A photoheterotrophic bacterium from Iceland has adapted its photosynthetic machinery to the long days of polar summer.

During their long evolution, anoxygenic phototrophic bacteria have inhabited a wide variety of natural habitats and developed specific strategies to cope with the challenges of any particular environment. Expression, assembly, and safe operation of the photosynthetic apparatus must be regulated to prevent reactive oxygen species generation under illumination in the presence of oxygen. Here, we report on the photoheterotrophic Sediminicoccus sp. strain KRV36, which was isolated from a cold stream in north-western Iceland, 30 km south of the Arctic Circle. In contrast to most aerobic anoxygenic phototrophs, which stop pigment synthesis when illuminated, strain KRV36 maintained its bacteriochlorophyll synthesis even under continuous light. Its cells also contained between 100 and 180 chromatophores, each accommodating photosynthetic complexes that exhibit an unusually large carotenoid absorption spectrum. The expression of photosynthesis genes in dark-adapted cells was transiently downregulated in the first 2 hours exposed to light but recovered to the initial level within 24 hours. An excess of membrane-bound carotenoids as well as high, constitutive expression of oxidative stress response genes provided the required potential for scavenging reactive oxygen species, safeguarding bacteriochlorophyll synthesis and photosystem assembly. The unique cellular architecture and an unusual gene expression pattern represent a specific adaptation that allows the maintenance of anoxygenic phototrophy under arctic conditions characterized by long summer days with relatively low irradiance.IMPORTANCEThe photoheterotrophic bacterium Sediminicoccus sp. KRV36 was isolated from a cold stream in Iceland. It expresses its photosynthesis genes, synthesizes bacteriochlorophyll, and assembles functional photosynthetic complexes under continuous light in the presence of oxygen. Unraveling the molecular basis of this ability, which is exceptional among aerobic anoxygenic phototrophic species, will help to understand the evolution of bacterial photosynthesis in response to changing environmental conditions. It might also open new possibilities for genetic engineering of biotechnologically relevant phototrophs, with the aim of increasing photosynthetic activity and their tolerance to reactive oxygen species.

PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1A redox states alleviate photoinhibition during changes in light intensity.

Plants have evolved photosynthetic regulatory mechanisms to maintain homeostasis in response to light changes during diurnal transitions and those caused by passing clouds or by wind. One such adaptation directs photosynthetic electron flow to a cyclic pathway to alleviate excess energy surges. Here, we assign a function to regulatory cysteines of PGR5-like protein 1A (PGRL1A), a constituent of the PROTON GRADIENT REGULATION5 (PGR5)-dependent cyclic electron flow (CEF) pathway. During step increases from darkness to low light intensity in Arabidopsis (Arabidopsis thaliana), the intermolecular disulfide of the PGRL1A 59-kDa complex was reduced transiently within seconds to the 28-kDa form. In contrast, step increases from darkness to high light stimulated a stable, partially reduced redox state in PGRL1A. Mutations of 2 cysteines in PGRL1A, Cys82 and Cys183, resulted in a constitutively pseudo-reduced state. The mutant displayed higher proton motive force (PMF) and nonphotochemical quenching (NPQ) than the wild type (WT) and showed altered donor and acceptor dynamic flow around PSI. These changes were found to correspond with the redox state of PGRL1A. Continuous light regimes did not affect mutant growth compared to the WT. However, under fluctuating regimes of high light, the mutant showed better growth than the WT. In contrast, in fluctuating regimes of low light, the mutant displayed a growth penalty that can be attributed to constant stimulation of CEF under low light. Treatment with photosynthetic inhibitors indicated that PGRL1A redox state control depends on the penultimate Fd redox state. Our results showed that redox state changes in PGRL1A are crucial to optimize photosynthesis.

Plants cope with fluctuating light by frequency-dependent nonphotochemical quenching and cyclic electron transport.

In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light. Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2-2, and pgrl1ab impaired in different pathways of the cyclic electron transport. The fastest was the PsbS-regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de-epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics. Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.

The Small RNA-Binding Protein CcaF1 Promotes Formation of Photosynthetic Complexes in Rhodobacter sphaeroides.

In natural habitats, bacteria frequently need to adapt to changing environmental conditions. Regulation of transcription plays an important role in this process. However, riboregulation also contributes substantially to adaptation. Riboregulation often acts at the level of mRNA stability, which is determined by sRNAs, RNases, and RNA-binding proteins. We previously identified the small RNA-binding protein CcaF1, which is involved in sRNA maturation and RNA turnover in Rhodobacter sphaeroides. Rhodobacter is a facultative phototroph that can perform aerobic and anaerobic respiration, fermentation, and anoxygenic photosynthesis. Oxygen concentration and light conditions decide the pathway for ATP production. Here, we show that CcaF1 promotes the formation of photosynthetic complexes by increasing levels of mRNAs for pigment synthesis and for some pigment-binding proteins. Levels of mRNAs for transcriptional regulators of photosynthesis genes are not affected by CcaF1. RIP-Seq analysis compares the binding of CcaF1 to RNAs during microaerobic and photosynthetic growth. The stability of the pufBA mRNA for proteins of the light-harvesting I complex is increased by CcaF1 during phototrophic growth but decreased during microaerobic growth. This research underlines the importance of RNA-binding proteins in adaptation to different environments and demonstrates that an RNA-binding protein can differentially affect its binding partners in dependence upon growth conditions.

Arabidopsis mutants lacking DLDG1 and non-photochemical quenching-related proteins reveal the regulatory role of DLDG1 in chloroplast pH homeostasis.

The control of pH in chloroplasts is important to regulate photosynthesis, although details of the precise regulatory mechanisms of H+ homeostasis in chloroplasts are not fully understood. We recently found that the cyanobacterial PxcA homolog DLDG1 is involved in plastidial pH control. PxcA and DLDG1 have been thought to control light-dependent H+ extrusion across the cyanobacterial cytoplasmic and chloroplast envelope membranes, respectively. To investigate DLDG1-dependent pH control in chloroplasts, we crossed the dldg1 mutant with various mutants lacking known non-photochemical quenching (NPQ)-related proteins, such as fluctuating-light acclimation protein 1 (FLAP1), PsbS/NPQ4, and proton gradient regulation 5 (PGR5). Phenotypes of these double mutants revealed that PsbS works upstream of DLDG1, PGR5 affects NPQ independently from DLDG1, and the ΔpH regulation by FLAP1 and DLDG1 are independent of each other.

High cyclic electron transfer via the PGR5 pathway in the absence of photosynthetic control.

The light reactions of photosynthesis couple electron and proton transfers across the thylakoid membrane, generating NADPH, and proton motive force (pmf) that powers the endergonic synthesis of ATP by ATP synthase. ATP and NADPH are required for CO2 fixation into carbohydrates by the Calvin-Benson-Bassham cycle. The dominant ΔpH component of the pmf also plays a photoprotective role in regulating photosystem II light harvesting efficiency through nonphotochemical quenching (NPQ) and photosynthetic control via electron transfer from cytochrome b6f (cytb6f) to photosystem I. ΔpH can be adjusted by increasing the proton influx into the thylakoid lumen via upregulation of cyclic electron transfer (CET) or decreasing proton efflux via downregulation of ATP synthase conductivity (gH+). The interplay and relative contributions of these two elements of ΔpH control to photoprotection are not well understood. Here, we showed that an Arabidopsis (Arabidopsis thaliana) ATP synthase mutant hunger for oxygen in photosynthetic transfer reaction 2 (hope2) with 40% higher proton efflux has supercharged CET. Double crosses of hope2 with the CET-deficient proton gradient regulation 5 and ndh-like photosynthetic complex I lines revealed that PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET is the major pathway contributing to higher proton influx. PGR5-dependent CET allowed hope2 to maintain wild-type levels of ΔpH, CO2 fixation and NPQ, however photosynthetic control remained absent and PSI was prone to photoinhibition. Therefore, high CET in the absence of ATP synthase regulation is insufficient for PSI photoprotection.

Distinct contribution of two cyclic electron transport pathways to P700 oxidation.

Cyclic electron transport (CET) around Photosystem I (PSI) acidifies the thylakoid lumen and downregulates electron transport at the cytochrome b6f complex. This photosynthetic control is essential for oxidizing special pair chlorophylls (P700) of PSI for PSI photoprotection. In addition, CET depending on the PROTON GRADIENT REGULATION 5 (PGR5) protein oxidizes P700 by moving a pool of electrons from the acceptor side of PSI to the plastoquinone pool. This model of the acceptor-side regulation was proposed on the basis of the phenotype of the Arabidopsis (Arabidopsis thaliana) pgr5-1 mutant expressing Chlamydomonas (Chlamydomonas reinhardtii) plastid terminal oxidase (CrPTOX2). In this study, we extended the research including the Arabidopsis chlororespiratory reduction 2-2 (crr2-2) mutant defective in another CET pathway depending on the chloroplast NADH dehydrogenase-like (NDH) complex. Although the introduction of CrPTOX2 did not complement the defect in the acceptor-side regulation by PGR5, the function of the NDH complex was complemented except for its reverse reaction during the induction of photosynthesis. We evaluated the impact of CrPTOX2 under fluctuating light intensity in the wild-type, pgr5-1 and crr2-2 backgrounds. In the high-light period, both PGR5- and NDH-dependent CET were involved in the induction of photosynthetic control, whereas PGR5-dependent CET preferentially contributed to the acceptor-side regulation. On the contrary, the NDH complex probably contributed to the acceptor-side regulation in the low-light period but not in the high-light period. We evaluated the sensitivity of PSI to fluctuating light and clarified that acceptor-side regulation was necessary for PSI photoprotection by oxidizing P700 under high light.

High Yield of B-Side Electron Transfer at 77 K in the Photosynthetic Reaction Center Protein from Rhodobacter sphaeroides.

The primary electron transfer (ET) processes at 295 and 77 K are compared for the Rhodobacter sphaeroides reaction center (RC) pigment-protein complex from 13 mutants including a wild-type control. The engineered RCs bear mutations in the L and M polypeptides that largely inhibit ET from the excited state P* of the primary electron donor (P, a bacteriochlorophyll dimer) to the normally photoactive A-side cofactors and enhance ET to the C2-symmetry related, and normally photoinactive, B-side cofactors. P* decay is multiexponential at both temperatures and modeled as arising from subpopulations that differ in contributions of two-step ET (e.g., P* → P+BB- → P+HB-), one-step superexchange ET (e.g., P* → P+HB-), and P* → ground state. [HB and BB are monomeric bacteriopheophytin and bacteriochlorophyll, respectively.] The relative abundances of the subpopulations and the inherent rate constants of the P* decay routes vary with temperature. Regardless, ET to produce P+HB- is generally faster at 77 K than at 295 K by about a factor of 2. A key finding is that the yield of P+HB-, which ranges from ∼5% to ∼90% among the mutant RCs, is essentially the same at 77 K as at 295 K in each case. Overall, the results show that ET from P* to the B-side cofactors in these mutants does not require thermal activation and involves combinations of ET mechanisms analogous to those operative on the A side in the native RC.

Influence of Polar Mutations on the Electronic and Structural Properties of QA-· in Bacterial Reaction Centers.

Reaction centers from Rhodobacter sphaeroides with residue M265 mutated from isoleucine to threonine, serine, and asparagine (M265IT, M265IS, and M265IN, respectively) in the QA-· state are studied by high-resolution electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance spectroscopy methods to investigate the structural characteristics of these mutants influencing the redox properties of the QA site. All three mutants decrease the redox midpoint potential (Em) of QA by ∼0.1 V, yet the mechanism for this drop in Em is unclear. In this work, we examine (i) the hydrogen bonding interactions between QA-· and residues histidine M219 and alanine M260, (ii) the electron spin density distribution of the semiquinone, and (iii) the orientations of the ubiquinone methoxy substituents. 13C measurements show no significant contribution of methoxy dihedral angles to the observed decrease in Em for the QA mutants. Instead, 14N three-pulse ESEEM data suggest that electrostatic or hydrogen bond formation between the mutated M265 side chain and His-M219 Nδ may be involved in the observed lowering of the QA midpoint potential. For mutant M265IN, analysis of the proton hyperfine couplings reveals a weakened hydrogen bond network, resulting in an altered QA-· spin density distribution. The magnetic resonance study presented here is most consistent with an electrostatic or structural perturbation of the His-M219 Nδ hydrogen bond in these mutants as a mechanism for the ∼0.1 V decrease in QA Em.

Commonalities and specialties in photosynthetic functions of PROTON GRADIENT REGULATION5 variants in Arabidopsis.

The PROTON GRADIENT REGULATION5 (PGR5) protein is required for trans-thylakoid proton gradient formation and acclimation to fluctuating light (FL). PGR5 functionally interacts with two other thylakoid proteins, PGR5-like 1 (PGRL1) and 2 (PGRL2); however, the molecular details of these interactions are largely unknown. In the Arabidopsis (Arabidopsis thaliana) pgr5-1 mutant, the PGR5G130S protein accumulates in only small amounts. In this work, we generated a knockout allele of PGR5 (pgr5-Cas) using CRISPR-Cas9 technology. Like pgr5-1, pgr5-Cas is seedling-lethal under FL, but photosynthesis and particularly cyclic electron flow, as well as chlorophyll content, are less severely affected in both pgr5-Cas and pgrl1ab (which lacks PGRL1 and PGR5) than in pgr5-1. These differences are associated with changes in the levels of 260 proteins, including components of the Calvin-Benson cycle, photosystems II and I, and the NDH complex, in pgr5-1 relative to the wild type (WT), pgr5-Cas, and pgrl1ab. Some of the differences between pgr5-1 and the other mutant lines could be tentatively assigned to second-site mutations in the pgr5-1 line, identified by whole-genome sequencing. However, others, particularly the more pronounced photosynthetic defects and PGRL1 depletion (compared to pgr5-Cas), are clearly due to specific negative effects of the amino-acid substitution in PGR5G130S, as demonstrated by complementation analysis. Moreover, pgr5-1 and pgr5-Cas plants are less tolerant to long-term exposure to high light than pgrl1ab plants. These results imply that, in addition to the previously reported necessity of PGRL1 for optimal PGR5 function, PGR5 is required alongside PGRL1 to avoid harmful effects on plant performance.

Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria.

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivax gelatinosus and Rhodobacter sphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria.

Two magnesium transporters in the chloroplast inner envelope essential for thylakoid biogenesis in Arabidopsis.

Magnesium (Mg2+ ) serves as a cofactor for a number of photosynthetic enzymes in the chloroplast, and is the central atom of the Chl molecule. However, little is known about the molecular mechanism of Mg2+ transport across the chloroplast envelope. Here, we report the functional characterization of two transport proteins in Arabidopsis: Magnesium Release 8 (MGR8) and MGR9, of the ACDP/CNNM family, which is evolutionarily conserved across all lineages of living organisms. Both MGR8 and MGR9 genes were expressed ubiquitously, and their encoded proteins were localized in the inner envelope of chloroplasts. Mutations of MGR8 and MGR9 together, but neither of them alone, resulted in albino ovules and chlorotic seedlings. Further analysis revealed severe defects in thylakoid biogenesis and assembly of photosynthetic complexes in the double mutant. Both MGR8 and MGR9 functionally complemented the growth of the Salmonella typhimurium mutant strain MM281, which lacks Mg2+ uptake capacity. The embryonic and early seedling defects of the mgr8/mgr9 double mutant were rescued by the expression of MGR9 under the embryo-specific ABI3 promoter. The partially rescued mutant plants were hypersensitive to Mg2+ deficient conditions and contained less Mg2+ in their chloroplasts than wild-type plants. Taken together, we conclude that MGR8 and MGR9 serve as Mg2+ transporters and are responsible for chloroplast Mg2+ uptake.

Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the ∼4-ps population, P+HA- forms through a two-step process, P*→ P+BA-→ P+HA-, while in the ∼20-ps population, it forms via a one-step P* → P+HA- superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA- intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P+BA- along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA- formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.

Identification of a Novel Mutation Exacerbated the PSI Photoinhibition in pgr5/pgrl1 Mutants; Caution for Overestimation of the Phenotypes in Arabidopsis pgr5-1 Mutant.

PSI photoinhibition is usually avoided through P700 oxidation. Without this protective mechanism, excess light represents a potentially lethal threat to plants. PGR5 is suggested to be a major component of cyclic electron transport around PSI and is important for P700 oxidation in angiosperms. The known Arabidopsis PGR5 deficient mutant, pgr5-1, is incapable of P700 oxidation regulation and has been used in numerous photosynthetic studies. However, here it was revealed that pgr5-1 was a double mutant with exaggerated PSI photoinhibition. pgr5-1 significantly reduced growth compared to the newly isolated PGR5 deficient mutant, pgr5hope1. The introduction of PGR5 into pgr5-1 restored P700 oxidation regulation, but remained a pale-green phenotype, indicating that pgr5-1 had additional mutations. Both pgr5-1 and pgr5hope1 tended to cause PSI photoinhibition by excess light, but pgr5-1 exhibited an enhanced reduction in PSI activity. Introducing AT2G17240, a candidate gene for the second mutation into pgr5-1 restored the pale-green phenotype and partially restored PSI activity. Furthermore, a deficient mutant of PGRL1 complexing with PGR5 significantly reduced PSI activity in the double-deficient mutant with AT2G17240. From these results, we concluded that AT2G17240, named PSI photoprotection 1 (PTP1), played a role in PSI photoprotection, especially in PGR5/PGRL1 deficient mutants.

Accurate prediction of mutation-induced frequency shifts in chlorophyll proteins with a simple electrostatic model.

Photosynthetic pigment-protein complexes control local chlorophyll (Chl) transition frequencies through a variety of electrostatic and steric forces. Site-directed mutations can modify this local spectroscopic tuning, providing critical insight into native photosynthetic functions and offering the tantalizing prospect of creating rationally designed Chl proteins with customized optical properties. Unfortunately, at present, no proven methods exist for reliably predicting mutation-induced frequency shifts in advance, limiting the method's utility for quantitative applications. Here, we address this challenge by constructing a series of point mutants in the water-soluble chlorophyll protein of Lepidium virginicum and using them to test the reliability of a simple computational protocol for mutation-induced site energy shifts. The protocol uses molecular dynamics to prepare mutant protein structures and the charge density coupling model of Adolphs et al. [Photosynth. Res. 95, 197-209 (2008)] for site energy prediction; a graphical interface that implements the protocol automatically is published online at http://nanohub.org/tools/pigmenthunter. With the exception of a single outlier (presumably due to unexpected structural changes), we find that the calculated frequency shifts match the experiment remarkably well, with an average error of 1.6 nm over a 9 nm spread in wavelengths. We anticipate that the accuracy of the method can be improved in the future with more advanced sampling of mutant protein structures.

Cryo-EM structure of the monomeric Rhodobacter sphaeroides RC-LH1 core complex at 2.5†Å.

Reaction centre light-harvesting 1 (RC-LH1) complexes are the essential components of bacterial photosynthesis. The membrane-intrinsic LH1 complex absorbs light and the energy migrates to an enclosed RC where a succession of electron and proton transfers conserves the energy as a quinol, which is exported to the cytochrome bc1 complex. In some RC-LH1 variants quinols can diffuse through small pores in a fully circular, 16-subunit LH1 ring, while in others missing LH1 subunits create a gap for quinol export. We used cryogenic electron microscopy to obtain a 2.5 Šresolution structure of one such RC-LH1, a monomeric complex from Rhodobacter sphaeroides. The structure shows that the RC is partly enclosed by a 14-subunit LH1 ring in which each αß heterodimer binds two bacteriochlorophylls and, unusually for currently reported complexes, two carotenoids rather than one. Although the extra carotenoids confer an advantage in terms of photoprotection and light harvesting, they could impede passage of quinones through small, transient pores in the LH1 ring, necessitating a mechanism to create a dedicated quinone channel. The structure shows that two transmembrane proteins play a part in stabilising an open ring structure; one of these components, the PufX polypeptide, is augmented by a hitherto undescribed protein subunit we designate as protein-Y, which lies against the transmembrane regions of the thirteenth and fourteenth LH1α polypeptides. Protein-Y prevents LH1 subunits 11-14 adjacent to the RC QB site from bending inwards towards the RC and, with PufX preventing complete encirclement of the RC, this pair of polypeptides ensures unhindered quinone diffusion.

Antagonistic Effects of Point Mutations on Charge Recombination and a New View of Primary Charge Separation in Photosynthetic Proteins.

Light-induced electron-transfer reactions were investigated in wild-type and three mutant Rhodobacter sphaeroides reaction centers with the secondary electron acceptor (ubiquinone QA) either removed or permanently reduced. Under such conditions, charge separation between the primary electron donor (bacteriochlorophyll dimer, P) and the electron acceptor (bacteriopheophytin, HA) was followed by P+HA- → PHA charge recombination. Two reaction centers were used that had different single amino-acid mutations that brought about either a 3-fold acceleration in charge recombination compared to that in the wild-type protein, or a 3-fold deceleration. In a third mutant in which the two single amino-acid mutations were combined, charge recombination was similar to that in the wild type. In all cases, data from transient absorption measurements were analyzed using similar models. The modeling included the energetic relaxation of the charge-separated states caused by protein dynamics and evidenced the appearance of an intermediate charge-separated state, P+BA-, with BA being the bacteriochlorophyll located between P and HA. In all cases, mixing of the states P+BA- and P+HA- was observed and explained in terms of electron delocalization over BA and HA. This delocalization, together with picosecond protein relaxation, underlies a new view of primary charge separation in photosynthesis.

PGRL2 triggers degradation of PGR5 in the absence of PGRL1.

In plants, inactivation of either of the thylakoid proteins PGR5 and PGRL1 impairs cyclic electron flow (CEF) around photosystem I. Because PGR5 is unstable in the absence of the redox-active PGRL1, but not vice versa, PGRL1 is thought to be essential for CEF. However, we show here that inactivation of PGRL2, a distant homolog of PGRL1, relieves the need for PGRL1 itself. Conversely, high levels of PGRL2 destabilize PGR5 even when PGRL1 is present. In the absence of both PGRL1 and PGRL2, PGR5 alters thylakoid electron flow and impairs plant growth. Consequently, PGR5 can operate in CEF on its own, and is the target of the CEF inhibitor antimycin A, but its activity must be modulated by PGRL1. We conclude that PGRL1 channels PGR5 activity, and that PGRL2 triggers the degradation of PGR5 when the latter cannot productively interact with PGRL1.

Cooperation of chloroplast ascorbate peroxidases and proton gradient regulation 5 is critical for protecting Arabidopsis plants from photo-oxidative stress.

High-light (HL) stress enhances the production of H2 O2 from the photosynthetic electron transport chain in chloroplasts, potentially causing photo-oxidative damage. Although stromal and thylakoid membrane-bound ascorbate peroxidases (sAPX and tAPX, respectively) are major H2 O2 -scavenging enzymes in chloroplasts, their knockout mutants do not exhibit a visible phenotype under HL stress. Trans-thylakoid proton gradient (∆pH)-dependent mechanisms exist for controlling H2 O2 production from photosynthesis, such as thermal dissipation of light energy and downregulation of electron transfer between photosystems II and I, and these may compensate for the lack of APXs. To test this hypothesis, we focused on a proton gradient regulation 5 (pgr5) mutant, wherein both ∆pH-dependent mechanisms are impaired, and an Arabidopsis sapx tapx double mutant was crossed with the pgr5 single mutant. The sapx tapx pgr5 triple mutant exhibited extreme sensitivity to HL compared with its parental lines. This phenotype was consistent with cellular redox perturbations and enhanced expression of many oxidative stress-responsive genes. These findings demonstrate that the PGR5-dependent mechanisms compensate for chloroplast APXs, and vice versa. An intriguing finding was that the failure of induction of non-photochemical quenching in pgr5 (because of the limitation in ∆pH formation) was partially recovered in sapx tapx pgr5. Further genetic studies suggested that this recovery was dependent on the NADH dehydrogenase-like complex-dependent pathway for cyclic electron flow around photosystem I. Together with data from the sapx tapx npq4 mutant, we discuss the interrelationship between APXs and ∆pH-dependent mechanisms under HL stress.

Effect of Detergents and Osmolytes on Thermal Stability of Native and Mutant Rhodobacter sphaeroides Reaction Centers.

Photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides is one of the most well-studied transmembrane pigment-protein complexes. It is a relatively stable protein with established conditions for its isolation from membranes, purification, and storage. However, it has been shown that some amino acid substitutions can affect stability of the RC, which results in a decrease of the RCs yield during its isolation and purification, disturbs spectral properties of the RCs during storage, and can lead to sample heterogeneity. To optimize conditions for studying mutant RCs, the effect of various detergents and osmolytes on thermal stability of the complex was examined. It was shown that trehalose and, to a lesser extent, sucrose, maltose, and hydroxyectoin at 1 M concentration slow down thermal denaturation of RCs. Sodium cholate was found to have significant stabilizing effect on the structure of native and genetically modified RCs. The use of sodium cholate as a detergent has several advantages and can be recommended for the storage and investigation of the unstable mutant membrane complexes of purple bacteria in long-term experiments.