Chromium oxalate: A new spin label broadening agent for use with thylakoids

Potassium tris(oxalato)chromate(III) trihydrate (chromium oxalate) has been shown to be a more useful broadening agent than potassium ferricyanide for the spin label 2,2,6,6-tetramethylpiperidine-N-oxy-4-amine (Tempamine) in thylakoid suspensions. Our data show that chromium oxalate is less permeable than ferricyanide, does not inhibit thylakoid electron transport or photophosphorylation, and is not photoreduced by thylakoids.     

Spin label motion in the internal aqueous compartment of spinach thylakoids.

We have measured the motion of the spin label TEMPAMINE (2,2,6,6-tetramethyl piperidine-N-oxyl-4-amine) in the internal aqueous compartment of spinach thylakoids by using potassium ferricyanide (80 mm) to remove TEMPAMINE signals eminating from the external aqueous regions. We found (1): that ferricyanide does not inhibit phosphorylation or electron transport at the concentrations required for TEMPAMINE broadening, but TEMPAMINE acts as a potent uncoupler of electron transport; (2) that TEMPAMINE does not bind detectably to the thylakoid membrane or thylakoid components during the time course of a typical electron spin resonance experiment, but that some binding does occur over a 48-h period to intact thylakoids; (3) that tightly packed intact thylakoids or thylakoids which have been disrupted in a 20% Triton X-100 do not hinder the motion of TEMPAMINE by more than a factor of 1.9; (4) that TEMPAMINE in the presence of 80 mm potassium ferricyanide gives rise to a signal characteristic of TEMPAMINE tumbling isotropically in an aqueous environment with a bulk viscosity of about 10 cP; and (5) that, although ferricyanide leaks slowly into the thylakoid interior, it does not alter the measurement of TEMPAMINE rotation. We conclude that the thylakoid interior is more viscous than bulk water. This may have functional significance regarding transport of electrons from Photosystem II and I to the ATP synthetase.     

The oxidation of C-550 by exogenously added oxidants in the presence of dichlorophenyldimethylurea: The localization of the primary electron acceptor of Photosystem II in the thylakoid membrane

The oxidation of C-550 by exogenously added oxidants in spinachchloroplasts and digitonin-treated chloroplasts was studiedin the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inan attempt to elucidate localization of the primary electronacceptor of Photosystem II in the thylakoid membrane. C-550was directly oxidized by various oxidants such as ferricyanide,N-methylphenazonium methosulfate (PMS) and quinones with redoxpotentials higher than that of C-550. Among the oxidants used,dibromothymoquinone was the most effective oxidant of C-550,followed by PMS. In spite of its high redox potential, ferricyanidewas rather a poor oxidant. The rates of C-550 oxidation by quinoneswere linearly proportional to the oxidant concentration, whereasthe rates tapered off with increasing concentrations of polaroxidants within the ranges of concentration used. These resultsindicate that C-550 is located inside the thylakoid membraneand is covered by a lipophilic shield. Addition of cations,especially divalent cations, significantly affected C-550 oxidationby ferricyanide or PMS but not by nonpolar oxidants. C-550 oxidationby ferricyanide was accelerated at low pH. Thus the accessibilityof C-550 to polar oxidants appears to be affected by electrostaticinteractions between oxidant ions and the negative charges ofthe thylakoid surface. When electrostatic interaction was minimized,ferricyanide oxidized C-550 as rapidly as several quinones did.This suggests that C-550 is located close to the surface ofthe membrane. The evidence indicates that the rates of C-550oxidation depend not only on the accessibility of C-550 to addedoxidants, but also on the reactivity between them. Reduction of cytochrome f by added reductants shows featuressimilar to those of C-550 oxidation by added oxidants, indicatingthat properties of the shield covering cytochrome f are similarto those of the shield covering C-550. (Received March 8, 1977; )     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Light Activation of Purified Aconitase by Washed Thylakoid Membranes of Pea (Pisum sativum L.)

Purified aconitase, an iron-sulfur protein, from either beef heart mitochondria or pig heart can be activated fully by light when combined with washed thylakoid membranes from pea (Pisum sativum L.) chloroplasts. The light activation of the enzyme does not require any other additive or cofactor and is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,6-dichlorophenol-indophenol, ferricyanide, and methyl viologen, indicating that the photoelectron transport system of the thylakoid membranes, and in particular, photosystem I, is involved in the process of activation. Light activation of the enzyme is also markedly inhibited when the thylakoid membranes are treated with sulfite or arsenite, and abolished totally when the membranes are treated with Zwittergent, suggesting that the light effect mediator involved in the light modulation of chloroplastic enzymes mediates the activation of purified aconitase also.     

Identification of the pheophytin-QA-Fe domain of the reducing side of the photosystem II as the Cu(II)-inhibitory binding site.

Oxygen evolution by photosystem II membranes was inhibited by Cu(II) when 2,6-dichlorobenzoquinone or ferricyanide, but not silicomolybdate, was used as electron acceptor. This indicated that Cu(II) affected the reducing side of the photosystem II. The inhibition curves of Cu(II), o-phenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), were compared; the inhibitory patterns of Cu(II) and o-phenanthroline were very similar and different in turn from that of DCMU. Cu(II) did not eliminate or modify the electron paramagnetic resonance signal at g = 8.1 ascribed to the non-heme iron of the photosystem II reaction center, indicating that the inhibition by Cu(II) was not the result of the replacement of the iron by Cu(II). Controlled trypsin digestion of thylakoid membranes inhibited oxygen evolution using 2,6-dichlorobenzoquinone, but had no effect when using ferricyanide or silicomolybdate. Using ferricyanide, oxygen evolution of trypsin-treated thylakoids was insensitive to DCMU but became even more sensitive to Cu(II) and o-phenanthroline than nontreated thylakoids; however, trypsinized thylakoids were insensitive to inhibitors in the presence of silicomolybdate. We conclude that Cu(II) impaired the photosystem II electron transfer before the QB niche, most probably at the pheophytin-QA-Fe domain.     

Time-dependent changes in the in vitro effects of sodium chloride on photosynthetic electron transport of isolated chloroplasts

The effects of a wide concentration range of NaCl and sorbitol on three photosynthetic electron transport reactions of Pisum sativum L. cv. Feltham First chloroplasts were examined as a function of time from thylakoid membrane isolation. Rates of electron flow from water to diaminodurene (DAD) and ferricyanide were determined polarographically, whilst photoreduction of 2,6-dichlorophenolindophenol (DCPIP) was monitored spectrophotometrically. Assay of thylakoids immediately after isolation showed that the rate of photoreduction of all three electron acceptors decreased with increasing salt concentration. However, 100 min after leaf homogenisation the response pattern of ferricyanide and DCPIP photoreduction to increasing NaCl, but not increasing sorbitol concentration, became significantly modified. This was not the case for DAD photoreduction. The results are discussed in relation to the assessment of the possible effect of salinity on photosynthetic electron transport in vivo.     

Determination of H+/e- ratios in chloroplasts with flashing light.

Using a rapid pH electrode, measurements were made of the flash-induced proton transport in isolated spinach chloroplasts. To calibrate the system, we assumed that in the presence of ferricyanide and in steady-state flashing light, each flash liberates from water one proton per reaction chain. We concluded that with both ferricyanide and methylviologen as acceptors two protons per electron are translocated by the electron transport chain connecting Photosystem II and I. With methyl viologen but not with ferricyanide as an acceptor, two additional protons per electron are taken up due to Photosystem I activity. One of these latter protons is translocated to the inside of the thylakoid while the other is taken up in H2O2 formation. Assuming that the proton released during water splitting remains inside the thylakoid, we compute H+/e- ratios of 3 and 4 for ferricyanide and methylviologen, respectively. In continuous light of low intensity, we obtained the same H+/e- ratios. However, with higher intensities where electron transport becomes rate limited by the internal pH, the H+/e- ratio approached 2 as a limit for both acceptors. A working model is presented which includes two sites of proton translocation, one between the photoacts, the other connected to Photosystem I, each of which translocates two protons per electron. Each site presents a approximately 30 ms diffusion barrier to proton passage which can be lowered by uncouplers to 6-10 ms.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl₂, an inhibitor of cytochrome b₆f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b₆f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans.

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Effect of temperature on electron transport activities of glutaraldehyde-fixed pea thylakoids

Temperature-induced changes in Hill activity of glutaraldehyde-fixed pea ( Pisum sativum L. cv. Alaska) thylakoids have been examined. Using ferricyanide as electron acceptor, a temperature-induced change occurred at ca 12–14°C for both control and fixed thylakoids. In contrast to the controls, fixed thylakoids not only showed a change in slope of the Arrhenius plots but also a discontinuity which has not been observed in previous studies. A drop in activity coincided with the decrease in slope: the extent of the reduction depended on the concentration of glutaraldehyde used for fixation. Using a lipophilic electron acceptor, a temperature-induced change also occurred at 12–14°C, but there was no reduction in activities of fixed thylakoids at temperatures above the change in slope.
The results indicate that a temperature-induced change in fixed thylakoids restricts the access of ferricyanide to its reductant(s) in the membrane but that fixation does not affect the temperature-induced change per se. The results confirm that temperature has a general effect on the functioning of thylakoid membranes. The data demonstrate that calculations of the extent of inhibition by glutaraldehyde of Hill activity with ferricyanide should take into account the temperature at which assays are performed.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Thylakoid volume, proton translocation and buffering capacity as measured with spin-label techniques

In model studies with lipid vesicles it is shown that the main population of the spin label Tempamine is bound to the membrane surfaces, the piperidine ring directed to the lipid phase. The signal of external label but not of label in the surface is broadened by chromium oxalate. Inner volumes of vesicles can be derived from the partially resolved polar part of the high-field line of Tempone or from the area of the Tempamine spectrum removed by making chromium oxalate enter the vesicles. If this membrane-associated population is corrected for, rotational correlation times for the label in the lumen can be obtained showing that hindrance of rotation is only by a factor of 3–5 instead of 10 as previously reported. In studies with thylakoids volumes of 1–3 μl/mg Chl were found with 0.3 M sorbitol as an osmoticum, 5 mM MgCl₂, 20 mM KCI, and 20 mM chromium oxalate. The internal buffering capacity and the magnitude of pH changes in the inner volume can be determined from flash-induced changes in the amine distribution. The buffering capacity is found to be 7–20 mM in buffer-permeable and approx. 100 mM in buffer-impermeable thylakoids, that is approx. 100 neq per mg Chl. The apparent H⁺/e⁻-value in impermeable preparations was found to be up to 0.7 and lower in permeabilized material. ΔpH per flash is 0.04–0.06 units. Possible sources of errors, particularly the presence of non-functional or non-thylakoid membranes, are discussed. Time-resolved signals are presented and several side effects and their suppression are discussed. The response time of the method is up to 2 ms, protons from the donor side of Photosystem II can be separated kinetically from those liberated by the intersystem chain. While transients with less than 2 ms and approx. 20 ms were found with ferricyanide as an electron acceptor in accordance with the results with neutral red, pronounced slow phases (t_1/2 is several hundred ms) were found without acceptors. Evidence is presented indicating that at least part of these responses do not originate from the thylakoid inner volume.     

Measurement of the relative electron-transport capacity of Photosystem I and Photosystem II in spinach chloroplasts

The ratio of Photosystem (PS) II to PS I electron-transport capacity in spinach chloroplasts was compared from reaction-center and steady-state rate measurements. The reaction-center electron-transport capacity was based upon both the relative concentrations of the PS II_α, PS II_β and PS I centers, and the number of chlorophyll molecules associated with each type of center. The reaction-center ratio of total PS II to PS I electron-transport capacity was about 1.8:1. Steady-state electron-transport capacity data were obtained from the rate of light-induced absorbance-change measurements in the presence of ferredoxin-NADP⁺, potassium ferricyanide and 2,5-dimethylbenzoquinone (DMQ). A new method was developed for determining the partition of reduced DMQ between the thylakoid membrane and the surrounding aqueous phase. The ratio of membrane-bound to aqueous DMQH₂ was experimentally determined to be 1.3:1. When used at low concentrations (200 μM), potassium ferricyanide is shown to be strictly a PS I electron acceptor. At concentrations higher than 200 μM, ferricyanide intercepted electrons from the reducing side of PS II as well. The experimental rates of electron flow through PS II and PS I defined a PS II/PS I electron-transport capacity ratio of 1.6:1.     

An evaluation of paramagnetic broadening agents for spin probe studies of intact mammalian cells.

Six transition metal ion complexes have been examined for their effects on the cell survival as well as their effectiveness in inducing the broadening of the electron spin resonance (ESR) spectra of nitroxide spin probes. These paramagnetic species are Ni(EDTA), Ni(DTPA), potassium tris(oxalato) chromate (chromium oxalate), K3Fe(CN)6, Cu(DTPA), and NiCl2. At 100 mM concentration, the typical concentration used in cell studies to broaden the extracellular nitroxide ESR signal, only Ni(EDTA) and Ni(DTPA) are found to be non-toxic to Chinese hamster ovary cells. The relative cytotoxicities of the six metal ion complexes are Cu(DTPA) greater than K3Fe(CN)6 greater than NiCl2 greater than chromium oxalate greater than Ni(DTPA) greater than Ni(EDTA). Thus, potassium ferricyanide and NiCl2, two most commonly used paramagnetic broadening agents, are relatively toxic to the cell. In contrast, among the six paramagnetic species tested here, chromium oxalate appears to be the most effective agent at non-toxic concentrations in inducing the broadening of the ESR spectra of both cationic and neutral nitroxide spin probes. By considering both their cytotoxicity and their effectiveness in causing line broadening of the nitroxide ESR spectra, chromium oxalate is a good paramagnetic broadening agent for spin probe studies of intact mammalian cells.     

Effects of bicarbonate depletion on secondary acceptors of Photosystem II

In thylakoid membranes incubated in the dark with ferricyanide, an auxiliary acceptor (Q400) associated with Photosystem II becomes oxidized. It has been reported that, based on oxygen flash-yield data, electron flow to Q400 did not occur in ‘bicarbonate-depleted’ (formate-pretreated) samples. Contrary to this earlier report, we find, based on oxygen flash-yield data and chlorophyll a fluorescence-transient measurements, that Q400 is active as an electron acceptor in formate-pretreated samples. It is concluded that the effect of formate pretreatment is on the flow of electrons between Q, B and the plastoquinone pool and not the flow to Q400. We also believe that another auxiliary acceptor of Photosystem II exists under conditions of formate pretreatment and pH larger than 7.0. This belief is based on increased double advancement in the oxygen flash-yield pattern and increased area above the chlorophyll a fluorescence-rise curve. The double advancement in the oxygen pattern shows a second-order dependence on flash intensity. These effects are eliminated by bicarbonate addition or shifts to lower values of pH such as 6.8. This new acceptor is believed to be different from Q400.     

THE REACTIONS OF DENATURED EGG ALBUMIN WITH FERRICYANIDE

The following facts have been established experimentally. 1. In the presence of the synthetic detergent, Duponol PC, there is a definite reaction between dilute ferricyanide and denatured egg albumin. 0.001 mM of ferrocyanide is formed by the oxidation of 10 mg. of denatured egg albumin despite considerable variation in the time, temperature, and pH of the reaction and in the concentration of ferricyanide. 2. If the concentration of ferricyanide is sufficiently high, then the reaction between ferricyanide and denatured egg albumin in Duponol solution is indefinite. More ferrocyanide is formed the longer the time of reaction, the higher the temperature, the more alkaline the solution, and the higher the concentration of ferricyanide. 3. Denatured egg albumin which has been treated with formaldehyde or iodoacetamide, both of which abolish the SH groups of cysteine, does not reduce dilute ferricyanide in Duponol PC solution. 4. Cysteine is the only amino acid which is known to have a definite reaction with ferricyanide or which is known to react with dilute ferricyanide at all. The cysteine-free proteins which have been tried do not reduce dilute ferricyanide in Duponol PC solution. 5. Concentrated ferricyanide oxidizes cystine, tyrosine, and tryptophane and proteins which contain these amino acids but not cysteine. The reactions are indefinite, more ferrocyanide being formed, the higher the temperature and the concentration of ferricyanide. 6. The amount of ferrocyanide formed from denatured egg albumin and a given amount of ferricyanide is less in the absence than in the presence of Duponol PC. 7. The amount of ferrocyanide formed when denatured egg albumin reacts with ferricyanide in the absence of Duponol PC depends on the temperature and ferricyanide concentration throughout the whole range of ferricyanide concentrations, even in the low range of ferricyanide concentrations in which ferricyanide does not react with amino adds other than cysteine. The foregoing results have led to the following conclusions which, however, have not been definitely proven. 1. The definite reaction between denatured egg albumin in Duponol PC solution and dilute ferricyanide is a reaction with SH groups whereas the indefinite reactions with concentrated ferricyanide involve other groups. 2. The SH groups of denatured egg albumin in the absence of Duponol PC react with iodoacetamide and concentrated ferricyanide but they do not all react rapidly with dilute ferricyanide. 3. Duponol PC lowers the ferricyanide concentration at which the SH groups of denatured egg albumin react with ferricyanide. The SH groups of denatured egg albumin, however, are free and accessible even in the absence of Duponol PC.     

Studies on oxygen evolution of inside-out thylakoid vesicles from mangroves: Chloride requirement, pH dependence and polypeptide composition

Inside-out thylakoid vesicles from the halophyte Avicennia marina were isolated by the aqueous polymer phase partition method. Oxygen-evolution activity measured with ferricyanide and phenyl-p-benzoquinone was absolutely dependent on added chloride, since the vesicles were almost completely depleted of the 23 and 16 kDa polypeptides of the O₂-evolving complex. Addition of the spinach 23 kDa protein to the mangrove inside-out vesicles lowered their chloride requirement for O₂ evolution at least 50-fold. In the absence of added chloride, the mangrove vesicles were very sensitive to inhibition by light, which could be prevented by high chloride or low chloride plus added purified spinach 23 kDa protein. The preparations were also inactivated by neutral or alkaline pH (greater than 7.2) in the absence of high chloride concentrations. This inactivation was not significantly influenced by addition of spinach 23 kDa protein. Chloride binding and alkaline inhibition may therefore be closely related, either directly via the manganese centers or, more likely, via pK_a changes in as yet unidentified proteins.     

Influence of unsaturated fatty acids in chloroplasts. Shift of the pH optimum of electron flow and relations to deltapH, thylakoid internal pH and proton uptake.

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to deltapH, the proton pump and the pH of the inner thylakoid space (pHi). The deltapH and pHi were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6-dichlorophenolindophenol) coupled or non-coupled; (b) photosystem II (from H2O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the deltapH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the deltapH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosytems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the deltapH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo. As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5-5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), deltapH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtile equilibrium between the factors is disturbed.     

Oxygenic photoreduction of methyl viologen and nicotinamide adenine dinucleotide phosphate without the involvement of photosystem I during plastid development

Studies on the appearance of various electron transport functions were followed during greening of etiolated cucumber cotyledons. Appearance of dichlorodimethoxy-p-benzoquinone, dimethyl quinone, tetramethyl-p-phenylenediamine, dichlorophenol indophenol and ferricyanide Hill reactions were observed after 8h of greening. However, photoreduction of methyl viologen (MV) and nicotinamide adenine dinucleotide phosphate (NADP) was observed from 2h of greening. Variable fluorescence, which is a direct indication of water-splitting function, was observed from 2h of greening in cotyledons, thylakoid membranes and photosystem II (PSII) particles. The decrease in variable fluorescence in the presence of MV (due to rapid reoxidation of Q-) observed from early stages of greening confirmed the photoreduction of MV by PSII. The early development of water-splitting function was further confirmed by the abolition of variable fluorescence in thylakoid membranes and PSII particles by heat treatment and concomittant loss of light dependent oxygen uptake in the presence of MV in heat treated chloroplasts. However, the photoreduction of MV and NADP was insensitive to intersystem electron transport inhibitors, dichlorophenyl dimethylurea or dibromomethyl isopropyl-p-benzoquinone till 8h of greening. Though the oxidation of intersystem electron carrier cytochrome f was observed from early stages of greening, the reduction of cytochrome f was not observed till 8h of greening. All these observations confirm that during early stages of greening MV and NADP are photoreduced by PSII without the involvement of intersystem electron carriers or the collaboration of PSI. Since these observations are at variance with the currently prevalent concept (Z-Scheme) of the photosynthetic generation of reducing power, which requires definite collaboration of the two photosystems, an alternate electron flow pathway is proposed.