The effect of norflurazon on protein composition and chlorophyll organization in pigment–protein complex of Photosystem II

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides in the 29.5–21 kDa region. The Chl a forms at 668, 676, and 690 nm that belong to LHC and antenna part of PS I disappear completely after treatment. The intensity of the Chl b form at 648 nm is sharply decreased in treated seedlings grown under 30 or 100 lx light intensity. The bands of carotenoid absorption at 421, 448 (Chl a), 452, 480, 492, 496 (β-carotene), and 508 nm also disappear. The band shift from 740 to 720 nm and decrease in its intensity relative to the 687 nm emission peak in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chla form at 710–712 nm.     

Modeling bacterial immune systems: Strategies for expression of toxic – but useful – molecules

Protection of bacterial cells against virus infection requires expression of molecules that are able to destroy the incoming foreign DNA. However, these molecules can also be toxic for the host cell. In both restriction–modification (R–M), and the recently discovered CRISPR/Cas systems, the toxicity is (in part) avoided through rapid transition of the expression of the toxic molecules from “OFF” to “ON” state. In restriction–modification systems the rapid transition is achieved through a large binding cooperativity, and low translation rate of the control protein. On the other hand, CRISPR array expression in CRISPR/Cas systems involves a mechanism where a small decrease of unprocessed RNAs leads to a rapid increase of processed small RNAs. Surprisingly, this rapid amplification crucially depends on fast non-specific degradation of the unprocessed molecules by an unidentified nuclease, rather than on large cooperativity in protein binding. Furthermore, the major control elements that are responsible for fast transition of R–M and CRISPR/Cas systems from “OFF” to “ON” state, are also directly involved in increased stability of the steady states of these systems. We here discuss mechanisms that allow rapid transition of toxic molecules from the unproductive to the productive state in R–M and CRISPR/Cas systems. The main purpose of this discussion is to put relevant theoretical and experimental work in a perspective that points to general similarities in otherwise mechanistically very different bacterial immune systems.     

A model of the protein–pigment baseplate complex in chlorosomes of photosynthetic green bacteria

In contrast to photosynthetic reaction centers, which share the same structural architecture, more variety is found in the light-harvesting antenna systems of phototrophic organisms. The largest antenna system described, so far, is the chlorosome found in anoxygenic green bacteria, as well as in a recently discovered aerobic phototroph. Chlorosomes are the only antenna system, in which the major light-harvesting pigments are organized in self-assembled supramolecular aggregates rather than on protein scaffolds. This unique feature is believed to explain why some green bacteria are able to carry out photosynthesis at very low light intensities. Encasing the chlorosome pigments is a protein-lipid monolayer including an additional antenna complex: the baseplate, a two-dimensional paracrystalline structure containing the chlorosome protein CsmA and bacteriochlorophyll a (BChl a). In this article, we review current knowledge of the baseplate antenna complex, which physically and functionally connects the chlorosome pigments to the reaction centers via the Fenna–Matthews–Olson protein, with special emphasis on the well-studied green sulfur bacterium Chlorobaculum tepidum (previously Chlorobium tepidum). A possible role for the baseplate in the biogenesis of chlorosomes is discussed. In the final part, we present a structural model of the baseplate through combination of a recent NMR structure of CsmA and simulation of circular dichroism and optical spectra for the CsmA–BChl a complex.     

Lymphadenovarix in the axilla – an unusual presentation of filariasis

Clinical manifestations of lymphatic filariasis depend on the area of lymphatic involvement and the duration of infection. A 21 year old man, resident in a filariasis endemic region, presented with multiple matted lymph nodes with cystic areas forming a large mass in his left axilla. An ultrasound scan of the axilla using a 7.5 MHz transducer revealed grossly dilated lymphatics but no filarial dance sign. Fine needle (21 G) aspiration cytology (FNAC) from the dilated lymphatics and solid areas in the lymph node mass revealed multiple microfilariae in a background of reactive lymphoid cells. Peripheral blood smears revealed microfilaremia with significant eosinophilia. Diagnosis of left axillary Bancroftian lymphadenovarix was made. On the administration of oral diethylcarbamazine, the diameter of the lymphatic vessels in the lymphadenovarix reduced considerably in size and microfilaremia disappeared. We report this case because axillary lymphadenovarix is a rare presentation of filariasis. This case is also unique since microfilariae were demonstrated in the fluid aspirated from the dilated lymphatics of the lymphadenovarix in the absence of live adult worms.     

Configuration of the carotenoid in the reaction centers of photosynthetic bacteria. Comparison of the resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C with those of cis-trans isomers of β-carotene

The resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C as well as those of the cis-trans isomers of β-carotene (all-trans, 9-cis, 13-cis, 15-cis and 9-cis, 13-cis- (or 9-cis, 13′-cis)) have been recorded at liquid N₂ temperature by use of the 457.9, 488.0 and 514.5 nm excitation lines. Comparison of the spectra indicated that the carotenoid in the reaction center takes the 15-cis configuration.     

Synthetic analogues of the histidine–chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex

Mg(II)–porphyrin–ligand and (bacterio)chlorophyl–ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. ¹H, ¹³C and ¹⁵N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9–5.2 ppm ¹H, 0.2–5.5 ppm ¹³C and 2.1–27.2 ppm ¹⁵N coordination shifts were measured for selectively labeled [1,3-¹⁵N]-Im, [1,3-¹⁵N,2-¹³C]-Im and [1,3-¹⁵N,1,2-¹³C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the ¹H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a–histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.Abbreviations (B)Chl a (bacterio)chlorophyll a - HMBC heteronuclear multiple bond correlation - Im imidazole - LH light-harvesting - 1-MeIm 1-methylimidazole - Mg(II)-Por Mg(II)-porphyrin macrocycle - OEP 2,3,7,8,12,13,17,18-octaethylporphyrin     

NaCl induced changes in photosystem stoichiometry and photosynthetic activity of the cyanobacterium Microcystis aeruginosa Kütz

Exposure to 0.4 M NaCl resulted in higher PS I/PS II stoichiometry and increase in the rate of photosynthesis in planktonic cyanobacterium M. aeruginosa. Altered ratios of PS I/PS II as well as photosynthesis and respiration were stabilized within 72 hr of exposure to salt, leading to adaptation of the organism to the changed conditions.     

Volume changes and electrostriction in the primary photoreactions of various photosynthetic systems: estimation of dielectric coefficient in bacterial reaction centers and of the observed volume changes with the Drude–Nernst equation

Photoacoustics (PA) allows the determination of enthalpy and volume changes of photoreactions in photosynthetic reaction centers on the 0.1–10 μs time scale. These include the bacterial centers from Rb. sphaeroides, PS I and PS II centers from Synechocystis and in whole cells. In vitro and in vivo PA data on PS I and PS II revealed that both the volume change (–26 A³) and reaction enthalpy (–0.4 eV) in PS I are the same as those in the bacterial centers. However the volume change in PS II is small and the enthalpy far larger, –1 eV. Assigning the volume changes to electrostriction allows a coherent explanation of these observations. One can explain the large volume decrease in the bacterial centers with an effective dielectric coefficient of ∼4. This is a unique approach to this parameter so important in estimation of protein energetics. The value of the volume contraction for PS I can only be explained if the acceptor is the super- cluster (Fe₄S₄)(Cys₄) with charge change from –1 to –2. The small volume change in PS II is explained by sub-μs electron transfer from Y_Z anion to P₆₈₀ cation, in which charge is only moved from the Y_Z anion to the Q_A with no charge separation or with rapid proton transfer from oxidized Y_Z to a polar region and thus very little change in electrostriction. At more acid pH equally rapid proton transfer from a neighboring histidine to a polar region may be caused by the electric field of the P₆₈₀ cation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: I. A review

The purpose of the review is to show that the tetrameric (bacterio)chlorophyll ((B)Chl) structures in reaction centers of photosystem II (PSII) of green plants and in bacterial reaction centers (BRCs) are similar and play a key role in the primary charge separation. The Stark effect measurements on PSII reaction centers have revealed an increased dipole moment for the transition at approximately 730 nm (Frese et al., Biochemistry 42:9205-9213, 2003). It was found (Heber and Shuvalov, Photosynth Res 84:84-91, 2005) that two fluorescent bands at 685 and 720 nm are observed in different organisms. These two forms are registered in the action spectrum of Q(A) photoreduction. Similar results were obtained in core complexes of PSII at low temperature (Hughes et al., Biochim Biophys Acta 1757: 841-851, 2006). In all cases the far-red absorption and emission can be interpreted as indication of the state with charge transfer character in which the chlorophyll monomer plays a role of an electron donor. The role of bacteriochlorophyll monomers (B(A) and B(B)) in BRCs can be revealed by different mutations of axial ligand for Mg central atoms. RCs with substitution of histidine L153 by tyrosine or leucine and of histidine M182 by leucine (double mutant) are not stable in isolated state. They were studied in antennaless membrane by different kinds of spectroscopy including one with femtosecond time resolution. It was found that the single mutation (L153HY) was accompanied by disappearance of B(A) molecule absorption near 802 nm and by 14-fold decrease of photochemical activity measured with ms time resolution. The lifetime of P(870)* increased up to approximately 200 ps in agreement with very low rate of the electron transfer to A-branch. In the double mutant L153HY + M182HL, the B(A) appears to be lost and B(B) is replaced by bacteriopheophytin Phi(B) with the absence of any absorption near 800 nm. Femtosecond measurements have revealed the electron transfer to B-branch with a time constant of approximately 2 ps. These results are discussed in terms of obligatory role of B(A) and Phi(B) molecules located near P for efficient electron transfer from P*.     

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g., Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+Q-A or P+Q-B, respectively; (b) the kinetics of electron transfer from Q-A to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (tau = 3.5 ps), followed by reduction of the bacteriopheophytin (tau = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (tau = 410 +/- 30 and 47 +/- 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type).(ABSTRACT TRUNCATED AT 400 WORDS)     

Symmetry-related mutants in the quinone binding sites of the bacterial reaction center – the effects of changes in charge distribution

To probe the structural elements that contribute to the functional asymmetries of the two ubiquinone₁₀ binding pockets in the reaction center of Rhodobacter capsulatus, we targeted the L212Glu–L213Asp (near Q_B) and the M246Ala-M247Ala (near Q_A) pairs of symmetry-related residues for site-specific mutagenesis. We have constructed site-specific mutants that eliminate the sequence differences at these positions (L212Glu–L213AspAla-Ala or M246Ala–M247AlaGlu-Asp), and have reversed that asymmetry by constructing a quadruple-mutant strain, RQ (L212Glu–L213Asp-M246Ala–M247AlaAla-Ala-Gl u-Asp). The mutations were designed to change the charge distribution in the quinone-binding region of the reaction center; none of the strains is capable of photosynthetic growth. In photocompetent phenotypic revertants of the RQ strain, second-site mutations which affect Q_B function are coupled to mutations in the Q_A site which restore an Ala or substitute a Tyr at the M247 site; one strain carries an additional MetLeu substitution at M260 near Q_A. All of the RQ revertants retain the engineered M246AlaGlu mutation in the Q_A site as well as the L212Ala–L213Ala mutations in the Q_B site. Kinetic characterization of the RQ revertants will give us an idea of what structural and functional elements are important for restoring efficiency to electron and proton transfer pathways in the RQ RC, which is far from native. To date, these preliminary results underscore the importance of an asymmetric distribution of polar amino acids in the quinone binding pockets and its influence on the functional properties of the reaction center.     

Light requirement and photosynthetic cell cultivation – Development of processes for efficient light utilization in photobioreactors

Although the potential of photosyntheticmicroorganisms for production of various metabolitesand in environmental bioremediation is recognized,their practical application has been limited by thedifficulty in supplying light efficiently tophotobioreactors. Various types of photobioreactorwith high illumination to volume ratios have beenproposed, but most are limited by cost, mass transfer,contamination, scale-up or a combination of these.The problem of light supply to photobioreactorscan be solved by developing photosynthetic cellcultivation systems where light is either substitutedor supplemented. Many strains of photosynthetic cellsare capable of heterotrophic growth under darkconditions and their heterotrophic culture can be usedfor efficient production of biomass and somemetabolites. However, light is absolutely required forefficient production of some metabolites. In suchcases, there is a need to supplement the heterotrophicwith photoautotrophic metabolism. Inphotoheterotrophic (mixotrophic) culture, thephotoautotrophic and heterotrophic metabolisms can beexploited for efficient production of usefulmetabolites but it has many problems such as processoptimization in terms of making a balance between thephotoautotrophic and heterotrophic metabolism. Another promising system is the sequentialheterotrophic/ photoautotrophic cultivation system,where the cells are cultivated heterotrophically tohigh concentrations and then passed through aphotobioreactor for accumulation of the desiredmetabolite(s). Furthermore, cyclicphotoautotrophic/heterotrophic cultivation system canbe used to achieve continuous cell growth underday/night cycles. This involves cultivating thecells photoautotrophically using solar light duringthe day and then adding controlled amount of organiccarbon source during the night for heterotrophicgrowth. In this review, these various systems arediscussed with some specific examples.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).     

The unusual extended C-terminal helix of the peroxisomal α/β-hydrolase Lpx1 is involved in dimer contacts but dispensable for dimerization

The yeast peroxisomal hydrolase Lpx1 belongs to the α/β-hydrolase superfamily. In the absence of Lpx1, yeast peroxisomes show an aberrant vacuolated morphology similar to what is found in peroxisomal disorder patients. Here, we present the crystal structure of Lpx1 determined at a resolution of 1.9 ?. The structure reveals the complete catalytic triad with an unusual location of the acid residue after strand β6 of the canonical α/β-hydrolase fold. A four-helix cap domain covers the active site. The interface between the α/β-hydrolase core and the cap domain forms the potential substrate binding site, which may also comprise the tunnel that leads into the protein interior and widens into a cavity. Two further tunnels connect the active site to the protein surface, potentially facilitating substrate access. Lpx1 is a homodimer. The α/β-hydrolase core folds of the two protomers form the dimer contact site. Further dimerization contacts arise from the mutual embracement of the cap domain of one protomer by the non-canonical C-terminal helix of the other, resulting in a total buried surface area of some 6000 ?2. The unusual C-terminal helix sticks out from the core fold to which it is connected by an extended flexible loop. We analyzed whether this helix is required for dimerization and for import of the dimer into peroxisomes using biochemical assays in vitro and a microscopy-based interaction assay in mammalian cells. Surprisingly, the C-terminal helix is dispensable for dimerization and dimer import. The unusually robust self-interaction suggests that Lpx1 is imported into peroxisomes as dimer.     

Energy dissipation in photosynthesis: Does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?

Dissipation of light energy was studied in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst., and in leaves of Spinacia oleracea L. and Arabidopsis thaliana (L.) Heynh., using chlorophyll fluorescence as an indicator reaction. Maximum chlorophyll fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated spinach leaves, as produced by saturating light and studied between +5 and −20 °C, revealed an activation energy ΔE of 0.11 eV. As this suggested recombination fluorescence produced by charge recombination between the oxidized primary donor of photosystem II and reduced pheophytin, a mathematical model explaining fluorescence, and based in part on known characteristics of primary electron-transport reactions, was developed. The model permitted analysis of different modes of fluorescence quenching, two localized in the reaction center of photosystem II and one in the light-harvesting system of the antenna complexes. It predicted differences in the relationship between quenching of variable fluorescence F _v and quenching of basal, so-called F ₀ fluorescence depending on whether quenching originated from antenna complexes or from reaction centers. Such differences were found experimentally, suggesting antenna quenching as the predominant mechanism of dissipation of light energy in the moss Rhytidiadelphus, whereas reaction-center quenching appeared to be important in spinach and Arabidopsis. Both reaction-center and antenna quenching required activation by thylakoid protonation but only antenna quenching depended on or was strongly enhanced by zeaxanthin. De-protonation permitted relaxation of this quenching with half-times below 1 min. More slowly reversible quenching, tentatively identified as so-called q _I or photoinhibitory quenching, required protonation but persisted for prolonged times after de-protonation. It appeared to originate in reaction centers. Received: 8 April 2000 / Accepted: 31 August 2000     

On the role of excitonic interactions in carotenoid–phthalocyanine dyads and implications for photosynthetic regulation

In two recent studies, energy transfer was reported in certain phthalocyanine–carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S₁) and phthalocyanines (Pcs) in the direction Pc → Car S₁ (Kloz et al., J Am Chem Soc 133:7007–7015, 2011) as well as in the direction Car S₁ → Pc (Liao et al., J Phys Chem A 115:4082–4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S₁ is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole–carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.

     

Redundancy of stomatal control for the circadian photosynthetic rhythm in Kalanchoë daigremontiana Hamet et Perrier

In continuous light, the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perrier has a circadian rhythm of gas exchange with peaks occurring during the subjective night. The rhythm of gas exchange is coupled to a weak, reverse phased rhythm of quantum yield of photosystem II (Phi (PSII)). To test if the rhythm of Phi (PSII) persists in the absence of stomatal control, leaves were coated with a thin layer of translucent silicone grease which prevented CO2 and H2O exchange. In spite of this treatment, the rhythm of Phi (PSII) occurred with close to normal phase timing and with a much larger amplitude than in uncoated leaves. The mechanism underlying the Phi (PSII) rhythm in coated leaves can be explained by a circadian activity of phosphoenolpyruvate carboxylase (PEPC). At peaks of PEPC activity, the small amount of CO2 contained in the coated leaf could have become depleted, preventing the carboxylase activity of Rubisco and causing decreases in electron transport rates (observed as deep troughs of Phi (PSII) at 23-h in LL and at ca. 24-h intervals afterwards). Peaks of Phi (PSII) would be caused by a downregulation of PEPC leading to improved supply of CO2 to Rubisco. Substrate limitation of photochemistry at 23 h (trough of Phi (PSII)) was also suggested by the weak response of ETR in coated leaves to stepwise light enhancement. These results show that photosynthetic rhythmicity in K. daigremontiana is independent of stomatal regulation and may originate in the mesophyll.