Chlorophyll: an efficient detector of electronically excited species in biochemical systems

Micelle-solubilized chlorophyll efficiently detects electronically excited species generated in enzymatic systems. In most, if not all, systems the chemiexcited species is formed in the triplet state; chlorophyll fluorescence is observed as result of energy transfer. Red emission can also be elicited from chlorophyll in chloroplasts or bound to microsomes.     

Direct and Indirect Mechanisms of Deaggregation by Fatty Acids in Chilorophyll-Containing Systems

The ability of an exogenous long-chain unsaturated fatty acid (linolenic acid) to induce changes in the circular dichroism (C.D.) spectra of chlorophyllous systems of various levels of organization is demonstrated and attributed to its deaggregating influence. In the case of chlorophyll in solution (CCl₄ or CCl₄-hexane), deaggregation is by direct action on the chromophore. Evidence is also given for an indirect mechanism when chlorophyll is attached to protein (e.g., in HP-700 complexes); in this case, deaggregation results from a conformational change in the protein. Interpretations are given for the differences in C.D. spectra of nonmembranous and membranous chlorophyll-containing systems. (The latter include “digitonin-isolated” system I particles, subchloroplast particles obtained by means of sonication, and specially prepared intact chloroplasts.)     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature.

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715--740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50--4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the light-harvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (Fv) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm. From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emitting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

Correlation between cation-induced formation of heavy subchloroplast fractions and cation-induced increase in chlorophyll a fluorescence yield in Tricine-washed chloroplasts

A good correlation exists between the extent of thylakoid aggregation (grana reconstitution) and the increase in the chlorophyll a fluorescence yield (F_DCMU; DCMU = 3-(3′,4′-dichlorophenyl)-1, 1-dimethyl urea) caused by the addition of monovalent or divalent cations to low-salt disorganized (agranal) chloroplasts. The extent of grana stacking was monitored by the yield of heavy subchloroplast fractions after digitonin disruption of chloroplasts. A good correlation of the cation effect on both parameters was also found in light subchloroplast fractions (10,000g supernatants) obtained from sonicated “low-salt” Tricine-suspended pea chloroplasts. Addition of cations to the agranal protochloroplasts of etiolated pea or bean leaves exposed to periodic light-dark cycles, suspended in low-salt Tricine buffer, does not affect formation of heavy subchloroplast fractions, nor does it affect their chlorophyll a fluorescence yield level (F_DCMU). The cation effect on the increase of the chlorophyll a fluorescence yield level seems to be due to the cation-induced thylakoid structural changes leading to grana stacking.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715–740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50-4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the lightharvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (F_v) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm.From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emittting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

The light-harvesting chlorophyll a/b protein acts as a torque aligning chloroplasts in a magnetic field

Displacement of particles from the purified light-harvesting chlorophyll a/b protein aggregate (LHC) was studied in magnetic fields of various strengths (0 to 1.6 T) by polarized fluorescence measurements. Macromolecular aggregates of LHC have a considerable magnetic susceptibility which enables the particles to rotate and align with their nematic axes parallel with H. As LHC is embedded in a transmembrane direction thylakoids should align perpendicular to H, the mode of alignment experimentally observed in thylakoids. The value of the magnetic susceptibility could be estimated by relating it to the integral susceptibility of the chlorophyll molecules in LHC. The fitting of this value with the field strength dependency of the fluorescence polarization ratio (FP) revealed a relationship between the LHC content of various photosynthetic membranes and their capacity for alignment, which suggested that LHC might be the torque ordering chloroplasts in a magnetic field.Abbreviations LHC light-harvesting chlorophyll a/b protein - FP fluorescence polarization ratio, Iz/Iy     

Alignment of biological microparticles by a polarized laser beam

The optical alignment of biological samples is of great relevance to microspectrometry and to the micromanipulation of single particles. Recently, Bayoudh et al. (J. Mod. Opt. 50:1581–1590, 2003) have shown that isolated, disk-shaped chloroplasts can be aligned in a controlled manner using an in-plane-polarized Gaussian beam trap, and suggested that this is due to their nonspherical shape. Here we demonstrate that the orientation of various micrometer-sized isolated biological particles, trapped by optical tweezers, can be altered in a controlled way by changing the plane of linear polarization of the tweezers. In addition to chloroplasts, we show that subchloroplast particles of small size and irregular overall shape, aggregated photosynthetic light-harvesting protein complexes as well as chromosomes can be oriented with the linearly polarized beam of the tweezers. By using a laser scanning confocal microscope equipped with a differential polarization attachment, we also measured the birefringence of magnetically oriented granal chloroplasts, and found that they exhibit strong birefringence with large local variations, which appears to originate from stacked membranes. The size and sign of the birefringence are such that the resulting anisotropic interaction with the linearly polarized laser beam significantly contributes to the torque orienting the chloroplasts.     

Polarized fluorescence spectroscopy of oriented isolated spinach Photosystem I particles

The fluorescence anisotropy of Photosystem I (PS I) particles, isolated from spinach chloroplasts and containing approximately 200 chlorophyll molecules per reaction center, is investigated at low temperatures. The particles are oriented by squeezing in polyacrylamid gels with different macroscopic deformation parameters. Fluorescence anisotropy is measured upon steady state excitation with a laser line at 632.8 nm. A formula for the fluorescence anisotropy in oriented Photosystem I particles is applied for a different polarization of the linearly polarized exciting light. Our calculations are based on the consideration of the Photosystem I complex as a triple-chromophore complex: the absorbing chlorophyll molecules (chl), belonging to the light-harvesting complex of PS I (LHC), and two fluorophores, emitting at 720 nm (F720) and at 735 nm (F735), respectively. Using polarized fluorescence spectroscopy with a different polarization of the linearly polarized exciting light, the experimental dependence of the fluorescence anisotropy on this polarization is obtained. Based on this dependence and applying the derived formula, as a first approximation, both the orientation of the photosynthetic pigments with respect to the membrane and their mutual orientation are determined in PS I particles. As the most probable average orientational angles in PS I particles, we obtained the values 35°÷ 50°, 50°÷ 60°, and 65°÷ 67° for the absorbing dipoles of chl and for the emission dipoles of F720 and F735, respectively, with the normal of the plane of the membrane. For their mutual orientation, the following limits are determined: 10°÷ 20°, 40 ± 2°, 20°÷ 30° for the angles between chl and F720, chl and F735; and F720 and F735, correspondingly. Of course, the values of the angles estimated as a result of our study are an average value of all angles of the excited transitions and must be considered as their first approximation valid for the idealized case when all PS I particles are oriented in gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Qualitative measurements of the mitochondrial membrane potential in situ in Ehrlich ascites tumour cells using the safranine method.

Radiative and nonradiative processes were investigated in subchloroplast particles highly enriched in P-700 (1 P-700 to 10 chlorophyll molecules) according to the method of classical fluorescence and of photoacoustical spectroscopy. The envelope of fluorescence spectrum divided into three Gaussian bands and their quantum yields of fluorescence were calculated. Indpendently the quantum yield of fluorescence was determined from the spectral course of the photoacoustical signal. Finally, the estimate of the photochemical activity of P-700, based upon the measured fluorescence quantum yield and upon the measured nonradiative losses of excitation energy, was done.     

Excitation energy transfer in the light-harvesting chlorophyll a/b.protein

The "light-harvesting chlorophyll a/b.protein" described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600-700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitiation dependence of the fluorescence polarization shows a minimum polarization of 1.9% at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8% at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a double structure in the chlorophyll b absorption band which suggest an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the So leads to S1 transition moments of the chlorophyll molecules within the protein.     

Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism

1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10–13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes.

2. The FP ratio is typically in the range of 1.2–1.9 in Chlorella, 1.20–1.25 in Scenedesmus and 1.4–1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Q_y transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented.

3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720–730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615–617).

4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01–0.06) determined by previous workers with unoriented cell suspensions.

5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered.

6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Q_y transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane.

7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena.

8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.     

Identification of the 120 mus phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH.

After a 500 mus laser flash a 120 mus phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Bio-phys. Acta 376, 329-344, indicates that their EPR signal IIf which they suppose to be due to Z+, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 mus phase. We explain our 120 mus phase by the intrinsic back reaction of the excited reaction center in the presence of Z+, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333-342. The redox state of Z+ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the mus region are compared with those obtained in the ms region.     

Anisotropy of photosynthetic membranes and the degree of fluorescence polarization

The degree of fluoresence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400–680 nm) and emission wavelengths (675–750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, P_IN and P_AN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization P_IN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Q_y (≈ 660−680 nm) absorption band of chlorophyll in vivo. With 670–680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts, P_IN is shown to be +5±1%. The excitation wavelength dependence studies of P_IN indicate that the carotenoid and chlorophyll Q_y transition moments tend to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles II. Energy conversion in chloroplasts and different types of subchloroplast particles

The light-induced absorbance change at 515 nm, light-inducedhydrogen ion uptake and ATP formation were compared in chloroplastsand different types of sonicated subchloroplast particles. Noparallel relationship among the activities for ATP formation,hydrogen ion uptake and the 515-nm change was observed in differenttypes of preparations. NH₄Cl inhibited ATP formation in chloroplastsbut had little effect on subchloroplast particles. In contrast,the light-induced hydrogen ion uptake was inhibited by NH₄Clin a similar manner. Tetraphenylboron (TPB), at 1 µM, inhibited ATP formationby about 30% in both chloroplasts and subchloroplast particles.In the presence of TPB, ATP formation in chloroplasts was stronglyinhibited by NHC₄Cl, but in subchloroplast particles the additionalinhibitory effect of NH₄Cl was small. A synergistic inhibitionof photophosphorylation by valinomycin plus NH₄Cl was much clearer.Although acceleration of the recovery of the 515-nm change byNH₄Cl or valinomycin was moderate, the 515-nm change virtuallydisappeared when NH₄Cl and valinomycin were added simultaneously. Although the membrane potential has a major role as the principaldriving force for ATP formation in subchloroplast particles,the simultaneous abolishment of the pH gradient and membranepotential may be required to uncouple ATP formation. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. ²Present address: Ryukyu University, Naha, Okinawa 903, Japan. (Received February 5, 1974; )     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles I. Ion transport, ionic environment and the light-induced 475-515 nm absorbance change

The light-induced absorbance change at 515 nm and the light-inducedhydrogen ion uptake in chloroplasts are sensitive to physicaltreatment and to changes in ionic environment. High concentrationsof salts (chlorides) were inhibitory to the 515-nm absorbancechange. This inhibition was stronger in chloroplasts than insubchloroplast particles. In subchloroplast particles, NH₄Clwas slightly stimulatory for the 515-nm change at low concentrations({small tilde}0.5 mM), as was the case with photophosphorylation. Tetraphenylboron (TPB), as a permeant anion, inhibited the 515-nmchange and the rate of hydrogen ion uptake. Tetraphenylarsonium(TPA) and tetraphenylphosphonium (TPP), both permeant cations,diminished the 515-nm change but did not affect the hydrogenion uptake. These results are analyzed in connection with adiscussion of the significance of the membrane potential andhydrogen ion gradient in the energy conversion of chloroplastsand subchloroplast particles. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. (Received February 5, 1974; )     

Excitation energy transfer in the light-harvesting chlorophyll

The “light-harvesting chlorophyll a/b · protein” described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600–700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitation dependence of the fluorescence polarization shows a minimum polarization of 1.9 % at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8 % at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a doublet structure in the chlorophyll b absorption band which suggests an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the S₀→S₁ transition moments of the chlorophyll molecules within the protein.     

Model for fluorescence quenching in light harvesting complex II in different aggregation states

Low-temperature (77 K) steady-state fluorescence emission spectroscopy and dynamic light scattering were applied to the main chlorophyll a/b protein light harvesting complex of photosystem II (LHC II) in different aggregation states to elucidate the mechanism of fluorescence quenching within LHC II oligomers. Evidences presented that LHC II oligomers are heterogeneous and consist of large and small particles with different fluorescence yield. At intermediate detergent concentrations the mean size of the small particles is similar to that of trimers, while the size of large particles is comparable to that of aggregated trimers without added detergent. It is suggested that in small particles and trimers the emitter is monomeric chlorophyll, whereas in large aggregates there is also another emitter, which is a poorly fluorescing chlorophyll associate. A model, describing populations of antenna chlorophyll molecules in small and large aggregates in their ground and first singlet excited states, is considered. The model enables us to obtain the ratio of the singlet excited-state lifetimes in small and large particles, the relative amount of chlorophyll molecules in large particles, and the amount of quenchers as a function of the degree of aggregation. These dependencies reveal that the quenching of the chl a fluorescence upon aggregation is due to the formation of large aggregates and the increasing of the amount of chlorophyll molecules forming these aggregates. As a consequence, the amount of quenchers, located in large aggregates, is increased, and their singlet excited-state lifetimes steeply decrease.     

Delayed light studies on photosynthetic energy conversion. VII. Effect of the high energy state, coupled to 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow, on millisecond emission from chloroplasts and digitonin subchloroplast particles

The effect of 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow on millisecond delayed light emission from chloroplasts has been compared to the effect on subchloroplast particles. Non-cyclic electron flow of both chloroplasts and subchloroplast particles was blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow increased the millisecond delayed emission by 2–4 times in both chloroplasts and subchloroplast particles. Uncoupling conditions which collapse only the pH gradient component of the proton motive force reduced the 2,3,5,6-tetramethyl p-phenylenediamine stimulation of delayed light in chloroplasts but not in particles. The 2,3,5,6-tetramethyl p-phenylenediamine stimulation of delayed light in particles was sensitive to uncoupling conditions which are presumed to destroy the transmembrane potential. Energy transfer inhibitors were without effect on the 2,3,5,6-tetramethyl p-phenylenediamine stimulation in both chloroplasts and particles.

The 2,3,5,6-tetramethyl p-phenylenediamine stimulation of millisecond delayed emission appears to reflect the particular form of the proton motive force; in chloroplasts it seems to be correlated with the proton concentration gradient, whereas in particles it is more closely correlated with the transmembrane potential.     

Composition of the photosystems and chloroplast structure in extreme shade plants

Chloroplasts were isolated from leaves of three species of tropical rainforest plants, Alocasia macrorrhiza, Cordyline rubra and Lomandra longifolia; these species are representative of extreme “shade” plants. It was found that shade plant chloroplasts contained 4–5 times more chlorophyll than spinach chloroplasts. Their chlorophyll a/chlorophyll b ratio was 2.3 compared with 2.8 for spinach. Electron micrographs of leaf sections showed that the shade plant chloroplasts contained very large grana stacks. The total length of partitions relative to the total length of stroma lamellae was much higher in Alocasia than in spinach chloroplasts. Freeze-etching of isolated chloroplasts revealed both the small and large particles found in spinach chloroplasts.

Despite their increased chlorophyll content, low chlorophyll a/chlorophyll b ratio, and large grana, the shade plant chloroplasts were fragmented with digitonin to yield small fragments (D-144) highly enriched in Photosystem I, and large fragments (D-10) enriched in Photosystem II. The degree of fragmentation of the shade plant chloroplasts was remarkably similar to that of spinach chloroplasts, except that the subchloroplast fragments from the shade plants had lower chlorophyll a/chlorophyll b ratios than the corresponding fragments from spinach. The D-10 fragments from the shade plants had chlorophyll a/chlorophyll b ratios of 1.78-2.00 and the D-144 fragments ratios of 3.54–4.07. We conclude that Photosystems I and II of the shade plants have lower proportions of chlorophyll a to chlorophyll b than the corresponding photosystems of spinach. The lower chlorophyll a/chlorophyll b ratio of shade plant chloroplasts is not due to a significant increase in the ratio of Photosystem II to Photosystem I in these chloroplasts.

The extent of grana formation in higher plant chloroplasts appears to be related to the total chlorophyll content of the chloroplast. Grana formation may simply be an means of achieving a higher density of light-harvesting assemblies and hence a more efficient collection of light quanta.