Wavelength-dependent quantum yield of ATP synthesis and NADP reduction in normal and dichlorodimethylphenylurea-poisoned chloroplast

At short wavelengths (525–690 mμ) the direct measurement of the quantum yield of the photoreduction of NADP⁺ in normal O₂-evolving spinach chloroplasts is constant ( approx. 0.3 equiv/hv). At short wavelengths (<690 mμ) the quantum yield for NADP⁺ reduction in 3(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts supplied with the ascorbate-2,6-dichlorophenolindophenol couple (donor system) is approx. half as efficient as the normal system. At long wavelengths the quantum yield of NADP⁺ reduction in the donor system increases by a factor of 2 ( approx. 0.3 equiv/hv) when compared with the corresponding yield for the donor system at short wavelengths ( approx. 0.15 equiv/hv).

Between 525 and 690 mμ, the phosphorylation yield for the normal system is constant ( = 0.15 ATP/hv), maintaining a constant P/2e ratio of unity. The P/2e ratios indicate a tight coupling between phosphorylation and electron transport encompassing a single phosphorylation site for the transfer of two electrons.

Between 525 and 680 mμ, the phosphorylation yield for the donor system is constant ( approx. 0.04 ATP/hv), maintaining a P/2e ratio of approx. 0.5. At longer wavelengths (>690 mμ) the phosphorylation yield of the donor system rises ( approx. 0.07–0.08 ATP/hv) concomitant with the rise in the yield of electron flow.

These experiments suggest the possibility that two types of phosphorylation processes operate in chloroplasts, (1) a short-wavelength process coupled to the normal O₂-evolving activity, and (2) a long-wavelength process coupled to the electron-donor activity of reagents such as DCIP.     

Acid phosphatases bind to the main high density lipoprotein apolipoprotein A-I

Light-dependent Ca²⁺ efflux via the Ca²⁺/H⁺ antiport in the photosynthetic purple sulfur bacterium Chromatium vinosum was inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca²⁺ uptake by C. vinosum in the dark nor any effect on the light-dependent efflux of either Na⁺ or Tl⁺ catalyzed, respectively, by the C. vinosum Na⁺/H⁺ or K⁺/H⁺ antiports. Ruthenium red and LaCl₃, neither of which inhibited light-dependent Ca²⁺ efflux in C. vinosum, markedly inhibited Ca²⁺ uptake in the dark by C. vinosum cells. Ruthenium red had no effect on the uptake of either Na⁺or the K⁺ analog T1⁺ by C. vinosum cells in the dark. These results have been interpreted in terms of two separate Ca²⁺ transport systems in C. vinosum: (i) a phenothiazine-sensitive and ruthenium red, La³⁺-insensitive Ca²⁺/H⁺ antiport responsible for Ca²⁺ efflux in the light; and (ii) a ruthenium red and La³⁺-sensitive but phenothiazine-insensitive Ca²⁺ uptake system.     

Wavelength-dependent quantum yields of chloroplast phosphorylation catalyzed by phenazine methosulphate

Studies of phenazine methosulphate (PMS)-catalyzed O₂ exchange and phosphorylation in spinach chloroplasts reveal that at short wavelengths (<680 mμ) PMS acts at a reduced quantum efficiency as an oxidant for O₂ evolution with concomitant phosphorylation. The quantum yield profile of phosphorylation obtained with PMS differs markedly from the yield profile of phosphorylation for normal chloroplasts with NADP⁺ or ferricyanide as oxidant. Between 525 and 680 mμ the quantum yield of phosphorylation (ATP) catalyzed by PMS is less than half the constant maximum ATP of the normal system. The maximum ATP value for the normal system is approx. 0.16 ATP/hv. With the PMS system a peak in the yield at 690 mμ is obtained approaching the ATP value of the normal system. This yield falls again at longer wavelengths (>700 mμ).

The addition of ascorbate to the PMS phosphorylating system decreases the short-wavelength (<680 mμ) phosophorylation activity but increases the long-wavelength (>690 mμ) phosphorylation activity. The quantum yield profile of this system, showing a long-wavelength rise in phosphorylation efficiency is obtained with or without the addition of 3(3,4-dichlorophenyl)-1, 1-dimethylurea.

These experiments have been interpreted as indicating two separate electrontransfer processes catalyzed by PMS, one in which PMS acts at a reduced efficiency as a Hill oxidant, and the other in which PMS acts as an electron donor and acceptor in a cyclic fashion in sensitizing and essentially long-wavelength phosphorylation process.     

Effect of dicyclohexylcarbodiimide on the proton conductance of thylakoid membranes in intact chloroplast

The effects of dicyclohexylcarbodiimide, a potent inhibitor of chloroplast ATPase, on the light-induced electric potential changes in intact chloroplasts of Peperomia metallica and of a hornwort Anthoceros sp. were investigated by means of glass microcapillary electrodes. The characteristics of potential changes induced by flashes or continuous light in chloroplasts of both species are similar except for the phase of potential rise in continuous light, which is clearly biphasic in Anthoceros chloroplasts. Dicyclohexylcarbodiimide at concentration 5 · 10⁻⁵ M completely abolishes the transient potential undershoot in the light-off reaction but has little effect on the peak value of the photoelectric response. The membrane conductance in the light and in the dark was tested by measuring the decay kinetics of flash-generated potential in dark-adapted and preilluminated chloroplasts. In the absence of dicyclohexylcarbodiimide, preillumination causes a significant acceleration of the potential decay. The light-induced changes in the decay kinetics of flash-induced responses were abolished in the presence of dicyclohexylcarbodiimide, whereas the rate of potential decay in dark-adapted chloroplasts was not altered by dicyclohexylcarbodiimide. The results are consistent with the notion that dicyclohexylcarbodiimide diminishes H⁺ conductance of energized thylakoid membranes by interacting with the H⁺ channel of ATPase. The occurrence of a lag (approx. 300 ms) on the plot of potential undershoot (diffusion potential) versus illumination time might suggest the increase in H⁺ permeability coefficient of thylakoid membrane during illumination.     

Light-induced absorbance changes in chloroplasts mediated by Photosystem I and Photosystem II at low temperature

Stable light-induced absorbance changes in chloroplasts at −196 °C were measured across the visible spectrum from 370 to 730 nm in an effort to find previously undiscovered absorbance changes that could be related to the primary photochemical activity of Photosystem I or Photosystem II. A Photosystem I mediated absorbance increase of a band at 690 nm and a Photosystem II mediated absorbance increase of a band at 683 nm were found. The 690-nm change accompanied the oxidation of P₇₀₀ and the 683-nm increase accompanied the reduction of C-550. No Soret band was detected for P₇₀₀.

A specific effort was made to measure the difference spectrum for the photooxidation of P₆₈₀ under conditions (chloroplasts frozen to −196 °C in the presence of ferricyanide) where a stable, Photosystem II mediated EPR signal, attributed to P₆₈₀⁺ has been reported. The difference spectra, however, did not show that P₆₈₀⁺ was stable at −196 °C under any conditions tested. Absorbance measurements induced by saturating flashes at −196 °C (in the presence or absence of ferricyanide) indicated that all of the P₆₈₀⁺ formed by the flash was reduced in the dark either by a secondary electron donor or by a backreaction with the primary electron acceptor. We conclude that P₆₈₀⁺ is not stable in the dark at −196 °C: if the normal secondary donor at −196 °C is oxidized by ferricyanide prior to freezing, P₆₈₀⁺ will oxidize other substances.     

Responses of apical ion fluxes to NaCl stress in Elaeagnus angustifolia seedlings

Aims Elaeagnus angustifolia is one of the most salt-tolerant species. The objective of this study was to understand the mechanisms of ion transporation in E. angustifolia exposed to different salt concentrations through manipulations of K⁺/Na⁺ homeostasis.
Methods Seedlings of two variants of the species, Yinchuan provenance (YC, salt-sensitive type) and the Alaer provenance (ALE, salt-tolerant type), were treated with three different NaCl application modes, and the ion fluxes in the apical regions were measured using non-invasive micro-test technology (NMT). In mode 1, Na⁺ and K⁺ fluxes were measured after 150 mmol·L^-1 NaCl stress lasted for 24 h. In mode 2, K⁺ and H⁺ fluxes were quantified with a transient stimulation of NaCl solution. In mode 3, Amiloride (Na⁺/H⁺ antiporters inhibitor) and tetraethylammonium (TEA, K⁺ channel inhibitor) was used to treat apical regions of E. angustifolia seedlings after NaCl stress for 24 h, respectively.
Important findings Under NaCl stress for 24 h, net effluxes of Na⁺ and K⁺ were increased significantly. The net Na⁺ effluxes of YC provenance seedlings (720 pmol·cm^-2•s^-1) were lower than that of ALE provenance (912 pmol·cm^-2·s^-1), but the net K⁺ efflux was higher in YC provenance. Under the instantaneous NaCl stimulation, net K⁺ efflux was remarkably increased, with the net K⁺ efflux of YC provenance always higher than that of ALE provenance. Interestingly, H⁺ at the apical regions was found from influx to efflux, with the net H⁺ efflux of ALE provenance greater than that of the YC provenance. Under the NaCl and NaCl + Amiloride treatment, the net Na⁺ efflux of ALE provenance seedlings was higher than that of YC provenance, while the net K⁺ efflux was less in ALE provenance seedlings. On the other hand, the differences in net Na⁺ and K⁺ effluxes were insignificant between the two provenances under the control group and NaCl + TEA treatment. In conclusion, NaCl stress caused Na⁺ accumulation and K⁺ outflows of E. angustifolia seedlings; The E. angustifolia seedlings utilize Na⁺/H⁺ antiporters to reduce Na⁺ accumulation by excretion; and the maintenance of K⁺/Na⁺ homeostasis in salt-tolerant E. angustifolia provenance seedlings roots accounted for a greater Na⁺ extrusion and a lower K⁺ efflux under NaCl stress. Results from this study provide a theoretical basis for further exploring salt-tolerant E. angustifolia germplasm resource.     

Kinetics of ATP formation and proton efflux by acid-base transition in chloroplasts

The relationship between the kinetics of ATP formation and proton release in chloroplast suspensions by acid-base transition were studied by means of a stopped-flow spectrophotometer. The time course of ATP synthesis shows two-phase kinetics, fast and slow, corresponding to the two-phase efflux of protons from the chloroplasts. Under certain conditions of the experiments, about 50% of the H⁺ gradient is constantly utilized for ATP formation in both phases. However, the ratio of ATP formed to the amount of protons leaked out, changes depending on the rate constants of proton efflux.     

Pigment systems and electron transport in chloroplasts I. Quantum requirements for the two light reactions in spinach chloroplasts

From studies of electron-transport reactions of isolated spinach chloroplasts, we observe the following quantum requirements: (A) For the photoreduction of NADP⁺, measured both aerobically and anaerobically, in a 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) poisoned system with ascorbate and reduced 2,6-dichlorophenolindophenol (DCIPH₂) present as electron donors, the quantum requirements are 1.0 ± 0.05 at wavelengths longer than 700 nm of actinic light, and 1.5–2.5 for wavelengths between 620 and 680 nm. (B) For the photoreduction of 2,6-dichlorophenolindophenol (DCIP) with water as the electron donor, the quantum requirements are 1.0 ± 0.05 in the range 630–660 nm. (C) For the photoreduction of NADP⁺ with water as the electron donor, the quantum requirements are 2.0 ± 0.1 in the wavelength range 640–678 nm of actinic light, increasing to 6 or greater at wavelengths beyond 700 nm. These results are shown to be inconsistent with the “separate package” model for the two pigment systems in higher plant photosynthetic electron transport. The evidence is most easily interpreted using a “controlled spillover” model, in which the transfer of electronic excitation energy from one pigment system to the other is under the control of incompletely identified factors in the reaction mixture.

At moderate light intensities the steady state rate of the [ascorbate + DCIPH₂ → NADP⁺] reaction (A) in the presence of DCMU and added ferredoxin can be increased more than 3 times when saturating amounts of plastocyanin and ferredoxin-NADP reductase are added to the chloroplasts. Similarly, the steady-state rate of the [H₂O → DCIP] Hill reaction (B) is increased about 3-fold by added MgCl₂ and plastocyanin, but added ferredoxin or ferredoxin-NADP reductase have no effect on this reaction. Plastocyanin appears to be the electron transport component which couples to DCIP, either in the oxidized or in the reduced form, in the reaction media. The steady-state rate of the [H₂O → NADP⁺] reaction (C) with saturating amounts of ferredoxin can be further increased more than 3-fold when MgCl₂, plastocyanin and ferredoxin-NADP reductase are added.     

Probing fluorescence induction in chloroplasts on a nanosecond time scale utilizing picosecond laser pulse pairs

The fluorescence induction and other fluorescence properties of spinach chloroplasts at room temperature were probed utilizing two 30-ps wide laser pulses (530 nm) spaced Δt (s) apart in time (Δt = 5–110 ns). The energy of the first pulse (P₁) was varied (10¹²–10¹⁶ photons · cm⁻²), while the energy of the second (probe) pulse (P₂) was held constant (5 · 10¹³ photons · cm⁻²). A gated (10 ns) optical multichannel analyzer-spectrograph system allowed for the detection of the fluorescence generated either by P₁ alone, or by P₂ alone (preceded by P₁). The dominant effect observed for the fluorescence yield generated by P₁ alone is the usual singlet-singlet exciton annihilation which gives rise to a decrease in the yield at high energies. However, when the fluorescence yield of dark-adapted chloroplasts is measured utilizing P₂ (preceded by pulse P₁) an increase in this yield is observed. The magnitude of this increase depends on Δt, and is characterized by a time constant of 28 ± 4 ns. This rise in the fluorescence yield is attributed to a reduction of the oxidized (by P₁) reaction center P-680⁺ by a primary donor. At high pulse energies (P₁ = 4 · 10¹⁴ photons · cm⁻²) the magnitude of this fluorescence induction is diminished by another quenching effect which is attributed to triplet excited states generated by intense P₁ pulses. Assuming that the P₁ pulse energy dependence of the fluorescence yield rise reflects the closing of the reaction centers, it is estimated that about 3–4 photon hits per reaction center are required to close completely the reaction centers, and that there are 185–210 chlorophyll molecules per Photosystem II reaction center.     

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.     

A new macrocyclic tetraamine, 2,4-dinitrophenylcyclen (= 1-(2,4-dinitrophenyl)-1,4,7,10-tetraazacyclododecane):synthesis, cation reporter properties, and recognition of 1-methylthymine by its zinc(II) complex

A new functional macrocyclic ligand, 2,4-dinitrophenylcyclen (= 1-(2,4-dinitrophenyl)-1,4,7,10-tetraazacyclododecane), has been synthesized and isolated as its trihydrochloric acid salt (L·3HCl). The protonation constants (log K_n) for three secondary nitrogens of L were determined by potentiometric pH titration to be 10.10, 7.33 and <2 with I = 0.10 (NaNO₃) at 25°C. The 2,4-dinitrophenylaniline chromophore was proven to be a good reporter signaling proton- and metal-binding events in the macrocyclic cavity. The UV absorption band (λ_max 370 nm, 8200) of the 2,4-dinitrophenylaniline moiety at pH ≥ 9 becomes quenched as pH is lowered (to pH 3.1, where the major species is L·2H⁺), due to the strong protonation effect extended to the aniline moiety within the macrocyclic cavity. This is in sharp contrast to the pH-independent UV absorption (λ_max 390 nm, 14 000) of a reference compound, N,N-diethyl-2,4-dinitroaniline. The UV absorption band of L is shifted to lower wavelengths with Zn²⁺ (λ_max 320 nm), Cd²⁺ (λ_max 316 nm) and Pb²⁺ (λ_max 317 nm), while it almost disappears with Cu²⁺ and Ni²⁺. The 1:1 Zn²⁺ and Cu²⁺ complexes with L were isolated and characterized. The Zn²⁺ complex recognizes 1-methylthymine anion (MT⁻) in aqueous solution at physiological pH to yield a stable ternary complex ZnL-MT⁻. The X-ray crystal structure of ZnL-MT⁻ showed that Zn²⁺ is four-coordinate with three secondary nitrogens of L and the deprotonated imide anion that is cofacial to the 2,4-dinitrophenyl ring.     

Cyanogen-induced gamma-glutamyl to imidazole cross-link in carbonic anhydrase. A unique mode of inhibition

The release of protons from intact cells of Rhodopseudomonas capsulata after either 4μs flashes or during brief periods of continuous illumination has been measured with the indicator, cresol red. The half-time for H⁺-release after a flash was 35 ms and the extent, 1H⁺ per 134 bacteriochlorophyll. Myxothiazol completely inhibited the flash-induced H⁺-release and antimycin A reduced it by 37%. The proton-releasing reaction is discussed with reference to the protonmotive Q-cycle. During continuous illumination the rapid phase of H⁺ release is followed by a lag and then by another period of acidification, suggesting that other protolytic reactions may be in operation.     

Light-induced proton slip and proton leak at the thylakoid membrane

A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H⁺/e⁻-ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF₁-γ of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF₁-γ was only 36%, which indicates that in vivo the reduction of CF₁-γ could be limited by fast reoxidation and/or restricted accessibility of CF₁-γ to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.     

The study of the state [P870 B800] in bacterial reaction centers by selective picosecond and low-temperature spectroscopies

The selective picosecond excitation of Rhodopseudomonas sphaeroides (R-26) reaction centers (RCs) at 870 nm induces the formation of the transient state within <1 ps followed by the conversion into the state P^F (P^± Bph^±− during 7 ± 2 ps at both 293 K and 110 K. The transient state including the intense bleaching at 800 nm has been shown not to be due: (a) to photon excitation at 870 nm; (b) the excitation of P⁺; (c) photoselection effects. The transient state is interpreted as the state ¹[P⁺B⁻] in agreement with earlier works. The primary formation of the state ¹P⁺B⁻] and the big effective singlet-triplet splitting in this state correspond to the spectral splitting of the P band at 900 nm in R-26 RCs and at 1000 nm in Rhodopseudomonas viridis RCs found at 4.2 K and attributed to the optical transition to both ¹P and ¹[P⁺B⁻] states.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

Light-induced movement of magnesium ions in intact chloroplasts. Spectroscopic determination with Eriochrome Blue SE

The metallochromic indicator Eriochrome Blue SE was used to measure light-induced internal movement of Mg²⁺ in intact chloroplasts. By dual-wavelength spectroscopy (measuring wavelength 554 nm, reference 592 nm) a light-induced, dark-reversible absorbance increase of Eriochrome Blue in samples of isolated intact chloroplasts was observed. The light/dark difference spectrum of Eriochrome Blue between 550 and 590 nm (reference wavelength 562 nm) indicated that this absorbance increase was caused by an increased concentration of free Mg²⁺ in a neutral or slightly alkaline chloroplast compartment.

The signal was seen only with intact, but not with broken, envelope-free chloroplasts, which had lost most of their divalent cations. This is interpreted to show that the indicator responds to an increase of Mg²⁺ concentration in the chloroplast stroma, which represents an efflux of Mg²⁺ from the intra-thylakoid space caused by light-dependent proton pumping.

As calculated from corrected values of the absorbance increase of Eriochrome Blue, the light-induced internal release of Mg²⁺ was close to 100 nequiv per mg chlorophyll at pH 7.6 and 250 nequiv at pH 7.1. This corresponds to a light-dependent increase in the concentration of free Mg²⁺ in the stroma of about 2 and 5 mM, respectively.     

Rb transport in Leishmania infantum promastigotes under various in vitro culture conditions

The survival of Leishmania, which encounter drastic changes of environment during their life-cycle, requires regulation and control of ionic concentrations within the cell. We analysed the influence of growth stage, ionic composition of the medium, heat and acidic stress on ⁸⁶Rb⁺ influx in L. infantum promastigetes. Proliferating promastigotes exibited faster and higher ⁸⁶Rb⁺ uptake than stationary cells. Cl⁻ anion did not have any effect, but in the presence of physiological concentration of HCO₃⁻, ⁸⁶Rb⁺ uptake was significantly increased. This enhancing effect was only partially inhibited by N,N′-dicyclohexylcarbodiimide (DCCD), a blocker of ion-translocating ATPases. ⁸⁶Rb⁺ influx was abolished by N-ethylmaleimide (NEM), indicating a major contribution of plasma membrane transporters. Heat shock and acidic shock notably decreased ⁸⁶Rb⁺ influx. Our data provide indirect evidence that an energy-dependent system which brings K⁺ in, such as K⁺/H⁺-ATPase evidenced by Jiang et al. (1994), is active in Leishmania in different environments. Mechanism(s) other than ion-translocating ATPase occur, at least in the presence of HCO₃⁻, and their contribution to K⁺ pathways varies in different environmental conditions.     

Nitrate transporter NRT1.1 and anion channel SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity

Ammonium (NH₄⁺) and nitrate (NO₃⁻) are major inorganic nitrogen (N) sources for plants. When serving as the sole or dominant N supply, NH₄⁺ often causes root inhibition and shoot chlorosis in plants, known as ammonium toxicity. NO₃⁻ usually causes no toxicity and can mitigate ammonium toxicity even at low concentrations, referred to as nitrate-dependent alleviation of ammonium toxicity. Our previous studies indicated a NO₃⁻ efflux channel SLAH3 is involved in this process. However, whether additional components contribute to NO₃⁻-mediated NH₄⁺ detoxification is unknown. Previously, mutations in NO₃⁻ transporter NRT1.1 were shown to cause enhanced resistance to high concentrations of NH₄⁺. Whereas, in this study, we found when the high-NH₄⁺ medium was supplemented with low concentrations of NO₃⁻, nrt1.1 mutant plants showed hyper-sensitive phenotype instead. Furthermore, mutation in NRT1.1 caused enhanced medium acidification under high-NH₄⁺/low-NO₃⁻ condition, suggesting NRT1.1 regulates ammonium toxicity by facilitating H⁺ uptake. Moreover, NRT1.1 was shown to interact with SLAH3 to form a transporter-channel complex. Interestingly, SLAH3 appeared to affect NO₃⁻ influx while NRT1.1 influenced NO₃⁻ efflux, suggesting NRT1.1 and SLAH3 regulate each other at protein and/or gene expression levels. Our study thus revealed NRT1.1 and SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity through regulating NO₃⁻ transport and balancing rhizosphere acidification.     

The site of manganese function in photosynthetic electron transport system

The effects of Mn²⁺ on aerobic photobleaching of carotenoids, on photoreduction of 2,6-dichlorophenolindophenol (DCIP) and on fluorescence above 600 mμ of spinach chloroplasts washed with 0.8 M Tris-HC1 buffer were investigated. Carotenoids (mostly carotenes, lutein and violaxanthin) in the Tris-washed chloroplasts were irreversibly bleached by illumination with red light, while carotenoids in normal chloroplasts prepared with a low concentration of Tris-HC1 underwent no bleaching upon illumination. The photobleaching of carotenoids observed with Tris-washed chloroplasts was inhibited by Mn²⁺ (MnCl₂ or MnSO₄) as well as by some inhibitors of the Hill reaction such as dichlorophenyl-1,1-dimethylurea (DCMU), methylthio-4,6-bis-isopropylamino-s-triazine and o-phenanthroline or by reducing agents such as ascorbate plus tetramethyl-p-phenylene diamine (TMPD). DCIP photoreduction, which was deactivated by Tris, was reactivated to 50–80% of the rate for normal chloroplasts upon addition of Mn²⁺. The restored photoreduction of DCIP was inhibited by DCMU and carbonylcyanide m-chlorophenylhydrazone (CCCP). The steady-state fluorescence yield of normal chloroplasts measured at room temperature was lowered by Tris treatment, and the decreased yield was restored by adding Mn²⁺ as well as ascorbate plus TMPD. CCCP also lowered the yield; the yield was recovered by adding ascorbate plus TMPD. Determination of manganese in normal and Tris-washed chloroplasts showed that 30% of the manganese in chloroplast was removed with Tris. It was postulated that Mn²⁺ functions in the electron transport on the oxidizing side of Photosystem II at a site between water and an electron carrier (Y). CCCP as well as Tris inhibits the reduction of Y⁺ by Mn²⁺, and carotenoids are oxidized by Y⁺ which is reduced by ascorbate plus TMPD.