1
Peculia p ope ies o chlo ophyll he moluminescence emission o au o ophically o
mixo ophically g own Chlamydomonas einha d ii
Jean-Ma c Duc ue 1,*, Au elio Se ano2, Me cedes Roncel2, José M. O ega2
1 G oupe de Biospec oscopie Végé ale, Ecophysiologie Végé ale, ESE, Bâ . 362, Uni e si é
Pa is-Sud-O say, 91400 O say. F ance.
2 Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa-CSIC, A da.
Amé ico Vespucio 49, 41092 Se illa, Spain.
* Co esponding au ho . Tel: 33 6 87 70 36 72; ax: 33 1 69 15 73 53, E-mail add ess: jean-
[email p o ec ed]
ABSTRACT
The mic oalgae Chlamydomonas einha d ii and Chlo ella sp. CCAP 211/84 we e g own
au o ophically and mixo ophically and hei he moluminescence emissions we e eco ded
a e exci a ion by 1, 2 o 3 xenon lashes o by con inuous a - ed ligh . An oscilla ion o he
B band in ensi y acco ding o he numbe o lashes was always obse ed, wi h a maximum
a e 2 lashes, accompanied by a downshi o he B band empe a u e maximum in
mixo ophic compa ed o au o ophic g own cells, indica i e o a da k s able pH g adien .
Mo eo e , new lash-induced bands eme ged in mixo ophic Chlamydomonas g own cells, a
empe a u es highe han ha o he B band. In con as o he a e glow band obse ed in
highe plan s, in Chlamydomonas hese bands we e no inducible by a - ed ligh , we e ully
supp essed by 2 µM an imycin A, and peaked a di e en empe a u es depending on he lash
J Pho ochem Pho obiol B. 2011 Jul-Aug;104(1-2):301-7. doi: 10.1016/
j.jpho obiol.2011.02.014.
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numbe and g ow h s age, wi h highe empe a u e maxima in cells a a s a iona y compa ed
o an exponen ial g ow h s age. These di e ences a e discussed acco ding o he pa icula
p ope ies o cyclic elec on ans e pa hways in Chlamydomonas einha d ii.
Keywo ds
The moluminescence
Mic oalga
Cyclic elec on low
Pho osys em II
Chlo o espi a ion
Abb e ia ions
AG band a e glow he moluminescence band
B band he moluminescence band due o S2/3 QB- ecombina ion
CCCP/FCCP chlo o/ luo o ca bonyl cyanide phenylhyd azone
FQR Fe edoxine-plas oQuinone-Reduc ase cyclic/Chlo o espi a o y pa hway
NDH NAD(P)H DeHyd ogenase cyclic/Chlo o espi a o y pa hway
TL The moluminescence
Tm Tempe a u e o he maximum o a TL band; Tm(B) o B band..
3
1. In oduc ion
Pho osyn hesis elies on he con e sion o a quan um o ligh ene gy in o a cha ge
sepa a ion wi hin pho osys em I (PSI) and pho osys em II (PSII) eac ion cen e s, c ea ing
cha ge pai s s abilized on elec on ca ie s by ac i a ion ene gy ba ie s ha limi cha ge
ecombina ion, i.e. he was e ul back eac ion o he o wa d cha ge sepa a ion [1]. In PSII
cen e s, cha ge ecombina ion, limi ed o a slow a e a physiological empe a u es, p oceeds
ollowing di e en pa hways [2], one o hese leading o he ec ea ion a a low yield o an
exci on in he chlo ophyll an enna, wi h a p obabili y o deac i a e as luo escence. This
delayed luo escence emi ed in da kness ollowing an illumina ion is also called PSII
luminescence. The moluminescence (TL) is a echnique o s udy luminescence emission ha
consis s in illumina ing he sample a a empe a u e su icien ly low o make negligibly small
he ecombina ion a e o he cha ge pai s unde in es iga ion, hen o e eal hem
successi ely as TL bands by a p og essi e wa ming. Due o he s ong empe a u e
dependency o cha ge ecombina ion, TL has a g ea e esol ing powe han luminescence
mul iphasic decays eco ded a a cons an empe a u e immedia ely a e an illumina ion. In
uns essed da k-adap ed pho osyn he ic ma e ial, one o ew lashes gi e ise essen ially o a
so-called B band, loca ed be ween 25 and 40 °C depending on species and condi ions, which
esul s om a ecombina ion o an elec on s o ed on he seconda y quinonic accep o QB and
a posi i e cha ge s o ed on he S2 o S3 s a es o he oxygen e ol ing complex o PSII (OEC).
Analysis o luminescence signals becomes mo e complex o in ac pho osyn he ic
sys ems, in which a delayed luminescence bounce appea s supe imposed o he exponen ial
decay phases o he di e en ypes o cha ge pai s ini ially s abilized on PSII elec on ca ie s
[3]. This delayed emission called "a e glow" can be ela ed o a da k elec on back- ans e
om s oma o he accep o side o PSII [4]. The a e glow (AG) emission, highly dependen
on empe a u e, can be op imally eco ded as a sha p TL band peaking a abou 45 °C a a 0.5
4
°C/s wa ming a e in plan lea es [5]. Complex luminescence decays a cons an empe a u es,
some imes exhibi ing one o wo successi e delayed luminescence bu s s, ha e been epo ed
in some algal species [6,7,8]. The kine ics obse ed we e s ongly dependen on he g ow h
condi ions, e.g. low o high CO2, phospha e de iciency and ionic composi ion.
G een mic oalgae a e conside ed by plan biologis s as aluable unicellula model
sys ems o pho osyn hesis s udies. In pa icula , Chlamydomonas einha d ii (C. einha d ii),
a lagella ed pho osyn he ic p o is ha belongs o he class Chlo ophyceae, has been widely
used o biochemical, physiological and gene ic s udies. This cosmopoli e mic oalga is able o
g ow h in many di e en en i onmen s, no mally de i ing ene gy om oxygenic
pho osyn hesis. Chlamydomonas exhibi s a ema kable me abolic lexibili y and can also
h i e in o al da kness wi h ace a e as an al e na i e ca bon sou ce. I s adap abili y and quick
gene a ion ime ha e made i an impo an model o ganism o biological esea ch and s udies
on his mic oalga ha e p o ided majo esea ch con ibu ions in di e se a eas o cell and
molecula biology, like pho osyn hesis, pho o axis, cell mo ili y, ino ganic nu ien
assimila ion, abio ic s ess, e c. [9]. Mo eo e , i s ecen ly sequenced nuclea genome has
ad anced he unde s anding o he ances al euka yo ic cell o animals and plan s [10].
He e we show ha TL emission p ope ies in C. einha d ii a y widely depending on
g ow h s age and nu i i e medium and a e qui e di e en om hose obse ed in Chlo ella
CCAP 211/84, ano he well known chlo ophycean mic oalga, o in highe plan s. Addi ional
TL bands peaking a empe a u es abo e he B band ha appea in mixo ophic g own cells,
can be asc ibed o an “a e glow” mechanism o elec on ans e wi h, howe e , no iceable
disc epancies compa ed o wha is obse ed in highe plan s: namely, occu ence o se e al
bands peaking a a iable empe a u es depending on cul u e g ow h s age, sensi i i y o
an imycine A, an inhibi o o he Fe edoxine-plas oQuinone-Reduc ase (FQR) and ailu e o
induce hese bands by a a - ed illumina ion.
5
2. Ma e ial and Me hods
2.1. Algal s ains and g ow h condi ions
The unicellula g een algae (Chlo ophyceae) C. einha d ii 21g and Chlo ella sp.
CCAP 211/84 we e g own in Sueoka liquid mine al medium (pH 7.1) unde pho oau o ophic
condi ions (con inuous whi e ligh , 100 moles m-2 s-1) as p e iously desc ibed [11]. When
indica ed Chlamydomonas and Chlo ella cul u es we e supplemen ed wi h 12 mM sodium
ace a e o 25 mM glucose, espec i ely, and main ained unde con inuous illumina ion
(mixo ophic condi ions). G ow h o he algal ba ch cul u es was moni o ed by measu ing he
chlo ophyll cell con en a e me hanol ex ac ion. Exponen ial g ow h phase and pla eau
g ow h phase cells we e collec ed a e wo-day and i e-days cul u ing, espec i ely.
2.2. The moluminescence
TL emission was eco ded as p e iously desc ibed [5,12,13,14]. B ie ly, empe a u e
egula ion, signal eco ding and lash sequences we e d i en by a compu e h ough a
Na ional Ins umen DAQ-Pad1200 in e ace, using dedica ed so wa e. Tempe a u e
egula ion was pe o med by means o a Ma low he moelec ic "Pel ie " elemen powe ed by
a a iable (0 o 5 A) compu e -d i en powe supply. Luminescence emission was de ec ed by
a H5701-50 Hamama su pho omul iplie module. Illumina ion was pe o med h ough a ligh
guide pa allel o he pho omul iplie , bo h o hem being a ached o he same s and sliding
ho izon ally om he illumina ion o he measu ing posi ion. Single u n-o e lashes we e
p o ided by a xenon whi e ligh (Walz XST-103). Fa - ed was p o ided by an Epi ex LED

6
emi ing a 735 nm wi h negligibly small in ensi y below 700 nm. TL signals we e analyzed
wi h a dedica ed nume ical simula ion so wa e desc ibed in Duc ue [12].
Algal suspensions we e main ained in he da kness o unde dim g een ligh in
E lenmeye essels (abou 5 mm laye hickness) ha we e all shaken ho izon ally be o e
each TL eco ding. Samples we e hen kep a 25 °C 2 minu es in comple e da kness in he TL
measu ing cell, hen cooled o 1 °C and submi ed o lash o a - ed illumina ion; he TL
eco ding was s a ed immedia ely.
3. Resul s
3.1. Va ia ions o TL emission depending on au o ophic o mixo ophic g ow h condi ions
(Figu e 1)
In da k-adap ed au o ophically o mixo ophically g own Chlo ella cells a s a iona y
phase, single u n-o e lash sequences induced a single TL band (Fig. 1) eaching a
maximum in ensi y a e 2 lashes and oscilla ing wi h a pe iod 4, as classically obse ed also
in plan lea es o he B band [15,16]. The maximal empe a u e Tm(B) o he B band a e 2
o 3 lashes occu ed a a lowe empe a u e in mixo ophic compa ed o au o ophic g own
cells.
A simila unique TL band was gene ally obse ed in Chlamydomonas cells om
au o ophic cul u es in he exponen ial g ow h phase (Fig. 2A). Howe e , when cells om a
cul u e ha eached he s a iona y g ow h phase we e analyzed, 2 o 3 lashes induced a
no iceable shoulde a >30 °C ha is supp essed by an imycin A, in addi ion o he B band
cen e ed nea 25 °C (see Fig.5, C l au o).
(Figu e 2)
7
A clea ly di e en emission pa e n was obse ed in mixo ophically g own cells a a
s a iona y s age (Fig 2B). The B band peaking a 25 °C a e 1 lash was downshi ed o lowe
empe a u es and b oadened a e 2 o 3 lashes. In addi ion, new bands eme ged abo e 30 °C.
A band a ised a 50 °C a e 1 lash (luminescence emi ing cen e s a e 100% S2QB-) and
ano he one a abou 40 °C o e lapping he smalle 50 °C band a e 2 lashes (mix u e o
S2QB-, S3QB-). In e es ingly, 3 lashes (mos ly S3QB-) induced a band a 32 °C, no a 40 °C as
obse ed a e 2 lashes, wi h a shoulde owa ds high empe a u es sugges ing ha he 50 °C
band obse ed a e 1 lash is s ill p esen a e 3 lashes, despi e he low amoun o S2QB-;
his was u he con i med by i s sensi i i y o an imycin A (Fig. 3C). As a consequence he
bands obse ed a 50 °C, 40 °C and 32 °C canno be simply asc ibed o he p opo ion o S2
and S3 s a es in luminescence-emi ing cen e s. A b ie 5 s a - ed illumina ion p oduced a
sha p band a abou 25 °C bo h in au o ophic (Fig. 2A) and mixo ophic (Fig. 2B) g own
cells, simila o a B band, wi hou any o he >30 °C non-B bands induced by lashes. I is
wo h no icing ha he Tm o his band is abou 3 °C lowe in mixo ophic compa ed o
au o ophic g own cells.
The s ongly downshi ed B band (Fig. 2B) due o an acidic da k-s able lumen pH is
also b oadened and la ened inc easingly wi h he S3/S2 a io, om 1 o 3 lashes. In hese
condi ions, he appa en B band is no a genuine TL band bu becomes an en elop o
elemen a y B bands dependen on a ied local pHs [17], making a simula ion impossible.
Ne e heless, disca ding he B band, he main band peaking a 42°C a e 2 lashes can be
en a i ely decomposed in o 2 componen s, including a 48°C componen simila o ha ound
a e 1 lash. (Fig. 2B, do s). This con i ms ha analyzing he main band a e 2 lashes as he
sum o 2 elemen a y TL band is possible, al hough his does no exclude a highe numbe o
componen s ha also p o ide a sa is ac o y i .
8
In mixo ophically g own Chlamydomonas a an exponen ial g ow h s age (Fig. 2C),
he same gene al pa e n o lash-induced TL bands was obse ed wi h, howe e , wo
di e ences compa ed o mixo ophic g own cells a a s a iona y phase (Fig. 2B):
- he downshi o he B bands (a ows in Fig. 2) was no so p onounced a exponen ial s age
(Fig. 2C) compa ed o s a iona y s age (Fig. 2B), he “ elaxed” posi ion o he B band being a
25 °C as obse ed in au o ophic exponen ial samples (Fig. 2A).
- he 50 °C, 40 °C and 32 °C bands ound in mixo ophic g own cells a s a iona y s age (Fig.
2B) we e had hei peak empe a u e Tm de c eased o 44 °C, 33 °C and 25 °C, espec i ely,
in cells a exponen ial g ow h s age (Fig. 2C).
The TL signal a e 3 lashes was en a i ely decomposed in o 3 elemen a y
componen s, including he B band (Fig. 2C, do s). Unexpec edly, he in ensi y o he highes
empe a u e componen a 42°C did no dec ease wi h lash numbe , as i should i i was due
o S2 cen e s.
P eillumina ion du ing 20 min wi h blue ligh (480 nm) did no change signi ican ly
he Tm o he 30-35 °C band induced by 2 o 3 lashes (da a no shown).
3.2. E ec s o inhibi o s o pho osyn he ic cyclic pa hways
(Figu e 3)
The o igins o he non-B lash-induced bands was u he in es iga ed by adding
an imycin A, a speci ic inhibi o o he FQR cyclic pa hway a sub-mic omola concen a ions
[18,19]. I should be no iced ha an imycin A had a sligh inhibi o y e ec on he B band in
au ophic (Fig. 3A) as well as in mixo ophic condi ions (Fig. 3B).
In a mixo ophic condi ions, a a s a iona y phase, he band induced by 2 lashes
peaking nea 40 °C (Fig. 3B, 2F) was sha pe in p esence o 1% e hanol han ha shown in
9
Fig. 2B wi hou e hanol. This 40 °C band was clea ly supp essed by an imycin A wi h a 0.4
µM hal -inhibi o y concen a ion (Fig. 3B). In mixo ophic g own cells a an exponen ial
s age (Fig. 3C), he band a 45 °C a e 1 lash was also p esen as a shoulde a e 3 lashes,
and was in bo h cases supp essed by 2 µM an imycin A. This sugges s an in ol emen o he
FQR pa hway in all he non-B bands induced by lashes in mixo ophically g own
Chlamydomonas. The main band a e 3 lashes appea ed unique and was loca ed a abou 25
°C (Fig. 2C, 3C), bu an imycin A e ealed a smalle B band a 18 °C, con i ming he 25 °C
band was o mo e han a hal o non-B o igin and ela ed o FQR pa hway.
Myxo hiazol and HQNO, o he inhibi o s o he FQR pa hway [19], also supp essed
he >30 °C TL bands (da a no shown).
3.3. E ec o phospho yla ion uncouple s
(Figu e 4)
No iceably, nige icine was ac i e a leas in p esence 3% e hanol (Fig. 4). E hanol in
he 0.5% o 3% ange modi ied he 32-40 °C band(s) induced by 2 o 3 lashes, wi h a
maximum sha pening and enhancing e ec a 1% (Fig. 3B, Fig. 4), ollowed by a sligh
downshi a highe e hanol concen a ions. This e hanol enhancing e ec was less e iden
when he non-B bands induced by 2 o 3 lashes we e peaking a lowe empe a u es
(mixo ophic Chlamydomonas cells a exponen ial g ow h s age, con ol shown in Fig. 2C,
Fig. 3C). 6 µM nige icine in 3% e hanol caused a usion o he wo bands o con ol in o one
single band a an in e media e posi ion, wi h a Tm upshi o he B band and a downshi o
he 40 °C band, as also obse ed in lea es o highe plan s in il a ed by nige icine [5].
(Figu e 5)
CCCP, ano he uncouple and also an ADRY eagen ha impai s he oxygen e ol ing
complex [20], caused a comple e supp ession o he TL signal in au o ophic cul u es, due o
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[1] E. Tyys jä i, I. Vass, Ligh emission as a p obe o cha ge sepa a ion and ecombina ion in
he pho osyn he ic appa a us: ela ion o p omp luo escence o delayed ligh emission
and he moluminescence, in: G.C. Papageo giou, Go indjee, (Eds.), Chlo ophyll a
Fluo escence: A Signa u e o Pho osyn hesis, Ad ances in Pho osyn hesis and
Respi a ion, Sp inge , Do d ech , 2003, Volume 19, pp. 363-388.
[2] F. Rappapo , A. Cuni, L. Xiong, R. Say e, J. La e gne, Cha ge ecombina ion and
he moluminescence in pho osys em II, Biophysical J. 88 (2005) 1948-1958.
[3] W.F. Be sch, J. R. Azzi, A ela i e maximum in he decay o long- e m delayed ligh
emission om he pho osyn he ic appa a us, Biochim. Biophys. Ac a 94 (1965)15-26.
[4] L.G. Sundblad, W.P. Sch öde , H.E. Ake lung, S-s a e dis ibu ion and edox s a e o QA
in ba ley in ela ion o luminescence decay kine ics, Biochim. Biophys. Ac a 973 (1988)
47-52.
[5] T. Mi anda, J.M. Duc ue , Cha ac e iza ion o he chlo ophyll he moluminescence
a e glow in da k-adap ed o a - ed-illumina ed plan lea es, Plan Physiol. Biochem.
33 (1995) 689-699.
[6] S. Mell ig, J. S. Tillbe g, T ansien peaks in he delayed luminescence om Scenedesmus
ob usiusculus induced by phospho ous s a a ion and ca bon dioxide de iciency,
Physiol. Plan . 28 (1986) 180-188.
[7] K. Palmq is , L. G. Sundblad, G. Samuelsson, E. Sundbom, A co ela ion be ween
changes in luminescence decay kine ics and he appea ance o a CO2-accumula ing
mechanism in Scenedesmus obliquus, Pho osyn . Res. 10 (1986) 113-123.
[8] W. Schmid , H. Senge , Long- e m delayed luminescence in Scenedesmus obliquus,
Biochim. Biophys. Ac a 891 (1987) 15-27.
[9] E.H. Ha is, The Chlamydomonas Sou cebook (2nd Edi ion, Volume 1), Academic
P ess/Else ie , Ams e dam (2009).

17
[10] Me chan , S, SE P ochnik, O Vallon, EH Ha is, SJ Ka powicz, GB Wi man, A Te y,
A Salamo , LK F i z-Laylin, L Ma échal-D oua d, WF Ma shall, L-H Qu, DR Nelson,
AA Sande oo , MH Spalding, e al., DS Rokhsa , AR G ossman, The Chlamydomonas
genome e eals e olu iona y insigh s in o key animal and plan unc ions, Science 318
(2007) 245-250.
[11] M.R. Gómez-Ga cía, M. Losada, A. Se ano, A no el sub amily o monome ic ino ganic
py ophospha ases in pho osyn he ic euka yo es, Biochem. J. 395 (2006) 211-221.
[12] J.M. Duc ue , Chlo ophyll he moluminescence o lea discs: simple ins umen s and
p og ess in signal in e p e a ion open he way o new ecophysiological indica o s, J.
Exp. Bo . 54 (2003) 2419-2430.
[13] M. Roncel, J.M. O ega, A e glow he moluminescence as a possible ea ly indica o o
changes in he pho osyn he ic elec on anspo in lea es, Pho osyn h. Res 84 (2005)
167-172.
[14] J.L. Zu i a, M. Roncel, J.M. O ega, M. Ba ón, J.M. Duc ue , Changes in pho osyn he ic
me abolism induced by obamo i us in ec ion in Nico iana ben hamiana s udied in
i o by chlo ophyll he moluminescence, New Phy ol. 175 (2007) 120-130.
[15] A.W. Ru he o d, A.R. C o s, Y. Inoue, The moluminescence as a p obe o
Pho osys em II pho ochemis y: he o igin o he lash-induced glow peaks, Biochim.
Biophys. Ac a 689 (1983) 457-465.
[16] A.W. Ru he o d, Go indjee, Y. Inoue, Cha ge accumula ion and pho ochemis y in
lea es s udied by he moluminescence and delayed ligh emission, P oc. Na l. Acad.
Sci. USA 81 (1984) 1107-1111.
[17] T. Mi anda, J. M. Duc ue , E ec s o da k- and ligh -induced p o on g adien s in
hylakoids on he Q and B he moluminescence bands, Pho osyn h. Res. 43 (1995) 251-
262.
18
[18] K. Tagawa, H.Y. Tsujimo o, D.I. A non, Role o chlo oplas e edoxin in he ene gy
con e sion p ocess o pho osyn hesis, P oc. Na l. Acad. Sci. USA 49 (1963) 567-572.
[19] J. Ra enel, G. Pel ie , M. Ha aux, The cyclic elec on pa hway a ound pho osys em-I in
Chlamydomonas einha d ii as de e mined in i o by pho oacous ic measu emen s o
ene gy s o age, Plan a (1994) 193, 251-259.
[20] G. Renge , Reac ion o CCCP in pho osyn hesis on an in e media e be ween chlo ophyll-
a and wa e , Na u wissenscha en 56 (1969), 370.
[21] P. Jolio , A. Jolio , Dependence o delayed luminescence upon adenosine iphospha ase
ac i i y in Chlo ella, Plan Physiol. 65 (1980) 691-696.
[22] J. La e gne, F. Rappapo , Redox and ATP con ol o pho osyn he ic cyclic elec on
low in Chlamydomonas einha d ii (I) ae obic condi ions, Biochim. Biophys. Ac a
1797 (2010) 44-51.
[23] J. La e gne, W. Jünge, P o on elease du ing he edox cycle o he wa e oxidase,
Pho osyn h. Res 38 (1992) 279-296.
[24] P. Bennoun, E idence o a espi a o y chain in he chlo oplas . P oc. Na l. Acad. Sci.
USA 79 (1982) 4352-4356.
[25] G. Pel ie , L. Cou nac, Chlo o espi a ion, Annual Re iew Plan Biol. 53 (2002) 523-550.
[26] P. Tapie, Y. Choque , J. B e on, P. Delepelai e, F.A. Wollman, O ien a ion o
pho osys em I pigmen s. In es iga ion by low- empe a u e linea dich oism and
pola ized luo escence emission, Biochim. Biophys. Ac a 767 (1984) 57-69.
[27] D.S. Bendall, R.S. Manasse, Cyclic pho ophospho yla ion and elec on anspo ,
Biochim. Biophys. Ac a 1229 (1995) 23-38.
[28] M. Ha aux, D. Rumeau, J.M. Duc ue , P obing he FQR and NDH ac i i ies in ol ed in
cyclic elec on anspo a ound Pho osys em I by he ‘a e glow’ luminescence,
Biochim. Biophys. Ac a 1709 (2005) 203-213.
19
[29] T. Shikanai, Cyclic elec on anspo a ound Pho osys em I: Gene ic app oaches, Ann.
Re . Plan Biol. 58 (2007) 199-217.
[30] M. Ma in, B. Saba e , Plas id ndh genes in plan e olu ion, Plan Physiol. Biochem. 48
(2010) 636-645.
[31] G. Pel ie , D. Tolle e , E. Billon, L. Cou nac, Auxilia y elec on anspo pa hways in
chlo oplas s in mic oalgae, Pho osyn h. Res. 106 (2010) 19-31.
[32] N. Bukho , R. Ca pen ie , Al e na i e Pho osys em-I d i en elec on anspo ou es:
mechanisms and unc ions, Pho osyn h. Res. 82 (2004) 17-33.
[33] J. Al ic, Cyclic elec on low a ound pho osys em I in unicellula g een algae,
Pho osyn h. Res. 106 (2010) 47-56.
[34] J.M. Duc ue , L. Ko acs, G. Ga ab, The moluminescence bands in un ozen cells o
Chlamydomonas einha d ii, in: R. Ca pen ie (Ed.), P oceedings o he 13 h
In e na ional Cong ess o Pho osyn hesis, Mon éal, 2004.
[35] G. Pel ie , G.W. Schmid , Chlo o espi a ion: an adap a ion o ni ogen de iciency in
Chlamydomonas einha d ii, P oc. Na . Acad. Sci. USA 88 (1991) 4791-4795.
[36] D. Pe ou sos, A.M. Te aushi, A. Busch, I. Hi schmann, S.S. Me chan , G. Finazzi, M.
Hipple , PGRL1 pa icipa e in i on-induced emodelling o he pho osyn hesis appa a us
and in ene gy me abolism in Chlamydomonas einha d ii, J. Biol. Chem. 284 (2009)
32770-32781.
[37] J.M. Duc ue , I. Vass,. The moluminescence: expe imen al. Pho osyn h. Res. 101 (2009)
195 – 204.
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Legends
Fig. 1 - The moluminescence emission cu es o Chlo ella sp. CCAP 211/84 cells g own
au o ophically (line) o mixo ophically (do s) a he s a iona y phase. Cells we e exci ed by
1, 2, o 3 lashes a 1 °C.
Fig. 2 - The moluminescence emission cu es o C. einha d ii cells. A: au o ophic g ow h,
exponen ial phase. B: mixo ophic g ow h, s a iona y phase. C: mixo ophic g ow h,
exponen ial phase. Exci a ion by 1, 2, 3 lashes (lines) o 5 s a ed-ligh (dashes). The a ows
poin a he maximum o he B band. Symbol ? indica es he es ima ed posi ion o B band.
Simula ed signal o 2 lashes in hicks do s, componen s in hin do s (B: 2 componen s,
excluding B band. C: 3 componen s, including B band).
Fig. 3 - E ec o an imycin A on he moluminescence o C. einha d ii cells. A: au o ophic
g ow h, a e 3 lashes in samples supplemen ed wi h 1% e hanol (line, C l); a e 3 lashes
wi hou e hanol (do s) and wi h 2 µM an imycin A and 1% e hanol (dashed). B: mixo ophic
g ow h, s a iona y phase, a e 2 lashes in samples wi h 1% e hanol (2F), 0.4 µM, 1 µM o 2
µM an imycin A and 1% e hanol. C: mixo ophic g ow h, exponen ial phase, a e 1 o 3
lashes in samples wi h 1% e hanol (lines) o 2 µM an imycin A and 1% e hanol (dashed and
do lines, espec i ely).
Fig. 4 - E ec o nige icine on he moluminescence o C. einha d ii. Cells g own
mixo ophically a s a iona y phase. A e 2 lashes wi hou addi ion ( hin line), wi h 1%
e hanol (do s), 3% e hanol ( hick line, C l) o 3% e hanol and 6 µM nige icine (dashed line,
Nig.).
21
Fig. 5 - E ec o CCCP on he he moluminescence o C. einha d ii. Au o ophically ( hin
and dashed lines, au o) and mixo ophically ( hick and dashed lines, mixo) cells g own, a
s a iona y s age. Whe e indica ed 1 µM CCCP was added.
Fig. 6 - E ec o phospha e de iciency on he moluminescence o C. einha d ii. Cells
g ow h mixo ophically, a exponen ial phase. P e ious o TL expe imen s cells we e
incuba ed h ee days wi h (A) and wi hou phospha e (B). Fo he expe imen wi hou
phospha e, cells we e g own in Sueoka liquid mine al medium in which phospha e sal s we e
subs i u ed by po assium ni a e (see Ma e ials and Me hods). TL emission cu es we e
eco ded a e 3 lashes. Con ol wi hou addi ion (do s); wi h 1% e hanol (lines); wi h 1%
e hanol and 2 µM an imycin A (dashed lines).