Reductive titration of photosystem I and differential extinction coefficient of P700 at 810-950 nm in leaves

We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e⁻) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e⁻ carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) [V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1-15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch (Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e⁻ generated in PSII by the flash was measured as four times O₂ evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700⁺ and PC⁺ components using the abovementioned oxidative titration method. The P700⁺ component was related to the absolute number of e⁻ that reduced the P700⁺ to calculate the extinction coefficient. The effective differential extinction coefficient of P700⁺ at 810-950 nm was 0.40±0.06 (S.D.)% of transmittance change per μmol P700⁺ m⁻² or 17.6±2.4 mM⁻¹ cm⁻¹. The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (±14% S.D.) is mainly caused by light-scattering properties of the leaf.     

Dark inactivation of ferredoxin-NADP reductase and cyclic electron flow under far-red light in sunflower leaves

The oxidation kinetics under far-red light (FRL) of photosystem I (PSI) high potential donors P700, plastocyanin (PC), and cytochrome f (Cyt f) were investigated in sunflower leaves with the help of a new high-sensitivity photometer at 810 nm. The slopes of the 810 nm signal were measured immediately before and after FRL was turned on or off. The same derivatives (slopes) were calculated from a mathematical model based on redox equilibrium between P700, PC and Cyt f and the parameters of the model were varied to fit the model to the measurements. Typical best-fit pool sizes were 1.0–1.5 μmol m⁻² of P700, 3 PC/P700 and 1 Cyt f/P700, apparent equilibrium constants were 15 between P700 and PC and 3 between PC and Cyt f. Cyclic electron flow (CET) was calculated from the slope of the signal after FRL was turned off. CET activated as soon as electrons accumulated on the PSI acceptor side. The quantum yield of CET was close to unity. Consequently, all PSI in the leaf were able to perform in cycle, questioning the model of compartmentation of photosynthetic functions between the stroma and grana thylakoids. The induction of CET was very fast, showing that it was directly redox-controlled. After longer dark exposures CET dominated, because linear e⁻ transport was temporarily hindered by the dark inactivation of ferredoxin-NADP reductase.     

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH(2)) through the cytochrome b(6)f complex (Cyt b(6)f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light-dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700(+) and PC(+) was recorded as leaf transmission measured differentially at 810-950 nm. The signal was deconvoluted into PC(+) and P700(+) components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1-15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225-234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b(6)f to the PSI donors. A significant down-regulation of Cyt b(6)f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b(6)f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light-dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e(-) transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.     

Equilibrium or disequilibrium? A dual-wavelength investigation of photosystem I donors

Oxidation of photosystem I (PSI) donors under far-red light (FRL), slow re-reduction by stromal reductants and fast re-reduction in the dark subsequent to illumination by white light (WL) were recorded in leaves of several C₃ plants at 810 and 950 nm. During the re-reduction from stromal reductants the mutual interdependence of the two signals followed the theoretical relationship calculated assuming redox equilibrium between plastocyanin (PC) and P700, with the equilibrium constant of 40 ± 10 (ΔE _m = 86–99 mV) in most of the measured 24 leaves of nine plant species. The presence of non-oxidizable PC of up to 13% of the whole pool, indicating partial control of electron transport by PC diffusion, was transiently detected during a saturation pulse of white light superimposed on FRL or on low WL. Nevertheless, non-oxidizable PC was absent in the steady state during fast light-saturated photosynthesis. It is concluded that in leaves during steady state photosynthesis the electron transport rate is not critically limited by PC diffusion, but the high-potential electron carriers PC and P700 remain close to the redox equilibrium.     

Inhibition of photosystems I and II and enhanced back flow of photosystem I electrons in cucumber leaf discs chilled in the light

Pre-illumination of cucumber leaf discs at a chilling temperature in low-irradiance white light resulted in accelerated re-reduction of P700(+) [the special Chl pair in the photosystem I (PSI) reaction centre] when the far-red measuring light was turned off. Measurements (in +/- methyl viologen or +/- DCMU conditions) of the re-reduction half time suggest that accelerated re-reduction of P700(+) appeared to be predominantly due to charge recombination and only partly due to reductants sustained by previous cyclic electron flow around PSI. Apparently, charge recombination in PSI was greatly enhanced by inhibition of forward, linear electron flow. Inhibition of PSII electron transport was observed to occur to a lesser extent than that of PSI, but only if the measurement of PSII functionality was free from complications due to downstream accumulation of electrons in pools. We suggest that promotion of controlled charge recombination and cyclic electron flow round PSI during chilling of leaves in the light may partly prevent further damage to both photosystems.     

The role of plastocyanin in the adjustment of the photosynthetic electron transport to the carbon metabolism in tobacco

We investigated adaptive responses of the photosynthetic electron transport to a decline in the carbon assimilation capacity. Leaves of different ages from wild-type tobacco (Nicotiana tabacum) L. var Samsun NN and young mature leaves of tobacco transformants with impaired photoassimilate export were used. The assimilation rate decreased from 280 in young mature wild-type leaves to below 50 mmol electrons mol chlorophyll(-1) s(-1) in older wild-type leaves or in transformants. The electron transport capacity, measured in thylakoids isolated from the different leaves, closely matched the leaf assimilation rate. The numbers of cytochrome (cyt)-bf complexes and plastocyanin (PC) decreased with the electron transport and assimilation capacity, while the numbers of photosystem I (PSI), photosystem II, and plastoquinone remained constant. The PC to PSI ratio decreased from five in leaves with high assimilation rates, to values below one in leaves with low assimilation rates, and the PC versus flux correlation was strictly proportional. Redox kinetics of cyt-f, PC, and P700 suggest that in leaves with low electron fluxes, PC is out of the equilibrium with P700 and cyt-f and the cyt-f reoxidation rate is restricted. It is concluded that the electron flux is sensitive to variations in the number of PC, relative to PSI and cyt-bf, and PC, in concert with cyt-bf, is a key component that adjusts to control the electron transport rate. PC dependent flux control may serve to adjust the electron transport rate under conditions where the carbon assimilation is diminished and thereby protects PSI against over-reduction and reactive oxygen production.     

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH₂) through the cytochrome b₆f complex (Cyt b₆f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light-dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700⁺ and PC⁺ was recorded as leaf transmission measured differentially at 810-950 nm. The signal was deconvoluted into PC⁺ and P700⁺ components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1-15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225-234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b₆f to the PSI donors. A significant down-regulation of Cyt b₆f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b₆f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light-dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e⁻ transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.     

Electron flow to photosystem I from stromal reductants in vivo: the size of the pool of stromal reductants controls the rate of electron donation to both rapidly and slowly reducing photosystem I units

Electron donation from stromal reductants to photosystem I (PSI) was studied using the kinetics of P700(+) (the oxidized primary donor of PSI) reduction in the dark after irradiation of barley ( Hordeum vulgare L.) leaves. The leaves were treated with diuron and methyl viologen to abolish both the electron flow from PSII and PSI-driven cyclic electron transport. The redox state of P700 was monitored using the absorbance changes at 830 nm (Delta A(830)). Two exponentially decaying components with half-times of about 3 s (the slow component) and about 0.6 s (the fast one) were distinguished in the kinetic curves of Delta A(830) relaxation after a 1-s pulse of far-red light. The complex kinetics of P700(+) reduction thus manifested two types of PSI unit differing in the rate of electron input from stromal reductants. The rates of both kinetic components assayed after 1-s pulses were increased about 20-fold by a short (2-5 min) heat-pretreatment of leaves, indicating the accelerated input of electrons to both types of PSI unit. The increased rates of electron flow to P700(+) were even observed 1.5 h after the action of heat had been completed. Both kinetic components were dramatically slowed down upon irradiation of heat-treated leaves for 20-30 s. Their rates were restored after a short (20-30 s) period of darkness. A 5-min leaf exposure at 38 degrees C was sufficient to stimulate by severalfold the reduction of P700(+) pre-oxidized by a brief light pulse. In contrast, the acceleration of P700(+) reduction after a 1-min irradiation was observed only if leaves were subjected to temperatures above 40 degrees C. Neither heat treatment of leaves nor light-dark modulations in the rates of the fast and the slow components of P700(+) dark reduction influenced the relative magnitudes of the two kinetic components, providing strong additional evidence in favor of two distinct types of PSI existing per se in barley leaves. The key role in the control of the activity of electron donation to P700(+) in both rapidly and slowly reducing PSI units was attributed to the amount of stromal reductants available for P700(+) reduction. The latter was expected to be reduced under illumination in the presence of methyl viologen, while increased again in the dark. The regeneration of the pool of stromal reductants in the dark was likely provided by starch breakdown within the chloroplast stroma, but not by import of reducing equivalents from the cytosol. This was evidenced by much lower rates, compared with 1-h dark-adapted leaves, of dark reduction of both components of P700(+) in leaves stored for 24 h in the dark and thus depleted of starch but containing large amounts of glucose, the respiratory substrate.     

Measurement of photochemical quenching of absorbed quanta in photosystem I of intact leaves using simultaneous measurements of absorbance changes at 830 nm and thermal dissipation

The relationship between the redox state of the photosystem (PS) I primary donor, P700, and thermal energy dissipation in PSI were examined in intact leaves using simultaneous measurements of absorbance changes at 830 nm and variations of thermal emission monitored by photoacoustic (PA) spectroscopy, respectively. A strict proportionality (close to a 1:1 ratio) was found between the magnitudes of P700 oxidation and a positive variable PA signal induced by far-red light of various irradiances under conditions favoring effective electron donation from PSII to PSI. The proportionality was observed also between the ratio of reduced P700 to the total P700 content and the ratio of the variable component to the total PA signal measured with modulated light of 695 nm. Those findings clearly revealed that in intact leaves, variable thermal dissipation in PSI is determined by the fraction of P700 in the reduced state. Diuron-treated leaves exposed to 45 degrees C in which PSI received electrons not from PSII, but from soluble reductants localized in the chloroplast stroma were also used. In such leaves, the linear relationship between the ratio of reduced P700 to the total P700 content and the ratio of the variable component to the total PA signal measured with modulated light of 700 nm has been found as well, but its slope was twice smaller than in untreated leaves. This is probably related to an increased contribution of thermal emission from inactive PSII to the steady-state level of the PA signal in diuron-treated leaves exposed to high temperatures. The results demonstrated that the yield of variable thermal dissipation is strictly dependent on the redox pressure applied to the photosystem. The above illustrates the strong photochemical energy quenching occurring when the reaction centers are in open state (reduced P700).     

Xanthophyll Cycle and Inactivation of Photosystem Ⅱ Reaction Centers Alleviating Reducing Pressure to Photosystem Ⅰ in Morning Glory Leaves under Short-term High Irradiance

investigated through chlorophyll fluorescence parameters in morning glory (Ipomoea setosa) leaves, which were dipped into water, dithiothreitol (DTT) and lincomycin (LM), respectively. During the stress, both the xanthophyll cycle and D1 protein turnover could protect PSI from photoinhibition. In DTT leaves, non-photochemical quenching (NPQ) was inhibited greatly and the oxidation level of P700 (P700+) was the lowest one. However, the maximal photochemical efficiency of PSII (Fv/Fm) in DTT leaves was higher than that of LM leaves and was lower than that of control leaves. These results suggested that PSI was more sensitive to the loss of the xanthophyll cycle than PSII under high irradiance. In LM leaves, NPQ was partly inhibited, Fv/Fm was the lowest one among three treatments under high irradiance and P700+ was at a similar level as that of control leaves. These results implied that inactivation of PSII reaction centers could protect PSI from further photoinhibition. Additionally, the lowest of the number of active reaction centers to one inactive reaction center for a PSII cross-section (RC/CSo), maximal trapping rate in a PSII cross-section (TRo/CSo), electron transport in a PSII cross-section (ETo/CSo) and the highest of 1-qP in LM leaves further indicated that severe photoinhibition of PSII in LM leaves was mainly induced by inactivation of PSII reaction centers, which limited electrons transporting to PSI. However, relative to the LM leaves the higher level of RC/CSo, TRo/CSo, Fv/Fm and the lower level of 1-qP in DTT leaves indicated that PSI photoinhibition was mainly induced by the electron accumulation at the PSI acceptor side, which induced the decrease of P700+ under high irradiance.     

Inactivation and deficiency of core proteins of photosystems I and II caused by genetical phylloquinone and plastoquinone deficiency but retained lamellar structure in a T-DNA mutant of Arabidopsis

Phylloquinone, a substituted 1,4-naphthoquinone with an 18-carbon-saturated phytyl tail, functions as a bound one-electron carrier cofactor at the A1 site of photosystem I (PSI). A Feldmann tag line mutant, no. 2755 (designated as abc4 hereafter), showed pale-green young leaves and white old leaves. The mutated nuclear gene encoded 1,4-dihydroxy-2-naphtoic acid phytyltransferase, an enzyme of phylloquinone biosynthesis, and high-performance liquid chromatography analysis revealed that the abc4 mutant contained no phylloquinone, and only about 3% plastoquinone. Photooxidation of P700 of PSI in the abc4 mutant was not observed, and reduced-versus-oxidized difference spectroscopy indicated that the abc4 mutant had no P700. The maximum quantum yield of photosystem II (PSII) in the abc4 mutant was much decreased, and the electron transfer from PSII to PSI in the abc4 mutant did not occur. For the pale-green leaves of the abc4 mutant plant, the ultrastructure of the chloroplasts was almost the same as that of the wild-type plant. However, the chloroplasts in the albino leaves of the mutant were smaller and had a lot of grana thylakoids and few stroma thylakoids. The amounts of PSI and PSII core subunits in the abc4 mutant were significantly decreased compared with those in the wild type. These results suggested that a deficiency of phylloquinone in PSI caused the abolishment of PSI and a partial defect of PSII due to a significant decrease of plastoquinone, but did not influence the ultrastructure of the chloroplasts in young leaves.     

A rapid, whole-tissue determination of the functional fraction of PSII after photoinhibition of leaves based on flash-induced P700 redox kinetics

Assaying the number of functional PSII complexes by the oxygen yield from leaf tissue per saturating, single-turnover flash, assuming that each functional PSII evolves one oxygen molecule after four flashes, is one of the most direct methods but time-consuming. The ratio of variable to maximum Chl fluorescence yield (F_v/F_m) in leaves can be correlated with the oxygen yield per flash during a progressive loss of PSII activity associated with high-light stress and is rapid and non-intrusive, but suffers from being representative of chloroplasts near the measured leaf surface; consequently, the exact correlation depends on the internal leaf structure and on which leaf surface is being measured. Our results show that the average F_v/F_m of the adaxial and abaxial surfaces has a reasonable linear correlation with the oxygen yield per flash after varied extents of photoinactivation of PSII. However, we obtained an even better linear correlation between (1) the integrated, transient electron flow (Σ) to P700⁺, the dimeric Chl cation in PSI, after superimposing a single-turnover flash on steady background far-red light and (2) the relative oxygen yield per flash. Leaves of C3 and C4 plants, woody and herbaceous species, wild-type and a Chl- b -less mutant, and monocot and dicot plants gave a single straight line, which seems to be a universal relation for predicting the relative oxygen yield per flash from Σ. Measurement of Σ is non-intrusive, representative of the whole leaf tissue, rapid and applicable to attached leaves; it may even be applicable in the field.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

Adjustment of leaf photosynthesis to shade in a natural canopy: reallocation of nitrogen

The present study was performed to investigate the adjustment of the constituents of the light and dark reactions of photosynthesis to the natural growth irradiance in the leaves of an overstorey species, Betula pendula Roth, a subcanopy species Tilia cordata P. Mill., and a herb Solidago virgaurea L. growing in a natural plant community in Järvselja, Estonia. Shoots were collected from the site and properties of individual leaves were measured in a laboratory, by applying a routine of kinetic gas exchange and optical measurements that revealed photosystem II (PSII), photosystem I (PSI), and cytochrome b₆f densities per leaf area and the distribution of excitation (or chlorophyll, Chl) between the two photosystems. In parallel, N, Chl and ribulose-bisphosphate carboxylase-oxygenase (Rubisco) content was measured from the same leaves. The amount of N in photosynthetic proteins was calculated from the measured contents of the components of the photosynthetic machinery. Non-photosynthetic N was found as the residual of the budget. Growth in shade resulted in the decrease of leaf dry mass to a half of the DW in sun leaves in each species, but the total variation, from the top to the bottom of the canopy, was larger. Through the whole cross-section of the canopy, leaf dry weight (DW) and Rubisco content per area decreased by a factor of four, N content by a factor of three, but Chl content only by a factor of 1.7. PSII density decreased by a factor of 1.9, but PSI density by a factor of 3.2. The density of PSI adjusted to shade to a greater extent than the density of PSII. In shade, the distribution of N between the components of the photosynthetic machinery was shifted toward light-harvesting proteins at the expense of Rubisco. Non-photosynthetic N decreased the most substantially, from 54% in the sun leaves of B. pendula to 11% in the shade leaves of T. cordata. It is concluded that the redistribution of N toward light-harvesting Chl proteins in shade is not sufficient to keep the excitation rate of a PSII centre invariant. Contrary to PSII, the density of PSI – the photosystem that is in immediate contact with the carbon assimilation system – shade-adjusts almost proportionally with the latter, whereas its Chl antenna correspondingly increases. Even under N deficiency, a likely condition in the natural plant community, a substantial part of N is stored in non-photosynthetic proteins under abundant irradiation, but much less under limiting irradiation. At least in trees the general sequence of down-regulation due to shade adjustment is the following: (1) non-protein cell structures and non-photosynthetic proteins; (2) carbon assimilation proteins; (3) light reaction centre proteins, first PSI; and (4) chlorophyll-binding proteins.     

The stoichiometry of the two photosystems in higher plants revisited

The stoichiometry of Photosystem II (PSII) to Photosystem I (PSI) reaction centres in spinach leaf segments was determined by two methods, each capable of being applied to monitor the presence of both photosystems in a given sample. One method was based on a fast electrochromic (EC) signal, which in the millisecond time scale represents a change in the delocalized electric potential difference across the thylakoid membrane resulting from charge separation in both photosystems. This method was applied to leaf segments, thus avoiding any potential artefacts associated with the isolation of thylakoid membranes. Two variations of this method, suppressing PSII activity by prior photoinactivation (in spinach and poplar leaf segments) or suppressing PSI by photo-oxidation of P700 (the chlorophyll dimer in PSI) with background far-red light (in spinach, poplar and cucumber leaf segments), each gave the separate contribution of each photosystem to the fast EC signal; the PSII/PSI stoichiometry obtained by this method was in the range 1.5-1.9 for the three plant species, and 1.5-1.8 for spinach in particular. A second method, based on electron paramagnetic resonance (EPR), gave values in a comparable range of 1.7-2.1 for spinach. A third method, which consisted of separately determining the content of functional PSII in leaf segments by the oxygen yield per single turnover-flash and that of PSI by photo-oxidation of P700 in thylakoids isolated from the corresponding leaves, gave a PSII/PSI stoichiometry (1.5-1.7) that was consistent with the above values. It is concluded that the ratio of PSII to PSI reaction centres is considerably higher than unity in typical higher plants, in contrast to a surprisingly low PSII/PSI ratio of 0.88, determined by EPR, that was reported for spinach grown in a cabinet under far-red-deficient light in Sweden [Danielsson et al. (2004) Biochim. Biophys. Acta 1608: 53-61]. We suggest that the low PSII/PSI ratio in the Swedish spinach, grown in far-red-deficient light with a lower PSII content, is not due to greater accuracy of the EPR method of measurement, as suggested by the authors, but is rather due to the growth conditions.     

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.     

The stoichiometry of the two photosystems in higher plants revisited

The stoichiometry of Photosystem II (PSII) to Photosystem I (PSI) reaction centres in spinach leaf segments was determined by two methods, each capable of being applied to monitor the presence of both photosystems in a given sample. One method was based on a fast electrochromic (EC) signal, which in the millisecond time scale represents a change in the delocalized electric potential difference across the thylakoid membrane resulting from charge separation in both photosystems. This method was applied to leaf segments, thus avoiding any potential artefacts associated with the isolation of thylakoid membranes. Two variations of this method, suppressing PSII activity by prior photoinactivation (in spinach and poplar leaf segments) or suppressing PSI by photo-oxidation of P700 (the chlorophyll dimer in PSI) with background far-red light (in spinach, poplar and cucumber leaf segments), each gave the separate contribution of each photosystem to the fast EC signal; the PSII/PSI stoichiometry obtained by this method was in the range 1.5-1.9 for the three plant species, and 1.5-1.8 for spinach in particular. A second method, based on electron paramagnetic resonance (EPR), gave values in a comparable range of 1.7-2.1 for spinach. A third method, which consisted of separately determining the content of functional PSII in leaf segments by the oxygen yield per single turnover-flash and that of PSI by photo-oxidation of P700 in thylakoids isolated from the corresponding leaves, gave a PSII/PSI stoichiometry (1.5-1.7) that was consistent with the above values. It is concluded that the ratio of PSII to PSI reaction centres is considerably higher than unity in typical higher plants, in contrast to a surprisingly low PSII/PSI ratio of 0.88, determined by EPR, that was reported for spinach grown in a cabinet under far-red-deficient light in Sweden [Danielsson et al. (2004) Biochim. Biophys. Acta 1608: 53-61]. We suggest that the low PSII/PSI ratio in the Swedish spinach, grown in far-red-deficient light with a lower PSII content, is not due to greater accuracy of the EPR method of measurement, as suggested by the authors, but is rather due to the growth conditions.     

Induction changes in photosystems I and II in plant leaves upon modulation of membrane ion transport

The steady-state regime of linear photosynthetic electron transport implies concerted operation of photosystems I and II (PSI and PSII) in plant leaves. Acidification of the thylakoid lumen is known to cause down-regulation of PSII photochemical activity but it is not yet clear how the proton accumulation in the lumen affects the PSI activity and coordinated operation of the two photosystems in intact leaves. Chlorophyll fluorescence and absorbance of oxidized chlorophyll P700 in the near-infrared region ΔA _810–870 (ΔA ₈₁₀) are convenient noninvasive indicators of the redox state of PSII and PSI components, respectively. Simultaneous measurements of chlorophyll fluorescence and ΔA ₈₁₀ in pea leaves revealed that some kinetic stages in the induction curves occur synchronously both in dark-adapted and preilluminated leaves. After the treatment of leaves with ionophores promoting or inhibiting the light-induced thylakoid pH gradient (valinomycin, nigericin, monensin), the induction curves of ΔA ₈₁₀ and chlorophyll fluorescence were consistently modified. The results suggest that characteristic stages of ΔA ₈₁₀ induction curve, representing the second and the third waves of P700 photooxidation, are closely related to ΔpH generation, although the bases of ΔpH dependence differ for these two stages. The second wave of ΔA ₈₁₀ depends presumably on stroma alkalinization as a precondition for photoactivation of electron flow from PSI to terminal acceptors. The third wave of ΔA ₈₁₀ is apparently due to retardation of electron flow between PSII and PSI upon acidification of the lumen.     

Photosynthetic electron transport and specific photoprotective responses in wheat leaves under drought stress

The photosynthetic responses of wheat (Triticum aestivum L.) leaves to different levels of drought stress were analyzed in potted plants cultivated in growth chamber under moderate light. Low-to-medium drought stress was induced by limiting irrigation, maintaining 20 % of soil water holding capacity for 14 days followed by 3 days without water supply to induce severe stress. Measurements of CO₂ exchange and photosystem II (PSII) yield (by chlorophyll fluorescence) were followed by simultaneous measurements of yield of PSI (by P700 absorbance changes) and that of PSII. Drought stress gradually decreased PSII electron transport, but the capacity for nonphotochemical quenching increased more slowly until there was a large decrease in leaf relative water content (where the photosynthetic rate had decreased by half or more). We identified a substantial part of PSII electron transport, which was not used by carbon assimilation or by photorespiration, which clearly indicates activities of alternative electron sinks. Decreasing the fraction of light absorbed by PSII and increasing the fraction absorbed by PSI with increasing drought stress (rather than assuming equal absorption by the two photosystems) support a proposed function of PSI cyclic electron flow to generate a proton-motive force to activate nonphotochemical dissipation of energy, and it is consistent with the observed accumulation of oxidized P700 which causes a decrease in PSI electron acceptors. Our results support the roles of alternative electron sinks (either from PSII or PSI) and cyclic electron flow in photoprotection of PSII and PSI in drought stress conditions. In future studies on plant stress, analyses of the partitioning of absorbed energy between photosystems are needed for interpreting flux through linear electron flow, PSI cyclic electron flow, along with alternative electron sinks.     

NaCl胁迫加重强光胁迫下超大甜椒叶片的光系统II和光系统I的光抑制

快速叶绿素荧光动力学可以在无损情况下探知叶片光合机构的损伤程度, 快速叶绿素荧光测定和分析技术(JIP-test)将测量值转化为多种具有生物学意义的参数, 因而被广泛应用于植物光合机构对环境的响应机制研究。该文研究了超大甜椒(Capsicum annuum)幼苗在强光及不同NaCl浓度胁迫下的荧光响应情况。与单纯强光胁迫相比, NaCl胁迫引起了叶绿素荧光诱导曲线的明显改变, 光系统II (PSII)光抑制加重, 同时PSII反应中心和受体侧受到明显影响, 而且高NaCl浓度胁迫下PSII供体侧受伤害明显, 同时PSI反应中心活性(P700⁺)在盐胁迫下明显降低。这些结果表明, NaCl胁迫会增强强光对超大甜椒光系统的光抑制, 并且浓度越高抑制越明显, 但对PSI的抑制作用低于PSII。高NaCl浓度胁迫易对PSII供体侧造成破坏, 且PSI光抑制严重。