 domain-con aining ansla ion egula o y ac o (os a05g02330). Bo om, phase-aligned p o ein abundance (do ed blue line) and gene
exp ession (dashed ligh blue line) p o iles. E) His og ams showing he dis ibu ion o he numbe o p o eins wi h speci ic o se s be ween ansc ip
and p o ein abundance phases ( ime poin s o maximum ansc ip /p o ein abundance) unde LD condi ion (le , blue) and SD condi ion ( igh , ed).
F) His og ams showing he dis ibu ion o he numbe o p o eins wi h phase o maximum abundance a speci ic ime poin s unde LD condi ion (le ,
blue) and SD condi ion ( igh , ed). G) Tempo al o ganiza ion o biological p ocesses based on he ime poin when p o eins in ol ed in he
co esponding p ocess each i s maximum abundance unde LD condi ion, le in blue, and SD condi ion, igh in ed.
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dec easing as ligh in ensi y inc eased, concomi an wi h an accu-
mula ion o bo h lu ein and dihyd olu ein (Fig. 10B). The enzymes
in ol ed in he in e con e sion o hese ca o enoids emain o be
iden i ied, and he compa ison o hei gene exp ession and p o-
ein accumula ion was no easible.
Ni a e assimila ion
In his sec ion, ni a e assimila ion in Os eococcus is examined as
an example o a physiological p ocess ha exhibi ed long empo-
al o se s be ween ansc ip s and p o eins. Ni a e assimila ion
is a undamen al p ocess in he me abolism o pho osyn he ic o -
ganisms as i con e s a ully oxidized ni ogen o m in o o ganic
educed ni ogen, ap o he syn hesis o essen ial biomolecules
o allow cell g ow h and biomass p oduc ion (Sanz-Luque e al.
2015). To analyze he adap i e esponse o ni a e assimila ion
o pho ope iodic a ia ions in diel cycles and i s implica ions o
op imizing nu ien up ake and me abolism, he ansc ip and
p o ein abundance p o iles co esponding o his pa hway in
Os eococcus we e analyzed (Fig. 11). Genes in his pa hway only
main ained hy hmici y unde LL being ep essed unde DD a e
bo h LD and SD en ainmen . This sugges s ha , al hough he ci -
cadian clock con ols pa ially hese genes, he e exis s a s ong
egula ion exe ed by ligh o e hem.
Ni a e assimila ion s a s wi h he up ake o ni a e media ed
by Ni a e T anspo e s 2 and 3 (NRT2, os a10g00950 and NRT3,
Figu e 11. In eg a ion o enzyma ic ac i i y p o iles wi h ansc ip and p o ein abundances o anspo e s and enzymes in ol ed in ni a e
assimila ion unde LD and SD condi ions. A) G aphical ep esen a ion o he ni a e assimila ion pa hway in Os eococcus. Fo each anspo e and
enzyme, i s co esponding p o ein and ansc ip abundance unde LD ( op in blue) and SD (bo om in ed) condi ions a e ep esen ed using hea maps.
Black ep esen s low, blue medium, and yellow high abundances. Whi e ec angles ep esen pho ope iods (ligh pe iods o days), blue- and ed- illed
ec angles co espond o sko ope iods (da k pe iods o nigh s) unde LD and SD, espec i ely. B) Mean scaled ansc ip (dashed line) and p o ein
(con inuous line) abundance p o iles o 3 consecu i e days o NR (le ) and GS ( igh ) unde LD ( op, blue) and SD (bo om, ed) condi ions. Ve ical lines
ep esen SE. Ac i i y p o iles o each enzyme unde LD and SD condi ions a e ep esen ed using hea maps, whe e black co esponds o low ac i i y and
yellow o high.
16 | The Plan Cell, 2025, Vol. 37, No. 2
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os a10g00940), ollowed by i s educ ion o ni i e by ni a e educ ase
(NR, os a10g00920). Ni i e is u he educed o ammonium by ni-
i e educ ase (NIR, os a10g00930). The cen al pa o ammonium
assimila ion is played by he glu amine syn he ase (GS, os a01g05020)
and glu amine oxoglu a a e amino ans e ase (GOGAT, os a14g01900)
cycle, which inco po a es ino ganic ammonium ni ogen i s
in o glu amine and hence in o glu ama e, he cen al p ecu so
o he biosyn hesis o all o he ni ogen-con aining biomolecules
(Fig. 11A; Supplemen a y Fig. S11).
Unde LD condi ions, NRT2/3, NR, and NIR gene exp ession
eached hei maximum a dawn (ZT0), while hei p o ein abun-
dances peaked 8 h la e a midday (ZT8), coinciding wi h he ime
poin o maximum i adiance. GS and GOGAT gene exp ession and
p o ein abundance peaked a he beginning o he day (ZT4) wi h-
ou no iceable empo al o se s. Howe e , a sligh inc ease in p o-
ein abundance was de ec ed a he end o he day (ZT12) o bo h
GS and GOGAT (Fig. 11A; Supplemen a y Fig. S11). In con as ,
unde SD condi ions, all genes encoding he anspo e s and
enzymes in ol ed in ni a e assimila ion showed hei maximum
exp ession a midnigh (ZT12–ZT16), p eceding hei p o ein
abundance peaks, eached a dawn (ZT0) o midday (ZT4),
by 8 h o mo e. No ably, GOGAT gene exp ession displayed a bimo-
dal pa e n unde SD condi ions, main aining he peak obse ed
unde LD a ZT4 besides a new peak a ZT16 (Fig. 11A;
Supplemen a y Fig. S11).
To alida e hese empo al o se s be ween ansc ip s and
p o eins, NR and GS ac i i ies we e measu ed h oughou com-
ple e diel cycles unde bo h LD and SD condi ions. Enzyme ac i i y
p o iles we e signi ican ly hy hmic (Supplemen a y Table S9). NR
maximal ca aly ic ac i i y eached i s peak a ZT8 unde bo h LD
and SD condi ions, coinciding wi h he p o ein abundance peak
unde LD and exhibi ing a sho o wa d empo al o se unde
SD. Simila ly, GS ac i i y peaked a ZT0 unde bo h LD and SD,
concomi an wi h he p o ein abundance peaks (Fig. 11B).
Discussion
Seasonali y plays a key ole in he na u al geog aphical g ow h dy-
namics o ma ine phy oplank on, including he model picoalga
Os eococcus (Bolaños e al. 2020). Ne e heless, he molecula
hy hms unde pinning i s esponses o pho ope iodic a ia ions e-
mained o be cha ac e ized. In his s udy, a mul iomics app oach
has been adop ed o un a el he plas ici y in he o ches a ion be-
ween ansc ip ome and p o eome hy hms go e ning cyclic phys-
iological esponses o changes in pho ope iod leng h.
T ansc ip omic analysis e ealed ha pho ope iodic a ia ions
had no e ec o e he iden i y o hy hmic genes, which comp ised
80% o he ansc ip ome. A hy hmic genes we e associa ed wi h
s ess esponses and exhibi ed almos comple e ep ession. These
genes could become hy hmic once highly exp essed unde he
co esponding s ess condi ion esul ing in a ull hy hmic an-
sc ip ome. Ou esul s a e in ag eemen wi h p e ious s udies
(Monnie e al. 2010; Zones e al. 2015) indica ing ha ansc ip ome
hy hmici y in chlo ophy e phy oplank on is much highe han
in o he o ganisms such as A. haliana 30% o 50% (Bläsing e al.
2005), Solanum ube osum 18% o 45% (Hoopes e al. 2022),
D osophila melanogas e 24% (Ma e al. 2021), o Mus musculus 3% o
10% (Mille e al. 2007). Ne e heless, only a small ac ion o he
ansc ip ome was hy hmic unde LL and DD ee- unning condi-
ions. These genes we e signi ican ly in ol ed in pho osyn hesis
and chlo oplas o ganiza ion, indica ing a p edominan con ol by
he au onomous ci cadian clock. Gene hy hmici y is s ongly in lu-
enced by al e na ing ligh /da k cycles in his picoalga. Fo ins ance,
DNA eplica ion genes main ained hy hmici y unde LL bu we e
ep essed unde DD, while ibosome biogenesis genes exhibi ed
hy hmici y unde DD bu los i unde LL. Gene ally, LL had a
mo e de imen al e ec on ansc ip ome hy hmici y han DD, in
ag eemen wi h s udies in plan s such as Medicago unca ula
(Wang e al. 2021), S. ube osum (Hoopes e al. 2022), and Ho deum ul-
ga e (Mülle e al. 2020). No ably, hy hmic pa e ns unde ee-
unning condi ions di e ed signi ican ly om hose unde diel
cycles. LL condi ions educed hy hmic ampli ude, likely due o de-
c eased cellula synch ony (Supplemen a y Fig. S13), as obse ed in
A abidopsis lea es (Yaki e al. 2011; Wenden e al. 2012), consis en
wi h he cell-au onomous cha ac e o ci cadian clocks. Typical e-
sponses o “noc u nal cha ac e we e ound, such as ad anced and
delayed phases unde DD and LL, espec i ely (Hundahl e al. 2012;
Imai e al. 2020). Indeed, mos ansc ip s peaked du ing he nigh ,
consis en wi h he “Escape om ligh heo y (Pi end igh 1993).
Du ing ansc ip ion, DNA is unwound lea ing i in an exposed s a e
mo e suscep ible o ligh -induced damage. To mi iga e his isk,
Os eococcus migh concen a e ansc ip ion p edominan ly du ing
he nigh .
Al hough hy hmic genes we e almos iden ical unde LD and
SD condi ions, signi ican e ec s on hei p o iles we e obse ed.
Speci ically, ad anced phases and educ ions in ampli ude unde
SD we e ound, as demons a ed a he le el o indi idual genes in
he model chlo ophy e mic oalgae Chlamydomonas einha d ii
(Se ano e al. 2009). Simula ions showed ha wo- hi ds o he
hy hmic genes wi h a single peak espond o changes in pho o-
pe iod by g adually adjus ing hei phases while he emaining
hi d adjus hei phases acco ding o a mo e complex mecha-
nism. Ano he esponse o pho ope iod sho ening was he eme -
gence o hy hmic bimodal gene exp ession p o iles. This ype
o hy hmici y has been sugges ed o be induced by he cyclic
occu ence o ides in coas al en i onmen s esul ing in ci ca idal
hy hms in ben hic dia oms (Bilcke e al. 2021). Howe e , ou
expe imen al design did no simula e idal condi ions and
Os eococcus is desc ibed as a plank onic picoalga. Mo eo e , he
exac pe iod o ci ca idal hy hms is 12.4 h, which would esul
in a daily phase shi o ∼1 h, which was no obse ed in ou
da a. I hese exp ession pa e ns we e uly ci ca idal, bimodali y
should ha e been main ained unde ee- unning condi ions.
Howe e , hey disappea ed esul ing in he main enance o only
one o he peaks unde LL and he o he one unde DD. This sug-
ges s he combina ion o 2 dis inc p o iles, 1 dependen on ligh
and he o he 1 on da k. Model simula ions showed how, unde
LD condi ions, he 2 dis inc p o iles o e lap in ime, p oducing
a single peak, while as he pho ope iod sho ens hey become
ou o phase p oducing a bimodal p o ile unde SD condi ions.
Bimodal hy hmic gene exp ession p o iles ha e also been iden i-
ied in plan s (Filichkin e al. 2011) and animals (Wege e al. 2021).
To u he explo e he seasonal esponses in Os eococcus a he
molecula le el, p o eomic da a we e gene a ed and in eg a ed
wi h he ansc ip omic da a. Ou 48% p o eome co e age ep e-
sen ed an imp o emen compa ed o p e ious s udies, such as
12% in A. haliana (Sea on e al. 2018), 30% in D. melanogas e
(Wang e al. 2020), and 9% in M. musculus (Chiang e al. 2014),
and lies be ween published s udies in Os eococcus wi h 27% in Le
Bihan e al. (2011) and 85% in Kay e al. (2021). In con as o he
high ansc ip ome hy hmici y, a d as ic educ ion in p o eome
hy hmici y was ound consis en wi h (Kay e al. 2021). In gene -
al, no coincidences be ween hy hmic p o ein and ansc ip
abundance p o iles we e obse ed wi h empo al o se s o se e al
hou s be ween hem. This obse a ion poin ed o he decoupling
o ansc ip ion and ansla ion and o he exis ence o a
Pho ope iodic plas ici y in ma ine phy oplank on hy hms | 17
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signi ican egula ion o e ansla ion ini ia ion (F eeney e al.
2016; Kay e al. 2021). Tempo al o se leng hs we e no uni o m
sugges ing he exis ence o a di e en ial pos ansc ip ional eg-
ula ion o each speci ic biological p ocess. Simila empo al shi s
be ween ansc ip s and p o eins ha e been epo ed in o he
o ganisms, such as M. musculus (Robles e al. 2014). No ably, a
pho ope iodic e ec on ansc ip /p o ein empo al o se s was
de ec ed, wi h longe o se s unde SD han LD. Mo eo e , in
SD-en ained cul u es, signi ican ly longe empo al o se s o
ansc ip s peaking du ing he sko ope iod we e ound.
Physiological measu emen s we e in eg a ed wi h ansc ip-
ome and p o eome dynamics, aiming o elucida e he ull empo-
al o ches a ion a di e en molecula le els unde lying he
esponses o changes in pho ope iod leng h. Cell-cycle p og es-
sion, pho osyn hesis, s a ch accumula ion, ca o enoid biosyn he-
sis, and ni a e assimila ion p esen ed ad anced hy hmic
pa e ns unde SD compa ed o LD condi ions, in ag eemen
wi h ansc ip ome and p o eome esponses o pho ope iod
sho ening. In line wi h he desc ibed Os eococcus algal blooms
in sp ing and summe (Bolaños e al. 2020; Chen e al. 2021;
Mondal and Bane jee 2022), in LD-en ained cul u es, inc eased
numbe o cells en e ing he S phase, enhanced pho osyn he ic
ac i i y, g ea e s a ch accumula ion, and a mo e ac i e xan ho-
phyll cycle we e pa en .
While empo al o se s o se e al hou s ha e been obse ed
be ween ansc ip s and p o eins, almos coinciden p o ein
abundance p o iles and hy hmic physiological measu emen s
occu . These empo al o se s shed ligh on he physiological sig-
ni icance o he obse ed ansc ip ional p og ams o e com-
ple e diel cycles. Fo ins ance, conside ing he empo al o se
o 8 h unde LD be ween he ansc ip and p o ein abundances
o NR, an enzyme ha equi es ligh o i s ac i a ion, peaking
o i s ansc ip a dawn (ZT0) su ices o make he p o ein peak
coincide wi h maximum ligh i adiance (ZT8). Howe e , unde
SD, wi h a pho ope iod o only 8 h, o ensu e ha he p o ein
peaks a ZT4 ( he poin o maximum ligh i adiance), he co e-
sponding ansc ip mus each i s maximum exp ession le el
a ound midnigh (ZT12–ZT16). This sugges s he exis ence o
molecula mechanisms ha allow Os eococcus o adjus i s an-
sc ip ome iming depending on he pho ope iod leng h, conside -
ing he empo al o se s be ween ansc ip s and p o eins o
ensu e ha p o eins a e a ailable a he app op ia e momen
o he day. Such pho ope iodic plas ic o ches a ion be ween
ansc ip ome, p o eome, and physiological iming migh play a
c ucial ole in he abili y o Os eococcus o h i e and op imize
i s physiological p ocesses acco dingly unde pho ope iodic a -
ia ions in diel cycles.
A ne wo k model iden i ied speci ic ansc ip ion ac o (TF) am-
ilies as po en ial key egula o s o he hy hmic physiology de ec ed
in Os eococcus such as he amilies MYB, DOF, bZIP, and CPP. This
was u he suppo ed by he TFBS en ichmen analysis, which
iden i ied signi ican DNA mo i s associa ed o hese TF amilies
in speci ic gene clus e s (Fig. 4). No ably, he CCA1 o hologue, os -
a06g2340, was ound o ha e a signi ican ly high p edic i e powe
o e s a ch con en , cell-cycle phases, and ca o enoid con en sug-
ges ing a egula o y ole o e he co esponding biological p oc-
esses (Fig. 12A). Fu he mo e, he EE mo i , ecognized by his
ansc ip ion ac o in A abidopsis, was ound signi ican ly en iched
in genes peaking a he end o he day unde LD condi ions, some o
which encode key p o eins in s a ch me abolism, cell-cycle p og es-
sion, and ca o enoid biosyn hesis (Fig. 12B). This indica es a con-
se ed ole o his ansc ip ion ac o in egula ing he co e o
he ci cadian clocks in Os eococcus and A abidopsis.
Ma e ials and me hods
Cul u e condi ions
O. au i (Os eococcus) sequenced s ain RCC4221 was used o all
expe imen s. The g ow h medium was p epa ed using a i icial
sea wa e (24.55 g NaCl, 0.75 g KCl, 4.07 g MgCl
2
·6H
2
O, 1.47 g
CaCl
2
·2H
2
O, 6.04 g MgSO
4
·7H
2
O, and 0.21 g NaHCO
3
pe 1 L dis-
illed wa e ) supplemen ed wi h 1 mL o Solu ion I (100 g NaNO
3
in 250 mL dis illed wa e ), 1 mL o Solu ion II (700 mg Na
2
HPO
4
and 2.5 g K
2
HPO
4
in 250 mL dis illed wa e ), 1 mL o Solu ion III
wi h ace me als (2.68 g NH
4
Cl, 5.2 g Fe-EDTA, 37.2 g Na
2
-EDTA,
23 mg ZnSO
4
, 14 mg CoSO
4
, 7.89 mg Na
2
MoO
4
·2H
2
O, 2.5 mg
CuSO
4
, 1.7 mg H
2
SeO
3
, and 180 mg MnCl
2
·4H
2
O in 500 mL dis illed
wa e ), and 1 mL o /2 i amin solu ion.
Expe imen s we e pe o med in pho ochemos a s consis ing o
wa e jacke ed bubble columns wi h 2 L capaci y (7 cm diame e
and 50 cm heigh ) con aining 1.8 L o cell suspension con inuously
spa ged wi h ai o ensu e cul u e homogeniza ion. The low o
wa e h ough he jacke , om an ex e nal cooling de ice, main-
ained empe a u e a 20 °C. A pH p obe was subme ged in o
he cul u e and connec ed o a pH me e se ing as inpu o a
LabJack ha con olled an elec o al e, allowing on demand in-
jec ion o CO
2
in o he ai s eam en e ing he cul u e o main ain
he pH a 8.0. Each pho ochemos a was illumina ed du ing he
co esponding ligh pe iod using 6 Phillips PL-32 W/840/4p whi e-
ligh luo escen lamps. The illumina ing sys em simula ed he
p og essi e ligh in ensi y inc ease and dec ease du ing sola day-
ligh cycles wi h a maximum ligh i adiance o 1,500 μE m
−2
s
−1
.
Unde LL condi ions ligh in ensi y was main ained o his maxi-
mum le el. Each pho ochemos a was kep wi hin a wooden
case and co e ed by a comple ely opaque ab ic, o ensu e ha
he illumina ion is only p o ided by he luo escen lamps. The il-
lumina ion egime was 16 h o ligh and 8 h o da kness o LD
condi ion and 8 h o ligh and 16 h o da kness o SD condi ion.
Pho ochemos a s we e ope a ed in con inuous mode adding esh
medium con inuously du ing he ligh pe iod a a low a e o
45 mL h
−1
wi h a pe is al ic pump. Since he pho ope iod unde
LD is wice as long as unde SD, he dilu ion a es we e 0.4 d
−1
unde LD and 0.2 d
−1
unde SD. Excess cul u e was emo ed a
he same a e by he o e low in o de o keep a cons an olume.
Fo he compa a i e analysis o s a ch con en , Os eococcus
cells we e g own ollowing he me hodology ou lined in O’-Neill
e al. (2011).
Sample collec ion, RNA ex ac ion, and
pu i ica ion
Samples we e collec ed o 3 consecu i e days e e y 4 h a ZT0,
ZT4, ZT8, ZT12, ZT16, and ZT20 whe e Zei gebe ime N (ZTN)
ma ks he ime poin N hou s a e he beginning o he pho ope -
iod simula ing dawn. Speci ically, ZT0 was collec ed immedia ely
a e ligh s we e on; and ZT8 (unde SD condi ions) and ZT16
(unde LD condi ions) we e collec ed immedia ely a e ligh
we e o . Subsequen ly, cul u es we e ans e ed o ee- unning
condi ions consis ing o LL and DD. No samples we e collec ed
du ing he i s day o allow cul u es acclima ion, and hen sam-
ples we e collec ed o 2 consecu i e days e e y 4 h a CT0, CT4,
CT8, CT12, CT16, and CT20 whe e Ci cadian ime N (CTN) deno es
he ime poin N hou s a e he subjec i e dawn. Fo each ime
poin , 50 mL o cul u e we e collec ed o RNA ex ac ion. Cells
we e washed wi h phospha e-bu e ed saline (PBS) solu ion using
cen i uga ion o 1 min a 13,000 ×g and 4 °C. A e supe na an
emo al, cells we e immedia ely lash ozen in liquid ni ogen
and s o ed a −80 °C.
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F ozen pelle s we e esuspended in 400 μL o dis up ion bu e
(Ga cía-Domínguez and Flo encio 1997) and added o a 1.5 mL
Eppendo ubes (RNAse ee and phenol-p oo ) con aining
400 μL o phenol:chlo o o m 1:1 and 100 μL o acid washed glass
beads (0.25 o 0.3 mm diame e ; B aun, Melsungen, Ge many).
Mechanical dis up ion was pe o med o 30 min by al e na ing
cycles o o exing o 60 s and ice incuba ion o 60 s. Ex ac s
we e cen i uged a 4 °C o 15 min a 13,000 ×g, he uppe aque-
ous phase was collec ed and mixed wi h 400 μL o phenol:chlo o-
o m 1:1 and cen i uged in he same condi ions o 5 min. This
p ocess was epea ed a o al o 3 imes. In he las wash, only
chlo o o m was used o emo e phenol om he samples. The
supe na an was incuba ed o e nigh a −20 °C in a solu ion o
80 μL 10 M LiCl and 550 μL 100% E OH o RNA p ecipi a ion.
Subsequen ly, samples we e cen i uged o 10 min a 13,000×g
a 4 °C. Pelle s we e d ied o a oid E OH con amina ion. Fu he
RNA pu i ica ion was pe o med using he Isola e II RNA Plan
Ki (Bioline) ollowing he manu ac u e ins uc ions. RNA con-
cen a ion and in eg i y we e measu ed using a Bioanalyze
2100 (Agilen RNA 6000 Nano Ki ).
RNA-seq da a gene a ion and analysis
Lib a y p epa a ion was ca ied ou ollowing he manu ac u e ’s
ins uc ions and sequencing was pe o med on an Illumina
Nex Seq500 sequence o 3 eplica es o each ime poin unde
LD and SD condi ions. App oxima ely 10 million 75 n long single
end eads we e gene a ed o each sample. RNA-seq da a we e
analyzed using ou pipeline Mic oAlgae RNA-seq and Chip-seq
AnalysiS (MARACAS) (Rome o-Losada e al. 2022). Speci ically,
he high quali y o he sequencing da a was assessed using
he so wa e package FASTQC. The O. au i genome sequence
and anno a ion 3.0 (h ps://phycocosm.jgi.doe.go /Os a4221_
3/Os a4221_3.home.h ml) we e used as e e ence genome.
Reads we e mapped o he e e ence genome wi h HISAT2 (Kim
e al. 2019). T ansc ip assembly and gene exp ession es ima ion
measu ed as agmen s pe kilobase o exon and millions o
mapped eads (FPKM) we e pe o med using S ingTie2 (Ko aka
e al. 2019) and he bioconduc o R package Ballgown (F azee
e al. 2015). PCA and HC we e pe o med using he R package
Fac oMineR (Lê e al. 2008). The R code o his analysis is a ailable
om he Gi Hub eposi o y SANDAL (h ps://gi hub.com/ an-
ome o-campe o/SANDAL).
RT-qPCR o CCA1 was pe o med using he o wa d p ime
(CTAGTACGTCGTCGAGC) and he e e se p ime (CCACGAACG
GACTCAT), as in e nal e e ence we used EF1 alpha wi h o wa d
p ime (GACGCGACGGTGGATCAA) and e e se p ime (CGACT
GCCATCGTTTTACC) ollowing he me hodology desc ibed p e i-
ously in Co ellou e al. (2009).
Sample collec ion and p o ein ex ac ion
Sample collec ion was pe o med as desc ibed o RNA analysis
o 3 consecu i e days unde LD and SD condi ions. Fo cell dis-
up ion, 1 mL o TRIsu e, 100 μL o acid washed glass beads (0.25
o 0.3 mm diame e ) and 40 μL o p o ein inhibi o cock ail (25×)
we e added on o ozen pelle s, ollowed by 3 dis up ion cycles
(60 s agi a ion—60 s incuba ion on ice) using a Mini-Beadbea e
(BioSpec P oduc s). P o eins we e ex ac ed using TRIsu e
Reagen (Sigma-Ald ich), acco ding o he manu ac u e ’s in-
s uc ions. The esul ing p o eins pelle s we e esuspended wi h
2 mL o 0.3 M guanidine solu ion in 95% E OH using 10 sonica ion
cycles (30 s sonica ion—30 s o incuba ing a 4 °C) and hen cen i-
uged a 4 °C du ing 5 min a 8,000×g. This washing p ocess
was epea ed wice, ollowed by 2 addi ional washes using 90%
E OH. The inal pelle s we e esuspended in NH
4
HCO
3
50 mM/
0.2% Rapidges (Wa e s) and o al p o eins we e quan i ied using
a Qubi de ice. Fo each sample, 50 μg o p o eins we e incuba ed
wi h di hio h ei ol ( inal concen a ion 4.5 mM) o 30 min a 60 °C.
Iodoace amide was added o a inal concen a ion o 10 mM and
incuba ed o 30 min, unde o al da kness a oom empe a u e.
An o e nigh ypsin ea men was done a 37 °C in a 1:40 ypsin:
Figu e 12. In eg a ion o ansc ip ion ac o ansc ip omic da a and physiological measu emen s. A) Ne wo k ep esen a ion o a sPLS model
in eg a ing ansc ip ion ac o gene exp ession as p edic o a iables wi h physiological measu emen s as esponse a iables. Ci cula nodes
ep esen genes encoding ansc ip ion ac o s om di e en amilies, indica ed by dis inc colo s. Rounded squa e nodes ep esen physiological
measu emen s: S and G1 cell cycle phases (in g een), S a ch con en (in whi e) and ca o enoid con en (in pink) p asinoxan hin (P asi), iolaxan hin
(Vio), an he axan hin (An he), zeaxan hin (Zea), neoxan hin (Neo), mic omonal (Mic o), and u iolide (U i). An edge be ween a ansc ip ion ac o node
and a physiological measu emen node is d awn when he exp ession p o ile o he co esponding ansc ip ion ac o has a signi ican ly high
p edic i e powe o e he co esponding physiological measu emen , sugges ing a po en ial egula o y ole. The CCA1 o hologue (os a06g02340) is
iden i ied as a po en ial cen al egula o . B) Iden i ica ion o he EE mo i , ecognized by he ansc ip ion ac o CCA1, in he p omo e s o genes
encoding key p o eins in ol ed in s a ch me abolism, cell cycle p og ession, and ca o enoid biosyn hesis.
Pho ope iodic plas ici y in ma ine phy oplank on hy hms | 19
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p o ein. Subsequen ly, o mic acid was added and incuba ed a 37
°C o 1 h. Finally, 2% ace oni ile ( / ) we e added o each a con-
cen a ion o he diges ed sample o ∼0.5 μg o p o ein/μL.
Sequen ial windowed acquisi ion o all heo e ical
mass spec a p o eomics da a gene a ion and
analysis
We u ilized a label ee quan i ica ion pla o m ha employs
independen da a acquisi ion called Sequen ial Windowed
Acquisi ion o all THeo e ical Mass Spec a (SWATH-MS)
(Ludwig e al. 2018) using a ime-o - ligh (TOF) iple quad upole
hyb id mass spec ome e MS (5600 plus, Sciex) equipped wi h a
nanoelec osp ay sou ce coupled o a nano-HPLC Eksigen model
425. The Sciex so wa e Analys TF 1.7 was used o he equipmen
con ol and da a acquisi ion. Pep ides we e i s loaded on o a ap
column (Acclaim PepMap 100 C18, 5 µm, 100 Å, 100 µm id×
20 mm, The mo Fishe Scien i ic) unde isoc a ical o de in 0.1%
o mic acid/2% ace oni ile ( / ) a a low a e o 3 μL/min o
10 min. Subsequen ly, hey we e elu ed on a e e sed-phase ana-
ly ical column, Acclaim PepMap 100 C18, 3 µm, 100 Å, 75 µm id×
250 mm, The mo Fishe Scien i ic, coupled o a PicoTip emi e
(F360-20-10-N-20_C12 om New Objec i e). Fo mic acid 0.1%
( / ) was used as Sol en A and 2% ace oni ile wi h o mic acid
0.1% ( / ) as Sol en B. Pep ides we e elu ed wi h a linea
g adien o 5% o 35% ( / ) o Sol en B in 120 min a a low a e
o 300 nL/min. The sou ce ol age was selec ed a 2,600 V and
he empe a u e was main ained a 100 °C. Gas 1 was selec ed
a 20 PSI, Gas 2 a 0, and cu ain gas a 25 PSI.
Fo p o eins iden i ica ion, a TOF-MS wi h a scan window o 400
o 1,250 m/z (accumula ion ime o 250 ms) was used ollowed by
50 MS/MS wi h a scan window o 230 o 1,500 m/z (accumula ion
ime o 65 ms) and wi h a cycle ime o 2,574 s.
Spec al lib a ies o LD and SD condi ions we e cons uc ed by
making one un wi h a mix u e o he biological eplica es co e-
sponding o each ime poin . P o einPilo 5.0.1 so wa e (Sciex)
was used o iden i y he p o eins in he lib a y. A pooled sea ch
o all uns was pe o med. The pa ame e s o he Pa agon me hod
we e: ypsin as enzyme and iodoace amide as cys eine alkyla ing
agen . The O. au i anno a ed p o eome 3.0 ile om: h ps://
phycocosm.jgi.doe.go /Os a4221_3/Os a4221_3.home.h ml
linked o a Sciex Con aminan s da abase we e used in lib a y con-
s uc ion. A alse posi i e analysis (FDR) was pe o med and hose
wi h FDR<0.01 we e conside ed.
Fo each sample, he equi alen o 1 µg o diges ed p o ein was
injec ed in o each un. P e iously, he equipmen was sel -
calib a ed using a s anda d, MS syn he ic pep ide calib a ion ki
om Sciex, o con ol sensi i i y and ch oma og aphic condi ions.
P o ein iden i ica ion and quan i ica ion we e pe o med using
SWATH uns wi h 60 ms o accumula ion ime and 3.7 s o cycle
ime. Th ee echnical eplica es o each 1 o he 3 biological ep-
lica es we e analyzed esul ing in 9 eplica es pe ime poin .
The gene a ed lib a ies (1% FDR) we e analyzed using he
Sciex so wa e PeaKView 2.2 wi h he mic oapp SWATH 2.0, o-
ge he wi h he da a ob ained om he SWATH uns. Using his
so wa e, he ch oma og aphic aces o he ions we e ex ac ed
and dumped in o he Ma ke iew 1.2.1.1 p og am whe e he
lis o iden i ied p o eins wi h hei co esponding a eas was
gene a ed. The pa ame e s o ex ac ion o ions and a eas
we e: 10 pep ides pe p o ein, 7 ansi ions o each pep ide,
h eshold o con idence o he pep ides se a 90, and FDR 1%.
The R package No malyze DE (Will o ss e al. 2019) was used
o pe o m Quan ile no maliza ion. Da a we e impu ed wi h
mean impu a ion me hod be ween he 9 echnical and biologi-
cal eplica es.
Cell cycle da a acquisi ion and analysis
A olume o 1.5 mL o cell suspension was ha es ed o each ime
poin unde LD and SD condi ions and dilu ed 1:10 in PBS. Two
millili e o hese dilu ions we e cen i uged and cells in he pel-
le s we e ixed wi h 10 mL o 100% E OH be o e s o age a −20 °C
o , a leas , 24 h. A e ixa ion, cell suspensions we e cen i uged
o 5 min a 3,500×g ( oom empe a u e) and esuspended in 1 mL
o PBS, washed once wi h PBS and sonica ed o 3 min in an
Ul asonic Cleane (JSP, US21, ul asonic powe 50 W), in o de
o elimina e cell clumps and agg ega es be o e s aining. In he
s aining p ocess, 2 μL o he Vib an Dye Cycle G een (V35004,
The mo Fishe ) (10 μM inal s ain concen a ion) we e added o
each sample and incuba ed 30 min (37 °C) o selec i e DNA label-
ing. A e incuba ion, cells we e washed and ans e ed o low
cy ome y ubes o cell cycle analysis. Flow cy ome y acquisi-
ion was pe o med wi h a BD FACS Can o II (BD Biosciences)
whe e s ained DNA was exci ed by a 488 nm lase and emission
was collec ed in a 530/30 nm pho omul iplie ube (PMT). Flow
a e was low and linea ampli ica ion was es ablished o he ac-
quisi ion. Da a we e analyzed using FlowJo .10.6.1 (Bec on
Dickinson & Company BD). Analysis was pe o med using he
Wa son p agma ic algo i hm p o ided by FlowJo (Wa son e al.
1987) o adjus he da a o he model.
Con ocal mic oscopy
Con ocal mic oscopy images o Os eococcus cells we e acqui ed
using a spec al Lase Scanning Con ocal Mic oscope (Olympus
FLOUVIEW FV3000). Exci a ion was pe o med wi h a 488 nm
lase . Emission signals we e de ec ed wi hin he g een chan-
nel (500 o 540 nm, gain 504) and ed channel (650 o 750 nm,
gain 432).
Pho osyn he ic ac i i y: sample collec ion
and da a acquisi ion
F esh cul u es we e ha es ed o each ime poin unde LD and SD
condi ions. Samples we e dilu ed 1:1 wi h g ow h medium and in-
cuba ed a 20 °C in o al da kness du ing 10 min. In o de o analyze
pho osyn he ic pa ame e s, pulse-ampli ude-modula ion (PAM)
luo ome y measu emen s we e pe o med using a Wal z
DUAL-PAM-100. A e da kness incuba ion, non-ac inic modula ed
ligh (450 nm, 2.8 μE m
−2
s
−1
) was u ned on o measu e he luo es-
cence basal le el, F
0
. A sa u a ing ed ligh pulse (655 nm and
5,000 μE m
−2
s
−1
) was applied o 400 ms o de e mine he maxi-
mum luo escence le el, F
m
. F /Fm, he maximum po en ial quan-
um e iciency o PSII when all eac ion cen e s a e open, was
calcula ed as F /Fm=(F
m
−F
0
)/F
m
.
S a ch con en de e mina ion
A each ime poin , 50 mL o esh cul u e we e ha es ed, cen i-
uged a 7,000×g o 10 min. The pelle s we e washed wi h 1% am-
monium o mia e (p/ ) o elimina e sal s, and lyophilized.
App oxima ely 2 o 3 mg o lyophilized biomass we e added o
he me ic ubes con aining 1 mL o glass beads (0.25 o 0.3 mm di-
ame e ) and 2 mL o chlo o o m:me hanol (2:1). Th ee dis up ing
cycles (60 s agi a ion−60 s incuba ion on ice) we e applied using
Mini-Beadbea e (BioSpec P oduc s). Then, cell ex ac s we e sep-
a a ed om he beads and sa ed in new ubes. Cell ex ac s we e
cen i uged o 4 min a 13,000×g and he supe na an was dis-
ca ded. Pelle s we e washed wi h chlo o o m:me hanol (2:1) un il
20 | The Plan Cell, 2025, Vol. 37, No. 2
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hey became whi e ensu ing pigmen s and lipids emo al. Pelle s
we e d ied and he spec opho ome ic p o ocol desc ibed in
Ga cía-Cube o e al. (2018) was used o de e mine s a ch con en .
Ca o enoid con en de e mina ion
Fou millig ams o lyophilized biomass we e added o a he me ic
ube con aining 1 mL o glass beads (0.25 o 0.3 mm diame e ) and
1 mL o pu e ace one. Th ee dis up ing cycles (60 s agi a ion−60 s in-
cuba ion on ice) we e applied using Mini-Beadbea e (BioSpec
P oduc s). Ca o enoids ex ac ion was achie ed ollowing he p o o-
col de eloped by Del Campo e al. (2004). A Hi achi HPLC (Eli e
LaCh om), equipped wi h a pho odiode-a ay de ec o (Hi achi
L-2455) was used. Sepa a ion was pe o med on a Wa e s No aPak
C-18 (3.9 ×150 mm, 4 µm pa icle size, 60 Å po e size) column. The
eluen s used o c ea e a g adien h ough he mobile phase we e:
Eluen A (0.1 M ammonium ace a e and 15:85 / H
2
O–me hanol)
and Eluen B (44:43:13 / me hanol–ace oni ile–ace one).
Tempe a u e was main ained cons an (20 °C) du ing he whole
p ocess and eluen s lowed a 800 μL min
−1
. Di e en ca o enoids
we e iden i ied acco ding o e en ion imes and abso p ion p o iles
o known ca o enoids. Quan i ica ion was de e mined as a pe cen -
age o he o al peak a ea co esponding o ca o enoids.
Iden i ica ion o genes, p o eins and physiological
measu emen s exhibi ing hy hmic pa e ns and
hei s a is ical compa a i e analysis
The bioconduc o R package hy hmici y analysis inco po a ing
nonpa ame ic me hods (RAIN) (Thaben and Wes e ma k 2014)
was used o iden i y genes, p o eins, and physiological measu e-
men s exhibi ing hy hmic pa e ns. A Benjamini–Hochbe g co -
ec ed P- alue h eshold equal o 0.05 was used in all he cases
unde s udy. Rhy hmic pa e ns wi h a single peak pe day we e
iden i ied by se ing he pe iod pa ame e o 24 h. Rhy hmic pa -
e ns exhibi ing 2 peaks pe day we e iden i ied by se ing he pe-
iod pa ame e o 12 h.
Fo s a is ical compa ison, hy hmic pa e ns we e i ed o a
co-sinusoidal cu e cha ac e ized by 3 pa ame e s: meso , ampli-
ude, and phase. The s a is ical signi icance o he di e ences in
ampli ude and phase be ween di e en g oups was pe o med
using he R package Ci caCompa e (Pa sons e al. 2020). A
Benjamini–Hochbe g co ec ed P- alue h eshold o 0.05 was
used o de e mine s a is ically signi ican di e ences.
The nonpa ame ic me hod implemen ed in RAIN was used
i s since i sea ches o any ype o hy hmic pa e ns. The me h-
ods in Ci caCompa e we e also employed iden i ying app oxi-
ma ely he same hy hmici y. Once i was con i med ha ou
da a could be u he analyzed using he pa ame ic me hods in
Ci caCompa e, signi icance analysis o di e ences in phases and
ampli udes we e pe o med using Ci caCompa e.
The signi icance o he global di e ences in hese pa ame e s
was assessed using he Mann–Whi ney–Wilcoxon nonpa ame ic
es implemen ed in he R unc ion wilcox. es . Phase di e ences
be ween ansc ip and p o ein exp ession p o iles we e com-
pu ed assuming ha ansc ip peaks p ecede hose o p o eins.
Signi ican di e ences be ween speci ic ea u es no ela ed
o hy hmici y we e also assessed using he Mann–Whi ney–
Wilcoxon nonpa ame ic es implemen ed in he R unc ion
wilcox. es .
Func ional anno a ion o gene se s
Func ional en ichmen analysis o e di e en gene se s was pe -
o med using ou online ool AlgaeFUN, mic oALGAE FUNc ional
en ichmen ool (Rome o-Losada e al. 2022) ha in u n is based
on he bioconduc o packages clus e P o ile , en ichplo , and
pa h iew (Luo and B ouwe 2013; Wu e al. 2021) and he unc ion-
al anno a ion package de eloped by ou g oup o g.O au i.eg.db
o O. au i (h ps://gi hub.com/ an- ome o-campe o/AlgaeFUN/
ee/mas e /packages/anno a ion_packages).
TFBS en ichmen analysis
The iden i ica ion o plan speci ic TFBS signi ican ly en iched in
he p omo e s o he genes peaking a speci ic ime poin s was
pe o med using he so wa e o mo i disco e y and nex gene -
a ion sequencing analysis Hype geome ic Op imiza ion o Mo i
EnRichmen (Heinz e al. 2010). The unc ion indMo i sGenome.pl
was applied wi h he pa ame e o plan speci ic TFBS-mse plan s.
The leng h o he gene p omo e was ixed o 500 n ups eam o
he s a codon and he mo i leng hs o 6 and 8 n .
In eg a i e model based on spa se pa ial leas
squa es
The R package mixomics (Roha e al. 2017) was used o de elop a
ne wo k model o he in eg a ion o physiological and ansc ip-
ion ac o gene exp ession da a based on he mul i a ia e p ojec-
ion me hod spa se pa ial leas squa es (sPLS) (Lê Cao e al. 2009).
Physiological da a co esponding o cell cycle phases, s a ch and
ca o enoid con en s we e conside ed as esponse a iables and
gene exp ession da a o ansc ip ion ac o s as p edic o s. The
model was cons uc ed wi h he unc ion spls in eg ession
mode and keeping 3 componen s in he p ojec ion. The unc ion
ne wo k wi h a cu o o 0.45 was applied o gene a e he ne wo k
ep esen a ion o he model. The so wa e ool Cy oscape
(Shannon e al. 2003) was used o he g aphical ep esen a ion
o his ne wo k using he yFiles O ganic Layou .
Enzyme ac i i ies
A each ime poin , 1 mL o esh cul u e was used o measu e
enzyme ac i i ies. Speci ically, NR and GS ac i i ies we e meas-
u ed as p e iously desc ibed by He e o e al. (1981) and
Ga cía-Domínguez and Flo encio (1997), espec i ely.
Accession numbe s
The O. au i RCC4221 genome sequence used in his a icle can be
ound in he GenBank da a lib a ies unde accession numbe
GCA_000214015.2.
Acknowledgmen s
We would like o acknowledge Eloisa Andúja and Mónica Pé ez
om he CABIMER Genomics Uni o hei assis ance wi h high-
h oughpu sequencing, Rocío Rod íguez om he IBVF
P o eomics Uni s o guidance wi h SWATH-MS p o eomics,
Ca los Pa ejo om he IBVF Ch oma og aphy and Mass
Spec ome y Uni o his con ibu ion in ca o enoid con en de-
e mina ion, Alicia O ea om he IBVF Mic oscopy Se ice o
he help wi h mic oscopy imaging, José En ique F ías om he
Cic-Ca uja Cell Cul u e Uni o his assis ance wi h Os eococcus
au i cul i a ion, and José Mo eno Fe nández o se ing up
Os eococcus au i cul i a ion in pho ochemos a s o his p ojec .
We would like o hank Miguel G. Gue e o and Inmaculada
Couso o c i ical eading o his manusc ip .
Pho ope iodic plas ici y in ma ine phy oplank on hy hms | 21
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Au ho con ibu ions
A.B.R.-L., M.E.G.-G., M.M.-P., and M.G.-G. pe o med we lab ex-
pe imen s. A.B.R.-L., C.A., and F.J.R.-C. gene a ed and analyzed
RNA-seq ansc ip omic and SWATH-MS p o eomic da a.
A.B.R.-L., M.J.C.-P., and F.J.R.-C. gene a ed and analyzed cell-cycle
da a. A.B.R.-L. and F.J.R.-C. gene a ed and analyzed pho osyn-
he ic e iciency measu emen s. A.B.R.-L., M.E.G.-G., M.M.-P.,
and C.A. measu ed and analyzed s a ch accumula ion, ca o enoid
con en , and enzyme ac i i ies. C.A. pe o med RT-qPCR analysis.
G.O. and Y.P.C. pe o med expe imen s in ba ch mode wi h low ni-
a e medium. F.J.R.-C., M.G.-G., A.B.R.-L., and G.O. in e p e ed he
esul s and w o e he manusc ip . All au ho s ead and app o ed
he inal manusc ip .
Supplemen a y da a
The ollowing ma e ials a e a ailable in he online e sion o his
a icle.
Supplemen a y Figu e S1. Expe imen al design and ansc ip-
omic RNA-seq da a eliabili y.
Supplemen a y Figu e S2. Biological p ocesses signi ican ly en-
iched in he genes exhibi ing hy hmici y unde al e na ing ligh /
da k cycles and cons an ligh .
Supplemen a y Figu e S3. Biological p ocesses signi ican ly en-
iched in he genes exhibi ing hy hmici y unde al e na ing ligh /
da k cycles and cons an da k.
Supplemen a y Figu e S4. Gene exp ession p o iles o clock
componen s and ligh ecep o s.
Supplemen a y Figu e S5. Func ional en ichmen o e ci ca-
dian genes a e LD and SD en ainmen .
Supplemen a y Figu e S6. T ansc ip ional empo al p og am o
he dis ibu ion o biological p ocesses o e diel cycles unde LD
condi ions (16 h ligh /8 h da k).
Supplemen a y Figu e S7. T ansc ip ional empo al p og am o
he dis ibu ion o biological p ocesses o e diel cycles unde
sho day condi ions (8 h ligh /16 h da k).
Supplemen a y Figu e S8. Valida ion o p edic ed eme gence o
bimodal hy hmic p o iles unde neu al day condi ions.
Supplemen a y Figu e S9. P o eomic da a no maliza ion and
eliabili y.
Supplemen a y Figu e S10. T ansc ip /p o ein phase o se s
did no co ela e o any biochemical p ope ies compu ed om
p o ein sequences.
Supplemen a y Figu e S11. Hea maps in eg a ing ansc ip
and p o ein abundance unde LD and SD condi ions o di e en
biological p ocesses.
Supplemen a y Figu e S12. Gene exp ession and s a ch con en
unde cons an da k.
Supplemen a y Figu e S13. Reduc ions in ampli ude unde
ee- unning condi ions can be explained by a decline in cul u e
synch ony.
Supplemen a y Table S1. Rhy hmic genes unde di e en ligh
egimes.
Supplemen a y Table S2. Se o hy hmic genes peaking a each
ime poin unde long- and sho -day condi ions.
Supplemen a y Table S3. Se o hy hmic genes wi h 2 peaks.
Supplemen a y Table S4. P o ein abundances unde long- and
sho -day condi ions.
Supplemen a y Table S5. Rhy hmic p o eins unde long- and
sho -day condi ions.
Supplemen a y Table S6. Cell cycle da a unde long- and sho -
day condi ions.
Supplemen a y Table S7. S a ch con en da a unde long- and
sho -day condi ions.
Supplemen a y Table S8. Ca o enoid con en da a unde long-
and sho -day condi ions.
Supplemen a y Table S9. Ni a e educ ase and glu amine syn-
he ase ac i i ies unde long- and sho -day condi ions.
Funding
This wo k was suppo ed by g an s BIO2017-84066-R (MINOTAUR)
and PID2021-123984OB-I00 (ELECTRA) om he Spanish Minis y
o Science and Inno a ion. C.A. was suppo ed by Conse je ía de
Conocimien o, In es igación y Uni e sidad, Jun a de Andalucia
(G an PREDOC_00999). G.O. and Y.P.C. we e suppo ed by a
Wellcome T us Awa d (225212/Z/22/Z) and a BBSRC Awa d
(APP3975).
Con lic o in e es s a emen . The au ho s decla e no compe ing
in e es s.
Da a a ailabili y
RNA-seq da a gene a ed in his s udy a e eely a ailable om he
Gene Exp ession Omnibus da abase unde he accession numbe
GSE155535. The SWATH-MS p o eomics da a gene a ed in his
s udy ha e been deposi ed o he P o eomeXchange Conso ium
ia he PRIDE (Pe ez-Ri e ol e al. 2022) pa ne eposi o y wi h
he da ase iden i ie PXD046992. The da a analysis code de el-
oped using he s a is ical p og amming language R is eely a ail-
able om he ollowing Gi Hub eposi o y SANDAL: h ps://
gi hub.com/ an- ome o-campe o/SANDAL. The esul s p e-
sen ed in his pape can be u he explo ed using he online
ool MINOTAUR: h ps://g eenne wo k.us.es/MINOTAUR/.
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