Thylakoid Protein Phosphorylation in Higher Plant ...stn7 stn8 mutants under strongly ﬂuctuating light conditions. Several proteins of PSII and its light-harvesting antenna (LHCII)

Thylakoid Protein Phosphorylation in Higher PlantChloroplasts Optimizes Electron Transfer underFluctuating Light1[C][OA]

Mikko Tikkanen, Michele Grieco, Saijaliisa Kangasjarvi, and Eva-Mari Aro*

Plant Physiology and Molecular Biology, Department of Biochemistry and Food Chemistry, University ofTurku, FIN–20014 Turku, Finland

Several proteins of photosystem II (PSII) and its light-harvesting antenna (LHCII) are reversibly phosphorylated according tolight quantity and quality. Nevertheless, the interdependence of protein phosphorylation, nonphotochemical quenching, andefficiency of electron transfer in the thylakoid membrane has remained elusive. These questions were addressed byinvestigating in parallel the wild type and the stn7, stn8, and stn7 stn8 kinase mutants of Arabidopsis (Arabidopsis thaliana),using the stn7 npq4, npq4, npq1, and pgr5 mutants as controls. Phosphorylation of PSII-LHCII proteins is strongly anddynamically regulated according to white light intensity. Yet, the changes in phosphorylation do not notably modify therelative excitation energy distribution between PSII and PSI, as typically occurs when phosphorylation is induced by “state 2”light that selectively excites PSII and induces the phosphorylation of both the PSII core and LHCII proteins. On the contrary,under low-light conditions, when excitation energy transfer from LHCII to reaction centers is efficient, the STN7-dependentLHCII protein phosphorylation guarantees a balanced distribution of excitation energy to both photosystems. The importanceof this regulation diminishes at high light upon induction of thermal dissipation of excitation energy. Lack of the STN7 kinase,and thus the capacity for equal distribution of excitation energy to PSII and PSI, causes relative overexcitation of PSII underlow light but not under high light, leading to disturbed maintenance of fluent electron flow under fluctuating light intensities.The physiological relevance of the STN7-dependent regulation is evidenced by severely stunted phenotypes of the stn7 andstn7 stn8 mutants under strongly fluctuating light conditions.

Several proteins of PSII and its light-harvestingantenna (LHCII) are reversibly phosphorylated bythe STN7 and STN8 kinase-dependent pathways ac-cording to the intensity and quality of light (Bellafioreet al., 2005; Bonardi et al., 2005). The best-knownphosphorylation-dependent phenomenon in the thy-lakoid membrane is the state transition: a regulatorymechanism that modulates the light-harvesting capac-ity between PSII and PSI. According to the traditionalview, “state 1” prevails when plants are exposed to far-red light (state 1 light), which selectively excites PSI.Alternatively, thylakoids are in “state 2” when plantsare exposed to blue or red light (state 2 light), favoringPSII excitation. In state 1, the yield of fluorescencefrom PSII is higher in comparison with state 2 (for

review, see Allen and Forsberg, 2001). State transitionsare dependent on the phosphorylation of LHCII pro-teins (Bellafiore et al., 2005) and their association withPSI proteins, particularly PSI-H (Lunde et al., 2000).Under state 2 light, both the PSII core and LHCIIproteins are strongly phosphorylated, whereas thestate 1 light induces dephosphorylation of both thePSII core and LHCII phosphoproteins (Piippo et al.,2006; Tikkanen et al., 2006). In nature, however, suchextreme changes in light quality rarely occur. Theintensity of light, on the contrary, fluctuates frequentlyin all natural habitats occupied by photosyntheticorganisms, thus constantly modulating the extent ofthylakoid protein phosphorylation in a highly dy-namic manner (Tikkanen et al., 2008a).

The regulation of PSII-LHCII protein phosphoryla-tion by the quantity of light is much more complexthan the regulatory circuits induced by the state 1 andstate 2 lights. Whereas changes in light quality inducea concurrent increase or decrease in the phosphoryla-tion levels of both the PSII core (D1, D2, and CP43)and LHCII (Lhcb1 and Lhcb2) proteins, the changes inwhite light intensity may influence the kinetics of PSIIcore and LHCII protein phosphorylation in higherplant chloroplasts even in opposite directions (Tikkanenet al., 2008a). Indeed, it is well documented that lowlight (LL; i.e. lower than that generally experiencedduring growth) induces strong phosphorylation ofLHCII but relatively weak phosphorylation of the

1 This work was supported by the Academy of Finland (projectno. 118637), the Finnish Graduate School in Plant Biology, and theEuropean Union (FP7 Marie Curie Initial Training Network projectno. 215174).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Eva-Mari Aro ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[OA] Open Access articles can be viewed online without a sub-scription.

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PSII core proteins. Exposure of plants to high light(HL) intensities, on the contrary, promotes the phos-phorylation of PSII core proteins but inhibits theactivity of the LHCII kinase, leading to dephosphor-ylation of LHCII proteins (Rintamaki et al., 2000; Houet al., 2003).

Thylakoid protein phosphorylation induces dy-namic migrations of PSII-LHCII proteins along thethylakoid membrane (Bassi et al., 1988; Iwai et al.,2008) and modulation of thylakoid ultrastructure(Chuartzman et al., 2008). According to the traditionalstate transition theory, the phosphorylation of LHCIIproteins decreases the antenna size of PSII and in-creases that of PSI, which is reflected as a quenchedfluorescence emission from PSII. Alternatively, subse-quent dephosphorylation of LHCII increases the an-tenna size of PSII and decreases that of PSI, which inturn is seen as increased PSII fluorescence (Bennettet al., 1980; Allen et al., 1981; Allen and Forsberg,2001). This view was recently challenged based onstudies with thylakoid membrane fractions, revealingthat modulations in the relative distribution of excita-tion energy between PSII and PSI by LHCII phosphor-ylation specifically occur in the areas of granamargins, where both PSII and PSI function under thesame antenna system, and the energy distributionbetween the photosystems is regulated via a moresubtle mechanism than just the robust migration ofphosphorylated LHCII (Tikkanen et al., 2008b). It hasalso been reported that most of the PSI reaction centersare located in the grana margins in a close vicinity toPSII-LHCII-rich grana thylakoids (Kaftan et al., 2002),providing a perfect framework for the regulation ofexcitation energy distribution from LHCII to both PSIIand PSI.

When considering the natural light conditions, theHL intensities are the only known light conditions thatin higher plant chloroplasts specifically dephosphor-ylate only the LHCII proteins but not the PSII coreproteins. However, such light conditions do not lead toenhanced function of PSII. Instead, the HL conditionsstrongly down-regulate the function of PSII via non-photochemical quenching of excitation energy (NPQ)and PSII photoinhibition (for review, see Niyogi, 1999).On the other hand, after dark acclimation of leaves andrelaxation of NPQ, PSII functions much more effi-ciently when plants/leaves are transferred to LL de-spite strong phosphorylation of LHCII, as comparedwith the low phosphorylation state of LHCII upontransfer to HL conditions.

The delicate regulation of thylakoid protein phos-phorylation in higher plant chloroplasts according toprevailing light intensity is difficult to integrate withthe traditional theory of state transitions (i.e. theregulation of the absorption cross-section of PSII andPSI by reversible phosphorylation of LHCII). More-over, besides LHCII proteins, reversible phosphoryla-tion of the PSII core proteins may also play a role indynamic light acclimation of plants. Recently, wedemonstrated that the PSII core protein phosphoryla-

tion is a prerequisite for controlled turnover of the PSIIreaction center proteinD1 upon photodamage (Tikkanenet al., 2008a). This, however, does not exclude thepossibility that the strict regulation of PSII core proteinphosphorylation is also connected to the regulation oflight harvesting and photosynthetic electron transfer.Moreover, the interactions between PSII and LHCIIprotein phosphorylation, nonphotochemical quench-ing, and cyclic electron flow around PSI in the regu-lation of photosynthetic electron transfer reactionsremain poorly understood. To gain a deeper insightinto such regulatory networks, we explored the effectof strongly fluctuating white light on chlorophyll(chl) fluorescence in Arabidopsis (Arabidopsis thali-ana) mutants differentially deficient in PSII-LHCIIprotein phosphorylation and/or the regulatory systemsof NPQ.

RESULTS

STN7 and STN8 Kinases Have MutualRegulation Pathways

Figure 1 demonstrates how the phosphorylationpatterns of the PSII core (D1, D2, and CP43) andLHCII proteins are dynamically regulated by the STN7and STN8 kinases upon varying the light intensity.Wild-type and stn mutant plants were subjected tolight treatments changing in intensity from LL (30mmol photons m22 s21) to HL (1,000 mmol photons m22

s21) every 30 min for a total duration of 180 min.Phosphorylation level of the PSII core proteins (espe-cially CP43) was low under LL in the wild type and thestn8 mutant lacking the STN8 kinase and having onlythe STN7 functional, whereas the LHCII protein phos-phorylation was relatively strong. In contrast, alwayswhen HL was switched on, the phosphorylation ofLHCII repeatedly decreased and the phosphorylationlevel of the PSII core proteins increased. This demon-strated the presence of two differently regulatedSTN7-dependent phosphorylation pathways, one spe-cific for LHCII proteins and the other specific for CP43.The stn7 mutant, containing only a functional STN8,did not phosphorylate LHCII but phosphorylated thePSII core proteins more strongly than the wild type,especially under LL. This is in accordance with ourprevious finding indicating that, particularly underLL, the photosynthetic electron transfer chain (ETC) instn7 is more reduced than in the wild type (Tikkanenet al., 2006). As mentioned above, in the stn8 mutant,the STN7 kinase strongly phosphorylated LHCII butalso relatively well the PSII core proteins, especiallyCP43. However, the STN7 kinase in the stn8 mutantgradually lost its capacity for phosphorylation of thePSII core proteins in the course of the 180-min exper-iment, probably due to breakdown of the STN7 en-zyme (Lemeille et al., 2009). In contrast, the capabilityof the STN8 kinase to phosphorylate the PSII coreproteins in the stn7 mutant remained unaltered

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throughout the entire experiment under fluctuatinglight. It is worth noting that light conditions used inour experiments were not high enough (Pursiheimoet al., 2001) to induce the phosphorylation of minorPSII-LHCII proteins like CP29.Feeding of leaves with Glc in darkness activated

both the STN7 and STN8 kinases (Fig. 1B). Suchmetabolic manipulation of the kinase activities indarkness resulted in the wild type in a phosphoryla-tion pattern that was comparable to that induced by

the state 2 light (Fig. 1C), which strongly reduces thephotosynthetic ETC but does not induce the feedbackdown-regulation of the STN7 kinase by overreductionof chloroplast stroma (Rintamaki et al., 2000). Thisexperiment highlighted the fact that thylakoid proteinphosphorylation is not only regulated by the intensityand quality of light. Under natural conditions, thephosphorylation status of the thylakoid proteins is setby cooperative regulation by the prevailing light con-ditions and the metabolic status of the chloroplast(Hou et al., 2003).

Effect of Thylakoid Protein Phosphorylation on AntennaSystems of PSII and PSI

As discussed above, the modifications of PSII-LHCIIprotein phosphorylation in natural environments crit-ically differ from those occurring upon the well-documented state 1-state 2 transitions induced byartificial light conditions. This prompted us to explorewhether the changes in white light intensity, and theassociated modifications in PSII-LHCII protein phos-phorylation, are coupled to short-term changes infunctional properties of the PSI and PSII antennasystems. To this end, we first applied state 1-state 2lights to study the 77 K chl a fluorescence emissionfrom PSII (CP43 and CP47) and PSI in wild-typeplants. As reported earlier (Allen and Forsberg,2001), the state 2 light, which strongly activates thephosphorylation of both the PSII core and LHCIIproteins (Fig. 1C), strongly increased the relative fluo-rescence from PSI (Fig. 2A).

In order to investigate the effects of white light-induced phosphorylation on the antenna systems ofPSII and PSI, the dark-acclimated wild-type, stn7, stn8,stn7 stn8, and npq4 plants were subjected to LL and HLfor 60 min and the 77 K chl a fluorescence emissionfrom PSII and PSI was recorded from isolated thyla-koids (Fig. 2). Opposite to the effects induced by state1 and state 2 light conditions (Fig. 2A), the effects ofdifferent light intensities on the relative fluorescenceemission from PSII and PSI in wild-type plants wereonly minor (Fig. 2B), even though distinct modulationsin the phosphorylation of the PSII and LHCII proteinswere recorded under these conditions (Fig. 1A; Aroand Ohad, 2003). As compared with the wild type (Fig.2B), the stn7 single mutant and the stn7 stn8 doublemutant showed slightly reduced relative fluorescencefrom PSI both after dark acclimation and after illumi-nation with LL (Fig. 2, C and E). After exposure to HL,however, no differences from the wild type wererecorded (Fig. 2, C and E). The stn8 mutant behavedlike the wild type under all light intensities tested (Fig.2D). Interestingly, the npq4 mutant (Fig. 2F) showedsomewhat lower relative fluorescence from PSI thanthe wild type after treatment in the dark or under HL,whereas LL-exposed npq4 mutant did not differ fromthe wild type.

According to the state transition theory (Allen andForsberg, 2001), illumination by state 2 light induces

Figure 1. Regulation of thylakoid protein phosphorylation by the STN7and STN8 kinase pathways. A, Phosphorylation of thylakoid proteins inwild-type (WT) plants and the stn7, stn8, and stn7 stn8mutants exposedto 30 and 1,000 mmol photons m22 s21 in 30-min intervals. B,Phosphorylation of thylakoid proteins in wild-type plants and thestn7, stn8, and stn7 stn8 mutants after 16 h of dark incubation in theabsence (2) and presence (+) of 0.25 mM Glc. C, Phosphorylation ofthylakoid proteins in the wild type after 1 h of treatment with far-redlight (state 1) and red light (state 2). Phosphorylation of thylakoidproteins was determined by immunoblotting with P-Thr antibody, and0.2 mg of chl was loaded in each well. P-CP43, P-D2, P-D1, andP-LHCII represent phosphorylated forms of the PSII core proteins CP43,D2, and D1 and the LHCII proteins Lhcb1 and Lhcb2. Representativedata from three independent experiments are shown.

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the migration of phosphorylated (P)-LHCII from PSIIin the grana membranes to PSI in the stroma lamellae,which should be detectable by measuring the 77 K chla fluorescence excitation spectra of PSII (CP43 andCP47) and PSI. Fluorescence excitation spectra giveinformation about pigments associated with the exci-tation of PSII and PSI and reveal changes in theabsorption cross-section of PSII and PSI due to rear-rangements of the antenna system upon illuminationof leaves with different light qualities and quantities.Accordingly, the phosphorylation of LHCII and mi-gration into contact with PSI should be detected asenhanced relative fluorescence emission from PSI atwavelengths associated with light absorption byLHCII (chl b, 480 nm; carotenoids, ,480 nm) as com-pared with the nonphosphorylated state, where onlychl a with maximal absorption at 440 nm takes care ofthe excitation of PSI.

As expected, the state 1 light-acclimated wild-typeleaves showed nearly similar 77 K chl a fluorescenceexcitation spectra of PSII (CP43 and CP47) and PSI asleaves acclimated to darkness (Fig. 3). State 2 light, incontrast, drastically changed the excitation spectra,and accordingly the absorption cross-sections, of bothphotosystems. Despite the fact that illumination understate 2 light clearly increased the relative excitation ofPSI (i.e. the fluorescence emission from PSI; Fig. 2), theconcurrent analysis of the PSI 77 K chl a fluorescenceexcitation spectrum revealed no specific enhancementin the excitation by wavelengths absorbed by chl b(around 480 nm) and thus the LHCII antenna system(Fig. 3). This result contradicts the idea that the in-crease in PSI 77 K chl a fluorescence emission was onlydue to a specific migration of P-LHCII from PSII to PSI(for review, see Allen and Forsberg, 2001). Notewor-thy, the illumination by state 2 light changed in a

Figure 2. The 77 K chl a fluorescence emitted fromCP43 (685 nm), CP47 (695 nm), and PSI (730 nm).The 77 K chl a fluorescence was recorded in the wildtype (WT; B), stn7 (C), stn8 (D), stn7 stn8 (E), andnpq4 (F) after 16 h of dark incubation and subsequent1-h exposure to 30 or 1,000 mmol photons m22 s21.Wild-type plants were also exposed to far-red light(state 1) and red light (state 2) for 1 h following the16-h dark incubation (A). All thylakoid samples wereprepared in the presence of 10 mM NaFand diluted to10 mg chl mL21 before the measurements. Data aremeans of three independent measurements, and errorbars represent SD. [See online article for color versionof this figure.]

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similar way the 77 K chl a fluorescence excitationspectra of both photosystems, CP43 and CP47, as wellas PSI (Fig. 3). This provides evidence that the migra-

tion of a specific LHCII antenna fraction from onereaction center into contact with the other cannot bethe sole mechanistic basis for the red light-inducedtransition to state 2 (see “Discussion”).

The 77 K chl a fluorescence of wild-type leaves orany of the mutants acclimated to different white lightintensities for 1 h (dark, LL, and HL) did not revealremarkable changes in the 77 K chl a fluorescenceexcitation spectra of either PSII or PSI (Fig. 3), despitedrastic differences in PSII-LHCII protein phosphoryla-tion levels (Fig. 1). Only a slight increase in theexcitation of PSI by wavelengths associated with chlo-rophyll b (480 nm) absorption was observed at LL inthe wild type and stn8 and npq4 mutants, but not instn7 and stn7 stn8 mutants, indicating the dependenceon LHCII phosphorylation (Fig. 3). The excitationproperties of PSII and PSI in the npq4 mutant weresimilar to those in the wild type after treatment ofleaves in darkness and LL. However, after HL treat-ment, the total fluorescence yield was dramaticallyreduced (data not shown), the shape of the spectrumbecame less defined, and high variation between themeasurements was detected (data not shown). Thisindicates that excess light damages the structure andfunction of the light-harvesting machinery if the PsbSprotein is missing (npq4 mutant).

Dynamics of Fluorescence Parameters Fm# and Fs# underFluctuating Light in the Wild Type and in MutantsDiffering in Thylakoid Protein Phosphorylation, NPQ,and Cyclic PSI Electron Transfer

In order to clarify the roles of LHCII and PSII coreprotein phosphorylation in dynamic acclimation ofthe photosynthetic electron transfer reactions, we sub-jected Arabidopsis wild-type and mutant leaves tochanging light intensities. Under such conditions, thecapability to dynamically adjust the light-harvestingand electron transfer properties in response to a givenlight intensity is required. During the experiment, thecapability of the photosynthetic machinery for dy-namic light acclimation was monitored by measuringthe maximal and steady-state fluorescence in the light-acclimated state, denoted as Fm# and Fs#, respectively.The yield of Fm#wasmeasured in order to elucidate thequenching state of PSII (NPQ), with high Fm# indicatinghigh efficiency of excitation energy transfer from LHCto photosystems. Fs#was measured in order to estimatethe redox state of the ETC, with low Fs# indicatingfluent electron flow from PSII to PSI and further to thedownstream electron acceptors (Baker, 2008).

Measurements were conducted with the wild typeand the stn mutants (stn7, stn8, and stn7 stn8) but alsowith several other mutants characterized by knowndisabilities in the light acclimation processes. The npq4mutant was the most important control for the exper-iments. It lacks the feedback deexcitation of PSII (qE),which is the main component of NPQ (Niyogi et al.,2005). Another key control was the npq4 stn7 doublemutant (Frenkel et al., 2007), which besides the

Figure 3. The 77 K chl a fluorescence excitation spectra of CP43 (685nm), CP47 (695 nm), and PSI (730 nm) from the wild type (WT), stn7,stn8, stn7 stn8, and npq4 recorded after incubation of plants first for 16h in darkness and then for 1 h under 30 (LL) or 1,000 (HL) mmol photonsm22 s21. Wild-type plants were also exposed to far-red light (state 1)and red light (state 2) for 1 h after 16 h of dark incubation. All spectrahave been normalized to the excitation by chl a at 440 nm. The peak at480 nm represents the excitation of the fluorescence emitters by chl b,and the excitation by longer wavelengths represents the excitation bycarotenoids. Plant treatments and thylakoid isolation were performedas in Figure 2. [See online article for color version of this figure.]



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feedback deexcitation also lacks LHCII phosphoryla-tion and is thus deficient in regulatory eventsoriginating from cooperation between LHCII phos-phorylation and the PsbS-dependent qE. npq1, thethird NPQ mutant used here, lacks the deepoxidaseenzyme responsible for the conversion reaction ofviolaxanthin to zeaxanthin (Niyogi et al., 1998) andwas used to clarify the relation between the xantho-phyll cycle and the other acclimation processes inves-tigated. Regulation of light reactions by proteinphosphorylation is based on sensing the redox stateof the ETC chain (Aro and Ohad, 2003; Lemeille et al.,2009), whereas the regulation of qE is based on sensingthe lumenal protonation (Niyogi, 1999). The develop-ment of lumenal protonation can be uncoupled fromthe water-splitting activity of PSII by cycling electronsaround PSI. Such cyclic electron transfer (CET) pro-vides a flexible mechanism to regulate the amount ofNPQ independently of PSII function. For this reason,also the pgr5 (Munekage et al., 2002) mutant with im-paired CETreactions was included in themeasurements.

For the experiments, wild-type and mutant plantswere grown under 100 mmol photons m22 s21 (growthlight [GL]). The leaves were detached 2 h after onset ofthe photoperiod, placed in the measuring holder cov-ered with wet tissue, and acclimated in darkness for 10min. Thereafter, leaves were first subjected for 7 min toLL (67 mmol photons m22 s21), which was below theGL intensity and maintained efficient energy transferfrom LHCII to fulfill the requirements of metabolicelectron sinks. LL was followed by 2 min under HL(590 mmol photons m22 s21), inducing down-regulationof LHCII efficiency, and then again 7 min under LL,which restored the efficiency of LHCII in energytransfer to reaction centers. After this first illuminationcycle, the leaves were returned to darkness for 10 min.The second illumination cycle consisted of moderatelyHL (340 mmol photons m22 s21) for 7 min, very LL(37 mmol photons m22 s21) for 2 min, and moderatelyHL again for 7 min to further elucidate the relationshipbetween the efficiency of LHCII in energy transduc-tion and the fluency of electron flow upon varyinglight intensities and time scales. Fm# and Fs#, normal-ized to the initial Fm# and Fs# values, were recordedduring the illumination cycles.

Wild-type plants demonstrated a highly flexibleregulation of chl a fluorescence upon shifts in lightintensity, in agreement with earlier reports (for re-views, see Baker, 2008; Eberhard et al., 2008). Briefly,the shift of wild-type leaves from darkness to moder-ately low actinic light resulted in an approximately40% decrease in the yield of Fm#. This sudden dropwas followed by about 20% restoration of Fm#, afterwhich the Fm# gradually evened out to the steady-statelevel of fluorescence characteristic of this light inten-sity. The following 2-min HL peak further quenched30% from the remaining fluorescence. However, im-mediately after turning off the HL, Fm#was restored tothe level preceding the HL pulse. During the subse-quent 10-min dark incubation, the Fm# value recovered

to the initial level. After the dark period, when mod-erately high actinic light (340 mmol photons m22 s21)was turned on, the yield of Fm# was quenched withsimilar kinetics detected upon the first moderately LLphase of the experiment (67 mmol photons m22 s21),but due to a higher light intensity, the fluorescenceyield stabilized to a somewhat lower level. During asubsequent 2-min pulse of very LL (37 mmol photonsm22 s21), Fm# was mostly restored, but when themoderately HL was turned on, it dropped again tothe same level recorded earlier at this light intensity.Fs# remained fairly constant throughout the illumina-tion cycles of wild-type leaves. Collectively, theseexperiments demonstrated that the wild type main-tained fluent electron flow from PSII to downstreammetabolism under fluctuating light intensities, likelydue to intact mechanisms to control thermal dissipa-tion of excess energy and the excitation energy distri-bution between PSII and PSI.

Comparison of the npq4 mutant with the wild typeconfirmed that the dynamic regulation of Fm# is pri-marily dependent on the PsbS protein-dependent qE(Li et al., 2002). The npq4 mutant was nearly inert anddid not show dynamic regulation of Fm#. However,despite the incapability for rapid changes in Fm#, thenpq4 mutant could gradually decrease the Fm# duringthe experiment. This slow induction of quenching wasnot related to state transition (qT), because the npq4stn7 double mutant did not differ from the npq4 singlemutant in this respect. The yield of Fm# was similar innpq4 and npq4 stn7 throughout the entire experimentunder fluctuating light. This demonstrates that in theabsence of the PsbS protein-dependent feedback deex-citation, the phosphorylation of LHCII did not haveany effect on the quenching properties of Fm# (NPQ).Unlike the npq4 mutant, the npq1 mutant showed awild-type pattern in the regulation of Fm#, although theamplitudes of the changes in fluorescence yield werediminished in the mutant as compared with the wildtype (Fig. 4). This was a clear indication that thexanthophyll cycle is not required to induce the PsbS-dependent quenching but that it strongly modulatesthe strength of thermal dissipation, as was shownrecently (Kalituho et al., 2007).

The npq4 mutant maintained Fs# at a similar level asthe wild type when light intensity did not exceed theGL intensity, thus maintaining fluent electron flow.Moreover, when light intensity increased from 67 to590 mmol photons m22 s21 for 2 min, the npq4 mutantcould maintain Fs# stable, indicating no disturbance inelectron flow. However, when leaves were shiftedfrom darkness directly to light intensity above theGL, the npq4mutant could not maintain Fs# at a similarlevel as the wild type. Because Fs# remained at anequal level in the wild type and npq4 under LLconditions, and neither did the short shifts in lightintensity affect the level of Fs# in the npq4 mutant, it isconceivable that the light-harvesting efficiency of bothphotosystems is similarly effected by qE, and Fs# in-creased onlywhen electron flowwas limited by electron

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acceptors after PSI. Also, the npq1 mutant had difficul-tiesmaintaining Fs# at a similarly stable level as the wildtype upon prolonged exposure to light intensity abovethe GL. This emphasizes the importance under excesslight of both the PsbS protein-dependent and the xan-thophyll cycle-dependent mechanisms in regulating theefficiency of LHCII to maintain fluent electron flowfrom light reaction to downstream metabolism.The stn7, stn8, and stn7 stn8 kinase mutants were all

capable of efficiently quenching Fm# when the lightintensity suddenly increased (Fig. 4). The initialquenching of Fm#was even stronger in the stnmutantsthan in the wild type. However, despite rapid andefficient quenching of Fm# in all the kinase mutants, thestn7 and stn7 stn8 mutants had severe difficulties inmaintaining the low Fm# level after the rapid inductionphase of Fm# quenching. Intriguingly, the rapid andstrong quenching of Fm# coupled to the incapability ofmaintaining the induced quenching level severelyamplified the difference between the low and highquenching states of LHCII in the stn7 and stn7 stn8mutants as compared with the wild type and stn8.Upon the initial induction phase of Fm# quenching

(NPQ), the wild type and stn7, stn8, and stn7 stn8mutants showed equal levels of Fs#. However, imme-diately when Fm# became partially recovered, the stn7

mutant and even more clearly the stn7 stn8 mutantshowed distinctly higher Fs# as compared with thewild type and stn8. This demonstrated that the lack ofSTN7-dependent PSII-LHCII protein phosphorylationled to a redox imbalance of ETC, especially underLL when NPQ was low and the excitation energytransfer from LHCII to photosystems was highlyefficient. Thus, the importance of the LHCII proteinphosphorylation-dependent regulation of excitationenergy distribution to the two photosystems becomesmanifested under low NPQ and, accordingly, the highefficiency state of LHC.

The impact of cyclic electron flow on the regulationof Fm# and Fs# was tested with the pgr5 mutant(Munekage et al., 2002; DalCorso et al., 2008), whichhas reduced capacity to perform cyclic electron flowaround PSI as compared with the wild type. Eventhough the exact role of the PGR5 protein in cyclicelectron flow is not fully understood, the use of thepgr5 mutant was expected to provide important infor-mation concerning the roles of multiple electron trans-fer routes in the regulation of PSII via NPQ andthylakoid protein phosphorylation. Interestingly, thepgr5 mutant was nearly as inert as npq4 in performingrapid changes in Fm# levels in fluctuating light. How-ever, despite the enhanced efficiency of LHCII (high

Figure 4. Chl a fluorescence as a response tochanging light intensity. Intact leaves from wild-type (WT), stn7, stn8, and stn7 stn8 plants andnpq4, npq4 stn7, npq1, and pgr5 control plantsgrown under 120 mmol photons m22 s21 weredetached and placed in a leaf holder 2 to 4 h afterGL was turned on in the morning. The measure-ments were started after 10 min of dark incuba-tion by turning the light on, and the fluorescenceparameters Fs# and Fm# were monitored duringillumination of leaves with programmed changesin light intensity as indicated. Value 1 was givento the first measuring points of Fm# and Fs#, and allother values were normalized against this initialvalue. Fluorescence curves were highly repro-ducible both for wild-type and mutant plants.Representative curves from at least 10 measure-ments are shown for the wild type and the mu-tants. [See online article for color version of thisfigure.]



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Fm#) under LL, the level of Fs# in pgr5 was low,indicating oxidized ETC as compared with the wildtype under limiting light intensities. Even under HL,the induction of NPQ in pgr5 was very low, but incontrast to LL Fs# stabilized at a higher level in pgr5 ascompared with the wild type. These experimentsindicated that under LL, the wild-type plants requiredthe PGR5-dependent CET to reduce intersystem ETC.However, when the light intensity increased, PGR5-dependent activity was required for the developmentof NPQ, thus decreasing the rate of electron flow.

Phosphorylation-Dependent Regulation of Electron

Transfer Has a Great Impact on Plant Growth underFluctuating Light

None of the mutants used here showed any visiblegrowth phenotype under constant LL, moderate light,or HL conditions (data not shown). To test whether themisregulation of excitation energy distribution andelectron transfer, as observed for some mutants in theshort-term fluctuating light experiments (Fig. 4), hasany specific impact on the phenotype when plants aregrown under fluctuating light intensity, we placedwild-type plants and stn7, stn8, stn7 stn8, npq4, npq4stn7, npq1, and pgr5 mutant plants to grow under LL,which was interrupted by frequent HL peaks. Thepurpose of using such a growth condition was tomaximally challenge the dynamic regulation of theexcitation energy distribution and electron transferreactions and to relate the impact of these capacitieson the growth of plants. The stn7 and stn7 stn8 plantsgrew very slowly as compared with the wild type, andpgr5 showed a lethal phenotype (Fig. 5). In contrast, thestn8, npq4, and npq1 mutants did not show any visiblephenotype, and stn7 npq4 showed a less profoundphenotype than stn7 (Fig. 5). As shown in Figure 4,short interruptions of LL illumination phases with HLpulses strongly disturb the redox balance of ETC in stn7and stn7 stn8 but not in stn8, npq4, and npq1 mutants,providing evidence that the regulation of excitationenergy distribution by the STN7-dependent phosphor-ylation cascade has a vital role for plant growth underfluctuating light. The pgr5 mutant showed severe dif-ficulties in maintaining redox balance of electron trans-fer reactions, yet the influence of short HL pulses wasnot apparent. Nevertheless, the pgr5 mutant had alethal phenotype under fluctuating light, suggestingthat the genotype probably has more influence on thestromal redox state than on the redox state of ETC.

DISCUSSION

Reversible PSII-LHCII Protein Phosphorylation Induced

by White Light of Different Intensity Does Not Result inState Transitions

Photosynthetic organisms cope with constantlychanging light conditions via highly flexible regula-tion mechanisms that adjust the light absorption and

photosynthetic processes according to metabolic andenvironmental cues. On the one hand, plants havemechanisms to enhance light harvesting when photo-synthesis and growth are limited by the availability oflight. On the other hand, when the light intensityexceeds the capacity by which the reducing energy canbe consumed by metabolic reactions, the excess exci-tation energy becomes dissipated as heat via NPQ.Moreover, even though the balancing of the light-harvesting capacity between PSII and PSI via statetransitions has long been recognized as a regulatorymechanism to optimize the relative function of PSIIand PSI (Allen and Forsberg, 2001; Haldrup et al.,2001; Rochaix, 2007; Kargul and Barber, 2008), itsbiological significance under different natural lightconditions has remained controversial. The mechanis-tic basis of state transitions is not fully understood, yetrecent data (Tikkanen et al., 2008b) indicated thatregulation takes place in grana margins, where themajority of PSI reaction centers are located in close

Figure 5. A, Visual phenotype of wild-type (WT) plants and stn7, stn8,stn7 stn8, npq4, stn7 npq4, npq1, and pgr5mutant plants grown underfluctuating light for 50 d. B, Rosette diameter of wild-type, stn7, stn8,and stn7 stn8 plants measured 30, 40, and 50 d after sowing. Data aremeans of five plants, and error bars represent SD. Similar differences ingrowth rates of wild-type and mutant plants were obtained also in twoother growth experiments. Five minutes of moderate LL (50 mmolphotons m22 s21) and 1 min of NPQ-inducing HL (500 mmol photonsm22 s21) were continuously following each other during the lightperiod of 12 h followed by a 12-h dark period. [See online article forcolor version of this figure.]

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vicinity to PSII-LHCII-rich grana thylakoids (Kaftanet al., 2002).In early studies, the induction of state transitions by

differential excitation of the two photosystems wasfound to correlate with changes in the level of LHCIIprotein phosphorylation (Bennett et al., 1980; Allenet al., 1981). More recently, studies with the stt7 kinasemutant of Chlamydomonas reinhardtii and the stn7 ki-nase mutant of Arabidopsis demonstrated that LHCIIphosphorylation indeed is a prerequisite for statetransitions to occur (Depege et al., 2003; Bellafioreet al., 2005). These classical state transition studies,however, are based on selective excitation of the tworeaction centers by different qualities of light andtherefore are hard to integrate with studies on themodulation of light harvesting and excitation of thetwo photosystems under natural environments, wherethylakoid protein phosphorylation is dually controlledby the intensity of light and the downstreammetabolicreactions. Indeed, we found no correlation betweenwhite light-induced modulations in the level of LHCIIprotein phosphorylation and the extent of state tran-sition (Figs. 1 and 2).While the state 1 and state 2 lights have similar

effects on the phosphorylation of both the PSII coreand LHCII proteins, either decreasing or enhancingthe phosphorylation of both of them at the same time(Piippo et al., 2006; Tikkanen et al., 2006), the changesin light intensity sometimes have completely oppositeeffects on the PSII core and LHCII protein phosphor-ylation (Fig. 1), also suggesting that one phosphoryla-tion pathway may negatively regulate the other.Under white light, the strongest level of LHCII proteinphosphorylation is typically observed upon a shift ofplants from normal GL to LL conditions (Pursiheimoet al., 2001). On the contrary, when the light absorptionby pigments exceeds the capacity of carbon assimila-tion to utilize converted light energy, the stromal sideof PSI becomes overreduced and induces the down-regulation of LHCII phosphorylation at the time whenthe PSII core protein phosphorylation reaches maxi-mal intensity (Rintamaki et al., 2000; see model in Fig.6). Moreover, the stromal overreduction not only in-activates the STN7 kinase but also accelerates thedegradation of the enzyme (Lemeille et al., 2009),thus providing an additional level of regulation inlight harvesting.Despite of strong fluctuations in phosphorylation of

the PSII core and LHCII proteins with respect tochanges in light intensity (Fig. 1), the relative 77 Kchl a fluorescence emission (Fig. 2) and the 77 K chl afluorescence excitation spectra (Fig. 3) of PSII and PSIremained nearly unaffected, implying no induction ofstate transitions. In the stn7 and stn7 stn8 mutants, therelative 77 K chl a fluorescence from PSI as comparedwith the wild type was slightly reduced under LL, yetthe normalized 77 K chl a fluorescence excitationspectra remained quite similar to that in the wildtype. These findings demonstrate that in the wild type,when the proper control of the PSII-LHCII protein

phosphorylation is present, the relative distribution ofexcitation energy between the two photosystems isquantitatively rather similar in intact thylakoid sys-tems under any natural light condition with or withoutphosphorylation of LHCII. However, the abnormalphosphorylation of the PSII-LHCII proteins in the stn7and stn7 stn8 mutants decreases the excitation energyflow from the entire antenna system, covering a widerange of excitation wavelengths, to the PSI reactioncenter. Thus, the fluctuations in phosphorylation of thePSII-LHCII proteins, as induced by changes in lightintensity, do not remarkably change the relative exci-tation of PSII and PSI but more likely function tomaintain equal excitation of both reaction centers.

Role of the STN7 Kinase in the Regulatory Network ofExcitation Energy Distribution and ElectronTransfer Reactions

Using the stn kinase mutants, we demonstrate herethat under moderate light intensities, plants adjustNPQ to slightly different levels depending on thephosphorylation status of the PSII-LHCII proteins(Fig. 4). Thus, the phosphorylation is possibly in-volved in regulation of the extent of excitation energydonated from the light-harvesting antenna to bothreaction center complexes. Under natural fluctuatinglight environments, the efficiency of light reactionsneeds nonstop regulation in order to keep the balancewith the needs and restrictions of downstream metab-olism. This can occur either by regulation of lightabsorption efficiency or by regulation of the efficiencyby which the absorbed light energy is used in photo-synthesis. The PsbS protein-dependent NPQ gives themechanistic framework for regulation of the efficiencyto use the absorbed light energy (Niyogi, 1999).

Using the stn and npq mutants, we addressed thequestion of how the PSII-LHCII protein phosphoryla-tion-dependent balancing of excitation energy distri-bution between the reaction centers cooperates withthe PsbS-dependent quenching process (Fig. 6). UnderLL, the stn7 mutant has a high-light-mimicking phos-phorylation pattern of LHCII, which leads to a situa-tion where PSII excitation is favored over PSIexcitation (Fig. 6B) and plants exhibit a strongly re-duced intersystem ETC (Fs# in Fig. 4; Bellafiore et al.,2005; Tikkanen et al., 2006). In contrast to the stn7mutant, the stn8 mutant strongly phosphorylatesLHCII, weakly phosphorylates the PSII core proteins(Fig. 1), and has slightly less reduced ETC (Fs#) ascompared with the wild type (Fig. 4). Despite theopposing behavior of the stn single mutants, the stn7stn8 double mutant resembled the stn7mutant, but theincapability of maintaining the redox balance waseven more obvious, thus stressing the importance ofboth kinase pathways in the regulation of electrontransfer reactions.

The STN7-dependent regulation to balance the ex-citation energy distribution between PSII and PSI isrequired particularly under LL intensities, when ther-



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mal dissipation of excitation energy is low and LHCIItransfers excitation energy efficiently to reaction cen-ters (Fig. 6, A and B). When light intensity increases

and concomitantly the efficiency of LHCII becomesdown-regulated via NPQ, the STN7 kinase-dependentregulation loses its importance in regulation of excita-

Figure 6. Model depicting the differential roles of PSII-LHCII protein phosphorylation in the regulation of excitation energydistribution between PSII and PSI. Such regulation mostly occurs in grana margins where PSII and PSI are in close proximity(Tikkanen et al., 2008a) and depends on light conditions used for the induction of LHCII phosphorylation. A, Under LL, the PsbSprotein is deprotonated and the xanthophyll cycle is inactivated, allowing efficient transfer of excitation energy from LHCII toPSII and PSI. B, Under such conditions, the STN7-dependent high LHCII and low PSII core protein phosphorylation areprerequisites for balanced excitation energy distribution to PSII and PSI, and the lack of the STN7 kinase leads to overexcitationof PSII as compared with PSI. C, Upon increase in light intensity, PsbS gets protonated and the xanthophyll cycle is activated,leading to efficient thermal dissipation of excitation energy. LHCII is dephosphorylated under such a low efficiency state ofLHCII, and no STN7 kinase-dependent mechanism to maintain excitation balance between PSII and PSI is required due to thefact that the majority of the absorbed excitation energy becomes dissipated as heat. D, Under traditional state 1 light (far red),both the PSII core and LHCII proteins are dephosphorylated, PSII excitation by LHCII is favored, and thermal dissipation ofexcitation energy is at a minimum. Nevertheless, in natural conditions under the canopy, where far-red light is enriched, theexcitation of PSII and PSI is in balance due to the fact that PSI absorbs far-red light more efficiently than PSII. hv, Light energy. E,Shift to state 2 light (red) induces strong phosphorylation of both the PSII core and LHCII proteins, yet the efficiency of LHCII is ashigh as under state 1 light (low NPQ). This unnatural state with strong overall phosphorylation of both the PSII core and LHCIIproteins combined with high-efficiency LHCII leads to very efficient excitation energy transfer from LHCII to PSI and thus to atransfer to state 2, where PSI excitation is strongly favored over PSII.

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tion energy distribution (Fig. 6C). In the stn7 npq4mutant, incapable of down-regulating the antennaefficiency, the stn7 mutation-induced excitation imbal-ance is not dependent on light intensity and exists alsoat HL conditions.The absence of NPQ, as such, did not affect the

redox balance of ETC under LL or upon short HLpulses, indicating that NPQ modulates the efficiencyof both photosystems to the same extent and does notaffect the relative distribution of excitation energybetween PSII and PSI. However, when the exposure ofnpq4 to HL intensity is prolonged, the capacity ofmetabolic sinks to accept electrons becomes crucial forelectron flow and the situation of overreduction ofETC is easily attained (Fig. 4). This brings up aquestion about the “unbalancing” force that makesthe STN7 kinase-dependent regulation so necessary inmaintaining the balance of equal excitation energydistribution to both photosystems under low andmoderate photon fluence rates. It is well known thatthe distribution of excitation energy between PSII andPSI changes depending on salt concentration, whichalso modulates the stacking-unstacking-restacking dy-namics of the thylakoid membrane (Murata, 1969,1971). Changes in grana width and extent of stackingalso occur depending on light conditions and areclosely related to the kinetics of PSII core and LHCIIprotein phosphorylation (Rozak et al., 2002; Chuartzmanet al., 2008), yet the physiological significance of such arelationship has remained elusive. It is conceivablethat the changes in granal stacking in chloroplastsregulate the light absorption capacity of the thylakoidmembrane according to the light intensity. As anirrevocable consequence of such a modulation of thy-lakoid ultrastructure, the lateral segregation of PSII,LHCII, and PSI also changes and affects the excitationbalance between PSII and PSI. This is reflected in theredox state of the electron transfer components, whichin turn regulates the phosphorylation of the PSII-LHCII proteins (Vener et al., 1998; Zito et al., 1999;Aro and Ohad, 2003). It is likely that the dynamicregulation of PSII core and LHCII protein phosphor-ylation by the STN7 kinase facilitates the movementsof PSII, LHCII, and PSI-LHCI complexes along eachother and the thylakoid membrane, enabling the main-tenance of excitation balance between the two reactioncenters upon any white light-induced configuration ofthe thylakoid ultrastructure.Similar to the stn7 single mutant and stn7 stn8

double mutant, the pgr5 mutant with impaired cyclicelectron flow (Munekage et al., 2002; DalCorso et al.,2008) could not maintain the wild-type level of NPQ.However, besides low NPQ, the pgr5 mutant also hadvery low Fs#, indicating oxidized ETC as comparedwith the wild type. In contrast to this, stn7 and stn7stn8 had very high Fs#, indicating strongly reducedETC. Thus, these data suggest that the stn kinasemutants may not be impaired in PGR5-dependentcyclic electron flow; therefore, the phosphorylation-dependent modulation of NPQ is not likely to occur

by CET-dependent lumenal protonation but rathervia regulation of linear electron transfer or some othermechanism. Common for both the stn7 and pgr5mutants is a severe difficulty in managing the electrontransfer under fluctuating light intensity (Fig. 4), andboth mutants also show strongly retarded growthunder fluctuating light, pgr5 even showing a lethalphenotype. Slow growth of the stn7 and stn7 stn8mutants indicates that the regulation of electron trans-fer via the STN7-dependent mechanism is importantfor plant growth under fluctuating light. Proper dis-tribution of excitation energy from LHCII to bothphotosystems seems to have more drastic effects onplant growth than the regulation of light-harvestingefficiency of LHCII via NPQ. Indeed, the growth ofnpq4 and npq1 was not retarded under fluctuatinglight. The drastic phenotype of pgr5 is evidently notrelated to its inability to induce NPQ.

CONCLUSION

Plants grown under constant light conditions have ahigh capacity to compensate the consequences ofmutations in genes encoding the proteins requiredfor proper acclimation to different light intensities.Under continuously fluctuating light, however, thestraightforward acclimation is not possible for mu-tants that lack the protein(s) responsible for dynamicregulation of the light-harvesting system and energydistribution between the two photosystems. The stn7mutant lacking the STN7 LHCII kinase belongs tosuch mutants and has a strongly stunted phenotypeonly when grown under strongly fluctuating lightintensity.

It is well known that LHCII phosphorylationchanges the relative excitation of the two reactioncenters in the traditional state transition experimentinvolving different qualities of light to induce revers-ible LHCII phosphorylation. It is conceivable that intandem with LHCII protein phosphorylation, the PSIIcore protein phosphorylation also takes part in thisprocess (Fig. 6). Phosphorylation of LHCII and PSII isalso dynamically regulated according to light inten-sity. However, the physiological role of such “whitelight phosphorylation” differs from traditional statetransitions. We have demonstrated here that althoughthe white light-induced changes in thylakoid proteinphosphorylation are strong, they do not change therelative excitation of PSII and PSI but instead guaran-tee equal excitation of both photosystems under vary-ing light intensities. In line with this observation, thelack of LHCII phosphorylation in the stn7 and stn7 stn8mutants leads to overexcitation of PSII.

Efficient electron transfer reactions require a bal-anced excitation energy distribution between PSII andPSI, particularly in grana margin regions. Our resultsprovide evidence that the STN7-dependent PSII-LHCII protein phosphorylation functions in balancingthe excitation energy distribution from LHCII to PSII



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and PSI, thus ensuring the functional stoichiometrybetween the photosynthetic protein complexes, espe-cially under LL when NPQ is low and LHCII effi-ciently transfers energy to photosystems. Thermaldissipation of excitation energy (NPQ), as such, doesnot affect the excitation balance between PSII and PSI.However, in the absence of the STN7 kinase, the high-efficiency LHCII (lowNPQ) overexcites PSII relative toPSI, leading to strong reduction of ETC. Due to such adomination of PSII excitation by LHCII in stn7,changes in LHCII efficiency (NPQ) according to lightintensity also predominantly affect the function ofPSII. Thus, strongly fluctuating light that constantlychanges the efficiency of LHCII (NPQ) leads to drasticfluctuations in the redox state of ETC when the STN7kinase is missing, causing the stunted growth of thestn7 mutant.

MATERIALS AND METHODS

Growth of Plants and Short-Term Light and

Glc Treatments

Wild-type Arabidopsis (Arabidopsis thaliana) ecotype Columbia and the

stn7 (Tikkanen et al., 2006), stn8 (Vainonen et al., 2005), stn7 stn8 (Bonardi et al.,

2005), npq4 stn7 (Frenkel et al., 2007), npq4 npq1 (Breitholtz et al., 2005), and

pgr5 (Munekage et al., 2002) mutant plants were grown in a Phytotron under

120 mmol photons m22 s21, 8-h photoperiod, and relative humidity of 70%.

OSRAM PowerStar HQIT 400/DMetal Halide Lamps served as a light source

both upon plant growth and in HL and LL treatments. For the growth of

plants under fluctuating light, an electronically controlled shading systemwas

used. Five minutes of moderate LL (50 mmol photons m22 s21) and 1 min of

HL (500 mmol photons m22 s21) were continuously following each other

during the light period of 12 h followed by a 12-h dark period. In HL and LL

treatments, leaves were placed in a temperature-controlled chamber at 23�Cand light was passed through a heat filter. Mature rosette leaves from 4- to

6-week-old plants were used for experiments.

Wild-type plants were exposed to light favoring the excitation of PSII (state

2 light) and that of PSI (state 1 light) to ensure the maximal phosphorylation

and dephosphorylation of thylakoid proteins, respectively. A fluorescent tube

(GroLux F58W/GROT8; Sylvania) covered with an orange filter (Lee 105; Lee

Filters) served as state 2 light, and state 1 light was obtained from halogen

lamps (500 W) covered with an orange filter (Lee 105; Lee Filters) and a

median blue filter (Roscolux 83; Rosco Europe). Temperature was maintained

at 23�C by a water-cooled glass chamber between the fluorescence tube and

the plants.

Glc (0.25 M) treatment was given through cut petioles overnight in dark-

ness.

SDS-PAGE and Immunoblotting

Proteins were separated on 15% polyacrylamide gels with 6 M urea. The

content of chl was determined according to Porra et al. (1989). Samples

equivalent to 0.2 mg of chl were loaded in each well. After electrophoresis, the

polypeptides were transferred to an Immobilon-P membrane (Millipore), and

the membrane was blocked with 5% fatty acid-free bovine serum albumin

(Sigma-Aldrich). Western blotting was performed with standard techniques

(Vainonen et al., 2009) using D1-, D2-, CP43-, Lhcb1-, and Lhcb2-specific

antibodies and P-Thr antibodies (New England Biolabs), and for visualization,

the Phototope-Star Chemiluminescent kit was used (New England Biolabs).

PSII-LHCII phosphoproteins D1, D2, CP43, Lhcb1, and Lhcb2 were identified

both by protein-specific antibodies and by mass spectrometry as described

earlier (Tikkanen et al., 2006; Vainonen et al., 2009).

Chl Fluorescence Measurements

The 77 K chl a fluorescence emission and fluorescence excitation spectra

were determined as described (Tikkanen et al., 2008b). Briefly, plants were

light treated, and immediately after treatments the leaves were frozen in

liquid nitrogen. Thylakoids were isolated and diluted in buffer containing 50

mM HEPES/KOH, pH 7.5, 100 mM sorbitol, 10 mM MgCl2, and 10 mM NaF to a

chl concentration of 10 mg mL21 and immediately subjected to measurements

of 77 K chl a fluorescence emission spectra using a diode array spectropho-

tometer (S2000; Ocean Optics) equipped with a reflectance probe. To record

the 77 K fluorescence emission curves, the samples were excited with white

light below 500 nm, defined by LS500S and LS700S filters (Corion). The

emission between 600 and 800 nm was recorded, and the amplitudes of 685-,

695-, and 730-nm chl a fluorescence emission peaks were measured for Figure

2. To obtain the 77 K chl a fluorescence excitation spectra, the samples were

excited with wavelengths from 400 to 540 nm with 5-nm steps using f/3.4

Monochromator (Applied Photophysics).

Chl fluorescence parameters Fm# and Fs# were determined from intact

leaves exposed to actinic light of 67, 590, 340, and 37 mmol photons m–2 s–1

provided by green light-emitting diodes using the JTS-10 spectrometer (Bio-

Logic SAS). Fm#was induced by a 0.25-s saturating flash (7,900 mmol m–2 s–1) of

green light. Value 1 was given to the first measuring points of Fm# and Fs#, andall other values were normalized against this initial value. Fluorescence curves

were recorded from at least seven leaves of each mutant and wild-type plant

with high repeatability.

ACKNOWLEDGMENTS

We thank Prof. Krishna Niyogi for the kind gift of npq4 and npq1 mutants,

Prof. Jean-David Rochaix for the stn7 npq4 mutant, Prof. Dario Leister for the

stn7 stn8 mutant, Prof. Toshiharu Shikanai for the pgr5 mutant, and Prof.

Alexander Vener for the stn8 mutant. Mika Keranen is acknowledged for

valuable help in data processing of fluorescence curves.

Received October 31, 2009; accepted November 27, 2009; published December

4, 2009.

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Thylakoid Protein Phosphorylation in Higher Plant ...stn7 stn8 mutants under strongly ﬂuctuating light conditions. Several proteins of PSII and its light-harvesting antenna (LHCII)