The oligomeric states of the photosystems and the light-harvesting complexes in the Chl b-less mutant

The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.     

Evolution of light-harvesting complex proteins from Chl c-containing algae

Background  

Light harvesting complex (LHC) proteins function in photosynthesis by binding chlorophyll (Chl) and carotenoid molecules that absorb light and transfer the energy to the reaction center Chl of the photosystem. Most research has focused on LHCs of plants and chlorophytes that bind Chl a and b and extensive work on these proteins has uncovered a diversity of biochemical functions, expression patterns and amino acid sequences. We focus here on a less-studied family of LHCs that typically bind Chl a and c, and that are widely distributed in Chl c-containing and other algae. Previous phylogenetic analyses of these proteins suggested that individual algal lineages possess proteins from one or two subfamilies, and that most subfamilies are characteristic of a particular algal lineage, but genome-scale datasets had revealed that some species have multiple different forms of the gene. Such observations also suggested that there might have been an important influence of endosymbiosis in the evolution of LHCs.     

Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts

Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs.     

Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light-harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b-antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl-binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77-K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long-wavelength absorption and 730-nm fluorescence emission. In Lhcb1 with only Chl b long-wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long-wavelength properties.     

Synthesis of chlorophyll <Emphasis Type="Italic">b</Emphasis>: Localization of chlorophyllide <Emphasis Type="Italic">a</Emphasis>oxygenase and discovery of a stable radical in the catalytic subunit

Background  

Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly.     

NPQ activation reduces chlorophyll triplet state formation in the moss Physcomitrella patens

Plants live in variable environments in which light intensity can rapidly change, from limiting to excess conditions. Non-photochemical quenching (NPQ) is a regulatory mechanism which protects plants from oxidative stress by dissipating excess Chl singlet excitation. In this work, the physiological role of NPQ was assessed by monitoring its influence on the population of the direct source of light excess damage, i.e., Chl triplets ((3)Chl*). (3)Chl* formation was evaluated in vivo, with the moss Physcomitrella patens, by exploiting the high sensitivity of fluorescence-detected magnetic resonance (FDMR). A dark adapted sample was compared with a pre-illuminated sample in which NPQ was activated, the latter showing a strong reduction in (3)Chl* yield. In line with this result, mutants unable to activate NPQ showed only a minor effect in (3)Chl* yield upon pre-illumination.The decrease in (3)Chl* yield is equally experienced by all the Chl pools associated with PSII, suggesting that NPQ is effective in protecting both the core and the peripheral antenna complexes. Moreover, the FDMR results show that the structural reorganization in the photosynthetic apparatus, required by NPQ, does not lead to the formation of new (3)Chl* traps in the LHCs. This work demonstrates that NPQ activation leads to effective photoprotection, promoting a photosystem II state characterized by a reduced probability of (3)Chl* formation, due to a decreased singlet excited state population, while maintaining an efficient quenching of the (3)Chl* eventually formed by carotenoids.     

Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll b-deficient mutants

We have analyzed precursor pools in the chlorophyll (Chi) synthesis pathway for a set of eighteen well studied Chl b -defident mutants in monocotyledonous (barley, maize and wheat) and dicotyledonous plants ( Antirrhinum, Arabidopsis , soybean, tobacco and tomato) that form abnormal thylakoid membrane systems. All of these mutants have a partial block in Chl synthesis and nearly all of them accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The large number of mutants at several genetic loci affecting this critical branchpoint in tetrapyrrole biosynthesis suggests that the Mg-chelatase enzyme, catalyzing the first committed step of Chi biosynthesis, is a multimeric complex composed of the products of some of these genetic loci, and perhaps regulated by others. We hypothesize that these mutants are Chi b -deficient and have reduced amounts of light-harvesting antenna complexes (LHCs.) and develop abnormal thylakoid membranes as a direct result of limited Chl synthesis. The observed bottleneck in Chl synthesis can also explain the light-intensity-dependent and temperature-dependent expression of the mutant phenotype. This hypothesis offers a simple explanation for the wide variety of pbenotypes that have been reported for the many Chl-deficient mutants in the literature. Our findings are also consistent with the notion that Chl b is made from "left over" Chl a molecules and suggest that the Chi b -deficient mutants should be considered more appropriately as leaky Chl-deficient mutants.     

Hyperdiversity of Genes Encoding Integral Light-Harvesting Proteins in the Dinoflagellate Symbiodinium sp

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c ₂-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences.     

FUNCTIONAL BINDING OF DIATOM PIGMENTS TO A RHODOPHYTE LIGHT HARVESTING POLYPEPTIDE

A close relationship of light harvesting polypeptides (LHC) of rhodophytes, chromophytes and chlorophytes is inferred from the amino acid sequence similarity in three transmembrane helices, and from the conservation of 8 putative chlorophyll (Chl)-binding sites (Durnford et al. 1999, J. Mol. Evol. 48:59). Differences in Chl and carotenoid pigments have been a major classification feature. Thus, it was of interest to ascertain whether pigments from a diatom ( Thallasiosira fluviatilis ) could be functionally inserted into a red algal ( Porphyridium cruentum ) polypeptide. A recombinant polypeptide, LHCaR1, was reconstituted with pigment extracts from the diatom (Chls a and c , fucoxanthin, diadinoxanthin and β-carotene). The pigments were found attached to protein upon separation on sucrose gradients, and on non-denaturing gels. Absorption and fluorescence excitation spectra revealed individual peaks corresponding to the absorption maxima of Chl a at 438/672 nm; Chl c at 463/638 nm; and fucoxanthin at 493/540 nm. Fluorescence emission and CD spectra showed functional binding and suitable orientation for energy transfer from Chl c and carotenoids to Chl a. The LHCaR1 successfully folded in the presence of the heterologous pigments and bound 7 Chl a , 1 Chl c , 8 fucoxanthin, and 1.9 diadinoxanthin per polypeptide. By comparison, this polypeptide with P. cruentum pigments binds 8 Chl a , and 4 zeaxanthins, thus revealing its capability of functionally binding 8 Chls with variations in carotenoid numbers. Such a trait may have favored the diversification of a large family of LHCs and the successful radiation of photosynthetic eukaryotes into different light environments.     

Why is chlorophyll <Emphasis Type="Italic">b</Emphasis> only used in light-harvesting systems?

Chlorophylls (Chl) are important pigments in plants that are used to absorb photons and release electrons. There are several types of Chls but terrestrial plants only possess two of these: Chls a and b. The two pigments form light-harvesting Chl a/b-binding protein complexes (LHC), which absorb most of the light. The peak wavelengths of the absorption spectra of Chls a and b differ by c. 20 nm, and the ratio between them (the a/b ratio) is an important determinant of the light absorption efficiency of photosynthesis (i.e., the antenna size). Here, we investigated why Chl b is used in LHCs rather than other light-absorbing pigments that can be used for photosynthesis by considering the solar radiation spectrum under field conditions. We found that direct and diffuse solar radiation (PAR_dir and PAR_diff, respectively) have different spectral distributions, showing maximum spectral photon flux densities (SPFD) at c. 680 and 460 nm, respectively, during the daytime. The spectral absorbance spectra of Chls a and b functioned complementary to each other, and the absorbance peaks of Chl b were nested within those of Chl a. The absorption peak in the short wavelength region of Chl b in the proteinaceous environment occurred at c. 460 nm, making it suitable for absorbing the PAR_diff, but not suitable for avoiding the high spectral irradiance (SIR) waveband of PAR_dir. In contrast, Chl a effectively avoided the high SPFD and/or high SIR waveband. The absorption spectra of photosynthetic complexes were negatively correlated with SPFD spectra, but LHCs with low a/b ratios were more positively correlated with SIR spectra. These findings indicate that the spectra of the photosynthetic pigments and constructed photosystems and antenna proteins significantly align with the terrestrial solar spectra to allow the safe and efficient use of solar radiation.     

Organization of the pigment molecules in the chlorophyll a/b/c containing alga Mantoniella squamata (Prasinophyceae) studied by means of absorption, circular and linear dichroism spectroscopy

In order to obtain information on the organization of the pigment molecules in chlorophyll (Chl) a/b/c-containing organisms, we have carried out circular dichroism (CD), linear dichroism (LD) and absorption spectroscopic measurements on intact cells, isolated thylakoids and purified light-harvesting complexes (LHCs) of the prasinophycean alga Mantoniella squamata. The CD spectra of the intact cells and isolated thylakoids were predominated by the excitonic bands of the Chl a/b/c LHC. However, some anomalous bands indicated the existence of chiral macrodomains, which could be correlated with the multilayered membrane system in the intact cells. In the red, the thylakoid membranes and the LHC exhibited a well-discernible CD band originating from Chl c, but otherwise the CD spectra were similar to that of non-aggregated LHC II, the main Chl a/b LHC in higher plants. In the Soret region, however, an unusually intense (+) 441 nm band was observed, which was accompanied by negative bands between 465 and 510 nm. It is proposed that these bands originate from intense excitonic interactions between Chl a and carotenoid molecules. LD measurements revealed that the Q(Y) dipoles of Chl a in Mantoniella thylakoids are preferentially oriented in the plane of the membrane, with orientation angles tilting out more at shorter than at longer wavelengths (9 degrees at 677 nm, 20 degrees at 670 nm and 26 degrees at 662 nm); the Q(Y) dipole of Chl c was found to be oriented at 29 degrees with respect to the membrane plane. These data and the LD spectrum of the LHC, apart from the presence of Chl c, suggest an orientation pattern of dipoles similar to those of higher plant thylakoids and LHC II. However, the tendency of the Q(Y) dipoles of Chl b to lie preferentially in the plane of the membrane (23 degrees at 653 nm and 30 degrees at 646 nm) is markedly different from the orientation pattern in higher plant membranes and LHC II. Hence, our CD and LD data show that the molecular organization of the Chl a/b/c LHC, despite evident similarities, differs significantly from that of LHC II.     

Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type horizontal cells

Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or dihydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R-and G-cones to LHCs, respectively.     

Characterization of the secondary electron-transfer pathway intermediates of photosystem II containing low-potential cytochrome b 559

Beta-carotene (Car) and chlorophyll (Chl) function as secondary electron donors in photosystem II (PS II) under conditions, such as low temperature, when electron donation from the O(2)-evolving complex is inhibited. In prior studies of the formation and decay of Car(*+) and Chl(*+) species at low temperatures, cytochrome b(559) (Cyt b(559)) was chemically oxidized prior to freezing the sample. In this study, the photochemical formation of Car(*+) and Chl(*+) is characterized at low temperature in O(2)-evolving Synechocystis PS II treated with ascorbate to reduce most of the Cyt b(559). Not all of the Cyt b(559) is reduced by ascorbate; the remainder of the PS II reaction centers, containing oxidized low-potential Cyt b(559), give rise to Car(*+) and Chl(*+) species after illumination at low temperature that are characterized by near-IR spectroscopy. These data are compared to the measurements on ferricyanide-treated O(2)-evolving Synechocystis PS II in which the Car(*+) and Chl(*+) species are generated in PS II centers containing mostly high- and intermediate-potential Cyt b(559). Spectral differences observed in the ascorbate-reduced PS II samples include decreased intensity of the Chl(*+) and Car(*+) absorbance peaks, shifts in the Car(*+) absorbance maxima, and lack of formation of a 750 nm species that is assigned to a Car neutral radical. These results suggest that different spectral forms of Car are oxidized in PS II samples containing different redox forms of Cyt b(559), which implies that different secondary electron donors are favored depending on the redox form of Cytb(559) in PS II.     

Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type horizontal cells

Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (α-amino-3-hydroxy-5-methylisoxa-zole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or di-hydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R- and G-cones to LHCs, respectively.     

The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.     

Characterization of the Porphyridium cruentum Chl a-binding LHC by in vitro reconstitution: LHCaR1 binds 8 Chl a molecules and proportionately more carotenoids than CAB proteins

The Porphyridium cruentum light harvesting complex (LHC) binds Chl a, zeaxanthin and -carotene and comprises at least 6 polypeptides of a multigene family. We describe the first in vitro reconstitution of a red algal light-harvesting protein (LHCaR1) with Chl a/carotenoid extracts from P. cruentum. The reconstituted pigment complex (rLHCaR1) is spectrally similar to the native LHC I, with an absorption maximum at 670 nm, a 77 K fluorescence emission peak at 677 nm (ex. 440 nm), and similar circular dichroism spectra. Molar ratios of 4.0 zeaxanthin, 0.3 -carotene and 8.2 Chl a per polypeptide for rLHCaR1 are similar to those of the native LHC I complex (3.1 zeaxanthin, 0.5 -carotene, 8.5 Chl a). The binding of 8 Chl a molecules per apoprotein is consistent with 8 putative Chl-binding sites in the predicted transmembrane helices of LHCaR1. Two of the putative Chl a binding sites (helix 2) in LHCaR1 were assigned to Chl b in Chl a/b-binding (CAB) LHC II [Kühlbrandt et al. (1994) Nature 367: 614–21]. This suggests either that discrimination for binding of Chl a or Chl b is not very specific at these sites or that specificity of binding sites evolved separately in CAB proteins. LHCaR1 can be reconstituted with varying ratios of carotenoids, consistent with our previous observation that the carotenoid to Chl ratio is substantially higher in P. cruentum grown under high irradiance. Also notable is that zeaxanthin does not act as an accessory light-harvesting pigment, even though it is highly likely that it occupies the position assigned to lutein in the CAB LHCs.This revised version was published online in October 2005 with corrections to the Cover Date.     

Changes in French bean cotyledon composition associated with modulated life-span

The onset of Phaseolus vulgaris L. cotyledon senescence and its characteristics were modulated by irradiance (higher or lower than standard) and by epicotyl decapitation. The cotyledon life-span of 16 d was not influenced by irradiance while decapitation prolonged the life-span to 28 d. The fresh mass of cotyledons, an indicator of organ viability, decreased in a similar manner in all non-decapitated plants, though it was relatively slower in plants grown under a low irradiance (LI). Three days after decapitation the fresh mass of cotyledons increased by one third, a slight decrease was observed on the 21st d, and it lasted until the end of the life span. Deducing from the fall of chlorophyll (Chl) concentration expressed per unit protein, senescence started after the 10th day in non-decapitated plants. Decapitation postponed the onset of senescence until the 21st day. Expression of Chl amount per unit dry mass did not detect any changes in LI plants, hence this parameter can not be used for the assessment of senescence. The measurements of Chl a and b concentrations indicated that the light-harvesting complexes (LHCs) proliferated during ageing and were rapidly destroyed at the onset of senescence. Changes of the concentrations of carotenoids supported the hypothesis of free radicals involvement in senescence. The bean cotyledons responded to free radical production induced under higher irradiance by increased β-carotene synthesis. Oxidative damage to galactolipids during senescence was documented by fluorescence measurements. The changes in cotyledon composition were correlated to morphologic changes observed by electron microscopy.     

Changes in French bean cotyledon composition associated with modulated life-span

The onset of Phaseolus vulgaris L. cotyledon senescence and its characteristics were modulated by irradiance (higher or lower than standard) and by epicotyl decapitation. The cotyledon life-span of 16 d was not influenced by irradiance while decapitation prolonged the life-span to 28 d. The fresh mass of cotyledons, an indicator of organ viability, decreased in a similar manner in all non-decapitated plants, though it was relatively slower in plants grown under a low irradiance (LI). Three days after decapitation the fresh mass of cotyledons increased by one third, a slight decrease was observed on the 21st d, and it lasted until the end of the life span. Deducing from the fall of chlorophyll (Chl) concentration expressed per unit protein, senescence started after the 10th day in non-decapitated plants. Decapitation postponed the onset of senescence until the 21st day. Expression of Chl amount per unit dry mass did not detect any changes in LI plants, hence this parameter can not be used for the assessment of senescence. The measurements of Chl a and b concentrations indicated that the light-harvesting complexes (LHCs) proliferated during ageing and were rapidly destroyed at the onset of senescence. Changes of the concentrations of carotenoids supported the hypothesis of free radicals involvement in senescence. The bean cotyledons responded to free radical production induced under higher irradiance by increased -carotene synthesis. Oxidative damage to galactolipids during senescence was documented by fluorescence measurements. The changes in cotyledon composition were correlated to morphologic changes observed by electron microscopy.     

Quenching of excited states of chlorophyll molecules in submembrane fractions of Photosystem I by exogenous quinones

The ability of three substituted quinones, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichloro-p-benzoquinone (DCBQ), and tetramethyl-p-benzoquinone (duriquinone) to quench the excited states of chlorophyll (Chl) molecules in Photosystem I (PSI) was studied. Chl fluorescence emission measured with isolated PSI submembrane fractions was reduced following the addition of exogenous quinones. This quenching progressively increased with rising concentrations of the exogenous quinones according to the Stern-Volmer law. The values of Stern-Volmer quenching coefficients were found to be 3.28 x 10(5) M(-1) (DBMIB), 1.31 x 10(4) M(-1) (DCBQ), and 3.7 x 10(3) M(-1) (duroquinone). The relative quenching capacities of the various exogenous quinones in PSI thus strictly coincided to those found for the quenching of Fo level of Chl fluorescence in isolated thylakoids, which is emitted largely by Photosystem II (PSII) [Biochim. Biophys. Acta (2003) 1604, 115-123]. Quenching of Chl excited states in PSI submembrane fractions by exogenous quinones slowed down the rate of P700, primary electron donor of PSI, photooxidation measured at limiting actinic light irradiances thus revealing a reduced photochemical capacity of absorbed quanta. The possible involvement of non-photochemical quenching of excited Chl states by oxidized phylloquinones, electron acceptors of PSI, and oxidized plastoquinones, mobile electron carriers between PSII and the cytochrome b(6)/f complex, into the control of photochemical activity of PSI is discussed.     

Regulation of energy dissipation in photosystem I by the redox state of the plastoquinone pool

The effect of exogenous plastoquinone (PQ) on the different deexcitation pathways of photosystem I (PSI) was investigated. Addition of oxidized decyl-plastoquinone (dPQ) and PQ-2 strongly quenched the chlorophyll (Chl) emission spectra of PSI submembrane fractions over all wavelengths. This quenching increased with the concentration of exogenous PQ added and followed the modified Stern-Volmer law. The Stern-Volmer constants found for dPQ and PQ-2 were 1.25 x 10(6) M-1 and 0.55 x 10(6) M-1, respectively, and the fraction of fluorescence accessible to the quencher was 0.7 for both exogenous PQ. dPQ and PQ-2 also retarded the P700 photooxidation measured under limiting actinic light irradiances. Photoacoustic measurements showed that addition of dPQ increased the heat dissipation and decreased the photochemical capacity of PSI. From these results, exogenous oxidized PQ were shown to efficiently quench the Chl excited state in the PSI antenna and change the balance between Chl deexcitation pathways. Moreover, reduction of the endogenous PQ pool in whole thylakoid membranes by NADPH increased PSI fluorescence by 65%, indicating the importance of the redox state of the PQ pool on PSI energy dissipation.