Comparative effect of tenuazonic acid, diuron, bentazone, dibromothymoquinone and methyl viologen on the kinetics of Chl a fluorescence rise OJIP and the MR820 signal.

In this study, the comparative effect of TeA, DCMU, bentazone, DBMIB and MV on prompt fluorescence and the MR820 signal was simultaneously analyzed to provide an insight into how to elucidate their precise influence on Ageratina adenophora photosystems. The herbicides that interrupt electron transport beyond QA, such as TeA, DCMU and bentazone, mainly increased the J-step level of fluorescence rise kinetics as a result of accumulation of QA-, but showed differences in detail. The IP phase disappeared in the presence of DCMU and bentazone with a significant increase in FO value. TeA treatment retained the IP phase with lowering FM. As an inhibitor of plastoquinone re-oxidation, DBMIB increased the I-step (IP phase almost unnoticable) without changing FO and FM values. MV blocking PSI electron transfer through intercepting electrons from the FeS clusters suppressed the IP phase by decreasing the P level. Considering the WIP kinetics, TeA and DBMIB also affected PSI activity. After DCMU and MV treatment, the major change in the MR820 kinetics was the loss of the slow phase due to the complete prevention of electron movement from PSII to re-reduce PC+ and P700+. TeA, bentazone and DBMIB clearly suppressed the MR820 slow phase and decreased the re-reduction rate of PC+ and P700+ (Vred), significantly. However, there were still parts of electrons being donated to PC+ and P700+, showing a smaller slow phase and PC+ and P700+ re-reduction rate. Additionally, TeA and DBMIB also somewhat declined the fast phase and PC and P700 oxidation rate (Vox).

NADPH from the oxidative pentose phosphate pathway drives the operation of cyclic electron flow around photosystem I in high-intertidal macroalgae under severe salt stress.

Pyropia yezoensis (Bangiales, Rhodophyta) is a representative species of high-intertidal macroalgae, whose blades can tolerate extreme stresses, such as salt stress and desiccation. In this study, the photosystem (PS) responses of P. yezoensis blades under salt stress were studied. Our results showed that when the effective photochemical quantum yield of PS (Y) II decreased to almost zero under high salt stress, YI still had a relatively high activity rate. PSII was therefore more sensitive to salt stress than PSI. Furthermore, in the presence of 3-(3', 4'-dichlorophenyl)-1,1-dimethylurea (DCMU), YI rose as salinity increased. The YI values for DCMU-treated thalli decreased in the presence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PDH) inhibitor (glucosamine, Glucm). The YI values were ∼0.09 in the presence of methyl viologen (MV) and almost zero in the presence of dibromothymoquinone (DBMIB). These results demonstrated that under severe salt stress (120‰ salinity) PSI activity was driven from a source other than PSII, and that stromal reductants probably supported the operation of PSI. Under salt stress, the starch content decreased and soluble sugar levels increased. The G6PDH and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) activities increased, but cytosolic glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) activity decreased. Furthermore, the NADPH content increased, but NADH decreased, which suggested that soluble sugar entered the oxidative pentose phosphate pathway (OPPP). All these results suggested that NADPH from OPPP increases the cyclic electron flow around PSI in high-intertidal macroalgae under severe salt stress.

Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long-Term Response of Arabidopsis to Light Quality Shifts.

Natural illumination conditions are highly variable and because of their sessile life style, plants are forced to acclimate to them at the cellular and molecular level. Changes in light intensity or quality induce changes in the reduction/oxidation (redox) state of the photosynthetic electron chain that acts as a trigger for compensatory acclimation responses comprising functional and structural adjustments of photosynthesis and metabolism. Such responses include redox-controlled changes in plant gene expression in the nucleus and organelles. Here we describe a strategy for the identification of early redox-regulated genes (ERGs) in the nucleus of the model organism Arabidopsis thaliana that respond significantly 30 or 60 min after the generation of a reduction signal in the photosynthetic electron transport chain. By comparing the response of wild-type plants with that of the acclimation mutant stn7, we could specifically identify ERGs. The results reveal a significant impact of chloroplast redox signals on distinct nuclear gene groups including genes for the mitochondrial electron transport chain, tetrapyrrole biosynthesis, carbohydrate metabolism, and signaling lipid synthesis. These expression profiles are clearly different from those observed in response to the reduction of photosynthetic electron transport by high light treatments. Thus, the ERGs identified are unique to redox imbalances in photosynthetic electron transport and were then used for analyzing potential redox-responsive cis-elements, trans-factors, and chromosomal regulatory hot spots. The data identify a novel redox-responsive element and indicate extensive redox control at transcriptional and chromosomal levels that point to an unprecedented impact of redox signals on epigenetic processes.

Respiration accumulates Calvin cycle intermediates for the rapid start of photosynthesis in Synechocystis sp. PCC 6803.

We tested the hypothesis that inducing photosynthesis in cyanobacteria requires respiration. A mutant deficient in glycogen phosphorylase (∆GlgP) was prepared in Synechocystis sp. PCC 6803 to suppress respiration. The accumulated glycogen in ΔGlgP was 250-450% of that accumulated in wild type (WT). The rate of dark respiration in ΔGlgP was 25% of that in WT. In the dark, P700(+) reduction was suppressed in ΔGlgP, and the rate corresponded to that in (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)-treated WT, supporting a lower respiration rate in ∆GlgP. Photosynthetic O2-evolution rate reached a steady-state value much slower in ∆GlgP than in WT. This retardation was solved by addition of d-glucose. Furthermore, we found that the contents of Calvin cycle intermediates in ∆GlgP were lower than those in WT under dark conditions. These observations indicated that respiration provided the carbon source for regeneration of ribulose 1,5-bisphosphate in order to drive the rapid start of photosynthesis.

Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.

The thiol/disulfide redox network mediated by the thioredoxin (Trx) system in chloroplasts ensures light-responsive control of diverse crucial functions. Despite the suggested importance of this system, the working dynamics against changing light environments remains largely unknown. Thus, we directly assessed the in vivo redox behavior of chloroplast Trx-targeted thiol enzymes in Arabidopsis thaliana. In a time-course analysis throughout a day period that was artificially mimicked to natural light conditions, thiol enzymes showed a light-dependent shift in redox state, but the patterns were distinct among thiol enzymes. Notably, the ATP synthase CF(1-γ) subunit was rapidly reduced even under low-light conditions, whereas the stromal thiol enzymes fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and NADP-malate dehydrogenase were gradually reduced/re-oxidized along with the increase/decrease in light intensity. Photo-reduction of thiol enzymes was suppressed by the impairment of photosynthetic linear electron transport using DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but sensitivity to the impairment was uneven between CF(1-γ) and other stromal thiol enzymes. These different dependencies of photo-reduction on electron transport, rather than the redox state of Trx and the circadian clock, could readily explain the distinct diurnal redox behaviors of thiol enzymes. In addition, our results indicate that the cyclic electron transport around PSI is also involved in redox regulation of some thiol enzymes. Based on these findings, we propose an in vivo working model of the redox regulation system in chloroplasts.

A chloroplast retrograde signal regulates nuclear alternative splicing.

Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action, we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.

Subset of heat-shock transcription factors required for the early response of Arabidopsis to excess light.

Sunlight provides energy for photosynthesis and is essential for nearly all life on earth. However, too much or too little light or rapidly fluctuating light conditions cause stress to plants. Rapid changes in the amount of light are perceived as a change in the reduced/oxidized (redox) state of photosynthetic electron transport components in chloroplasts. However, how this generates a signal that is relayed to changes in nuclear gene expression is not well understood. We modified redox state in the reference plant, Arabidopsis thaliana, using either excess light or low light plus the herbicide DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), a well-known inhibitor of photosynthetic electron transport. Modification of redox state caused a change in expression of a common set of about 750 genes, many of which are known stress-responsive genes. Among the most highly enriched promoter elements in the induced gene set were heat-shock elements (HSEs), known motifs that change gene expression in response to high temperature in many systems. We show that HSEs from the promoter of the ASCORBATE PEROXIDASE 2 (APX2) gene were necessary and sufficient for APX2 expression in conditions of excess light, or under low light plus the herbicide. We tested APX2 expression phenotypes in overexpression and loss-of-function mutants of 15 Arabidopsis A-type heat-shock transcription factors (HSFs), and identified HSFA1D, HSFA2, and HSFA3 as key factors regulating APX2 expression in diverse stress conditions. Excess light regulates both the subcellular location of HSFA1D and its biochemical properties, making it a key early component of the excess light stress network of plants.

Re-evaluation of the side effects of cytochrome b6f inhibitor dibromothymoquinone on photosystem II excitation and electron transfer.

Dibromothymoquinone (DBMIB) has been used as a specific inhibitor of plastoquinol oxidation at the Q0 binding site of the cytochrome b6f complex for 40 years. It is thought to suppress electron transfer between photosystem (PS) II and I, as well as cyclic electron transfer around PSI. However, DBMIB has also been reported to act as a quencher of chlorophyll excited states. In this study, we have re-evaluated the effects of DBMIB on chlorophyll excited states and PSII photochemistry. The results show that DBMIB significantly quenches the chlorophyll excited states of PSII antenna even at low concentration (from 0.1 µM), lowering the effective excitation rate of the actinic light. It also acts as a potent PSII electron acceptor retarding the reduction of the plastoquinone pool with almost maximal potency at 2 µM. Altogether, these results suggest that experiments using DBMIB can easily be misinterpreted and stress on the importance of taking into account all these side effects that occur in the same range of DBMIB concentration used for inhibition of plastoquinol oxidation (1 µM).

The enhancement of cyclic electron flow around photosystem I improves the recovery of severely desiccated Porphyra yezoensis (Bangiales, Rhodophyta).

Porphyra yezoensis, a representative species of intertidal macro-algae, is able to withstand periodic desiccation at low tide but is submerged in seawater at high tide. In this study, changes in photosynthetic electron flow in P. yezoensis during desiccation and re-hydration were investigated. The results suggested that the cyclic electron flow around photosystem I (PSI) increased significantly during desiccation, continued to operate at times of severe desiccation, and showed greater tolerance to desiccation than the electron flow around PSII. In addition, PSI activity in desiccated blades recovered faster than PSII activity during re-hydration. Even though linear electron flow was suppressed by DCMU [3-(3',4'-dichlorophenyl)-1,1-dimethylurea], cyclic electron flow could still be restored. This process was insensitive to antimycin A and could be suppressed by dibromothymoquinone (DBMIB). The prolonged dark treatment of blades reduced the speed in which the cyclic electron flow around PSI recovered, suggesting that stromal reductants, including NAD(P)H, played an important role in the donation of electrons to PSI and were the main cause of the rapid recovery of cyclic electron flow in desiccated blades during re-hydration. These results suggested that cyclic electron flow in P. yezoensis played a significant physiological role during desiccation and re-hydration and may be one of the most important factors allowing P. yezoensis blades to adapt to intertidal environments.

Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803.

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30 min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme.

A unique regulation of the expression of the psbA, psbD, and psbE genes, encoding the 01, 02 and cytochrome b559 subunits of the Photosystem II complex in the chlorophyll d containing cyanobacterium Acaryochloris marina.

Photosynthetic electron transport, chromatic photoacclirnation and expression of the genes encoding the 01, 02, and cytochrome b559 subunits of the Photosystem II complex were studied in the chlorophyll d containing cyanobacterium Acaryochloris marina MBIC11017 under various environmental conditions. During oxygen deprivation and inhibition of photosynthetic electron transport by dibromothymoquinone the psbA1 gene encoding a 01' isoform was induced. All of the three psbA and one of the three psbD (psbD2) genes, encoding two different isoforms of the 01 and the abundant isoform of the 02 proteins, respectively were induced under exposure to UV-B radiation and high intensity visible light. Under far red light the amount of Photosystem II complexes increased, and expression of the psbE2 gene encoding the alpha-subunit of cytochrome b559 was enhanced. However, the psbF and psbE1 genes encoding the beta- and another isoform of alpha-cytochrome b559, respectively remained lowly expressed under all conditions. Far red light also induced the psbD3 gene encoding a 02' isoform whose primary structure is different from the abundant 02 isoform. psbD3 was also induced under low intensity visible light, when chromatic photoacclimation was indicated by a red-shifted absorption of chlorophyll d. Our results show that differential expression of multigene families encoding different isoforms of 01 and 02 plays an important role in the acclimation of A. marina to contrasting environmental conditions. Moreover, the disproportionate quantity of transcripts of the alpha and beta subunits of cytochrome b559 implies the existence of an alpha-alpha homodimer organization of cytochrome b559 in Photosystem II complexes.

Modulation of gene expression of carotene biosynthesis-related protein by photosynthetic electron transport for the acclimation of intertidal macroalga Ulva fasciata to hypersalinity and excess light.

A gene (UfCBR) encoding carotene biosynthesis-related (CBR) protein that potentially functions for the dissipation of excessive energy has been cloned from the intertidal green macroalga Ulva fasciata Delile. Hypersalinity and high light ≥300 µmol m(-2) s(-1) increased both UfCBR mRNA level and non-photochemical quenching (NPQ). The increase of UfCBR mRNA level and NPQ by high light was inhibited by treatment of photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but not by stigmatellin, an inhibitor that blocks electron transfer from quinol oxidase to iron-sulfur protein in cytochrome b(6) f complex. Treatment of dimethylthiourea, an H(2) O(2) scavenger, under 1200 µmol m(-2) s(-1) condition inhibited H(2) O(2) accumulation but did not affect UfCBR mRNA level, while treatment of H(2) O(2) in 150 µmol m(-2) s(-1) condition decreased UfCBR mRNA level. Thus, an reactive oxygen species-independent redox control via a more reduced state downstream the cytochrome b(6) f complex is involved in high light up-regulation of UfCBR expression in U. fasciata. The expression of UfCBR in U. fasciata against oxidative stress occurring in high light or high salinity in relation to NPQ is discussed.

Moderate heat stress induces state transitions in Arabidopsis thaliana.

The effect of temperature on the photosynthetic machinery is crucial for the fundamental understanding of plant physiology and the bioengineering of heat-tolerant varieties. In our study, Arabidopsis thaliana was exposed to mild (40°C), short-term heat stress in the dark to evaluate the heat-triggered phosphorylation and migration of light harvesting complex (LHC) II in both wild-type (wt) and mutant lacking STN7 kinase. The 77K emission spectra revealed an increase in PSI relative to PSII emission similar to increases observed in light-induced state I to state II transitions in wt but not in stn7 mutant. Immunoblotting results indicated that the major LHCII was phosphorylated at threonine sites under heat stress in wt plants but not in the mutant. These results support the proposition that mild heat stress triggers state transitions in the dark similar to light-induced state transitions, which involve phosphorylation of LHCII by STN7 kinase. Pre-treatment of Arabidopsis leaves with inhibitor DBMIB, altered the extent of LHCII phosphorylation and PSI fluorescence emission suggests that activation of STN7 kinase may be dependent on Cyt b(6)/f under elevated temperatures in dark. Furthermore, fast Chl a transient of temperature-exposed leaves of wt showed a decrease in the F(v)/F(m) ratio due to both an increase in F(o) and a decrease in F(m). In summary, our findings indicate that a mild heat treatment (40°C) induces state transitions in the dark resulting in the migration of phosphorylated LHCII from the grana to the stroma region.

Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803.

To identify optimal conditions for renewable hydrogen production from sunlight and water we have studied transcriptional changes of the hoxEFUYH genes encoding the bidirectional hydrogenase in the cyanobacterium Synechocystis PCC 6803. Transcript abundance detection by real time polymerase chain reaction was supplemented with variable chlorophyll fluorescence measurements to monitor redox changes of the photosynthetic electron transport chain. Our main observations are: (i) abundance of hox transcripts decreases in the dark and recovers in the light. (ii) Inhibition of the Calvin cycle by glycolaldehyde suppresses hox gene transcription, which can be restored by the addition of electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea and dibromothymoquinone. (iii) The transcript levels of all hox genes are increased in anoxia, with additional induction of hoxEF in darkness or in the presence of dibromothymoquinone. (iv) Plastoquinone pool redox changes are not correlated with hox transcript level changes. (v) Changes in the transcript levels of lexA and sll0359 genes, encoding putative regulators of hox genes, are only partly correlated with transcript changes of hox genes under different conditions. Our data demonstrate a previously unrecognized light- and oxygen-dependent regulation of hox gene transcription in Synechocystis PCC 6803, which is related to photosynthetic electron transport and to unidentified oxygen and redox sensors. We also conclude that neither LexA nor Sll0359 are likely to be exclusive regulators of hox gene transcription.

New feature of delayed luminescence: preillumination-induced concavity and convexity in delayed luminescence decay curve in the green alga Pseudokirchneriella subcapitata.

A new method for measuring delayed luminescence (delayed fluorescence) employs preillumination and a dark waiting period before normal excitation. The preillumination results in a concavity and a convexity in the decay curve in delayed luminescence in the green alga Pseudokirchneriella subcapitata. Formation of the concavity and the convexity is not affected by excitation wavelength (680 nm and 700 nm). However, the concavity and the convexity progressively decrease as the dark waiting period increases after preillumination. The formation of the concavity and the convexity was inhibited by exposure to the electron transport inhibitors DBMIB (644 microg/L, 2.0 microM) and Antimycin A (55 microg/L, 0.1 microM). Samples exposed to DBMIB exhibited noticeable reduction in the concavity, whereas samples exposed to Antimycin A exhibited pronounced reduction in the convexity. There is a possibility that the formation and disappearance of the concavity and the convexity are due to the reduction-oxidation state of the plastoquinone pool and the cyclic electron transport. We expect this method being useful in evaluating the effects of chemicals (particularly toxic chemicals) on photosynthetic reactions, and the method may also help to resolve questions regarding the source of long delayed luminescence.

Quinone sensing by the circadian input kinase of the cyanobacterial circadian clock.

Circadian rhythms are endogenous cellular programs that time metabolic and behavioral events to occur at optimal times in the daily cycle. Light and dark cycles synchronize the endogenous clock with the external environment through a process called entrainment. Previously, we identified the bacteriophytochrome-like circadian input kinase CikA as a key factor for entraining the clock in the cyanobacterium Synechococcus elongatus PCC 7942. Here, we present evidence that CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms, varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC during resetting of circadian phase by a dark pulse. The abundance of CikA varies inversely with light intensity, and its stability decreases in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The pseudo-receiver domain of CikA is crucial for sensitivity to DBMIB, and it binds the quinone directly, a demonstration of a previously unrecognized ligand-binding role for the receiver fold. Our results suggest that resetting the clock in S. elongatus is metabolism-dependent and that it is accomplished through the interaction of the circadian oscillator with CikA.

Influence of state-2 transition on the proton motive force across the thylakoid membrane in spinach chloroplasts.

The proton motive force (pmf) across the thylakoid membrane is composed of the proton gradient and the membrane potential, which promotes millisecond-delayed light emission (ms-DLE). In this study, the time courses of LHC II phosphorylation and ms-DLE were investigated in spinach chloroplast during State-2 transition. Red light illumination resulted in an exponential rise in LHC II phosphorylation and a biphasic time course of ms-DLE. The phospho-LHC II appeared upon approximately 1 min illumination. The phosphorylation level increased exponentially when illumination was elongated to 20 min. The t((1/2) )of saturated LHC II phosphorylation was estimated 4-5 min under present illumination. During this process, the amplitudes of ms-DLE increased transiently to a maximal amplitude within 0.5 min illumination, and the reached maximum of the fast phase of ms-DLE was approximately 140% of the dark control. Then, ms-DLE decreased from the maximum. After > or =3 min illumination, ms-DLE decreased to a lower level than the dark control. In the presence of uncouplers and inhibitors, the transient increase in the biphasic time course of ms-DLE was removed by nigericin and DCMU, and the sequential decrease was delayed by DCCD. The time course was not affected significantly by valinomycin and DBMIB. Moreover, the level of LHC II phosphorylation was enhanced by nigericin, valinomycin and DCCD, and was inhibited completely by DCMU and partially by DBMIB. Taken together, we proposed that the PS II photochemical activity remained unaffected even with a higher level of LHC II phosphorylation, which was reflected by the effect of DCCD on the time course of ms-DLE. Probably, the evidence of LHC II phosphorylation is the rearrangement of LHC II-PS II complex and the thylakoid, a feedback to light-exposure, rather than the redistribution of excitation energy from PS II to PS I.

Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light.

In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15-20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells.

Photosynthetic electron transport controls expression of the high light inducible gene in the cyanobacterium Synechococcus elongatus strain PCC 7942.

The hliA gene of Synechococcus, encoding a photoprotective high light inducible polypeptide, is up-regulated by high light (HL) or low intensity blue/UV-A light (BL). hliA expression was found to be up-regulated by KCN in low light (LL) (but not in the dark), and up-regulation in HL, BL, and LL (with KCN) was inhibited by 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone. A working hypothesis is proposed whereby up-regulation is in response to the reduced state of cytochrome b(6)f or a carrier beyond in photosynthesis. Modest up-regulation occurs in LL by treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but this is related to effects on hliA mRNA stability rather than on transcription.

The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex.

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.