TrendsLight capture at both photosystemsneeds to be tightly adjusted to the pre-vailing light supply to prevent an over-excitation of photosystems, causingphotodamage.

Control mechanisms preferred by plantsand microalgae reveal numerous differ-ences: A. thaliana, a sessile organism,possesses constitutive mechanisms forenergy-dependent quenching (qE) thatcan be activated rapidly. By contrast,the mobile organism C. reinhardtii usesa combination of state transitions (qT)and an inducible qE mechanism.

While qE and qT protect the photosyn-thetic apparatus without changing light-harvesting antenna composition, long-term acclimation fine-tunes antennastoichiometry.

Cytosolic translation control, in particu-lar, is emerging as a key process in fine-tuning antenna size and composition.

Antenna engineering in green algaeimproves the efficiency of light-to-bio-mass conversion.

1Bielefeld University, Faculty ofBiology, Center for Biotechnology(CeBiTec), Universitätsstrasse 27,33615, Bielefeld, Germany2Universita degli Studi di Verona,Department of Biotechnology, StradaLe Grazie 15, 37134 Verona, Italy

*Correspondence:olaf.kruse@uni-bielefeld.de (O. Kruse).

ReviewMulti-Level Light CaptureControl in Plants and GreenAlgaeLutz Wobbe,1 Roberto Bassi,2 and Olaf Kruse1,*

Life on Earth relies on photosynthesis, and the ongoing depletion of fossilcarbon fuels has renewed interest in phototrophic light-energy conversionprocesses as a blueprint for the conversion of atmospheric CO2 into variousorganic compounds. Light-harvesting systems have evolved in plants and greenalgae, which are adapted to the light intensity and spectral composition encoun-tered in their habitats. These organisms are constantly challenged by a fluctu-ating light supply and other environmental cues affecting photosyntheticperformance. Excess light can be especially harmful, but plants and microalgaeare equipped with different acclimation mechanisms to control the processingof sunlight absorbed at both photosystems. We summarize the current knowl-edge and discuss the potential for optimization of phototrophic light-energyconversion.

Improving Green Cell Factories for Light ConversionAmong the current societal challenges is the depletion of photosynthesis-derived fossil carbonstores. This calls for sustainable alternatives based on the ability of particular photosyntheticorganisms to convert CO2 into organic products using light energy [1]. In phototrophs, light-harvesting and energy-dissipation processes are primary key events for the efficient formation ofATP and the reducing equivalents necessary for CO2 fixation. Understanding the moleculardetails of light-conversion processes is therefore crucial for their (bio)-technological exploitation.Recently, major progress was made on the characterization of short- and long-term acclimationmechanisms which adjust the light-harvesting capacity in plants and microalgae to ever-changing environmental conditions [2–5]. A tight control of light capture is vital because ofthe janus-faced nature of light: it drives photosynthesis but is harmful when provided in excess.In this review we try to depict the complex regulatory network underlying the adjustment ofphotosynthetic light harvesting in plants and green microalgae, which operates on distinct time-scale and implicates different cellular compartments.

Light-Harvesting ProteinsWhile photosystem reaction center (see Glossary) proteins exhibit strong conservation in theirprimary sequence and their general supercomplex architecture (Figure 1A) and mechanisms [6],antenna systems have undergone a far higher level of diversification, particularly in aquaticsystems [7–9]. However, only organisms equipped with light-harvesting complex (LHC)-likeantenna proteins, which first appeared in green algae as an evolution from a cyanobacterialancestor [10], managed to survive in the subaerial environment characterized by high oxygen,low moisture, and strong irradiances, making life highly stressful for sessile photosyntheticorganisms. Phylogenetic analysis of the LHC protein family identified as many as 30 proteins[11], only a third of which are conserved through the chlorophyta as subunits of photosystems(PS) I and II. Many have as yet undefined functions likely related to photoprotection, pigment

Trends in Plant Science, January 2016, Vol. 21, No. 1 http://dx.doi.org/10.1016/j.tplants.2015.10.004 55© 2015 Elsevier Ltd. All rights reserved.

GlossaryCyclic electron transfer (CET):mode of photosynthetic electrontransport. Electrons are recycled fromreduced ferredoxin or NADPH to PQ.This generates a proton gradientacross the thylakoid membrane,enabling the exclusive production ofATP without simultaneous productionof NADPH. By contrast, linearelectron transport involves water-splitting at PSII and the generation ofNADPH.Exciton: view that electron excitationgenerates a quasi-particle capable ofmigrating.Non-photochemical quenching(NPQ): excited singlet chlorophyll(1Chl*) can have different fates in thephotosynthetic apparatus. Itsexcitation can be used to triggerphotochemical processes (qP; chargeseparation and photosyntheticelectron transport) or relax back tothe ground state by emittingfluorescence. It can also de-excite bydissipating heat (NPQ). NPQ is acollective term including severalquenching mechanisms (qE, qT, qI,qZ, and qM; see Box 1).Photosynthetic control: feedbackcontrol mechanism that restrainsphotosynthetic electron flow whenATP accumulation results from a highproton motive force (PMF).Photosynthetic electron transport isconnected to the translocation ofprotons from the stromal to theluminal side of the thylakoidmembrane, which acidifies thethylakoid lumen (DpH) and leads tothe accumulation of positive charges(DC), both contributing to a rise inthe proton motive force. Lumenacidification slows down PQoxidation by the cytochrome b6fcomplex, thus restricting electronflow and a further acidification of thelumen.Plastoquinone (PQ): lipid-solubleand diffusible electron carrier, whichconnects PSII and PSI via thecytochrome b6f complex as acomponent of the photosyntheticelectron transport chain.Reaction centers: core complexesof PSI and PSII which harbor so-called ‘special pair’ chlorophyll amolecules. Excitation energy istransferred from the light-harvestingantenna to these chlorophyllmolecules, causing charge separationas an initial event of photosyntheticelectron transport.

Arabidopsis thaliana

Chlamydomonas reinhard�i

Chl a 602

Chl a 612

Chl a 611

Chl a/b 613

Chl a/b 614

Chl b 601

Chl a 610 Chl a 603

Chl a 608

Chl b 609

Chl b 606

Chl b 605

Chl b 607Chl a/b 604

N1Neoxanthin

V1Violaxanthin

L1Lutein

L2Lut./Vio.

LHCII-L

LHCII-S

LHCII-M

LHCII-M

LHCII-L

LHCII-L

LHCII-L

LHCII-L

LHCII-S

LHCII-S

LHCII-M

LHCII-M

LHCII-L

LHCII-L

LHCII-L

LHCII-L

LHCII-L

LHCII-S

CP26

CP26CP24

CP29

CP29

CP29

CP26

CP29

CP26

CP24

CP29PSIIcore

PSIIcore

PSIIcorePSIcore

PSIcore

PSIIcore Lhca

Lhca

Lhca

Lhca

Lhca

LhcaLhca

Lhca

Lhca

Lhca2

Lhca1

Lhca

4

Lhca3

(A) (B)

Figure 1. Organization and Structure of Key Photosynthetic Components. (A) Supramolecular organization ofphotosystems (PS)I and PSII with their light-harvesting complex (LHC) antenna proteins in higher plants (upper panel) andthe green algae model system Chlamydomonas reinhardtii. Based on data from [112–114] for plants and [115–117] forChlamydomonas. The core components and the antenna system of photosystem II are shown in green and components ofthe PSI–LHCI supercomplex in blue. Upon phosphorylation part of the PSII antenna can connect to PSI. (B) Structure ofmonomeric LHCII according to crystallographic data [15]. Four binding sites for xanthophylls and 14 binding sites forchlorophylls are indicated. In other members of the LHC family the number of binding sites for chromophores can vary.Abbreviations: CP, chlorophyll-binding protein; Lut., lutein; Vio., violaxanthin.

metabolism, and biogenesis as assessed for early light-induced proteins (ELIPs) [12], light-harvesting complex stress-related (LHCSR) [13], and photosystem II chlorophyll-binding proteinS (PSBS) [14]. The most conserved and widespread LHC proteins include the subunits of thePSII antenna moiety, the trimeric LHCII-M, monomeric chlorophyll-binding proteins (CP)29 andCP26, and the dimeric LHCI subunits of PSI (Figure 1A and Table 1).

LHCs have a common architecture, as shown by available protein structures and homology[15–17]. Their major functional traits are associated to bound chromophores, which are as manyas 14 chlorophylls (Chl) and four xanthophylls for each �22 kDa polypeptide, and diverse,including two types of Chls and 4 xanthophyll species (Figure 1B). Chl b occupies moreperipheral binding sites and transfers excitation energy to an excitonically tightly connectedcentral Chl a cluster [18] which delocalizes excitons for further transfer towards reaction centers(Figure 1A, PSII core; 2A, D1/D2). Each Chl is in tight contact with one of the xanthophylls(Figure 1B) which are essential for triplet chlorophyll quenching (lutein; Figure 2B,C) andsinglet oxygen/superoxide scavenging (violaxanthin/neoxanthin; Figure 2D). By contrast,enhanced quenching/scavenging can be obtained by exchanging violaxanthin bound to siteL2 (Figure 1B) by zeaxanthin, which is produced under excess light conditions only.

Recent findings highlight that LHCII isoforms have additional photoprotective functions besidestheir primary role as efficient light-harvesting devices, and that isoforms differ in this regard(Table 1).

Short-Term Light-Acclimation MechanismsLight intensity and spectral quality undergo rapid variations depending on external factors suchas the time of day, weather, and shading from other cells in a dense algal culture or within thecanopy of land plants. Light capture and photochemistry must be optimized to cope with lightlimitation. Instead, photon capture is minimized, energy dissipation activated, and protectionagainst reactive oxygen species (ROS) enhanced when light is in excess.

56 Trends in Plant Science, January 2016, Vol. 21, No. 1

Reactive oxygen species (ROS):highly reactive and damagingbyproducts of oxygenicphotosynthesis which accumulate inthe chloroplast under stressconditions. The main ROS producedat PSII is singlet oxygen (1O2), whileelectron transfer from reducedferredoxin to oxygen createssuperoxide (O2

�) at PSI.Singlet oxygen (1O2): highly reactiveform of oxygen in an excited state,which can be generated from lessreactive triplet oxygen (3O2) in theground state. Triplet chlorophyll canact as a photosensitizer, whichfacilitates the conversion from 3O2 to1O2 in the reaction center or light-harvesting antenna of PSII.Triplet chlorophyll (3Chl*): canoriginate from excited singletchlorophyll states (1Chl*) by inversionof the electron spin (intersystemcrossing) or charge recombinationreactions (reversal of chargeseparation and electron transfer) inthe reaction center. The likelihood of3Chl* formation increases when theelectron transport chain is an over-reduced state.

Table 1. Light-Harvesting Proteins Found in the Two Model Organisms for Eukaryotic Photosynthesis –

Chlamydomonas reinhardtii and Arabidopsis thalianaa

Organism LHCIIProtein

UniProtKBAccessionNumber

Classb Proposed Function Refs

C.r. LHCB4(CP29)

A8J6D1 M In state II (S2), phosphorylation of CP26/29 triggersundocking of the peripheral antenna from PSII. Bothare part of the mobile LHCII antenna and CP29connects the mobile antenna to PSI.

[48,52,118]

C.r. LHCB5(CP26)

Q9FEK6 M

C.r. LHCBM1 A8JCU4 T(IV) Reduced qE capacity and sensitivity towards singletoxygen in mutant npq5 devoid of LHCBM1.

[51,61]

C.r. LHCBM2 Q93WE0 T(III) Simultaneous knockdown of LHCBM2 andLHCBM7 perturbs qT and reduces tolerancetowards superoxide.

[51]

C.r. LHCBM3 Q93WL4 T(I) LHCBM3 is the most abundant type I-isoform.Phosphorylation of type I-isoforms in S2 triggersdissociation of the PSII megacomplex.

[48,53]

C.r. LHCBM4 A8J264 T(I)

C.r. LHCBM5 Q9ZSJ4 T(II) LHCBM5 is part of the ‘extra’ LHCII population notconnected to the PSII core and migrates to PSIupon phosphorylation in S2.

[52–54]

C.r. LHCBM6 A8J287 T(I) See function proposed for other type I isoforms.Main target of long-term antenna size adjustmentvia NAB1-mediated translation control.

[4,48,70]

C.r. LHCBM7 Q9AXF6 T(III) Mature protein identical to LHCBM2. [51]

C.r. LHCBM8 A8J270 T(I) See function proposed for other type I isoforms. [48]

C.r. LHCBM9 Q8S3T9 T(I) Only expressed under stress conditions(sulfur/nitrogen limitation) to promote light energydissipation, thereby protecting PSII againstphotooxidative damage.

[62]

C.r. LHCSR1 P93664 SR Essential component for qE.Binds chlorophyll a, lutein,and violaxanthin. Binds DCCD.Activates qE upon protonationof lumen-exposed acidicresidues. Binds to thePSII–LHCII complex in aPSBR-dependent manner

Constitutivelyexpressed.

[13,31,119,120]

C.r. LHCSR3 A8J431 SR Accumulatesduring growthin excess light.

A.t. LHCB1.1LHCB1.2LHCB1.3LHCB1.4

P0CJ48Q8VZ87P04778Q39142

T(I) The major component of LHCII outer antenna.Involved in NPQ.

[50,121]

A.t. LHCB2.1LHCB2.2LHCB2.3

Q9SHR7Q9S7J7Q9XF87

T(II) A minor component of LHCII outer antenna. Involvedin state 1–state 2 transitions, but not in NPQ.Phosphorylated in low light by STT7 kinase.

A.t. LHCB3 Q9S7M0 T (III) Minor component of LHCII, found as a heterotrimerwith LHCBM1 and 2 in trimer M.

[122]

A.t. LHCB4.1(CP29)

Q07473 M Part of the C2S2 PSII supercomplex. Majorcomponent of fast-activated NPQ response. Site offormation of the zeaxanthin/lutein radical cation duringPSBS-dependent qE. Regulates Chl triplet quenching.

[123,124]

A.t. LHCB4.2 Q9XF88 M

A.t. LHCB4.3 Q9S7W1 ? Protein not found in thylakoids.

A.t. LHCB5(CP26)

Q9XF89 M Part of the C2S2 PSII supercomplex. Reversibly bindszeaxanthin in excess light. Major isoform responsiblefor qZ component of NPQ. Enhances Chl tripletquenching in HL.

[23]

Trends in Plant Science, January 2016, Vol. 21, No. 1 57

Table 1. (continued)

Organism LHCIIProtein

UniProtKBAccessionNumber

Classb Proposed Function Refs

A.t. LHCB6(CP24)

Q9LMQ2 M Part of the C2S2M2 PSII supercomplex. Reversiblybinds zeaxanthin in excess light. Dissociates fromC2S2 complexes in a PSBS-dependent event.Degraded upon growth in excess light.

[33,125]

A.t. PSBS Q9XF91 SR Constitutively expressed. Essential component for qE.Activates qE upon protonation of 2 Glu residues.Chlorophyll-binding residues not conserved.

[14,29,126]

A.t. ELIP2 Q94K66 SR Expressed in HL, decreases Chl level in leaves byscavenging Chl precursors.

[12]

A.t. LIL3:1LIL3:2

Q9SYX1Q6NKS4

SR Regulation of Chl biosynthesis under light stress. [127]

A.t. LHCA5 Q9C639 Regulation of LET versus CET. [128]

A.t. LHCA6 Q8LCQ4 Regulation of LET versus CET.

aAbbreviations: A.t., Arabidopsis thaliana; C.r., Chlamydomonas reinhardtii, M, monomeric LHCII proteins of the innerantenna layer; SR, stress-related chlorophyll a/b binding protein; T(), major LHCII protein of the outer trimeric antenna layer(type according to branch on phylogenetic tree).

bThree main classes of LHCII proteins can be distinguished.

LHCII-M

LHCII-S

CP29CP24

CP47

D1 D2D1 D2

CP43

CP26

1O2

O2

1O2O2ROS scavenging

HeatFluorescence Heat

Fluorescence

Phot

oche

mist

ry

Phot

oche

mist

ry

1 Chl∗

1Chl∗ quenching 3Chl∗ quenching

1Chl∗ quenching 3Chl∗ quenching

3 Chl∗

Xan

Chl

Chl

3Xan ∗

M

MMM

S

S

S

S

L

M

MMM

S

S

S

S

L

L L

L

L

L

L

PSI-LHCI

PSI-LHCI

PSI-LHCI

PSI-LHCI

PSI lightPSI light

PSII lightPSII light

PSIIco

rePSII

core

PSIIco

rePSII

core

L

S

S

M

M

S

S MELEL

(A) (B) (C)

(D)(E)

(F)

L

Figure 2. The Different Functions of Light-Harvesting Complex (LHC) Proteins. (A) Monomeric LHC (CP24/26/29)mediates excitation energy transfer between the trimeric LHCII complexes (LHCII-S/M) and the dimeric photosystem II (PSII)core (CP43/47 and D1/D2). (B,C) LHC proteins can undergo a transition from an unquenched state (B) with low heatdissipation to a quenched state (C) with higher heat dissipation and lower fluorescence yield. (D) Singlet oxygen is anunavoidable product of unquenched chlorophyll excited states, and xanthophylls have a constitutive function in quenchingchlorophyll triplets and scavenging singlet oxygen. (E) Over-reduction of the plastoquinone (PQ) pool triggers LHCIIphosphorylation upon unbalanced excitation of PSII. A mobile part of the LHCII antenna migrates to PSI, thereby balancingphoton capture at both photosystems. (F) LHCII, particularly the loosely-bound population, is degraded upon acclimation toexcess light (EL) to prevent overexcitation. Abbreviations: Chl, chlorophyll; CP, chlorophyll-binding protein; ROS, reactiveoxygen species; Xan, xanthophyll.

58 Trends in Plant Science, January 2016, Vol. 21, No. 1

Box 1. The Distinct Fates of Excited Chlorophyll States

The energy captured in an excited chlorophyll molecule can have several fates: it can be transferred to another pigment,used to drive the redox reactions of photosynthetic electron transfer (PET), dissipated thermally as heat, or re-emitted asfluorescence. Because these processes are in competition, the fluorescence yield will be modified by changes in any ofthe other pathways. Decreases in the yield of chlorophyll fluorescence that are not due to photosynthetic activity are thusdescribed as non-photochemical quenching (NPQ), which is attributed to several mechanisms. Upon exposure toexcess light, the most rapid process, operating in the range of seconds to minutes, is ascribed to energy dissipation (qE)and is regulated by the pH gradient across the thylakoid membrane. Low Calvin cycle activity leads to fast ADP plusNADP+ depletion and lumen acidification, which further increases owing to activation of CET (cyclic electron transport)caused by high NADPH levels. The equilibration of ATP and redox poise between chloroplast and mitochondria accountsfor the influence of metabolic conditions on the irradiance required to saturate photosynthesis [129–131]. Response tothe rapid PQ reduction obtained in these conditions is catalyzed by qE. This is the fastest-responding quenchingmechanism and is triggered within tens of seconds upon lumen acidification [37]. Slower components reflect theenhancement of energy dissipation via the accumulation of zeaxanthin (qZ) through the xanthophyll cycle, and, under veryhigh light intensities, photoinhibition and photodamage to the electron transfer chain (qI). In addition to these majorquenching components, others are specific for groups of organisms. State transitions (qT, see Figures 1A, 2E in maintext) appear to be equally important in algae [2], while in plants a slow component of fluorescence decay is due to theavoidance of photon absorption caused by chloroplast movement (qM) [109]. Changes in the spectral composition ofincident light easily occur under canopies, leading to an imbalance between PSI and PSII excitation. In water bodies, bluelight is transmitted far better than red light. Because of the need to avoid PQ over-reduction, the mechanisms of state 1–state 2 transitions (qT) re-equilibrate PET rates through both photosystems. Under state 2 conditions, PQH2 activates thekinase STT7 (STN7 in plants) [132,133] at the Qo site of the cytochrome b6f complex [77] which phosphorylates LHCII, asubset of which migrates out from grana membranes [134,135] leading to re-equilibration of PET rates between PSI andPSII [133].

Light is in excess when absorption exceeds the rate of energy utilization by downstreamreactions. Excess photons enhance the concentration of excited singlet (1Chl*) states, theprobability for triplet chlorophyll (3Chl*) and of 1O2 formation (Figure 2B–D). The definition of‘excess photons’ is not a constant, but depends on environmental conditions during actual andprevious growth. Because light reactions are essentially independent of temperature andsubstrate availability, changing environmental conditions form ever-changing bottlenecks indownstream reactions. In fact, the rate of downstream metabolic reactions, first by the Calvin–Benson cycle, is strongly modulated by CO2 availability and temperature, thereby controlling itscapacity for NADP+ and ADP + Pi regeneration. Electron acceptor limitations cause an over-reduction of the plastoquinone (PQ) pool (Figure 3) and favor charge recombination to P680*.Carotenoids cannot resist the strongly oxidizing (+1.1 volts) species generated at P680. 3Chlquenching (Figure 2B,C) is thus impaired, leading to 1O2 formation and photoinibition [19]. Inaddition, owing to the very shallow energy gradient within PSII antenna systems [20],unquenched 1Chl* states are delocalized, leading to 3Chl* formation [21–23]. The consequencesof ADP + Pi limitations are more complex: ATPase activity is limited by substrate availability andlumenal protons from water splitting and cyclic electron transfer (CET) cannot find their wayback to the stromal compartment, causing lumen acidification (Figure 3A,B). This, in turn, limitselectron transport owing to photosynthetic control [24] and triggers non-photochemicalquenching (NPQ; Box 1).

NPQ MechanismsTo avoid ROS formation, oxygenic organisms evolved photoprotection mechanisms, includingtriplet quenching [25,26], ROS scavenging [23,27], and alternative electron transport pathways[28]. Energy-dependent quenching (qE) is the fastest response to excess light among thedifferent NPQ mechanisms. The pH-dependence is conferred by the presence of acidic residueson the luminal surfaces of PSBS [29] and LHCSR3 [30], proteins essential for qE in plants andalgae, respectively, whose protonation induces conformational changes to a quenching stateeither in LHCSR3 itself [31] or in PSBS-interacting proteins such as CP29 [32,33]. LHCSR andPSBS differ in their pigment-binding capacity: while LHCSR3 forms a stable complex with Chlsand xanthophylls [31], in PSBS most of the Chl-binding residues are not conserved, suggestingthat it is not a pigment-binding protein [34]. Quenching mechanisms proposed include formation

Trends in Plant Science, January 2016, Vol. 21, No. 1 59

Transcrip�oncontrol

Transla�oncontrol

NAD(P)H

NADP+

6 H+

4 H+

4 H+

H2O ½ O2+ 2 H+

NADPH

Redox valves

ATP

Cytosol

NAB1

LHCSR3

LHCBM9

LHCBM

LHCBM6

NucleusNAB1

SX

OxThiolbuffers

LHCB

MLH

CBM

6

LHCI

I

PSII

e−

e−

e−

PQ

PQH2

cytb6f

PSILHCI

PC

Fd

FNR

CB HighCO2

ADP + Pi

Stroma

Lumen

Chloroplast

NAB1NAB1

LHCBM9

LHCSR3

LHCBM

LHCBM6SH

redThiol

buffers

NAD(P)H

NADPH CB

Redox valves

Over-satura�on ofphotosynthesis triggers

remodeling of the antenna

4 H+

4 H+

½ O2H2O

+ 2 H+

e−

e−

PQ

PQH2

cytb6f

PC

LHCB

M9

LHCI

I

PSII

LHCS

R3

1O2

PSILHCI

Fd

FNR

NADP+

CO2

ADP+ Pi

ATPLow

Low

(A)

(B)

Figure 3. Long-Term Light-Harvesting Control Mechanisms as Described for the Microalgal Model OrganismChlamydomonas reinhardtii. (A) Unperturbed photosynthesis. In the unstressed state [e.g., sub-saturating light supplyor high activity of the Calvin–Benson cycle (CB)] the photosynthetic electron transport (PET) chain is an oxidized state andthe thylakoid lumen is not acidified. In such conditions, all nuclear genes encoding major light-harvesting protein isoformsassociated with PSII (LHCBMs) are transcribed, except for isoform 9. LHCBM-encoding mRNAs (broken lines) aretranslated in the cytosol (indicated by black ribosomes) before the import of polypeptides into the chloroplast and insertioninto the thylakoid membrane, causing formation of the PSII–LHCII complex. The translation repressor NAB1, whose maintarget is isoform LHCBM6, is in a less active state because the cysteine activity switch is set to the ‘off’ position via thiolmodification (SX). This permits translation of LHCBM6. (B) The oversaturated state of photosynthesis. Excess light orinsufficient provision of CO2/nutrients (S, N) create a situation where the provision of ATP/NADPH by photosynthetic lightreactions exceeds their consumption by the Calvin–Benson cycle, and ATP as well as NADPH accumulate in thechloroplast. This results in an over-reduction of the PET chain (PQH2 > PQ). In addition, a reduced backflow of protonsinto the stroma, caused by ATP accumulation, acidifies of the thylakoid lumen. In this overexcited state of PSII only a smallfraction of the absorbed light energy can be dissipated by photochemical reactions, leading to the formation of harmfulsinglet oxygen. Long-term exposure to external conditions suboptimal for photosynthesis triggers a remodeling of thephotosynthetic antenna. This is achieved by a reduced rate of nuclear LHCBM transcription as well as by disassembly ofpolyribosomes, abolishing the de novo synthesis of LHCBM proteins. Translation repression of LHCBM6 is mediated byNAB1, which can be activated by reduction of a specific cysteine. NAB1 activation might involve export of excess reducingequivalents via chloroplast redox valves, eventually resulting in reduction of the cytosol. In contrast to all other isoforms,expression of LHCBM9 is induced under stress conditions (e.g., nutrient deprivation). Stress conditions also trigger theaccumulation of LHCSR3, which activates qE upon protonation of lumen-exposed acidic residues. Recruitment of LHCSR3or LHCBM9 promotes energy dissipation at PSII, thereby avoiding photodamage, while a reduced supply of other LHCBMswith a low quenching but high light-harvesting capacity helps in controlling PSII excitation during long-term acclimation.Abbreviations: cyt bf6, cytochrome b6f complex (plastoquinol-plastocyanin reductase); Fd, ferredoxin; FNR, ferredoxinNADP+ oxidoreductase; LHC, light-harvesting complex; NAB1, nucleic acid binding 1; Ox, oxidized; PC, plastocyanin; PET,photosynthetic electron transport; PQ, plastoquinone; PSI/II, photosystems I/II.

60 Trends in Plant Science, January 2016, Vol. 21, No. 1

of carotenoid radical cations [31,32,35] and energy transfer from 1Chla* state to lutein S1 followedby thermal dissipation [36]. In addition to the presence of lumen-exposed protonatable sites, CP29and LHCSR, the subunits for which a clear involvement in quenching was reported [13,31,37], arecharacterized by a low Chl b/a ratio, consistent with the stabilizing effect of Chl b on LHC proteinstructure through H-bonding [15]. In agreement, Chl b-rich LHCII has a single fluorescence lifetimeof about 4 ns while Chl b-poor CP29, CP26, and LHCSR show multiple lifetimes representingdistinct conformations with the short-living conformations being favored by zeaxanthin binding [38]or acidification [31]. A further important difference between antenna-type LHC proteins and thosecatalyzing quenching is the capacity to exchange violaxanthin in the L2 binding site (Figure 2B) forzeaxanthin that is synthesized in excess light conditions [39,40], which favors short-living, dissi-pative conformations, and strongly enhances NPQ [41,42]. Lumen acidification activates theviolaxanthin de-epoxidase enzyme (VDE), causing a fast de-epoxidation of violaxanthin to zea-xanthin, whereas the back-reaction by zeaxanthin epoxidase is slow. This causes sustainedfluorescence quenching that relaxes slowly within 1–3 hours following light stress and depends onthe release of zeaxanthin from antenna proteins. Formerly, this slowly-relaxing component wascompletely attributed to inhibitory quenching (qI) and was mechanistically explained by slowrecovery from photoinhibition based on repair cycle activity which implicates de novo synthesisof the reaction center protein D1 [43]. The effect of LHC-bound zeaxanthin is not merely todecrease the concentration of 1Chl* by �30%, but rather to prevent 3Chl* formation and toenhance 3Chl* and ROS scavenging [23], thus protecting against photodamage during repeatedlight-stress episodes. However, some algae do not show qE enhancement by zeaxanthin, and thisappears to be associated to the sessile lifestyle typical for biofilm-forming algae [31,44]. Anothersustained quenching effect was reported in monocots as the result of CP29 phosphorylation,which is slowly reversed during the recovery from excess light [45].

The importance of state transitions (qT) as an NPQ mechanism for photoprotection and energyquenching appears to be significantly different in plants versus microalgae. In Chlamydomonasreinhardtii most LHCBM proteins undergo phosphorylation, including the inner antenna proteinsCP29 and CP26 which associate with PSI in state 2 [46,47]. Owing to the requirement of minorantenna proteins for supercomplex assembly, algal PSII supercomplexes dissociate in state 2[48]. The quenching obtained in the process is large [46] and is photoprotective in excess light[2,5]. In plants, LHCII phosphorylation is inhibited already at moderate light levels [49], challeng-ing its significance for photoprotection and quenching in excess light. In plants and algae onlyspecific LHCII/LHCBM gene products are implicated in phosphorylation-mediated regulation.Indeed, only Lhcb2, a quantitatively minor antenna fraction, is involved in state transitions [50] inArabidopsis thaliana, similar to the case of LHCBM2/7 [51] and LHCBM5 [52–54] inChlamydomonas.

Long-Term Acclimation MechanismsPhototrophic eukaryotes adjust the stoichiometry of their photosynthetic machineries followinglong-term exposure to changes in external conditions via modulated gene expression ofindividual subunits (Figure 3), and LHC proteins represent a prime example [55].

Plants and microalgae respond to light quantity changes (Figure 2F) by either reducing (excesslight) or increasing (limiting light) the cellular LHC amount [56–58]. In Chlamydomonas a shift fromlower to higher light intensities decreases the cellular amount of LHCI and LHCII proteins[56,59,60], which does not result in an altered LHCII/PSII stoichiometry or functional PSIIantenna size. In contrast to LHCII proteins, the stress-related LHC proteins LHCSR3/PSBS(Table 1) accumulate and increase the overall NPQ capacity of plant and algal cells [56,57].

Similarly to C. reinhardtii, A. thaliana also downregulates the cellular LHCII amount uponacclimation to excess light. Here, however, the LHCII/PSII core stoichiometry changes, resulting

Trends in Plant Science, January 2016, Vol. 21, No. 1 61

in reduced PSII antenna size [57,58]. Interestingly, the three LHCB isoforms (Table 1) constitut-ing the major antenna are differentially affected, with LHCB1/2 levels being more stringentlyregulated than those of LHCB3. Similarly, expression of minor antenna protein LHCB6 (CP24;Table 1) is more stringently controlled than is the expression of LHCB4/5 [57,58]. The down-regulation of LHCB1–3 seen in A. thaliana plants exposed to high light levels mainly reduces theamount of LHCII-M trimers (Figure 1A), which are moderately bound to PSII and are not part ofthe C2S2M2 supercomplex. As a consequence, this stoichiometry modulation reduces PSIItrapping-time based on decreased time for excitation energy migration in a smaller antennacomplex [58], and thus the half-life of 1Chl* and singlet oxygen production.

It remains unclear whether microalgal LHCBM isoforms are also expressed differentially inresponse to excess light, which is suggested by their distinct involvement in short-termacclimation responses of C. reinhardtii [51,61,62] (Table 1). Whereas the LHCII/PSII core ratioundergoes changes during acclimation to high light levels in Arabidopsis, the respective LHCI/PSI ratios seem to be unaffected in both Arabidopsis and Chlamydomonas [57,58]. Recently, itwas demonstrated that binding of zeaxanthin to LHCI in A. thaliana causes a transition from thelight-harvesting into a quenched state, which reduces excitation energy transfer to the PSIreaction center without a need for stoichiometric adjustments [63].

Macronutrient or CO2 limitation decreases Calvin cycle activity, leading to photosyntheticelectron transport (PET) chain over-reduction and overexcitation of photosystems (Figure 3).In C. reinhardtii CO2 limitation reduces the LHCII/PSII core ratio and functional antenna size [4],while sulfur and nitrogen deprivation trigger the accumulation of isoform LHCBM9 (Table 1),which replaces other, constitutively expressed, isoforms within the PSII–LHCII complex(Figure 3). Recruitment of LHCBM9 to PSII–LHCII complexes protects PSII by inducing anenergy-dissipative state with reduced singlet oxygen formation [62].

The adjustments of PS antenna size and composition that occur during the long-term responserequire fine-tuned LHC expression. In green algae excess light causes a precipitous drop inLHCII and LHCI transcript levels, while light limitation has the opposite effect [59,64,65]. Thisstrong modulation of transcript levels is mainly achieved by controlling nuclear transcriptioninitiation rates [59,64], a view supported by the unchanged stability of LHCII transcripts in C.reinhardtii cells grown under high light conditions [59]. In Dunaliella tertiolecta the PQ redox stateand the trans-thylakoid membrane potential were shown to impact strongly on LHCII transcrip-tion [64,65], but in plants the involvement of the PQ pool [66] seems to be less clear [67].

Although LHCII mRNA levels are stringently controlled in response to elevated light, post-transcriptional mechanisms play at least an equally important role [4,59,68–73]. In Arabidopsis,long-term acclimation to excess light involves translation control of individual light-harvestingprotein isoforms, and repression/activation of translation seems to depend on their specificfunction within the PSII–LHCII complex. Isoforms whose primary function is light-harvesting,such as LHCB1 (Table 1), are translated at a reduced level in plants exposed to high light,whereas translation of isoforms having a photoprotective function (such as ELIPs) increases.Translational activation of pre-existing ELIP mRNAs precedes their de novo synthesis bypromoter induction, thus enabling rapid accumulation of proteins required for photoprotection[69].

In C. reinhardtii, the cytosolic RNA-binding protein NAB1 (nucleic acid binding 1) acts as atranslation repressor (Figure 3) by sequestrating LHCBM mRNAs into translationally silent mRNP(messenger ribonucleoprotein) complexes [70]. NAB1 prefers LHCBM6 transcripts, which is inline with a specific, non-redundant function of individual isoforms (Table 1). The extent of NAB1-mediated translation repression is controlled at multiple levels, which perfectly reflects the vital

62 Trends in Plant Science, January 2016, Vol. 21, No. 1

role that light-harvesting plays in a phototroph [4,74,75]. Limiting CO2 conditions trigger NAB1accumulation, which in turn reduces LHCII protein synthesis when antenna size reduction isrequired to maintain normal PSII excitation pressure despite lowered Calvin cycle activity. This isachieved via activation of the nuclear NAB1 promoter and can be abolished by blocking PET,suggesting the involvement of chloroplast retrograde signals [4]. NAB1 activity is regulated bytwo distinct post-translational modifications, with arginine methylation acting as a ‘masterswitch’ that adjusts activity to the prevailing metabolic mode [75]. In addition, activity isredox-controlled via cysteine modification, and the presence of a reduced cysteine residuedefines the ‘on’ state, whereas its modification reduces RNA-binding activity (Figure 3).

The Regulation of LHCII Requires Intense Inter-Compartmental CrosstalkControl of light harvesting at PSII occurs at various levels and distinct time-scales. Additionalcomplexity is added to the regulatory circuits, ensuring balanced PSII excitation, by the fact thatlight-harvesting proteins are encoded by nuclear genes, are translated in the cytosol, andeventually exert their function in the thylakoid membrane of the chloroplast [76] (Figure 3). For theshort- and long-term regulation of light-harvesting capacities, the redox state of the PET chainand especially that of the PQ pool were shown to play a central role [64,65,68,77,78]. Based ondirect sensing of the PQ redox state by state-transition kinases [77], they represent idealcandidates for sensors of the PET chain redox state and upstream components of plastid-to-nucleus signaling pathways.

Interestingly, the A. thaliana STN7 (STATE TRANSITION 7) and C. reinhardtii STT7 (statetransition 7) mutants are not only affected in the state-transition process, but also show aperturbed long-term acclimation response [4,79,80]. Similarly, state transitions and antenna sizecontrol via LHCII translation repression were recently shown to be linked intimately within theresponse to increased PSII excitation pressure in C. reinhardtii. During acclimation of C.reinhardtii cells to prolonged CO2 limitation, NAB1-mediated translation repression leads tostate transitions replacing excitation pressure control, which relaxes despite ongoing PET chainover-reduction. Intriguingly, this regulatory mechanism is affected in a mutant devoid of STT7 [4].

Recent studies in Arabidopsis propose a prominent role for plastid gene expression within thesignaling cascades which modulate LHCII gene transcription following changes in the plastidredox state [81–83]. A pathway containing the chloroplast-localized pentatricopeptide-repeat(PPR) protein GUN1 (GENOMES UNCOUPLED 1) [81], the nuclear transcription factor ABI4(ABA INSENSITIVE 1) [81] and a chloroplast envelope-bound protein PTM (PHD TYPE TRAN-SCRIPTION FACTOR WITH TRANSMEMBRANE DOMAIN) was demonstrated to be requiredfor the downregulation of LHCB transcription induced by high light [82]. Interestingly, processingof PTM under these conditions induces the migration of an N-terminal fragment from the plastidenvelop membrane to the nucleus [82].

In addition to transcription control, the modulation of cytosolic LHCII mRNA translation is a majorregulatory step [69,70], and there is evidence that redox crosstalk between chloroplast andcytosol regulates this process plants and algae [72,74].

The Design of Green Cell FactoriesStakeholders in bioeconomy focus on the biotechnological potential of microalgae as green cellfactories for the sustainable conversion of sunlight energy and CO2 into valuable products.However, the efficiency of photon energy conversion (PCE) into biomass in phototrophs is low,with theoretical maximum-achievable rates of about 10% [84]. Under real outdoor cultivationconditions PCE rates of plants and algae are below 2%, and are therefore considered to be oneof the key limiting factors for reaching high productivities [84–89]. Consequently, variousattempts to increase photosynthetic conversion efficiencies have been reported, either by

Trends in Plant Science, January 2016, Vol. 21, No. 1 63

Outstanding QuestionsWhat is the specific function of individ-ual light-harvesting protein isoformswithin light-harvesting and photopro-tection processes?

Which differences in the regulation ofplant and green algal LHC kinasesexplain the distinct photoprotectivesignificance of state transitions in thesesystems?

How are qE and qT mechanisms inter-twined in green algae, where bothmechanisms seem to be requiredwithin distinct phases of the responseto excess light?

How are short- and long-termresponses to photosynthetic perturba-tions mechanistically connected?

Does the isoform composition of theLHCII complex in green algae changeas part of the long-term response toexcess light?

Which protein factors, residing in thecytosol, modulate the synthesis of spe-cific light-harvesting protein isoforms inA. thaliana and other higher plants?

How is the rate of cytosolic LHC proteinsynthesis connected to the plastidredox state?

What are the signaling pathwaysenabling communication betweenchloroplast, cytosol, and nucleus?

How can we exploit our knowledgeabout photosynthetic light-harvestingto create novel microalgal strains withbespoke antennae to improve light con-version efficiency in photobioreactors?

enhancing the capacity of the major electron sink, the Calvin–Benson Cycle [90,91] or byoptimizing light harvesting and energy transfer between antenna and photosynthetic reactioncenters in crops and microalgae.

Structural and biochemical analyses of PSII–LHCII complexes in microalgae have revealed thatthey are equipped with large ‘oversized’ antenna systems [53,92] that are required for efficientlight harvesting in aqueous habitats.

In state-of-the-art photobioreactors (PBRs), insufficient light availability can be partly overcomeby the construction of systems with short optical path-lengths, such as thin plate PBRs, toincrease light penetration [93,94]. Even so, large light-harvesting antenna systems reduce lightpenetration, creating ‘dark zones’ in the PBR, where respiration rates exceed photosyntheticrates. Outer layer cells receive oversaturating irradiances; only a fraction of which can be used forphotochemistry, while the bulk part is dissipated as heat or re-emitted as fluorescence [95].

Antenna truncation was postulated as a suitable engineering approach to tune algal cells to theartificial environment in a PBR for optimal photon to biomass energy flow [85,96,97]. Severalscreening approaches were taken to identify truncated antenna mutants with a reduced pigmentcontent [96,98–104]. Antenna size reduction by the nonspecific downregulation of LHCII subunitsprovided a first proof of concept for the positive effect of antenna truncation on PCE rates inmicroalgae grown in high-light conditions [96,98], but simultaneously revealed negative conse-quences on phototrophic biomass generation in sub-saturating light. To resolve this discrepancy,new engineering strategies came to the fore, focusing on more elaborate and specific LHC antennamanipulations. Following the functional characterization of individual LHC subunits at PSII (Table 1),it was suggested that only LHC proteins with a predominant function in light harvesting could beconsidered as potential targets for efficient antenna engineering, while those involved in photo-protection should not be downregulated. Furthermore, downregulation of the overall cell opticaldensity rather than PSII antenna size controls light diffusion in PBR [88]. New strategies wereapplied to exploit knowledge about light utilization for the construction of new C. reinhardtii variants[99,101–103] with an enhanced photoautotrophic growth phenotype. This was achieved either bymiRNA-mediated knockdown of distinct LHCII subunits (Lhcbm1, Lhcbm2, Lhcbm3) [102], byselectively repressing translation of isoform LHCBM6 through manipulation of the LHCII translationrepressor NAB1 [99], by downregulation or random knockout of TLA1, TLA2, or TLA3 (truncatedlight-harvesting antenna 1–3) genes which are proposed to regulate the chlorophyll antenna size[101,105], or by targeting genes of the chloroplast signal-recognition particle (CpSRP) pathway[103]. Very recently another truncated antenna mutant (TAM-2) [104] was identified by a randommutagenesis approach in Chlorella sorokiniana, a microalga of high biotechnological potential, andshowed increased photon use efficiency on a laboratory-scale and in outdoor photobioreactors. Inaddition to antenna engineering, more subtle modifications can significantly modify the activationthreshold and profile of energy-dissipation triggers such as PSBS or LHCSR which functionallydepend on the protonation of lumen-exposed acidic residues. To survive stress conditions,phototrophs induce energy-dissipation mechanisms at the expense of biomass accumulation.Thus, decreasing the lhcsr copy number (and NPQ activity) in C. reinhardtii has significantlyincreased biomass accumulation (S. Berteotti et al., unpublished) in agreement with previousresults obtained with the PSBS-less npq4 mutant [106]. In addition, mutagenesis for changing thenumber and pK of protonatable residues is expected to be effective in tuning the activation profile ofenergy dissipation, minimizing activity in low light and maximizing it under potentially photo-damaging irradiances [107,108].

Concluding Remarks and Future OutlookPast research on mechanisms controlling light capture in the model organisms C. reinhardtii andA. thaliana has indicated that differences in the acclimation strategies deployed [56,58] might

64 Trends in Plant Science, January 2016, Vol. 21, No. 1

reflect the lifestyle of an organism (e.g., sessile vs non-sessile) and environmental cues experi-enced in its natural habitat (e.g., aqueous vs terrestrial). For instance, the photoprotectivesignificance of particular NPQ mechanisms differs remarkably between plants and microalgae,with qE and qT representing a prime example [2,5,39,44,45,49,109]. Although recent researchhas shed light upon the mechanistic details of photoprotection in plants [14,29] and microalgae[13,30,31], the list of the factors involved is still not comprehensive. New genome modificationtechniques such CRISPR/Cas [110] will open up novel opportunities to complete the list. Futureresearch should elucidate how chloroplast, cytosol, mitochondria, and nucleus communicate toprecisely adjust photosynthetic light capture both in the short and long term [4,74,75,79–83].The true biotechnological potential of current antenna engineering concepts needs to beexplored further [111], and novel strategies based on increased knowledge about LHCII proteinsshould be devised.

AcknowledgmentsThe authors thank the Deutsche Forschungsgemeinschaft (KR1586/5-2 to O.K.), the European Commission (FP7-

ACCLIPHOT 245070 to R.B.; FP7-SUNBIOPATH 3164 to O.K. and R.B.) and the Accademia dei Lincei (Roma, Italy,

to R.B.) for funding.

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