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sGranal stacking of thylakoid membranes in higher plantchloroplasts: the physicochemical forces at work and the functionalconsequences that ensue†

Wah Soon Chow,*a Eun-Ha Kim,a Peter Hortonb and Jan M. Andersona

a Photobioenergetics Group, Research School of Biological Sciences, GPO Box 475, Canberra,ACT 2601, Australia. E-mail: chow@rsbs.anu.edu.au; Fax: 61 2 6125 8056;Tel: 61 2 6125 3980

b Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank,Sheffield, S10 2TN, UK. E-mail: p.horton@sheffield.ac.uk; Fax: 44 114 222 2712;Tel: 44 114 222 4189

Received 23rd May 2005, Accepted 25th July 2005First published as an Advance Article on the web 24th August 2005

The formation of grana in chloroplasts of higher plants is examined in terms of the subtle interplay ofphysicochemical forces of attraction and repulsion. The attractive forces between two adjacent membranes comprise(1) van der Waals attraction that depends on the abundance and type of atoms in each membrane, on the distancebetween the membranes and on the dielectric constant, (2) depletion attraction that generates local order by granalstacking at the expense of greater disorder (i.e. entropy) in the stroma, and (3) an electrostatic attraction of oppositecharges located on adjacent membranes. The repulsive forces comprise (1) electrostatic repulsion due to the netnegative charge on the outer surface of thylakoid membranes, (2) hydration repulsion that operates at smallseparations between thylakoid membranes due to layers of bound water molecules, and (3) steric hindrance due tobulky protrusions of Photosystem I (PSI) and ATP synthase into the stroma. In addition, specific interactions mayoccur, but they await experimental demonstration. Although grana are not essential for photosynthesis, they areubiquitous in higher plants. Grana may have been selected during evolution for the functional advantages that theyconfer on higher plants. The functional consequences of grana stacking include (1) enhancement of light capturethrough a vastly increased area-to-volume ratio and connectivity of several PSIIs with large functional antenna size,(2) the ability to control the lateral separation of PSI from PSII and, therefore, the balanced distribution of excitationenergy between two photosystems working in series, (3) the reversible fine-tuning of energy distribution between thephotosystems by State 1–State 2 transitions, (4) the ability to regulate light-harvesting via controlled thermaldissipation of excess excitation energy, detected as non-photochemical quenching, (5) dynamic flexibility in the lightreactions mediated by a granal structure in response to regulation by a trans-thylakoid pH gradient, (6) delaying thepremature degradation of D1 and D2 reaction-centre protein(s) in PSII by harbouring photoinactived PSIIs inappressed granal domains, (7) enhancement of the rate of non-cyclic synthesis of adenosine triphosphate (ATP) aswell as the regulation of non-cyclic vs. cyclic ATP synthesis, and (8) the potential increase of photosynthetic capacityfor a given composition of chloroplast constituents in full sunlight, concomitantly with enhancement ofphotochemical efficiency in canopy shade. Hence chloroplast ultrastructure and function are intimatelyintertwined.

IntroductionChloroplasts in plants have a highly organized internal mem-brane system. Flattened disk-like sacs (thylakoids) adhere toform stacks (grana). The thylakoids within a stack are connectedto those in adjacent grana by non-appressed, stroma-facingmembranes (stromal lamellae). The internal aqueous space(lumen) of the thylakoids of a chloroplast is continuous,separated from the external aqueous phase (stroma) in which, forexample, the Benson–Calvin cycle enzymes and the chloroplastgenetic machinary reside. Various three-dimensional models ofgranal ultrastructure have been proposed; they are comparedwith the latest computer model based on electron micrographsfrom serial section of granum–stroma assemblies.1

The thylakoids in chloroplasts are the site of light-harvesting,photosynthetic electron transport, proton translocation, trans-duction of an electrochemical gradient in adenosine triphos-

† Dedicated to Professor James Barber on the occasion of his 65thbirthday.

phate (ATP) synthesis and repair of Photosystem II (PSII)after light-induced damage. Photochemical utilization of lightleads to the production of ATP and reduced nicotinamideadenine dinucleotide phosphate (NADPH), both required forphotosynthetic carbon assimilation and other biochemical reac-tions. The compact arrangement of internal membranes meansthat although chloroplasts have a relatively small volume, theycontain an enormous area of thylakoid membrane, with an area-to-volume ratio from 7 × 107 to 5 × 108 m−1, compared with 106

m−1 for a solid sphere of radius 3 lm; the large area-to-volumeratio greatly increases the ability of chloroplasts to capture light.2

What physicochemical forces shape the ultrastructure ofthe chloroplast? In this paper, we will consider the interplayof attractive and repulsive forces, the net effect of whichdetermines the organization of the internal membrane system ofchloroplasts. We will also consider the functional consequencesof granal formation that, although not essential for photo-synthesis, helps to fine-tune it under ever-changing conditions.Understanding the formation of grana is integral to elucidatingthe structure–function relationship of higher-plant chloroplasts.D

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Electrostatic repulsionSurface charge density

Thylakoid membranes carry net negative charges on theirsurfaces, as demonstrated by their electric mobility under anelectric field.3 An accurate determination of the net surfacecharge density (r) of thylakoids is not a trivial task, but anapproximate value of −0.025 C m−2 for the outer surface seemsa reasonable working value.4 In thylakoids isolated from leavesof Alocasia grown in deep shade, the measured magnitude ofr seemed to be particularly small; this is consistent with a lowelectrostatic repulsion and the formation of very large grana.5

Fig. 1 shows a large granum containing more than 160 pairs ofmembranes in the stack in an Alocasia chloroplast. Similarly,neutralizing carboxylate groups on thylakoid proteins decreasesthe magnitude of r, thereby promoting stacking at pH 4.0–4.5.6

In contrast, chemical modification of lysine amino residues withacetic anhydride causes a loss of positive charge and a gain ofnegative charge, resulting in an increase of net negative charge,and a decrease in the extent of stacking.6 These studies clearlydemonstrate the electrostatic influence that controls the stackingand unstacking of thylakoid membranes.7

Fig. 1 An electron micrograph of a chloroplast in a leaf of Alocasiamacrorrhiza grown under low light, showing a large granum that consistsof over 160 pairs of membranes. Adapted from ref. 5.

Electrostatic screening

Even for a given value of r on thylakoid membranes, the elec-trostatic repulsion can be modulated by the ionic compositionof the aqueous medium. Thus, suspension of envelope-freechloroplasts in a low-salt zwitterionic buffer (containing ca.20 mM Na+ from the NaOH used to adjust the pH) causesa loss of grana structure.8 The addition of divalent cations orhigh concentrations of monovalent cations causes re-stacking.9

Fig. 2(A) shows the re-stacking on increasing [MgCl2] in twobackground concentrations (10 or 50 mM) of KCl. In thisexperiment, the extent of stacking was assessed by treatingthe envelope-free chloroplasts with the non-ionic detergentdigitonin. Digitonin readily solubilized the stromal lamellae,leaving the granal stacks which were collected by centrifugationand assayed for the chlorophyll content.6,7 As [MgCl2] increased,the extent of thylakoid stacking increased. Significantly, therewas a “cross-over” of the two curves, so that a higher background[KCl] meant less stacking at a given high [MgCl2].10 Thecalculated electrostatic repulsion per unit membrane area (ata fixed separation between membranes) decreases with increasein [MgCl2], and the cross-over is also evident from the calculatedcurves (Fig. 2(B)), qualitatively consistent with experiment(Fig. 2(A)). In the absence of MgCl2, electrostatic repulsion ismuch stronger at 10 mM KCl than at 50 mM, but the observedextent of stacking was not very different; presumably, themembranes were completely unstacked, and stronger repulsiondid not have any further effect.

An interesting observation was that when unwashed thylakoidmembranes (in the stacked state) were suspended in an almostcation-free medium (containing ≤1 mM K+), they maintained

Fig. 2 Experimental determination (A, C) of the extent of thylakoidstacking as a function of ionic composition of the suspension mediumand the corresponding electrostatic repulsion calculated for each par-ticular ionic medium (B, D). Either the monovalent or divalent cationconcentration was varied while the other was kept constant. Re-drawnfrom ref. 10.

their granal stacking; addition of low concentrations of mono-valent cations brought about unstacking of the thylakoids, butas the monovalent cation concentration was raised to a highlevel, stacking occurred again.11 Fig. 2(C) gives an example ofthis “dip”; the minimum [KCl] required for complete unstackingvaries, depending on the background [MgCl2], which is typicallycarried over from the unwashed thylakoid stock suspension.10,12

The calculated electrostatic repulsion per unit membrane surfacearea at a fixed separation of the membranes shows a “crest”(Fig. 2(D)), consistent with the dip (Fig. 2(C)).

When two negatively charged membranes are at a certainseparation distance, each experiences an electrostatic repulsion.The net negative surface charge on each membrane attractscations but repels anions; the net concentration of charge ata point in the inter-membrane aqueous space is the space chargedensity (q, C m−3). Because of the accumulation of cations in

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the inter-membrane aqueous space, water molecules are draggedinto the space, and the electrostatic repulsion can be looked uponas being due to an increase in osmotic pressure in the inter-membrane aqueous space. Indeed, the positive space chargedensity was first used to explain the dip.13 Subsequently, anintegrated space charge density (r′

x, C m−2), with q integratedover distance from the membrane surface, was shown to bea more satisfactory measure of both short and long rangeeffects associated with electrostatic screening and double layerrepulsion (P) of charged surfaces; this was achieved by thederivation of a formal connection between r′

x and P.14 Takentogether, the above studies demonstrate that the electrostaticproperties of thylakoid membranes give rise to an importantcomponent of the forces between thylakoid membranes.

van der Waals attractionA crucial force between thylakoid membranes is the van derWaals or London dispersion force. Even in molecules thatpossess no net electric charge or permanent dipoles, instan-taneous dipoles exist as electrons move around the nucleus.Instantaneous electric dipoles induce instantaneous dipoles inneighbouring molecules; these electric dipoles interact to givea net attraction that is universally present and depends on thenumber and type of atoms involved, on the distance betweenthem and on the dielectric constant. Earlier models of membranestacking relied mainly on a consideration of electrostatic prop-erties of thylakoid membranes,4,5,10 and van der Waals attractionwas not emphasized. Although van der Waals force is consideredweak, its universal nature means that a multitude of weakforces combine to give a strong attraction.15 Thus, chloroplastswith an abundance of LHCII, light-harvesting chlorophyll a/b-protein complexes that serve Photosystem (PS) II, tend to exhibitenhanced thylakoid stacking. Alocasia chloroplasts from a deep-shade plant possess abundant LHCII which, together with adecreased magnitude of r, must have contributed to the largegrana stacks in Fig. 1. In contrast, the Chl b-less mutant of barleyhas no LHCII to confer van der Waals attraction, so it exhibitspoor thylakoid stacking despite a relatively low magnitude of r.15

There has been only one attempt to calculate the van derWaals attraction between thylakoid membranes.16 In that study,the van der Waals attraction was probably underestimated, forit was not sufficient to overcome the electrostatic repulsionwithout assuming an unrealistically low magnitude of r. Suchan underestimation could arise from the assumption of a lowdielectric constant (2–3) for the thylakoid membrane, a valuethat is not much higher than that of a pure lipid membrane. Theappropriate dielectric constant is likely to be higher because ofthe high protein content of thylakoid membranes, particularly inthe appressed membrane domains. A higher dielectric constantwould give a correspondingly larger van der Waals attraction.

Hydration repulsionIn early high-resolution electron micrographs of grana, therewas a separation between the appressed surfaces of two adjacentthylakoids (partition gap) of about 4 nm.17 More recent estimatesgive a partition gap of ca. 2 nm.18 This non-zero separationbetween the two surfaces is taken to suggest that a short-rangerepulsive hydration force operates to prevent closer approacheven when the van der Waals attraction exceeds the electrostaticattraction.16

The hydration force or “structural repulsion” presumablyoriginates from the water structure around charged groups ex-posed on membrane surfaces or on a hydrophilic neutral surfacesuch as the head groups of galactolipids. Close to the thylakoidsurface, water molecules assume preferred orientations. As twomembranes approach each other, however, such structured watermolecules have to be displaced. The displacement is energeticallyunfavourable (though strongly entropically favoured because the

displaced water molecules are free to move randomly), giving riseto a repulsive hydration force or more, correctly, a repulsion thatis associated with dehydration.19

A strong repulsive force was measured directly between twomica surfaces20 and lecithin bilayers.21 Further measurementsof repulsive forces between phospholipid bilayers indicated thatrepulsion is dominated by a strong hydration force,22 and thatthe force went through peaks and troughs as water moleculeswere displaced.23

Indeed, there is indirect evidence for the involvement of hydra-tion repulsion in grana formation. On lowering the pH from 7.7to 4.7 in a stacking medium containing 100 mM NaCl, the spacebetween membrane surfaces of adjacent thylakoids in the darkis markedly decreased.9 This can be interpreted as a decreasein hydration repulsion resulting from protonation of membranesurfaces and the loss of water-ordering charged groups.24

Depletion attraction: entropy-driven attractionbetween thylakoid membranesOn adequate lowering of electrostatic repulsion, thylakoidmembranes will stack under the influence of van der Waalsattraction, reaching a lower Gibbs free energy; that is, DG isnegative, and the process will occur spontaneously. The changein Gibbs free energy, however, can have a contribution fromchanges in enthalpy (H) or entropy (S) or both:

DG = DH − TDS

A negative DH will obviously help the stacking process, buteven when DH is positive, DG may be negative if DS is sufficientlypositive. Thus, a hypothesis was first put forward that a sufficientincrease in entropy overall will induce thylakoid stacking.25 Thatis, local order in the form of granal stacks can be generated atthe expense of an increase in entropy in the stroma.

Perhaps a simplest example of order from disorder is anisotropic suspension of thin hard rods. Upon being concen-trated, the originally isotropic suspension undergoes a transitionto a nematic phase in which the rods have preferred orientation.By aligning themselves more or less in a parallel manner, the thinhard rods are able to gain considerable translational entropyby getting out of one another’s way. Thus the loss of entropyassociated with orientational ordering is more than compensatedfor by the increased freedom for translational motion.26

In the generation of order from an overall increase indisorder, structures are formed by an attraction between thecomponents. Entropy-driven adhesion or aggregation, wherebysmall particles when mixed with large ones, maximize their ownentropy by pushing the large ones together, is called depletionattraction in physics or macromolecular crowding in biology.27 Itis a thermodynamic force that causes, for example, the attractionbetween large colloidal particles when small particles are alsopresent,28 the restraint of a colloidal particle from falling over theedge of a step,28 and the formation of ordered structures in a sus-pension of rod-like viruses and sphere-like polystyrene latex.27

To understand how an increase in entropy can assist thylakoidstacking, consider initially unstacked thylakoids (large particles)bathing in a medium in which the concentration of a macro-molecule (small particles, e.g. the ribulose 1,5-bisphosphatecarboxylase/oxygenase or Rubisco, the most abundant proteinin nature) is suddenly increased greatly. The large Rubiscooctamers at a high concentration are initially restricted in theirdiffusion. For simplicity, consider a Rubisco complex as a hardsphere (Fig. 3(A)). The surface of each sphere, at the closestapproach to a thylakoid membrane (non-filled area in Fig. 3),touches the ordered layers of water molecules (dotted area inFig. 3), which as described in the previous section are responsiblefor the short-range repulsion. That is, the centre of each sphere,unable to be any closer to the membrane than the dashed line,is excluded from the volume (depletion zone) indicated by thehatched area. When two adjacent thylakoids adhere, they are in

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Fig. 3 A scheme for the generation of more volume for free diffusionof macromolecules (e.g. Rubisco) by the appression of thylakoid mem-branes. In (A), each non-appressed thylakoid membrane non-shaded)is surrounded by a hydration layer of bound water molecules (dotted).The centre of a macromoleular complex such as the octameric Rubisco(represented by the circle) cannot approach the membrane beyond thedashed line. The “exclusion volume” is shown hatched. In (B), twomembranes adhere to contact each other at the hydration layer. Theexclusion volume (hatched) is now less, yielding increased volume forfree diffusion of the macromolecular complex. Adapted from ref. 25.

contact at the ordered water layers (Fig. 3(B)). In this case, thehatched area is smaller and the depletion zone is less, allowingmore volume for free diffusion of the spheres. Accordingly, eachsphere now has increased freedom for diffusion, resulting inan increase in entropy overall in the stroma, while generatinglocal order in the granal stacks. Therefore, addition of a highconcentration of a macromolecule should promote stacking,thereby relieving the restriction on macromolecular diffusion.

To experimentally test the hypothesis of entropy-assistedstacking of thylakoids,25 we added high concentrations of awater-soluble macromolecule to mimic the very high concen-trations of proteins and nucleic acids in the chloroplast stroma,particularly of Rubisco which is present at 300–400 mg ml−1. Wechose two very different macromolecules to test the generalityof the effect. One was bovine serum albumin (BSA), a globularprotein that carries net negative surface charge; the other wasdextran, an electrically neutral polysaccharide. We determinedthe extent of thylakoid stacking by the total cross-sectionalarea of grana seen in each chloroplast by transmission electronmicroscopy. In a basic buffer medium (containing a background

of 26 mM K+) the addition of 30 mM KCl did not lead to anysignificant re-stacking of the thylakoids, the cross-sectional areaof grana being only 0.15 lm2 per chloroplast29 (see Table 1).When either 5% BSA or 15% dextran was included, however,the cross-sectional area of grana in each chloroplast increasedby an order of magnitude29 (see Table 1). The cross-sectionalarea of grana in each chloroplast was at a maximum value inthe presence of either 5% BSA or 15% dextran; an increase ofadded [KCl] to 100 mM did not produce any further increase instacking. We interpret this result to mean that stacking releasesmore volume for free diffusion of the macromolecules andincreases the overall entropy, as explained above through theexample of Rubisco (Fig. 3).

We also estimated the increase in volume for free diffusionof Rubisco upon thylakoid stacking. This was done by takinghigh-resolution electron micrographs from the literature andestimating the total length of appressed pairs of membranes.A layer of thickness 6 nm (the approximate radius of eachRubisco octameric complex) adjacent to each membrane surfaceis excluded for the diffusion of Rubisco prior to stacking. Onstacking, the exclusion area decreases by an amount equal tothe total length of appressed membranes multiplied by thethickness 6 nm. This area is expressed as a percentage of thetotal cross-sectional area of the chloroplast cross-sectional area,excluding that of starch granules. The percentage decrease incross-sectional area represents the same percentage of decreasein exclusion volume since the areas are multiplied by the samedepth. From published electron micrographs of various plantchloroplasts in the literature, we obtained an average of 17%decrease in exclusion volume for Rubisco diffusion in the stromafollowing grana formation (Table 2). This is a significant increasein volume for Rubisco diffusion in the highly crowded stromalmilieu. Similarly, diffusion of the bulky stromal chloroplastribosomes to attach to non-appressed thylakoid domains whennew proteins are being synthesized should be also enhanced byan increase in stromal volume due to grana thylakoid stacking.

Steric hindranceThe structural differentiation of higher plant thylakoids intoappressed granal and non-appressed stromal lamellae is ac-companied by a functional differentiation. There is a well-defined lateral heterogeneity in the location of membrane proteincomplexes. PSI30 and ATP synthase31 are located exclusively innon-appressed membrane domains, which comprise the stromalthylakoids, and the margins and end membranes of granalstacks. PSII dimeric supercomplexes with associated trimericLHCII are located in appressed granal domains. Cytochrome bfdimers occur in both appressed and non-appressed domains.

Early ideas of what causes PSI to partition into non-appressed membrane domains were based on electrostaticconsiderations.2,7 When unstacked, thylakoid membranes havea random distribution of membrane proteins and lipids, thesurface electric potential is more or less uniform throughout themembrane system. It was thought that when two membranes

Table 1 The extent of grana formation in envelope-free chloroplasts

Treatment Total granal cross-sectional area per chloroplast/lm2 (±SE, n = 20 chloroplasts)

30 mM KCl 0.15 ± 0.0330 mM KCl + 5% BSA 1.22 ± 0.0830 mM KCl + 15% dextran 1.48 ± 0.08100 mM KCl 1.36 ± 0.10100 mM KCl + 5% BSA 1.38 ± 0.07100 mM KCl + 15% dextran 1.37 ± 0.07

The extent of thylakoid stacking was measured by the total cross-sectional area of grana in each of 20 chloroplasts. The basic buffer medium consistedof 300 mM sorbitol, 1 mM EDTA and 50 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (pH 7.6, adjusted with 26 mM KOH). To thisbackground [KCl] was added 30 or 100 mM KCl in the absence or presence of BSA or dextran. Taken from ref. 29.

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Table 2 Decrease in cross-sectional area for Rubisco diffusion upon thylakoid stacking

Decrease in exclusion area for Rubisco (% of total cross-sectional area) Plant Ref.

11.2 Spinach 9813.6 Maize 9915.7 Maize 10016.2 Barley 10116.4 Arabidopsis 10217.0 Spinach 10319.7 Peperomia 10424.1 Spinach 105

16.7 ± 1.4 (mean ± S.E.)

A number of high-resolution electron micrographs of grana and stroma were taken from the literature. The cross-sectional area excluded for thediffusion of the octameric Rubisco complex (taken to be a sphere of radius 6 nm) was calculated as the product of the total length of appressedmembranes and the radius of Rubisco, and expressed as a percentage of the total cross-sectional area of the grana and stroma in each micrograph,neglecting any cross-sectional area of cytosol and starch grains. This percentage represents the percentage gain in volume for free diffusion of Rubiscowhen both cross-sectional areas are multiplied by the same depth.

come together as electrostatic repulsion is decreased by cationscreening, the two electric diffuse layers on opposite membranesurfaces begin to overlap, the additive effect making the surfaceelectric potential of adhering membranes more negative. ByLe Chatelier’s principle, the membrane components with moresurface charges will diffuse into non-appressed membranedomains, thereby tending to restore a uniform distribution ofsurface electric potential over all the thylakoids.2,7 In this view,PSI, being located in non-appressed membranes, is expected tocarry more net negative surface charge.

Experimental evidence in favour of this idea was sought,but the results were equivocal. First, stromal vesicles derivedfrom stromal membranes did not readily aggregate under theinfluence of cation screening.32 Although consistent with ahigher postulated magnitude of r, the observation could also bedue to weaker van der Waals attraction because of the paucityof LHCII in stromal vesicles. Second, stromal vesicles, on anequal chlorophyll basis, were much more able to quench 9-aminoacridine fluorescence as compared with “average” vesi-cles derived from unstacked membranes; this “concentrationquenching” arises when the 9-aminoacridine cation is concen-trated in the diffuse layer adjacent to the thylakoid surface.33

Again, this observation is consistent with a higher postulatedmagnitude of r on stromal vesicles, but it could be argued thatstromal vesicles have less chlorophyll content and, therefore,greater quenching of 9-aminoacridine fluorescence per unit ofchlorophyll. Third, analysis of earlier data gave a magnitudeof r that was apparently 10 times greater in non-appressedmembranes compared with appressed membranes.34 However, insolving two simultaneous equations, it was assumed that r wasthe same for appressed membranes in both high-light and low-light-grown lettuce plants, which may not be valid. For all thesereasons, the evidence in favour of non-appressed membraneshaving a greater magnitude of r is only circumstantial.

Indeed, it is certain that steric factors play an important role inthe segregation of PSI and ATP synthase to non-appressed mem-brane domains. Early work by Staehelin31 demonstrated thatthe bulky “knob” of ATP synthase could not be accommodatedin the partition gap. More recently, X-ray crystallography35,36

revealed that the bulky protrusions of PSI at the stroma-facingmembrane surface could also not be accommodated in thenarrow partition gap. Segregating PSI and ATP synthase tonon-appressed membrane domains would allow the appressedmembranes to assume a closer separation under a net attractiveforce and therefore, a lower free energy level.

Specific interactionsSo far, we have considered general repulsive and attractiveforces without involving “lock-and-key” type interactions be-

tween specific molecular entities. Conceivably, such specificinteractions could occur in addition to the non-specific forcesdescribed above. Relevant to this possibility, purified LHCIIwas found to form planar sheets which became appressed inmultilamellar stacks on adding MgCl2; similar observationswere also made when the LHCII was incorporated into un-charged lipid vesicles.37 Pre-treatment of the purified LHCIIwith the proteolytic enzyme trypsin inhibited the multilamellarappression in the presence of MgCl2. It was proposed that thiscation-induced membrane appression is mediated by negativecharges on the stroma surface-exposed segments of LHCII, i.e.specific surface-localized regions of LHCII.37 Whether or notspecific interactions are involved, however, the elimination ofthe appression after trypsin treatment cannot be interpretedunequivocally. Trypsin treatment removes a 2 kDa segment(equivalent to about 19 amino acids) from the N-terminus ofLHCII, thereby changing the net charge of zero (neglectingthe charge on Asp-55 at the start of Helix 138) to −4. Thislarge increase in negative charge and the resultant boosting ofelectrostatic repulsion would have prevented stacking regardlessof whether the 2 kDa segment played a specific role or not.

Although the results of trypsin treatment are ambiguous,there remains a tantalising possibility of specific interactionsbetween LHCII, either among themselves or with other proteincomplexes across the partition gap. For example, it is noted thatthe negative charged groups on the surface exposed segment ofthe amino end of LHCII are located nearer to Helix 1, whilethe positive charged groups are located nearer to the N-terminalend. In principle, the amino terminal segments from oppositemembrane surfaces could inter-digitate so that positive chargeson one segment are counterbalanced by negative charges onanother from the opposing membrane (Trissl, personal com-munication). Indeed, diagrams alluding to this trans-partition-gap effect have been presented.37,38 Such a specific effect atclose range would greatly enhance the electrostatic attractionbetween opposite charges from opposite membranes containingLHCII. The repulsion between two opposite membranes dueto negative charges located at the loop between Helices II andIII, however, occurs at longer range, and so tends to be weaker.In spite of such plausible electrostatic interaction between twoappressed membranes, however, there seems to be no excitationenergy transfer across the partition gap.39

Another mechanism of electrostatic attraction across thepartition gap, somewhat non-specific, has been reported forpacking of LHCII in a crystal lattice.40 The pattern of positiveand negative charges on the “stromal” surface of LHCII trimerssuggests a ‘velcro’-like interaction, positive charges on one sheetbeing aligned with negative charges on the opposite sheet.Again, such attraction could occur at close range with enhancedstrength.

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Other specific interactions will also need to be soughtexperimentally in relation to thylakoid stacking, e.g. specificlipid-lipid interactions induced by ions and specific lipid-proteininteractions.41

The functional consequences of grana stackingLight not only drives photosynthesis, but also is perceived assignals for the regulation of expression of plastid and nucleargenes. It is hardly surprising then that the differences in the lightquality and irradiance of land and aquatic environments have ledto such a difference in the evolution of diverse light-harvestingantennae.

Most Chl b-containing higher plant chloroplasts have grana,but only the most evolved green algal group, the Charaphyta,have grana that are microscopically indistinguishable fromthose of land-plant chloroplasts.42–44 Other green algae, e.g.Chlamydomonous reinhardtii, have only a few membranes in eachgranal stack, but they are stacked over much longer lengths. Incontrast, cyanobacteria, red algae, and Chl c-containing algaehave single, unstacked thylakoids.42

Non-granal photosynthetic organisms such as cyanobacteriaobviously grow well in a variety of environmental conditions.Even in higher plant chloroplasts, grana are not essential forphotosynthetic electron transport or ATP synthesis. Indeed,partially developed thylakoid membranes that are largely devoidof granal stacks are able to perform photosynthesis.45 Why, then,are grana so ubiquitous in higher plants? Here we summarizevarious ways in which grana formation can help fine-tune pho-tosynthesis, photoprotection and adaptation to terrestrial envi-ronments, thereby allowing higher plants to thrive and survivein widely contrasting, ever-fluctuating light environments fromlimiting to saturating light and even under prolonged high light.

Spatial separation of PSI and PSII between stacked andunstacked membrane domains

It was shown that the distribution of energy between PSI andPSII could be altered artificially in isolated thylakoids of higherplant chloroplasts.46,47 When the suspending medium containedlow concentrations of a monovalent cation, the distributionof energy in favour of PSI at the expense of PSII (spillover)was maximum; on addition of salts, there was a decrease ofspillover.48,49 Associated with the high-spillover condition wereunstacked membranes8,9 and a randomization of membrane pro-tein complexes as seen in freeze-fracture electron microscopy.50,51

In-depth analysis of the effects of cations on chlorophyllfluorescence yield by Barber’s group led him to suggest thatsalt-induced changes in maximum chlorophyll fluorescence yieldresults from changes in spillover of energy from PSII to PSI,which in turn depends on the spatial separation between thetwo photosystems.52,53 Thus, unstacking of thylakoids results inrandomisation of the two photosystems, a decreased separationbetween them and, therefore, greater spillover. For this reason,there is a close relation between the extent of stacking and themaximum yield of chlorophyll fluorescence (Fm). For example,the same “cross-over” and “dip” were observed in Fm

10 as in theextent of stacking in Fig. 2(A) and (C). Further, analysis of therise of Fm during the course of re-stacking (at various tempera-tures) in terms of diffusional segregation of PSII from PSI wassuccessfully applied to obtain lateral diffusion coefficients forthe movement of PSII complexes in thylakoid membranes.54

However, the relation between stacking and the spatialseparation between the two photosystems should not be takentoo far; prevention of spillover does not require grana per se,only a degree of lateral segregation. Indeed, it was shown thatthylakoid stacking and segregation of the two photosystems aretwo independent phenomena with two different ion-dependentmechanisms.55 Thylakoid stacking involves relatively large dis-tances, while the spatial segregation of PSI and PSII produces a

pronounced effect even at small separations (see floor discussionin ref. 52). Almost certainly, thylakoid stacking is precededby the segregation of the two photosystems, whether inducedby lateral forces such as those that govern the formation ofLHCII-PSII macrodomains,1,56 interplay between electrostaticand lipid-mediated interactions57 or a spinodal decompositionmechanism.58

In 1980, an extreme spatial separation of the two photosys-tems was proposed, with PSI excluded from the stacked granalmembrane domains.30 If the bulk of antenna pigments of PSIIand PSI were in direct contact with the reactions centres of bothphotosystems, as favoured in early continuous array models,59

most of the excitation energy would end up in PSI which containslonger-wavelength absorbing pigments than PSII. In that case,light-driven linear electron flow through both photosystems inseries would be inefficient under limiting light, as in a leaf canopy.Therefore, there is a need to limit spillover of excitation energyfrom PSII to PSI.60,61

Energetic and kinetic considerations of PSII led to the conclu-sion that PSII is a slow photosystem, with a slow exciton trap-ping time of 300–500 ps, while PSI is a fast photosystem with atrapping time of 60–90 ps. Hence, thylakoid stacking is proposedas a way of keeping the fast and slow photosystems apart.62 Fine-tuning of the energy distribution between the two photosystemscan then occur via State 1–State 2 transitions (see below).

Light-harvesting

The granum provides PSII with an intricate macrostructurecomprising the reaction centre, core complexes and the variouslight-harvesting complexes.18 The LHCII-PSII supercomplexassociates with further LHCII trimers to form PSII megacom-plexes, which can assume domains of semicrystalline order inthe granum. The result of this macro-organization is a largefunctional antenna size, which facilitates energy transfer amongPSII complexes. This provides connectivity between a number ofPSII reaction centres, a sharing of their antenna in which energycan flow between them until it is trapped by an open one. Thisfeature can therefore be considered as a mechanism to enhancethe efficiency of energy trapping in PSII. The organization of thepigments in this macrostructure gives rise to an enhancementof the circular dichroism, known as psi-type CD,56 a propertythat can be found also in certain types of in vitro aggregates ofLHCII. The exact features of the granum that give rise to thisCD remain to be established, but it serves as a useful probe ofstructural flexibility of the thylakoid membrane. In particular, ithas been suggested to indicate a state whose conformation canbe directly affected by light.63 Indeed, light-induced decrease inthis CD has been found in vitro and in vivo.

There is evidence for a link between the trimeric stateof LHCII and formation of grana. When the main LHCIIcomplexes were removed by expression of antisense genes, therewere still grana, to the same extent as in the wild type plants.64

However, instead of LHCII, there was an increase in the contentof the normally monomeric minor complex, CP26, which wasassembled into trimers.65

Regulation of light-harvesting: state transitions

Fine-tuning of energy distribution between the photosystemscan occur via State 1–State 2 transitions.66 The discoveries thatphosphorylation of LHCII was the molecular basis for statetransitions67 and that it caused a change in the absorptioncross section of PSII rather than a change in spillover68,69

were consistent with the role of surface charge in controllinglateral segregation. Hence, phosphorylated LHCII would tendto be excluded from the PSII-enriched appressed regions anddistribute towards PSI in the non-appressed membranes, as wassubsequently demonstrated.70 The H subunit of PSI was found tobe necessary for state transitions,71 suggesting that the relativeaffinities of PSII and PSI for phosphoLHCII determines its

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equilibrium distribution between the appressed and unappressedmembranes. It has also been suggested that the effect ofphosphorylation on the conformation of the N-terminus domainof LHCII is of crucial importance in weakening its interactionwith PSII.72

Regulation of light-harvesting: nonphotochemical quenching

The dissipation of excess energy from PSII, detected as the non-photochemical quenching of chlorophyll fluorescence (NPQ)occurs within the light harvesting antenna of PSII. Specificfeatures of the configuration of some pigments in the structuralmodel of LHCII have been linked to the decrease in excitationlifetime in the crystal form.73 This shows that LHCII has anintrinsic ability to regulate energy dissipation and transfer bybeing able to switch between different conformational states.These conformational changes in Lhcb proteins may dependupon, or be amplified by, protein-protein interactions74–76 Sincedeletion of specific Lhcb proteins and bound pigments bymutation invariably causes reduction of NPQ, but never itselimination, it has been argued that NPQ requires a particularmacro-structure of the PSII antenna.77

Because NPQ occurs within the granal membranes, it hasbeen suggested that the unique features of the granum have arole in regulating this process.78 Here interactions in the planeof the membrane would be determined by peripheral featuressuch as the binding of the xanthophyll cycle carotenoids and thepresence of PsbS protein interactions in the plane of the mem-brane. Also, NPQ could be affected by the forces exerted betweenmembranes, both on the stromal and lumenal surfaces. Evidenceconsistent with this view comes from the observation that thepresence of ordered rows of PSII complexes on one membrane isassociated with a similar ordering on the opposing membrane.18

Structural dynamics of thylakoid membranes

A spectacular contraction of the grana stacks of isolatedthylakoids occurs in light vs. dark or under different pH’sequivalent to dark and light,9 the same conditions inducingNPQ. In the light there is a decrease in width of both thepartition gap and the lumen which was 30% less than indarkness; there was also a thinning of membrane width. Ifthis occurs in vivo it implies enormous reversible changes ofthe supramolecular organization of PSII megacomplexes in thegranal stacks between dark and light, which could thereforeunderlie NPQ. This results from alterations of neutralizationand screening surface charge during dynamic dark/light changesdue to the light-induced transport of protons across thylakoidsto the lumen, and the counterbalancing transport of Mg2+

from the lumen to the partition gap and the stroma. Thus,the extremely high effective two-dimensional concentrations ofsurface charges at the membrane surfaces allows robust dynamicflexibility in the light reactions governed by both electron andproton transfer reactions.77,79 The size of DpH is therefore ofutmost importance.74,77,79 DpH responds not only to the demandsof carbon assimilation but also it may be modulated by theextent of cyclic transfer around PSI, the activation state of theATP synthase, and the partitioning of the proton motive forcebetween DpH and the membrane electrical potential.80

Delay of premature degradation of D1 protein

It was postulated that during prolonged illumination withhigh light, the non-functional, photoinhibited PSII centres ofhigher plant thylakoids accumulate in stacked granal domainswhere they are prevented from disassembly by virtue of beinginaccessible to D1 protein degradation.81 Consistent with thishypothesis, the rate coefficient of photoinactivation of PSII inthe absence of repair was reported to be 20% lower in low-light-grown leaves (in which granal size is increased) compared tohigh-light-grown leaves.82

In the photoinactivation of PSII, D1 protein in the very heartof PSII complex is the primary target of the photodamage,and replacement of the damaged D1 protein is a prerequisitefor recovery of PSII function.83 Damage of D1 protein leadsto monomerization of the dimeric PSII complex84 which mayfacilitate the migration of PSII to non-appressed thylakoiddomains where degradation of D1 protein and its replacement bynewly-sythesized D1 protein take place. However, only a smallnumber of non-functional PSII centres can undergo repair ata time. Under conditions of rapid photoinactivation, therefore,most of the photoinactivated PS II complexes have to remainin the grana,85 with their D1 protein in a phosphorylatedstate to prevent premature disassembly of the damaged PSII.86

Only after migration of the damaged PSII to non-appressedmembrane regions and dephosphorylation of the PSII coreproteins does the damaged D1 protein become susceptible todegradation.87 Therefore, grana serve as a reservoir habouringphotoinactivated PSII complexes until repair can take place andrecovery of PSII function is feasible.

Enhancement of non-cyclic ATP synthesis

To compare the rate of ATP synthesis in stacked and unstackedthylakoids, a special assay medium had to be devised becauseATP synthesis requires inter alia Mg2+, which normally tendsto cause thylakoid stacking. We recall that a low backgroundof a divalent cation such as Mg2+ in a medium with a verylow concentration of monovalent cations maintains originally-stacked thylakoids in the stacked state11 (also Fig. 2(C)). Itwas realized the same medium could also maintain originallyunstacked thylakoids in the unstacked state because of theactivation energies involved in any transition from one structuralstate to the other88 Hence, by chelating the bulk of the Mg2+ withadenosine diphosphate (ADP), a low concentration of free Mg2+

could be maintained in a medium which allowed ATP synthesiswhile keeping thylakoid membranes in either the stacked orunstacked state.89

Using the same assay medium, it was demonstrated that thelight-saturated rate of non-cyclic ATP synthesis was twice ashigh in stacked thylakoids as in unstacked thylakoids.89 This wasso despite unstacked membranes having (1) a greater capacityfor uncoupled linear electron transport, (2) a greater capacity forcyclic ATP synthesis (mediated by phenazine methosulfate) thatis limited by the amount of ATP synthase enzyme, and (3) only aslight decrease in the efficiency of non-cyclic ATP synthase (theamount of ATP formed per pair of electrons being lowered byonly 3–30% depending on growth irradiance).90

How is non-cyclic ATP synthesis enhanced by thylakoidstacking? One explanation was based on the circuit of protonsdriving non-cyclic ATP synthesis.25 The proton circuit comprises(1) the deposition of protons in the thylakoid lumen on oxidationof water at PSII and of plastoquinol at the Cyt bf complex,(2) the efflux of protons from the lumen through the ATPsynthase in non-appressed membranes and (3) the uptake ofprotons in plastoquinone reduction, which in the case of stackedthylakoids occurs predominantly in the partition gap betweentwo appressed membranes. It was argued that the proton circuitwas a limiting factor in non-cyclic ATP synthesis, particularlyin unstacked thylakoids with non-overlapping electric diffuselayers. It was proposed that membrane appression enhancesproton mobility in the partition gap.

A specific mechanism by which this enhancement of protonmobility could come about is based on the inter-digitising ofthe N-termini of LHCII on adjacent thylakoid membranesdescribed above under the heading of specific interactions. Itis known that proton migration along the surface of bacteri-orhodopsin micelles is faster than transfer from the surface to thebulk solution.91 Conceivably, much of the proton circuit drivinglight-saturated ATP synthesis could occur along thylakoid mem-brane surfaces, in particular, along the surfaces of appressed

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membranes. Proton migration along the thylakoid membranesurface would be hindered by presence of protonatable groupsonto which protons bind and from which they dissociate. Indeed,the dwell time of a proton on a protonatable group depends onthe pKa of the group.91 However, the inter-digitising of the N-termini of LHCII on adjacent thylakoid membranes may allowthe negative charge on one N-terminus to be counter-balancedby positive charge from an opposite N-terminus. The resultis that the affinity of protons for negatively-charged groupsis lowered, and the dwell time of protons is decreased. Thishypothesis, however, has yet to be tested experimentally.

Regulation of non-cyclic vs. cyclic ATP synthesis

Regardless of the mechanism, an enhancement of both thecapacity and efficiency of non-cyclic ATP synthesis,90 due to thy-lakoid stacking, has an important bearing on plant performancein high light as well as low light. Non-cyclic ATP synthesis,unlike the cyclic pathway, is accompanied by the formation ofNADPH, the reducing power needed for photosynthetic carbonassimilation.

The lateral separation of PSII from both PSI and ATPsynthase allows dynamic regulation of linear vs. cyclic electrontransport92,93 and the associated ATP synthesis. There aresituations where cyclic ATP synthesis may need to be enhanced.For example, following sustained high light when PSII has beenseverely photoinactivated, cyclic electron transport may needto be enhanced to provide the ATP for recovery under lowlight. In another situation, during photosynthetic inductionfollowing dark adaptation, linear electron flow is slow; cyclicelectron flow and the coupled ATP synthesis are the mainmeans of utilizing the light energy94 which otherwise has to bedissipated harmlessly. Similarly, the functional integrity of thephotosynthetic apparatus of overwintering evergreens, duringthawing in winter and during recovery from winter in spring,are aided by cyclic ATP synthesis.95

Grana allow a potential increase in photosynthetic capacity for agiven chloroplast composition in full sunlight, together with theenhancement of photochemical efficiency

As mentioned above, thylakoid stacking increases the volumefor Rubisco diffusion by about 17% (Table 2), thereby increasingentropy. Given the extremely high fractional volume occupancyby stromal enzymes, particularly Rubsico octamers, neededfor a high rate of carbon assimilation, the overall rate of thereaction is likely to be limited by the rate with which reactantmolecules (e.g. Rubisco activase and Rubisco) encounter oneanother through diffusional motion.96 Hence, alleviation ofmacromolecular crowding in the stroma by thylakoid stackingshould also enhance photosynthetic capacity. In addition, anincrease in volume for diffusion may facilitate enzyme-catalyzedreactions of small molecules if the mechanism of catalysisinvolves significant conformational changes of the enzyme.96

Taken together, these effects of thylakoid stacking probably tendto enhance metabolic rates in the stroma.

In contrast to the aquatic light environment of most non-granal photosynthetic organisms where light at increasing waterdepth favours PSII excitation, only terrestrial plants and inter-tidal algae may receive direct sunlight. In particular, whenatmospheric CO2 levels declined 370 million years ago, stomatalnumbers in land plants rose in response; the greater evaporativecooling allowed primitive land plants to spread their leaves andsoak up sunlight.97 The greater exposure to direct sunlight meantthat land plants needed higher photosynthetic capacities (e.g. ona cytochrome bf basis) and hence greater abundance of stromalenzymes. Thus, there seems to be a need for ordered thylakoidstacking to generate more volume for macromolecular diffusionin the stroma of land plant chloroplasts that were exposed tofull sunlight, all else being equal.

In addition, land plants form canopies for maximum lightinterception. Associated with canopy shading of land plantsis an increase in far-red irradiance down through the leafcanopy, tending to favour PSI excitation as the light intensity isattenuated. However, in the aquatic environment with increasingwater depth, first far-red, then red, and then blue light isattenuated thereby favouring excitation of PSII: note that thisis the converse of canopy shading. Conveniently, as outlinedabove, the spatial separation of PSII from PSI by granal stackingdecreases spillover of excitation energy from PSII to PSI, andallows for a large, connected light-harvesting antenna for PSIImegacomplexes in stacked granal domains. At the same time,the highly dynamic properties of the PSII antenna allow rapidadjustment to the large changes in the intensity and spectralquality of incident light arising from sunflecks.79 Thus, wespeculate that the requirements of (1) high metabolic fluxes in fullsunlight, (2) decreased spillover of excitation energy from PSIIto PSI in canopy shade and (3) the increased dynamic flexibilityof light-harvesting and electron transport may have helped drivethe evolutionary selection for grana in higher plants.

Concluding remarksAlthough grana are not essential for photosynthesis, they areubiquitous in higher plants. We have reviewed the attractiveand repulsive physicochemical forces that determine the granalultrastructure of chloroplasts. It is seemingly paradoxical butmarvellous that while grana give a degree of stability to thechloroplast ultrastructure, they also confer dynamic flexibility inresponses to ever-changing environmental conditions; while theyrepresent local order generated in the membrane system, theirformation is assisted by an increase in entropy in the stroma;and while grana probably enhance photosynthetic rates in fullsunlight at a given composition of chloroplast constituents, theyalso confer advantages of improved photochemical efficiency incanopy shade. Elucidating the mechanism of grana formationreveals how closely structure and function are intertwined inchloroplasts.

AcknowledgementsW. S. C. thanks his PhD supervisor, Alex Hope, for introducinghim to the electrostatics of biomembranes, and his post-doctoralsupervisors (in chronological order), Keith Boardman, JimBarber and Jan Anderson under whose guidance he developedan interest in thylakoid stacking. In particular, he thanks JimBarber for a wonderful postdoc period in London (Jan 1979–Mar 1981). E.-H. K. is supported by an ANU/RSBS Scholar-ship. P. H. is supported by grants from the United KingdomBiotechnology and Biological Sciences Research Council andthe INTRO2 EU Marie Curie Research Training Network.

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