photosynthesis and photoprotection

Molecular design of the photosystem II light-harvestingantenna: photosynthesis and photoprotection

Peter Horton* and Alexander Ruban

Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank,Sheffield S10 2TN, UK

Received 21 April 2004; Accepted 2 September 2004

Abstract

The photosystem II (PSII) light-harvesting system car-

ries out two essential functions, the efficient collection

of light energy for photosynthesis, and the regulated

dissipation of excitation energy in excess of that which

can be used. This dual function requires structural and

functional flexibility, in which light-harvesting proteins

respond to an external signal, the thylakoid DpH, to

induce feedback control. This process, referred to as

non-photochemical quenching (NPQ) depends upon the

xanthophyll cycleand thePsbSprotein. Innature,NPQ is

heterogeneous in terms of kinetics and capacity, and

this adapts photosynthetic systems to the specific

dynamic features of the light environment. The molecu-

lar features of the thylakoid membrane which may en-

able this flexibility and plasticity are discussed.

Key words: Light-harvesting complex, non-photochemical

quenching, photoprotection, thylakoid membrane, xanthophyll

cycle.

Introduction

During the evolution of oxygenic photosynthesis, therehave been two conflicting demands placed upon themolecular machinery of photosystem II (PSII). On theone hand efficient collection of sunlight is needed to deliverexcitation energy to the photosynthetic reaction centre ata rate sufficient to drive electron transport at the highestsustainable rate. On the other hand, excited states ofchlorophyll and the presence of molecular oxygen providea potentially lethal cocktail that can irreversibly damage theproteins, lipids, and pigments of the photosynthetic mem-brane. To combat this, constitutive features of the photo-system, such as the presence of carotenoids to scavenge

singlet oxygen and trap triplet states of chlorophyll, arecombined with other photoprotective mechanisms whichare induced under high levels of illumination to bring aboutthe dissipation of excess chlorophyll excited singlet states.In this paper those features of molecular design thatpromote efficient light harvesting will be reviewed, andthen it will be shown how the dynamic nature of this systemallows for an inducible enhancement of the rate of dissi-pation of excited states. It will be shown how these featuresof design differ in different classes of organism, and thesedifferences will be rationalized in terms of evolution withinparticular ecological niches.

The molecular principles of light harvesting

The chlorophyll a and b molecules, bound to the pigment–protein complexes of the light-harvesting system, arearranged in three-dimensional space to serve as an antenna;gathering light over a large absorption cross-section, and‘funnelling’ the absorbed energy to the reaction centres.Energy transfer between pigment molecules, both betweenand within individual complexes is highly efficient. Equi-libration of energy within the antenna is rapid, and tendstowards the red-shifted lower energy excited states of thereaction centre core complex (van Amerongen and vanGrondelle, 2001). This highly efficient antenna dependsentirely on the specific structures of the apo-proteins of thelight-harvesting complexes: they must bind sufficient pig-ments to maximize absorption; they must provide for theoptimal orientation and configuration of pigments forenergy transfer; they must tune the excited state energylevels of the pigments to promote energy transfer; and theymust permit protein–protein contacts to allow appropriateroutes of energy transfer between complexes.

There are important constraints placed upon this design.Most notable is that the chlorophyll molecules must be

* To whom correspondence should be addressed. Fax: +44 (0)114 222 2787. E-mail: [email protected]

Journal of Experimental Botany, Vol. 56, No. 411, ª Society for Experimental Biology 2004; all rights reserved

Journal of Experimental Botany, Vol. 56, No. 411,

Light Stress in Plants: Mechanisms and Interactions Special Issue, pp. 365–373, January 2005

doi:10.1093/jxb/eri023 Advance Access publication 22 November, 2004

by on 18 October 2009 http://jxb.oxfordjournals.orgDownloaded from

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arranged so as to prevent the tendency for excited statesto dissipate by non-radiative decay. In the main light-harvesting complex, LHCII, the chlorophyll concentrationis approximately 0.6 M: if chlorophyll was dissolved in anorganic solvent at this concentration all absorbed energywould be dissipated in this way, a process described asconcentration quenching (Beddard and Porter, 1976). Thisoccurs via close chlorophyll–chlorophyll interactions, dim-ers or excimers. Clearly the apoprotein provides a specific‘solvent’, an environment that allows a high chlorophyllconcentration without the probability of quenching by suchassociations. This environment arises from the formation ofhydrophobic binding pockets, liganding of the chlorophyllMg atoms by amino acid residues of the alpha helices andhydrogen bonding to the porphyrin ring (Liu et al., 2004). Itis useful to regard chlorophylls as the ‘cofactors’ of the light-harvesting ‘enzymes’, and many parallels exist betweenthese and other proteins with essential cofactors, such ashaem proteins and flavoproteins.

PSII macro-organization

The basic structural unit of PSII is the so-called LHCII–PSIIsupercomplex (Hankamer et al., 1997). This is a macro-molecular dimer, consisting of the D1/D2/CP43/CP47 PSIIcore complex and several light-harvesting proteins, onecopy of the minor monomeric complexes CP26 and CP29,and one of the major trimeric complex, LHCII. In the granalmembrane of the chloroplast, the supercomplex is associ-atedwith further LHCII trimers and themonomeric CP24, toform ‘megacomplexes’, which frequently display a semi-crystalline order (Boekema et al., 2000).The subunits of the PSII antenna are therefore associated

together with increasing levels of complexity. For example,LHCII monomers form trimers, which are themselvesassociated, on the one hand, with the monomeric complexesand core complexes and on the other, with more LHCIItrimers, to form a 2-D organization in the thylakoid mem-brane. Finally, the stromal-facing surfaces ofmembranes areappressed together to form the grana, with contacts betweenLHCII and between LHCII and PSII cores of the adjacentmembranes. This three-dimensional macrostructure hasbeen referred to as the macrodomain (Garab and Mustardy,1999). The tendency for oligomerization is displayed byLHCII, which can in vitro self-assemble into 2-D and 3-Dstructures that have a strong resemblance to thylakoidmembranes (Burke et al., 1987). Equally, oligomers ofLHCII can be prepared following mild detergent treatmentof thylakoid membranes (Ruban et al., 1999). The trimericstate of LHCII appears to be essential for this organization ofPSII. When the main proteins that comprise LHCII areremoved by expression of antisense genes, it was found thatthe amount of the normally monomeric CP26 dramaticallyincreased in level and it became trimeric (Ruban et al.,2003). This change in expression of photosynthetic genes

must be a response to the LHCII deficiency, as the plantattempts to compensate for the loss of an essential protein.Amazingly, this response allows the wild-type oligomericPSII structure and the granal membrane to be maintained,emphasizing the importance and robustness of the thylakoidmacrostructure.

Molecular accessories for regulating lightharvesting

In addition to themain protein complexes, both the PSII coreand the light-harvesting system contain a number of otherproteins, pigments, and lipids (Hankamer et al., 1997).Whereas some of these may have essential roles in thestructure and function of the light-harvesting systems (e.g. aspecific phospholipid is required for LHCII trimerization;Remy et al., 1982) and DGDG may enable associationbetween trimers (Liu et al., 2004), others appear to haveanother role, that of tuning the properties of light-harvestingproteins for regulation. Specific lipids appear to be necessaryfor certain dynamic events within the 3-D organization ofLHCII (Simidjiev et al., 2000). Peripherally associated withLHCII are the carotenoids of the xanthophyll cycle (Rubanet al., 1999), which plays a vital role in photoprotectiveenergy dissipation (Demmig-Adams, 1990), observed as thenon-photochemical quenching of chlorophyll fluorescence(NPQ). This cycle is the reversible de-epoxidation ofviolaxanthin (found under light-limiting conditions) intozeaxanthin (found under light-saturating conditions) that iscorrelated with the induction of NPQ. Violaxanthin de-epoxidase catalyses this reaction, and this enzymemay havea specific docking site on LHCII (Liu et al., 2004). A groupof LHC-related proteins encoded by the members of the lhcgene super family (Jansson, 1999), including the ELIPS, theLil proteins, and PsbS are also involved in stress responses(Heddad and Adamska, 2002), with PsbS having a key rolein NPQ. Protein kinases also interact with LHCII (Bennett,1983) and CP29 (Croce et al., 1996), phosphorylating them.In the case of LHCII it is well-known that the redoxregulation of kinase activity provides the basis for regulatingthe distribution of absorbed energy between PSII and PSI(Horton and Black, 1980), phosphorylation modulating thestructure of the complex, and its interaction with otherphotosystem proteins (Allen and Forsberg, 2001). Phos-phorylation of CP29 appears to be activated under coldstress, causing a structural change in the protein, which mayhave a photoprotective role (Croce et al., 1996).

Changes in structure as the basis for regulationof light harvesting

Whereas the higher order structure of PSII was first thoughtto be important only in increasing the efficiency of lightharvesting, more recently it has been suggested that itprovides the essential dynamic properties involved in

366 Horton and Ruban



regulation (Horton, 1999). When isolated light-harvestingcomplexes self-assemble into oligomers in vitro, there isa decrease in fluorescence yield (Mullet and Arntzen, 1980;Ruban et al., 1992), resulting from a decrease in excitationlifetime by non-radiative decay. Since, in vivo, the com-plexes also exist in an oligomeric state (Dekker et al., 1999;Ruban et al., 1999), it could be proposed that increasedenergy dissipation would result. Indeed, it has been recog-nized that themaximumfluorescence lifetime of chlorophyllin vivo is always less than free chlorophyll or chlorophyll inunaggregated LHCII (Horton andRuban, 1994). Perhaps theadvantages of oligomeric organization outweigh the dis-advantage of some waste of absorbed excitation energy.

More interesting, however, is the notion that structuralchanges within the oligomeric antenna could providea physiological mechanism for regulating the partitioningof energy between utilization in photosynthesis and dissi-pation by NPQ (Horton et al., 1991, 1996). Indeed, thereare many remarkable similarities between NPQ and in vitrofluorescence quenching in LHCII and other antenna com-plexes (Horton et al., 1999). Whilst the exact mechanism ofenergy dissipation in these complexes remains to be de-termined, it almost certainly is equivalent to the process of‘concentration’ quenching discussed above. Hence, giventhat the precise 3-D structure of the protein prevents suchquenching, it is easy to imagine how (relatively subtle)changes in the structure could induce quenching. Examin-ation of the structural model of LHCII reveals the presenceof several candidate quenching pairs of chlorophyll mole-cules (Kuhlbrandt et al., 1994; Liu et al., 2004). Althoughthe pair of Chl a 613/614 has been put forward as a potentialquenching site (Liu et al., 2004), it has previously beensuggested that the Chl a 610/611/612/lut1 domain was thesite of quenching (Wentworth et al., 2003). It was shownhow the differing tendencies of trimeric and monomericLHCII to quench could be explained if the conformation ofquenching locus was controlled by the Lut 2 domain(Wentworth et al., 2003). This idea was consistent withearlier observations of altered fluorescence yield of com-plexes when the Lut 2 site binds different xanthophylls(Moya et al., 2001; Morosinotto et al., 2002; Formaggioet al., 2001; Caffarri et al., 2004).

As a guiding principle, it was suggested that NPQ wasa response of the structure of the PSII light-harvestingantenna, resulting in altered pigment interactions withinprotein subunits and caused by the change in externalenvironment and the binding of protons (because of theincrease in transthylakoidDpH) (Horton et al., 1991; Hortonand Ruban, 1992). Of course, the notion that proteinstructure and function can be altered by pH change is wellestablished and, extrapolating further, it is suggested that theoligomeric nature of the light-harvesting system may allowco-operativity in proton binding and, most importantly,allosteric regulation by other ligands (Ruban and Horton,1995a; Ruban et al., 2001; Horton et al., 2000).

A conceptual model was formulated, comprising fourconformational states of the light-harvesting system, withdifferent fluorescence yields, differing in de-epoxidationand protonation state (Horton et al., 1991). The action ofzeaxanthin was explained, it is an allosteric activator ofquenching binding to a peripheral site on LHCII or anotherlight-harvesting antenna protein. There is considerableexperimental support for this hypothesis, although directparticipation of zeaxanthin in the quenching mechanismcannot be excluded (Frank et al., 1994; Ma et al., 2003).Paramount in this evidence is the startlingly differentkinetic properties of qE observed in thylakoids isolatedfrom leaves pretreated to have different de-epoxidationstates (Ruban et al., 2001): qE forms more rapidly, ata lower DpH and with reduced co-operativity in thepresence of zeaxanthin, all characteristics of it being anallosteric activator. Such properties also explain the kineticsof NPQ formation upon illumination of dark-adaptedleaves, and the accelerated rate of formation upon a secondillumination following a brief dark interval in which DpH,but not de-epoxidation is relaxed (Ruban and Horton,1999). Recently it has been suggested that, rather thanbeing a peripheral site on an antenna protein, the internalLut 2 site discussed above provides the allosteric bindingsite for zeaxanthin (Formaggio et al., 2001). Indeed it hasbeen shown that when the Lut 2 site in CP26 is occupied byzeaxanthin, there is a decrease in fluorescence lifetime,compared with when violaxanthin is bound (Moya et al.,2001; Crimi et al., 2001). In this model, reversibleexchange of carotenoid binding to this site is proposed(Bassi and Caffarri, 2000), and in vitro this has been shownto be promoted at low pH (Morosinotto et al., 2002).

It is interesting, and rather unexpected, that there is noevidence for any one light-harvesting complex having anobligatory role in NPQ, at least in higher plants; wild-typelevels of NPQ are found in Arabidopsis plants expressingantisense genes for CP29, CP26, and LHCII (Anderssonet al., 2001, 2003). Similarly, chl b less mutants, whichhave only low levels of all Lhcb proteins show NPQ(Lokstein et al., 1993). This indicates that no single Lhcbprotein can be the unique site of either energy dissipation orzeaxanthin activation. Furthermore, it suggests that NPQcould be ‘shared’ by all antenna proteins, including those ofthe PSII core, since the above principle of control ofconcentration quenching can apply to them all. It remains tobe shown what the minimum structural unit for NPQ is.In vitro studies also show how each antenna subunit hasonly a small capacity for zeaxanthin-induced quenching(Wentworth et al., 2000; Crimi et al., 2001), but that this isamplified when subunit interactions are enabled (Rubanand Horton, 1992; Moya et al., 2001). Indeed, this suggeststhat the intra- and inter-subunit conformational changes actin a concerted manner, for example, a quenching locus inLHCII or CP26 could be controlled by the Lut 2 domain,which is in turn affected by the xanthophyll bound there,

Molecular design of the PSII light-harvesting antenna 367



the binding of xanthophylls at the more peripheral neo-xanthin (N1) and violaxanthin (V1) sites, and interactionswith neighbouring protein subunits.

The role of PsbS and zeaxanthin activation

Arabidopsis plants with mutation in the PsbS gene (npq4mutants) show none of the rapidly reversible type of NPQknown at qE, despite having wild-type levels of zeaxanthinand DpH, and unaltered levels of light-harvesting andreaction centre complexes (Li et al., 2000). It was sug-gested that PsbS provides the site of NPQ, by binding bothprotons and zeaxanthin, and interacting with a chlorophyll-containing complex to introduce a new quenching pathway.This hypothesis for the role of PsbS has been subject tointensive investigation. Evidence for proton binding hascome from the identification of binding sites for DCCD(Dominici et al., 2002), and from the inhibition of qE bysite-directed mutagenesis of potential proton binding resi-dues (Li et al., 2002a). The pigment-binding properties ofPsbS are unclear; purified PsbS appears to contain nochlorophyll or carotenoid.Finding the location of zeaxanthin binding sites has been

an important goal in understanding NPQ. It has been shownthat isolated LHCII and the minor complexes CP26 andCP29 contain violaxanthin (Ruban et al., 1994, 1999; Bassiand Caffarri, 2000). Only some of this is available forde-epoxidation, shown to include that loosely bound tothe complex (Ruban et al., 1999; Caffarri et al., 2001), andthere is evidence that violaxanthin bound to the Lut 2site on CP26 and CP24 could exchange for zeaxanthin(Morosinotto et al., 2002). However, such studies ofzeaxanthin binding sites could be misleading, since it islikely that only a small proportion of the zeaxanthin pool isinvolved in NPQ (Gilmore, 2001). This subpopulation ofzeaxanthin has been studied using a spectroscopic ap-proach. It is well known that qE is very strongly correlatedwith an absorption change at 535 nm (Bilger et al., 1988;Ruban et al., 1993), whilst it was considered to arise fromselective light scattering, its proximity to the carotenoidabsorption bands, suggested it may be electronic in origin.Indeed, the DA535 spectrum could be modelled as a redshift in 1–2 carotenoid molecules (Ruban et al., 2002).Resonance Raman spectroscopy has been used to identifythe absorption bands from each of the xanthophylls (Rubanet al., 2000). In leaves, induction of qE was associated withthe appearance of a zeaxanthin species selectively excited at528 nm. This signal was absent in the npq4 mutant. Thesedata not only proved the electronic nature of DA535, butalso showed that it most likely arose from a small popu-lation of red-shifted zeaxanthin molecules (Ruban et al.,2002). It is suggested that this was indicating the activationof zeaxanthin, required to fulfil its role in qE. The Ramansignal also indicated that this species was found in a ratherspecific configuration, indicating binding to a protein com-

ponent of the membrane. In order to test whether this sitecould be on PsbS, zeaxanthin was reconstituted witha purified sample of this protein; a red-shifted absorptionband was created with an identical Raman signature toDA535 (Aspinall-O’Dea et al., 2002).

Although specific binding to another protein site is notexcluded, these data suggest that the activator role ofzeaxanthin in qE requires that it be bound to PsbS. Thisbinding, in vivo, requires DpH, most likely because ofproton binding to PsbS, and confers upon zeaxanthin verynew configurational and excited state features. How theseproperties are related to its mechanism in qE is currentlyunknown. On the one hand, these features may be neededfor zeaxanthin to quench chlorophyll excited states ona neighbouring antenna complex directly (Ma et al., 2003).On the other hand, protonated PsbS may ‘deliver’ zeax-anthin to its active site in the antenna: in this case it againmay be a direct quencher, or as suggested above, it mayfulfil an allosteric activating role. Finally, a protonated zea/PsbS complex may have an allosteric role. In the latter twocases, PsbS would be regarded as the regulatory subunit ofthe light-harvesting antenna. Further work is needed todistinguish these possibilities.

Comparative biochemistry and physiologyof NPQ

NPQ is observed in all higher plants; it has been detected ina wide range of species in many different plant types, ina wide range of habitats (Demmig-Adams, 1998; Johnsonet al., 1993). It has also been found in lower plants(Campbell et al., 1998; El Bissati et al., 2000), green algae(Niyogi et al., 1997), and diatoms (Casper-Lindley andBjorkman, 1998; Arsalane et al., 1994). It seems reasonableto assume that the underlying molecular mechanisms arethe same in all cases. Nevertheless a comparative study ofNPQ in different species has proved to be rewarding,different aspects of NPQ appear to be enhanced in certainspecies, making them good models for further study.Moreover, in such cases, the knowledge obtained, can givenew approaches to gaining fundamental information on themechanism of NPQ.

NPQ has been described as being the sum of twodifferent processes (Horton et al., 1996). In addition tothe rapidly relaxing qE type discussed above, there isfurther NPQ that relaxes very slowly when excess light isremoved. This latter type, called qI, is usually much smallerthan qE, and is attributed to a more sustained effect ofexcess light. Therefore qI has been associated with ‘photo-inhibition’ rather than ‘regulation’, and was thought to arisefrom quenching by photodamaged PSII reaction centres.However, it was suggested that qI, like qE, may not be aneffect of excess light stress, but an adaptive response to it(Horton et al., 1988, 1996; Ruban et al., 1993). Thus,perhaps quenching by a photoinhibited reaction centre is




a useful response that provides photoprotection of theantenna and thylakoid membrane (Chow et al., 2002).Since strong evidence of the accumulation of damaged PSIIunder photoinhibitory conditions was absent, it was furthersuggested that qI occurs in the antenna, perhaps even by thesame or similar mechanism to qE (Demmig and Winter,1988; Ruban et al., 1993; Ruban and Horton, 1995b).

In order to determine the relationship between qE and qI,species that show particular NPQ characteristics have beensought. The epiphytic plant, Guzmania monostachia, ex-hibits a large NPQ capacity (Ruban et al., 1993). Both qEand qI are induced in excess light: successive periods ofillumination progressively ‘pump’ qE to itsmaximumvalue,and this is activation is correlated with an increase in qI.Thus, qI is the signature of the light ‘activation’ describedabove, and is therefore attributed to the de-epoxidation ofviolaxanthin. The large extent of activation and qI in thisspecies is also associatedwith a large xanthophyll cycle pooland a high de-epoxidation state. Spectroscopy carried out onGuzmania leaves upon the induction of qI showed theformation of red-shifted chlorophyll emission previouslyidentified in aggregated LHCII. A very stable type of NPQfound in plants acclimated to low temperature also has thisfeature, the ‘cold hard band’, suggesting that evergreenplants which can withstand freezing conditions over winterexist in a permanently photoprotected qI state of aggregatedLHCII (Gilmore and Ball, 2000). Biochemical data on thisstate suggests that factors such as LHCII phosphorylation(Ebbert et al., 2001) and accumulation of PsbS (Oquist andHuner, 2003) may play a role in establishing and/or stabiliz-ing this state of the PSII antenna.

NPQ found in some algae has different features comparedwith higher plants. In Chlamydomonas, NPQ is rather weak(about 50% of a typical value in higher plants), slow to form,and partially dependent upon zeaxanthin formation (Elradet al., 2002). Various NPQ-deficient mutants have beenisolated, including one with a mutation in a light-harvestinggene, not in PsbS (Elrad and Grossman, 2004). In manyrespects NPQ resembles the higher plant qI, rather than qE.

In diatoms, NPQ is similar to that found in plants, exceptthat it is large (sometimes about five times bigger) and it isvery slow to relax; it forms like qE but relaxes like qI(Lavaud et al., 2002). Diatoms have a xanthophyll cycle, butit is different from that found in plants. It is a one-step de-epoxidation of diadinoxanthin to diatoxanthin, equivalent tothe second step of the plant xanthophyll cycle, antherax-anthin–zeaxanthin conversion. The light-harvesting com-plexes in diatoms are different from those found in higherplants; they bind different carotenoids and chlorophylls, andhave very low sequence similarity. They are predicted3-helix proteins. The xanthophyll cycle carotenoids accountfor 20–40% of the carotenoid pool, and it is estimated thateach LHC binds 2–4 molecules of diadinoxanthin. PsbS isabsent from these organisms, although it is interesting thatsome similar PsbS protein sequences are found in their LHC

proteins. The DA535 is absent, although a light-inducedabsorbance change around 522 nm is observed, and may beindicative of some diatoxanthin activation. The formation ofNPQ isDpH-dependent and this appears to be not only due tostimulation of de-epoxidation at low lumen pH; collapse ofthe DpH by uncouplers reverses NPQ even though the levelof diatoxanthin in unchanged. Interestingly, when NPQ hasbeen completely formed, it is no longer uncoupler sensitive.

Previously an attempt was made to explain both qE andqI in terms of a 4-state model of the light-harvesting system(Horton and Ruban 1992); qI was considered to be a state inwhich de-epoxidation has taken place, but not yet pro-tonated, whereas qE is always a protonated state that wouldnormally also bind zeaxanthin. The pKa of the epoxidizedand de-epoxidized states, the strength and number ofxanthophyll cycle binding sites, the xanthophyll cycle poolsize, the level of PsbS and the pH of the thylakoid lumenwould determine the amounts of qE and qI observed indifferent plants. Extending this rationale to the green algae,perhaps the DpH is not large enough, and the zeaxanthinconcentration not high enough to cause protonation of theqE active sites in the absence of PsbS.

In diatoms, the large NPQ can be explained by eithera stronger quencher, or by a larger number of quenchingsites. There is currently insufficient data to distinguish these.The kinetic features indicate a qI-type process, but onewhichis nevertheless dependent upon protonation, and formedrapidly. The fact that formation of diatoxanthin only requiresone de-epoxidation, could explain the rapid activation ofquenching. Since, PsbS is absent from diatoms, NPQ mustoccur by direct effects on the light-harvesting complexes.The slow relaxation could arise from extreme stability of theprotonated state of these complexes when diatoxanthin ispresent, so that reversal of quenching absolutely dependsupon the rather slow kinetics of de-epoxidation. Thisstability may arise from particularly strong protein interac-tionswithin this antenna that ‘locks’ the quenched state. Thisblurring of the distinction between qE and qI leads to a newsuggestion about the role of the PsbS protein in plants:perhaps it provides control over NPQ, enabling not onlyinduction of NPQ, but also its rapid reversibility.

Comparative ecology of NPQ

The variation in character of NPQ in different organismssuggests that facets of this process have undergone evolu-tionary adaptation to particular ecological niches. In landplants, a major requirement for the regulation of lightharvesting is a rapid, dynamic response to changes in lightlevels. The activation by de-epoxidation, and triggering byprotonation, is adapted to the more rapid changes associ-ated with leaf movements and sunflecks, against a back-ground of diurnal and daily changes. The relatively slowkinetics of de-epoxidation act as a molecular memory of‘average’ light conditions, whereas protonation provides an




‘on/off switch’ with a response time of just a few seconds.This switch, apparently, requires a transition between twostates of the light-harvesting system with different excita-tion lifetimes, setting the upper limit on the magnitude ofNPQ. The amount of quenching depends upon the numberof units in each state. Plants acclimated, and adapted toparticular light environments, tune the dynamics of NPQ.Plants exposed consistently to large excesses of radiationare found to have the high NPQ capacity, driven by a highxanthophyll cycle pool and PsbS concentration, a lowcontent of LHCII and perhaps an increased capacity of DpHgeneration (by cyclic electron transfer). In overwinteringplants, these features appear to be accompanied by othermechanisms, which stabilize the quenched state even in theabsence of DpH.Diatoms are able to achieve a very high NPQ capacity.

This appears to be an adaptation to environment, with largebut relatively slow fluctuations in incident radiation, arisingfrom ocean currents. The requirement for a rapid off switchis quite low in this environment. It seems likely in any casethat it is not possible to have, at the same time, a very largeNPQ, and to have it rapidly reversible.

Evolution of NPQ

The diversity of NPQ characteristics suggests a strongevolutionary pressure upon the attainment of optimizedphotoprotection. This evolutionary process can be viewedwithin the context of the LHC gene super family (Green andPichersky, 1994; Jansson, 1999). The cyanobacterial HLIPand plant Lil 2 genes encode single-helix proteins thatresemble the progenitors of the Lhc proteins. The Lil3 genethen arose by duplication, to give a 2-helix protein. PsbS isthen considered as a further duplication, since it is a 4-helixprotein. ELIPS, which are 3-helix proteins, would thenhave evolved from a PsbS ancestor by deletion of a C-helixregion. All of these proteins are associated with stresstolerance (Heddad and Adamska, 2002), and probably donot bind chlorophylls. Instead, they may have evolved ascarotenoid protein complexes. For instance, the ELIPprotein found in abundance in some algae, Cbr, may bindcarotenoids (Levy et al., 1993). Accumulation of plantELIPS have also been reported to correlate closely with theamount of xanthophyll pigments (Montane et al., 1998).

Light-harvesting proteins can then be considered to haveevolved from these stress proteins, by binding chlorophylls,and excluding many carotenoids (Fig. 1). The trimeric stateof plant LHCII is probably the most evolved complex forlight harvesting (Wentworth et al., 2003). It is interesting toconsider the LHCF protein of diatoms; this complex bindschlorophyll, but has retained a high amount of carotenoidbinding. The level of this protein increases under condi-tions of high NPQ, and hence may be considered as trulybifunctional—operating equally in both photoprotectionand light harvesting, its two modes depending upon de-epoxidation. By contrast, in plants, photoprotection requiresPsbS, a protein not involved in light harvesting. However,this protein is able to confer upon LHCII (or any PSIIantenna complex) a photoprotective function, perhaps byproviding delivery and removal of the ‘active’ zeaxanthin.The presence of PsbS, therefore, allows the light-harvestingcomplex to function as an efficient light harvester, witha high absorption cross-section, its chlorophyll pigments notneeding to be diluted by binding high levels of carotenoid.The extra benefit from this mode of control is rapidity asreversal does not depend upon epoxidation of zeaxanthin.Moreover, once the appropriate de-epoxidation state hasbeen reached, NPQ can be turned on and off rapidly. Inthis context it is interesting to consider the effect of PsbSconcentration on NPQ. Manipulation of the amount of PsbSin Arabidopsis shows that its stoichiometry correlates withchanges in the amplitude of the qE component of NPQ (Liet al., 2002b).Mutantswhich lack PsbS altogether have onlya slowly relaxingNPQ; in the absence of PsbS the unassistedprocess is revealed, one which relies upon the passive massaction effect of the xanthophyll cycle and in which qE and qIare indistinguishable. The evolution of qE in plants, drivenby the need to respond rapidly to changing light intensity,resulted from the specification of an ancestral carotenoid–protein complex to a new role reversibly binding xantho-phyll cycle carotenoids and protons, and interacting with thePSII light-harvesting antenna proteins.

Conclusions

The principles that have guided the molecular design of thePSII light harvesting have been elucidated. Modifications tothis design are found in different organisms’ adaptations to

Fig. 1. Evolutionary and functional relationships between proteins involved in light harvesting and photoprotection. LHCII is seen as the end-product ofa process that maximizes light harvesting through high amounts of chlorophyll binding. These may have evolved from stress-responsive single-helixproteins such as HLIP and ELIPS, which bind only carotenoids. PsbS provides an essential function in plant photoprotection, with only minimal pigmentbinding. On the other hand, LHCF binds both chlorophyll and carotenoids in high amounts, functioning in both light harvesting and photoprotection.




specific environments. An essential feature is the structuralflexibility of the light-harvesting proteins, which allowcontrol over the transfer of excitation energy to particularquenching species, associates of chlorophyll or chlorophylland xanthophyll. The recent elucidation of the three-dimensional structure of LHCII has revealed the possiblemolecular mechanisms by which this may occur (Liu et al.,2004). High resolution electron microscopy and imageanalysis of wild-type and genetically altered plants areshowing how these structures are organized within theintact membrane (Yakushevska et al., 2003; Ruban et al.,2003) and new spectroscopic approaches to the investiga-tion of intact systems are beginning to reveal details of howsuch mechanisms operate (Robert et al., 2004).

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