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Are NADP-dependent isocitrate dehydrogenases and ferredoxin-dependent glutamate synthase co-regulated by the same photoreceptors?

Activities of NADP-dependent isocitrate dehydrogenases (cytosolic and plastidic isoforms, ICDH1 and ICDH2; EC 1.1.1.42) and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in turions of Spirodela polyrhiza were all stimulated by light. Single or repeated red light (R) pulses induced the activity of the enzymes and this effect was reverted by subsequent far-red light (FR) pulses. The enzymes are, therefore, co-regulated by the low-fluence response of phytochrome. For ICDH, this is reported here for the first time. Neither an effect of the very low-fluence response nor of the FR-mediated high-irradiance response was detectable. Irradiance with continuous R resulted in enhanced enzyme activities and protein levels (Western analysis using polyclonal antibodies against ICDH1 and Fd-GOGAT). These additional effects of continuous R (called a "non-induction effect") could be inhibited for ICDH1 and ICDH2 by the inhibitor of photosynthetic electron transport, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, and are therefore related to the effect of photosynthesis. In contrast, the non-induction effect of Fd-GOGAT was resistant against this inhibitor. Moreover, hourly R pulses did not replace the effect of continuous R. The non-induction effect of light on the activity and protein level of Fd-GOGAT was therefore tentatively classified as an R-mediated high-irradiance response. The activity of Fd-GOGAT but not that of ICDHs was additionally regulated by a specific blue-light receptor. It can be concluded that the levels of ICDHs and Fd-GOGAT were coordinated by light but were not co-regulated by the same photoreceptors. Nitrate is necessary for the light regulation of both enzymes, contributing to the coordinated expression of the relevant genes.

Amino Acid Oxidoreductases↗

Reduction of the thylakoid electron transport chain by stromal reductants--evidence for activation of cyclic electron transport upon dark adaptation or under drought.

The reduction of P700(+), the primary electron donor of photosystem I (PSI), following a saturating flash of white light in the presence of the photosystem II (PSII) inhibitor 3-(3.4-dichlorophenyl)-1,1-dimethylurea (DCMU), was examined in barley plants exposed to a variety of conditions. The decay kinetic fitted to a double exponential decay curve, implying the presence of two distinct pools of PSI. A fast component, with a rate constant for decay of around 0.03-0.04 ms(-1) was observed to be sensitive to the duration of illumination. This rate constant was slower than, but comparable to, that observed in non-inhibited samples (i.e. where linear flow was active). It was substantially faster than values typically reported for experiments where PSII activity is inhibited. The magnitude of this component rose in leaves that were dark-adapted or exposed to drought. This component was assigned to PSI centres involved in cyclic electron transport. The remaining slowly decaying P700(+) population (rate constant of around 0.001-0.002 ms(-1)) was assigned to centres normally involved in linear electron transport (but inhibited here because of the presence of DCMU), or inactivated centres involved in the cyclic pathway. Processes that might regulate the relative flux through cyclic electron transport are discussed.

Adaptation, Physiological↗

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.

Actins↗

Accumulation of heavy metals in epidermal glands of the waterlily (Nymphaeaceae).

This study investigates the anatomical aspects of heavy-metal accumulation in the waterlily (Nymphaea 'Aurora', Nymphaeaceae). Epidermal glands were identified by light microscopy on the abaxial side of the leaf laminae and on the epidermis of the rhizome; glandular trichomes were observed in the petiole epidermis. Glands were not observed in the roots. Accumulation of heavy metals in these glands was monitored using a scanning electron microscope equipped for energy-dispersive spectroscopy. Further experiments showed maximal cadmium and calcium accumulation in the mature leaf lamina in daylight, and this accumulation was inhibited by the herbicide 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. These results suggest that, in Nymphaea, heavy metals are accumulated primarily in association with glands found in plant organs that have direct contact with water or mud. Deposition and storage of heavy metals by these glands may represent a stage in the sequestration and detoxification of the metals. Our results raise the possibility of utilizing waterlilies for the removal of heavy metals from polluted environments.

Cadmium↗

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 and -20 degrees C, revealed an activation energy delta E 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 Fv and quenching of basal, so-called F0 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 qI or photoinhibitory quenching, required protonation but persisted for prolonged times after de-protonation. It appeared to originate in reaction centers.

Bryopsida↗

Ferric reduction by iron-limited Chlamydomonas cells interacts with both photosynthesis and respiration.

Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO2 production without affecting the rate of respiratory O2 consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939-944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO2 fixation at saturating irradiance, and almost 100% inhibition of CO2 fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 microM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO2 compensation point) resulted in a stimulation in the rate of gross photosynthetic O2 evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light.

Animals↗

The Antarctic psychrophile, Chlamydomonas subcaudata, is deficient in state I-state II transitions.

State I-State II transitions were monitored in vivo and in vitro in the Antarctic, psychrophillic, green alga, Chlamydomonas subcaudata, as changes in the low-temperature (77 K) chlorophyll fluorescence emission maxima at 722 nm (F722) relative to 699 nm (F699). As expected, the control mesophillic species, Chlamydomonas reinhardtii, was able to modulate the light energy distribution between photosystem II and photosystem I in response to exposure to four different conditions: (i) dark/anaerobic conditions, (ii) a change in Mg2+ concentration, (iii) red light, and (iv) increased incubation temperature. This was correlated with the ability to phosphorylate both of its major light-harvesting polypeptides. In contrast, exposure of C. subcaudata to the same four conditions induced minimum alterations in the 77 K fluorescence emission spectra, which was correlated with the ability to phosphorylate only one of its major light-harvesting polypeptides. Thus, C. subcaudata appears to be deficient in the ability to undergo a State I-State II transition. Functionally, this is associated with alterations in the apparent redox status of the intersystem electron transport chain and with higher rates of photosystem I cyclic electron transport in the psychrophile than in the mesophile, based on in vivo P700 measurements. Structurally, this deficiency is associated with reduced levels of Psa A/B relative to D1, the absence of specific photosystem I light-harvesting polypeptides [R.M. Morgan et al. (1998) Photosynth Res 56:303-314] and a cytochrome b6/f complex that exhibits a form of cytochrome f that is approximately 7 kDa smaller than that observed in C. reinhardtii. We conclude that the Antarctic psychrophile, C. subcaudata, is an example of a natural variant deficient in State I-State II transitions.

Algal Proteins↗

Potassium drives daily reversible thallus enlargement in the marine red alga Porphyra leucosticta (Rhodophyta).

Growth rate in terms of area expansion per 30 min was measured in the marine red algae Porphyra leucosticta under light/dark cycles of 6:6 h. Thalli grown in artificial seawater (ASW), under controlled ionic concentrations, showed a rapid thallus expansion just after light-on (morning-peak). Dark phase began with a significant thallus contraction (dark-peak) but no growth was observed until the next light phase. The removal of K+ from the medium inhibited the reversible growth peak that this species shows after light-on. On the other hand, the removal of Na+ did not have an apparent effect on growth pattern. Addition of Rb+ to K+-free ASW restored the morning-peak to 60% of its value in ASW, but addition of Li+ failed to restore the morning-peak. Intracellular ion analyses revealed that after light-on, the internal K+ content of the cells of this species increased 4-fold in 90 min, reaching an intracellular concentration of up to 300 micromol K+ per gram fresh weight, and that this value remained fairly constant over the light phase. Addition of 100 mM of tetraethylammonium, a specific K+-channel blocker, inhibited the morning-peak by 40%. The Na+ and Cl- contents in the cells increased rapidly in the first 45 min of the light phase, but then the internal concentrations of both ions decreased to their minimum values in the light phase. The carbonic anhydrase inhibitors acetazolamide and 6-ethoxyzolamide did not affect thallus expansion during the light phase. In contrast, thallus expansion in the light phase was completely inhibited by diethylstilbestrol, an inhibitor of the putative primary pump, and by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthesis, but not by sodium vanadate, a specific inhibitor of the plasma-membrane H+-ATPase. We suggest that the periodic oscillation of the short-term growth rate of P. leucosticta occurs in response to potassium fluxes, which control the osmotic pressure and eventually the relative cell volume. The possible effects of the loosening of the cell wall and the internal K+ concentration on the growth of this species are also discussed.

Acetazolamide↗

Effect of CO2 supply on formation of reactive oxygen species in Arabidopsis thaliana.

Light-induced generation of reactive oxygen species (ROS) in 2-week-old leaves of Arabidopsis thaliana was studied by means of the ROS-sensitive dyes nitroblue tetrazolium (NBT) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Superposition of pictures of chlorophyll fluorescence and DCF fluorescence indicated that the origin of ROS was in the chloroplasts. Experiments were done with zero, 0.1, or 10 mM NaHCO3 in the infiltration medium. Energy quenching in photosystem II was higher under low CO2 concentrations as measured by chlorophyll fluorescence. DCF fluorescence showed that CO2 deficiency led to an increase of ROS generation. In contrast, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced the light-induced increase of DCF fluorescence. This indicates that ROS production does not primarily result from over-reduction of photosystem II as caused by impeding electron flow in the electron transfer chain. More likely, it is an effect of diverting electron flux normally aimed at carboxylation in the Calvin cycle to other sinks more prone to the generation of toxic radicals. There was no significant effect of salicyl hydroxamate (a blocker of the alternative oxidase), showing that the mitochondrial electron transfer chain seems to play a minor role as already indicated by the superposition of chlorophyll and DCF fluorescence.

Arabidopsis↗

Regulation of gamete release in the economic brown seaweed Hizikia fusiforme (Phaeophyta).

Gamete release is an essential event in artificial seeding of the economic brown seaweed, Hizikia fusiforme. Mass egg release occurred in the dark, with few eggs being discharged in the light. Release of eggs was elicited with eight practical salinity units (one PSU identical with 1 g sea salts l(-1)) and was inhibited by salinity levels >32 PSU. Egg release was optimal at 23 degrees C, and was decreased by 72% in agitated seawater compared to unstirred seawater. Inhibitors of photosynthesis and ions channels suppressed egg release, indicating that this process was physiologically associated with photosynthetic activity and ion transport.

Acetazolamide↗

Effects of dark adaptation on light-induced electron transport through photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

Kinetics of the redox reactions in the reaction center (P700) of photosystem I (PSI) of the cyanobacterium Synechocystis sp. PCC 6803 have been studied by EPR spectroscopy. The redox kinetics were recorded based on accumulation of the EPRI signal when the final signal was the sum of individual signals produced in response to illumination of the cells. After prolonged (more than 3 sec) dark intervals between illuminations, the kinetic curve of the EPR signal from P700+ was multiphasic. After a sharp increase in the signal amplitude at the beginning of illumination (phase I), the amplitude rapidly (for 0.1-0.2 sec) decreased (phase II). Then the signal amplitude gradually increased (phase III) until the steady rate of electron transfer was established. With short-term (1 sec) dark intervals between the flashes and also in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the kinetics of the light-induced increase in the EPR signal from P700+ were monophasic. Inhibition with iodoacetamide of electron transport on the acceptor side of PSI under anaerobic conditions or an increase in the amount of respiration substrates on addition of glucose into a suspension of DCMU-treated wild-type cells increased the level of P700 reduction in phase III. The findings suggest that the kinetic curve of the EPR signal from P700+ is determined by both the electron entrance onto P700+ on the donor side of PSI and activity of electron acceptors of PSI.

Chlorophyll↗

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.

Dibromothymoquinone↗