Isoenzymes of ferredoxin-NADBΔ oxidoreductase from the cyanobacterium Nostoc strain MAC

Ferredoxin-NADP⁺ oxidoreductase from the cyanobacterium Nostoc strain MAC was separated into two fractions by ion-exchange chromatography. Both were purified to electrophoretic homogeneity and exhibited diaphorase and ferredoxin-dependent cytochrome c reductase activity. The activities with three different electron carriers in this latter assay were similar for the two fractions, as were the pH optima in both assays. Each fraction, however, could be resolved into several active components by isoelectric focusing, both after initial separation and following apparent purification by gel filtration on Sephadex G-150, further chromatography on DEAE-cellulose, and use of hydroxylapatite columns.Abbreviation DCIP = phenolindo-3,6-dichlorophenol>     

Influence of Ca2+ on the thylakoid lumen violaxanthin de-epoxidase activity through Ca2+ gating of H+ flux at the CFo H+ channel

Part of the chloroplast photoprotection response to excess light absorption involves formation of zeaxanthin (and antheraxanthin) via the violaxanthin deepoxidase enzyme, the activity of which requires lumen acidity near or below pH 6.0. Clearly, the violaxanthin de-epoxidase activity is strongly regulated because at equivalent energization levels (including the parameters of H⁺ accumulation and ATP formation rates), there can be either low or high violaxanthin de-epoxidase enzyme activity. This work shows that the factor or factors responsible for regulating the violaxanthin deepoxidase correlate directly with those which regulate the expression of membrane-localized or delocalized proton gradient (Δ~μ_H+) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca²⁺ binding to the lumen side of the thylakoid membrane; in particular, Ca²⁺ binding to the 8 kDA subunit III of the CF_o H⁺ channel. The activity of violaxanthin deepoxidase in pea (Pisum sativa) and spinach (Spinacea oleracea) thylakoids is shown here to be strongly correlated with conditions known from previous work to displace Ca²⁺ from the CF_o H⁺ channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence of the carotenoid lactucaxanthin in higher plant LHC II

The pigment composition of the light-harvesting complexes of Photosystem II (LHC II) has been determined for lettuce (Lactuca sativa). In common with other members of the composite, the photosynthetic tissues of this species may contain large amounts of the carotenoid lactucaxanthin (, -carotene-3,3'-diol) in addition to their normal compliment of carotenoids. The occurrence and distribution of lactucaxanthin in LHC II has been examined using isoelectric focusing of BBY particles followed by reversed-phase HPLC analysis of the pigments. The major carotenoids detected in LHC IIb, LHC IIa (CP29) and LHC IIc (CP26) purified from dark-adapted lettuce were lutein, violaxanthin, neoxanthin and lactucaxanthin. Lactucaxanthin has been shown to be a major component of PS II, accounting for 26% of total xanthophyll in both LHC IIb (23% total xanthophyll) and in the minor complexes (12–16%). In this study, LHC IIb was clearly resolved into four bands and their carotenoid composition determined. These four bands proved to be very similar in their pigment content and composition, although the relative amounts of neoxanthin and lutein in particular were found to increase from bands 1 to 4 (i.e. with increasing electrophoretic mobility). The operation of the xanthophyll cycle has also been examined in the LHC of L. sativa following light treatment. The conversion efficiency for violaxanthinzeaxanthin was nearly identical for each light-harvesting complex examined at 58–61%. Nearly half of the zeaxanthin formed in PS II was associated with LHC IIb, although the molar ratio of zeaxanthin:chlorophyll a was highest in the minor LHC.Abbreviations HPLC high performance liquid chromatography - IEF isoelectric focusing - LHCII light-harvesting complex associated with Photosystem II - PS II Photosystem II - qE pH-dependent nonphotochemical quenching of chlorophyll fluorescence     

Purification of membrane-bound ferredoxin: NADP+ oxidoreductase and of plastocyanin from a detergent extract of washed thylakoids

A method is described for the isolation and purification of ferredoxin-NADP⁺ oxidoreductase (FNR, E.C. 1.18.1.2) and plastocyanin from spinach thylakoids. FNR is recovered from pools which are loosely and tightly bound to the membrane, with minimal disruption of pigment-protein complexes; yields can thus be higher than from procedures which extract only the loosely bound enzyme.Washed thylakoid membranes were incubated with the dipolar ionic detergent CHAPS (3-(3-cholamidopropyl-dimethylammonio)-1-propane-sulfonate). This provided an extract containing FNR and PC as its principal protein components, which could be rapidly separated from one another by chromatography on an anion-exchange column. FNR was purified to homogeneity (as judged from sodium dodecyl sulfate gel electrophoresis and the ratio between protein and flavin absorption maxima), using chromatography on phosphocellulose followed by batchwise adsorption to, and elution from hydroxylapatite. Plastocyanin was further purified on a Sephadex G-75 molecular sieve column.A typical yield, obtained in 3–4 days from 1 kg of deveined spinach leaves, was 7 mg of pure FNR (a single protein of M_r=37,000) and 3.5 mg of plastocyanin.Abbreviations CHAPS- 3-(3-cholamidopropyl-dimethylammonio)-1-propanesulfonate) - Chl- chlorophyll - FNR- ferredoxin-NADP⁺ oxidoreductase - Mops- 3-(N-morpholino) propanesulfonic acid - PC- plastocyanin - PMSF- phenylmethanesulfonylfluoride - SDS- sodium dodecyl sulfate - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tricine- N-tris (hydroxymethyl) methylglycine     

Species-related differences in the electrophoretic behavior of CP 29 and CP 26: An immunochemical analysis

The monomeric chlorophyll-protein complexes, CP 29 and CP 26 seen in the Camm and Green (1980) and Dunahay and Staehelin (1986) green gels do not always migrate in the order of the apparent molecular weight of their apoproteins as determined by denaturing gel electrophoresis. In barley and corn they do, but in spinach they do not. In addition, in some higher plant species these chlorophyll-protein complexes comigrate on green gels causing confusion in the literature. To remedy this situation and circumvent future confusion, we propose that the CP 29 and CP 26 complexes be named according to the relative molecular weight of their apoproteins on denaturing gels. Our proposal is supported by the results obtained from four antibodies used on Western blot samples of whole thylakoids, grana membranes, and PS II preparations from different plants. The higher molecular weight proteins (proposed CP 29's) react strongly to one set of antibodies, and the lower molecular weight proteins (proposed CP 26's) react strongly to a different set. In spinach, CP 26 antibodies react also with CP 29, but the extent of the cross-reactivity depends critically on the gel electrophoresis system used. Accordingly, a lack of antibody reactivity under certain conditions may not indicate two proteins are unrelated, just simply that a particular epitope is no longer accessible following gel electrophoresis with a particular buffer system.Abbreviations CP Chlorophyll-protein - ammediol 1,2, amino-methyl-propanediol - Tris tris(hydroxymethyl)aminomethane - TBS Tris-buffered saline - MES 2-[N-Morpholino]ethane sulfonic acid - PS II Photosystem II - LHC II light harvesting polypeptides of PS II - BBY stacked membrane preparation of Berthold, Babcock and Yocum - HRP horseradish peroxidase - AP alkaline phosphatase - PVDF polyvinylidene fluoride - BSA bovine serum albumin - KLH keyhole limpet hemocyanin     

Chromosome locations of five genes encoding photosynthetic electron transfer proteins in Arabidopsis thaliana

The chromosome locations of nuclear genes encoding four photosynthetic electron transfer proteins have been determined by examining restriction fragment length polymorphisms in F₈recombinant inbred lines of Arabidopsis thaliana. The single-copy PetC gene encoding the chloroplast Rieske FeS protein was mapped to the top of chromosome 4, whereas the PetE and PetF genes encoding plastocyanin and ferredoxin, respectively, were mapped to different parts of chromosome 1. Two PetH genes encoding ferredoxin-NADP⁺oxidoreductase were mapped to the top of chromosome 1 and the bottom of chromosome 5.     

Molecular characterization and gene expression of lhcb5 gene encoding CP26 in the light-harvesting complex II of Chlamydomonas reinhardtii

We isolated and sequenced a cDNA clone encoding a minor chlorophyll a/b-binding protein, CP26, which is associated with the light-harvesting complex II of Chlamydomonas reinhardtii. Protein sequences of internal peptide fragments from purified CP26 were determined and used to identify a cDNA clone. The 1.1 kb lhcb5 gene codes for a polypeptide of 289 amino acids with a predicted molecular weight of 30713. The lhcb5 gene product could reconstitute with chlorophylls and xanthophylls to form a green band on a gel. Although the expression of many lhcb genes are strictly regulated by light, the lhcb5 gene was only loosely regulated. We propose that a plant acclimatizes itself to the light environment by quantitatively and qualitatively modulating the light-harvesting complex. Characterization of the primary structure and the implications of its unique expression are discussed.     

The effects of ultraviolet irradiation on P680+ reduction in PS II core complexes measured for individual S-states and during repetitive cycling of the oxygen-evolving complex

Flash-induced absorbance measurements at 830 nm on both nanosecond and microsecond timescales have been used to characterise the effect of ultraviolet light on Photosystem II core particles. A combination of UV-A and UV-B, closely simulating the spectrum of sunlight below 350 nm, was found to have a primary effect on the donor side of P680. Repetitive measurements indicated reductions in the nanosecond components of the absorbance decay with a concomitant appearance and increase in the amplitude of a component with a 10 s time constant attributed to slow reduction of P680⁺ by Tyr_z when the function of the oxygen evolving complex is inhibited. Single-flash measurements show that the nanosecond components have amplitudes which vary with S-state. Increasing UV irradiation inhibited the amplitude of these components without changing their S-state dependence. In addition, UV irradiation resulted in a reduction in the total amplitude, with no change in the proportion of the 10 s contribution.Abbreviations BBY- PS II membrane fragments - P680- primary electron donor of PS II - PS II- Photosystem II - Q_A and Q_B– primary and secondary quinone electron acceptors of PS II - S-state- redox state of the oxygenevolving complex - Tyr_z– tyrosine residue in PS II - UV-A- ultraviolet radiation 320–400 nm - UV-B- ultraviolet radiation 280–320 nm     

The sodium cycle: A novel type of bacterial energetics

The progress of bioenergetic studies on the role of Na⁺ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na⁺ can, under certain conditions, substitute for H⁺ as the coupling ion. Various primary Na⁺ pumps ( generators) are described, i.e., Na⁺-motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The formed is shown to be consumed by Na⁺ driven ATP-synthase, Na⁺ flagellar motor, numerous Na⁺, solute symporters, and the methanogenesis-linked reverse electron transfer system. InVibrio alginolyticus, it was found that , generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na⁺, solute symports), and mechanical (rotation of the flagellum). InPropionigenum modestum, circulation of Na⁺ proved to be the only mechanism of energy coupling. In other species studied, the Na⁺ cycle seems to coexist with the H⁺ cycle. For instance, inV. alginolyticus the initial and terminal steps of the respiratory chain are Na⁺ - and H⁺-motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na⁺ as well as of H⁺. In the alkalo- and halotolerantBacillus FTU, there are H⁺ - and Na⁺-motive terminal oxidases. Sometimes, the Na⁺-translocating enzyme strongly differs from its H⁺-translocating homolog. So, the Na⁺-motive and H⁺-motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H⁺-motive and Na⁺-motive terminal oxidases differ in that the former is ofaa ₃-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na⁺ and H⁺ can be translocated by one and the sameP. modestum ATPase which is of the F₀F₁-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary generator(s) and consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera:Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcin, etc. Thus, the sodium world seems to occupy a rather extensive area in the biosphere.     

Central-Cis isomers of lutein found in the major light-harvesting complex of Photosystem II (LHC IIb) of higher plants

Lutein (,-carotene-3,3-diol) is the major carotenoid of the light-harvesting systems of higher plants. Lutein was isolated at 4°C and in complete darkness from the bulk light-harvesting complex of Photosystem II of spinach (LHC IIb) and from BBY particles. Separation using normal-phase HPLC (with 2D detection) in comparison to the authentic isomers (prepared by iodine-sensitised isomerization) showed the presence of a number of geometrical isomers of this xanthophyll in PS II, namely all-trans (the major component); 13-cis, 13-cis and 15-cis-lutein. Iodine-sensitised photo-isomerization of all-trans lutein produced six geometrical isomers of lutein as determined by HPLC. The configuration of five of these isomers was determined by ¹H-NMR to be all-trans, 9-cis, 9-cis, 13-cis and 13-cis. In addition, small amounts of another isomer have been tentatively identified to be 15-cis lutein on the basis of its electronic absorption spectrum. The possible functional significance of the presence of cis-isomers of this carotenoid in LHC IIb is discussed.     

Juvenile hormone dependent phosphorylation of a 100 kDa polypeptide is mediated by protein kinase C in the follicle cells of Rhodnius prolixus

Summary

The addition of juvenile hormone I (JH I) to membrane preparations of the follicle cells from vitellogenic follicles of the insect Rhodnius prolixus causes a significant increase in the phosphorylation of a 100 kDa polypeptide; and ouabain, a specific inhibitor of Na⁺K⁺-ATPase, eliminates this effect. H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine), an inhibitor of protein kinase C (PKC), also eliminates the JH-dependent phosphorylation of this polypeptide. PDBU (phorbol-12, 13-dibutyrate), an activator of PKC, mimics the action of JH in increasing the phosphorylation of the 100 kDa polypeptide. Because these findings parallel the action of JH in causing the patency, the appearance of large spaces between the follicle cells through which vitellogenin gains access to the oocyte surface, they suggest that phosphorylation of one or more membrane proteins is a key event in the development of patency in response to JH. The 100 kDa polypeptide may represent the a-subunit of Na⁺K⁺-ATPase.     

Two sites of photoinhibition of the electron transfer in oxygen evolving and Tris-treated PS II membrane fragments from spinach

Photoinhibition was analyzed in O₂-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680⁺ formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a stable charge separation between P680⁺ and Q_A ^- within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from Y_Z to P680⁺. The quantum yield of site I photoinhibition (2–3×10^-7 inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O₂-evolving samples, the quantum yield of site II photoinhibition is about 10^-7 inhibited RC/quantum. After selective elimination of the O₂-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m²), the quantum yield is 10^-4 inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.Abbreviations A₈₃₀ absorption change at 830 nm - P680 primary electron donor of PS II - PS II photosystem II - Mes 2(N-morpholino)ethansulfonic acid - Q_A, Q_B primary and secondary acceptors of PS II - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbohydrazide - FWHM fullwidth at half maximum - Ph-p-BQ phenyl-p-benzoquinone - PFR photon fluence rate - Pheo pheophytin - RC reaction center     

Evidence of excited state absorption in PS II membrane fragments

Utilizing a two-beam technique in the frequency domain, the pumped absorption of PS II membrane fragments from spinach and of acetonic chlorophyll-a solutions was measured at room temperature. In a very narrow wavelength region (0.2 nm around the pump pulse wavelength) the relative test beam transmission exhibited either a decrease or an increase, respectively, dependent on the intensity of a strong pump beam. In contrast, the transmission changes of chl-a solutions were not affected by the wavelength mistuning between pump and test beam. The data obtained for PS II membrane fragments were interpreted in terms of excited state absorption of pigment-protein clusters within the light-harvesting complex of PS II. The interpretation of the small absorption band as a homogeneously broadened line led to a transversal relaxation time for chlorophyll in vivo of about 1 ps.Abbreviations PS I photosystem I of green plants - PS II photosystem II of green plants - P700 primary donor of PS I - P680 primary donor of PS II     

Participation of plastoquinone,cytochrome c 553 and ferrodoxin-NADP+ oxido-reductase in both photosynthesis and respiration in Spirulina maxima

Dark and light oxidation of NADPH was measured in Spirulina maxima thylakoid membranes. The dark reaction was more cyanide sensitive than the light reaction. In light, 83% of the electrons from NADPH produced H₂O₂ on reducing oxygen, whereas in the dark this number was only 36%. These results are explained by assuming the presence of an electron transport segment common to the photosynthetic and the respiratory chains, so that electrons flowing through the cyanide sensitive oxidase in the dark are diverted to the photosytem (PS) I reaction center (P700). In addition, cytochrome (cyt) c ₅₅₃ was found to be an electron donor for both cyt oxidase and P700. Half maximum reduction rates were obtained with 7 M cyt c ₅₅₃. The intrathylakoidal concentration of cyt c ₅₅₃ was determined to be 83 M. About 60% of the respiratory NADPH oxidation activity was lost by extracting the membranes with pentane and was restored by adding plastoquinone (the main photosythetic quinone). NADPH oxidation activity was also inhibited upon washing the membranes with a low salt buffer. This activity was restored by adding partially purified ferredoxin-NADP⁺ oxido-reductase (FNR). A model for the electron transport in thylakoids, in which cyt c ₅₅₃, plastoquinone and FNR participate in both photosynthesis and respiration is proposed.     

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680+ in PS II membrane fragments from spinach

The functional connection between redox component Y _z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680⁺ was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680⁺ reduction by Y _z under conditions where the catalytic site of water oxidation stays in the redox state S₁. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca²⁺. The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the Q_B-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680⁺ reduction by Y _z , and (c) Ca²⁺ could play a functional role for the electronic and vibrational coupling between the redox groups Y _z and P680. The electron transport from Y _z to P680⁺ is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - ANT 2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5 dinitrothiophene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2[N-Morpholino]ethanesulfonic acid - PS II photosystem II - Q_A, Q_B primary and secondary plastoquinone acceptor of photosystem II - S _i redox states of the catalytic site of water oxidation - Y _z redox active Tyr-161 of polypeptide D-1     

Chemiosmotic systems in bioenergetics: H+-cycles and Na+-cycles

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H⁺ cycle concept. The circulation of H⁺ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H⁺ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes.Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H⁺. It was found that, in ceertain bacteria, as well as in the outer membrane of the animal cell, Na⁺ effectively substitutes for H⁺ as the coupling ion (the chemiosmotic Na⁺ cycle). A precedent is set when the Na⁺ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H⁺ and Na⁺ cycles coexist in one and the same membrane (bacteria) or in two diffeerent membranes of one and the same cell (animals). The sets of and generators as well as and consumers found in different types of biomembranes, are listed and discussed.     

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na⁺/H⁺ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na⁺-resistance on an antiporter-deficient E. coli strain, nor did they confer Na⁺/H⁺ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.     

Competition between linear and cyclic electron flow in plants deficient in Photosystem I

Photosynthetic electron transport can involve either a linear flow from water to NADP, via Photosystems (PS) II and I or a cyclic flow just involving PSI. Little is known about factors regulating the relative flow through each of these pathways. We have examined photosynthetic electron transport through each system in plants of Arabidopsis thaliana in which either the PSI-D1 or PSI-E1 subunits of PSI have been knocked out. In both cases, this results in an imbalance in the turnover of PSI and PSII, such that PSII electron transport is limited by PSI turnover. Phosphorylation of light-harvesting complex II (LHCII) and its migration to PSI is enhanced but only partially reversible and not sufficient to balance photosystem turnover. In spite of this, cyclic electron flow is able to compete efficiently with PSI across a range of conditions. In dark-adapted leaves, the efficiency of cyclic relative to linear flow induced by far-red light is increased, implying that the limiting step of cyclic flow lies in the re-injection of electrons into the electron transport chain. Illumination of leaves with white light resulted in transient induction of a significant non-photochemical quenching in knockout plants which is probably high energy state quenching induced by cyclic electron flow. At high light and at low CO₂, non-photochemical quenching was greater in the knockout plants than in the wildtype. Comparison of PSI and PSII turnover under such conditions suggested that this is generated by cyclic electron flow around PSI. We conclude that, when the concentration of PSI is limiting, cyclic electron flow is still able to compete effectively with linear flow to maintain a high ΔpH to regulate photosynthesis.