Quantification of cyclic electron flow around Photosystem I in spinach leaves during photosynthetic induction

The variation of the rate of cyclic electron transport around Photosystem I (PS I) during photosynthetic induction was investigated by illuminating dark-adapted spinach leaf discs with red + far-red actinic light for a varied duration, followed by abruptly turning off the light. The post-illumination re-reduction kinetics of P700⁺, the oxidized form of the photoactive chlorophyll of the reaction centre of PS I (normalized to the total P700 content), was well described by the sum of three negative exponential terms. The analysis gave a light-induced total electron flux from which the linear electron flux through PS II and PS I could be subtracted, yielding a cyclic electron flux. Our results show that the cyclic electron flux was small in the very early phase of photosynthetic induction, rose to a maximum at about 30 s of illumination, and declined subsequently to <10% of the total electron flux in the steady state. Further, this cyclic electron flow, largely responsible for the fast and intermediate exponential decays, was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, suggesting an important role of redox poising of the cyclic components for optimal function. Significantly, our results demonstrate that analysis of the post-illumination re-reduction kinetics of P700⁺ allows the quantification of the cyclic electron flux in intact leaves by a relatively straightforward method.     

Cell-wall lectins during winter wheat cold hardening

The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5-and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37-and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses.     

Effect of cold hardening on the phenolic complex of winter wheat leaves

The effect of hardening on the composition of phenolic compounds in winter wheat (Triticum aestivum L.) leaves was studied. It was shown that green tissues contained mainly flavonoids, especially flavons (C-and O-glycosides of apigenin and luteolin), and also ferulic acid derivatives. Among flavons, derivatives of luteolin dominated, including isoorientin, which comprised approximately a half of the content of all identified phenolic compounds. Low temperature induced the accumulation of phenolic compounds in winter wheat leaves, whereas their qualitative composition was not practically changed.     

Genetic study of glutathione accumulation during cold hardening in wheat

The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots of wheat (Triticum aestivum L.) cv. Cheyenne (Ch, frost-tolerant) and cv. Chinese Spring (CS, moderately frost-sensitive), in a T. spelta L. accession (Tsp, frost-sensitive) and in chro- mosome substitution lines CS (Ch 5A) and CS (Tsp 5A). The fast induction of total glutathione accumulation was detected during the first 3 d of hardening in the shoots, especially in the frost-tolerant Ch and CS (Ch 5A). This observation was corroborated by the study of de novo GSH synthesis using [³⁵S]sulfate. In Ch and CS (Ch 5A) the total cysteine, γ-glutamylcysteine (precursors of GSH), hydroxymethylglutathione and GSH contents were greater during the 51-d treatment than in the sensitive genotypes. After 35 d hardening, when the maximum frost tolerance was observed, greater ratios of reduced to oxidised hydroxymethylglutathione and glutathione were detected in Ch and CS (Ch 5A) compared to the sensitive genotypes. A correspondingly greater glutathione reductase (EC 1.6.4.2) activity was also found in Ch and CS (Ch 5A). It can be assumed that chromosome 5A of wheat has an influence on GSH accumulation and on the ratio of reduced to oxidised glutathione as part of a complex regulatory function during hardening. Consequently, GSH may contribute to the enhancement of frost tolerance in wheat. Received: 24 March 1999 / Accepted: 19 July 1999     

Changes in the level of MACC during cold hardening and dehardening in winter wheat

Changes in the level of 1-(malonylamino)-cyclopropane-1-carboxylic acid (MACC) were determined in 6 winter wheat cultivars during cold hardening at 4°C. The cultivars differed by one degree of frost resistance within the range of degree II to VII of the COMECON scale. The time-course of changes in MACC level showed a similar pattern in all 6 cultivars; i.e. increase till day 6, no changes for the next 10 days, and then a steady decrease till the end of the hardening period. There was little difference between the final and the initial levels. The increase of MACC level, expressed as per cent of the original level, was not directly correlated with either the degree of frost resistance of the actual percentage of survival. In some cultivars. mean errors exceeded the difference in MACC accumulation between cultivars closest on the resistance scale.
The fate of MACC during the second half of hardening and after transfer of plants to 25°C was studied in cultivars Bezostaya and San Pastore. During the second half of the hardening period the level of MACC decreased in the leaves of both cultivars, but increased significantly in the roots. Within two days of transfer of the hardened plants to 25°C, the MACC level in leaves increased again, while that in the roots decreased. This finding, together with the preliminary evidence of very low MACC metabolism, strongly suggest that MACC accumulates in roots during the hardening period and when transferred to 25°C, it moves from roots to leaves.     

Properties of chloroplast membrane-bound ferredoxin--NADP + reductase during cold hardening of wheat. No indication of qualitative membrane changes during cold hardening

Kinetic parameters of the chloroplastbound ferredoxin-NADP⁺ reductase from two varieties of wheat (Triticum aestivum), hardy Kharkov 22 MC (winter wheat) and less hardy Rescue (spring wheat), were followed during induction of frost hardiness as a means of examining possible changes in chloroplast membranes during hardening. No changes were found in the Michaelis constants for NADPH and 2,6-dichlorophenol indophenol, inhibition constants for p-chloromercuriphenylsulfonate, and activation energy values of the enzyme in either variety. The data suggest that no qualitative changes occurred in the properties of wheat chloroplast membranes related to ferredoxin-NADP⁺ reductase during cold hardening.     

Antioxidant function of alternative oxidase in mitochondria of winter wheat during cold hardening

The activity of alternative oxidase (AOX) and generation of reactive oxygen species (ROS) in mitochondria of winter wheat Triticum aestivum L. isolated from seedlings subjected to one (7-day exposure to 2–3°C) and two (7-day exposure to 2–3°C and 2-day exposure to −2°C) phases of a cold hardening has been studied. The antioxidant role of AOX in the first phase of the cold hardening has been determined using inhibitors of respiratory chain. Exposure to low temperature was shown to lead to inhibition of cytochrome pathway in mitochondria, increase of ROS production, and switching of the electron transport to the alternative pathway. Decrease in succinate- and antimycin A-induced ROS generation was found during two phases of cold hardening. This fact may point out to functioning of uncoupling proteins under these conditions. Thus, antioxidant function of AOX during the first phase of cold hardening may be an important component of the cold adaptation mechanism in winter crops. The data suggest that ROS and free fatty acids may be signal molecules regulating the activity of two energy-dissipation systems (AOX and uncoupling proteins).     

Rates and roles of cyclic and alternative electron flow in potato leaves

Measurements of 810 nm transmittance changes in leaves, simultaneously with Chl fluorescence, CO(2) uptake and O(2) evolution, were carried out on potato (Solanum tuberosum L.) leaves with altered expression of plastidic NADP-dependent malate dehydrogenase. Electron transport rates were calculated: J(C) from the CO(2) uptake rate considering ribulose-1,5-bisphosphate (RuBP) carboxylation and oxygenation, J(O) from the O(2) evolution rate, J(F) from Chl fluorescence parameters and J(I) from the post-illumination re-reduction speed of PSI donors. In the absence of external O(2), J(O) equaled (1.005 +/- 0.003) J(C), independent of the transgenic treatment, light intensity and CO(2) concentration. This showed that nitrite and oxaloacetate reduction rates were very slow. The Mehler-type O(2) reduction was evaluated from the rate of electron accumulation at PSI after the O(2) concentration was decreased from 210 to 20 mmol mol(-1), and resulted in <1% of the linear flow. J(F) and J(I) did not differ from J(C) while photosynthesis was light-limited, but considerably exceeded J(C) at saturating light. Then, typically, J(F) = 1.2 J(C) and J(I) = 1.3 J(C), and J(F) -J(C) and J(I) -J(C) depended little on CO(2) and O(2) concentrations. The results showed that the alternative and cyclic electron flow necessary to compensate variations in the ATP/NADPH ratio were only a few percent of the linear flow. The data do not support the requirement of 14H(+)/3ATP by the chloroplast ATP synthase. We suggest that the fast PSI cyclic electron flow J(I) - J(C), as well as the fast J(F) - J(C) are energy-dissipating cycles around PSI and PSII at light saturation.     

Measurements of mesophyll conductance,photosynthetic electron transport and alternative electron sinks of field grown wheat leaves

Photosynthetic electron transport drives the carbon reduction cycle, the carbon oxidation cycle, and any alternative electron sinks such as nitrogen reduction. A chlorophyll fluorescence— based method allows estimation of the total electron transport rate while a gas-exchange-based method can provide estimates of the electron transport needed for the carbon reduction cycle and, if the CO₂ partial pressure inside the chloroplast is accurately known, for the carbon oxidation cycle. The gas-exchange method cannot provide estimates of alternative electron sinks. Photosynthetic electron transport in flag leaves of wheat was estimated by the fluorescence method and gasexchange method to determine the possible magnitude of alternative electron sinks. Under non-photorespiratory conditions the two measures of electron transport were the same, ruling out substantial alternative electron sinks. Under photorespiratory conditions the fluorescence-based electron transport rate could be accounted for by the carbon reduction and carbon oxidation cycle only if we assumed the CO₂ partial pressure inside the chloroplasts to be lower than that in the intercellular spaces of the leaves. To further test for the presence of alternative electron sinks, carbon metabolism was inhibited by feeding glyceraldehyde. As carbon metabolism was inhibited, the electron transport was inhibited to the same degree. A small residual rate of electron transport was measured when carbon metabolism was completely inhibited which we take to be the maximum capacity of alternative electron sinks. Since the alternative sinks were small enough to ignore, the comparison of fluorescence and gas-exchange based methods for measuring the rate of electron transport could be used to estimate the mesophyll conductance to CO₂ diffusion. The mesophyll conductance estimated this way fell as wheat flag leaves senesced. The age-related decline in photosynthesis may be attributed in part to the reduction of mesophyll conductance to CO₂ diffusion and in part to the estimated decline of ribulose 1,5-bisphosphate carboxylase amount.     

Regulation of photosynthetic cyclic electron flow pathways by adenylate status in higher plant chloroplasts

Cylic electron flow (CEF) around Photosystem I in photosynthetic eukaryotes is likely to be necessary to augment ATP production, rapidly- and precisely balancing the plastid ATP/NADPH energy budget to meet the demands of downstream metabolism. Many regulatory aspects of this process are unclear. Here we demonstrate that the higher plant plastid NADH/Fd:plastoquinone reductase (NDH) and proposed PGR5/PGRL1 ferredoxin:plastoquinone reductase (FQR) pathways of CEF are strongly, rapidly and reversibly inhibited in vitro by ATP with K_i values of 670 μM and 240 μM respectively, within the range of physiological changes in ATP concentrations. Control experiments ruled out effects on secondary reactions, e.g. FNR- and cytochrome b₆f activity, nonphotochemical quenching of chlorophyll fluorescence etc., supporting the view that ATP is an inhibitor of CEF and its associated pmf generation and subsequent ATP production. The effects are specific to ATP, with the ATP analog AMP-PNP showing little inhibitory effect, and ADP inhibiting only at higher concentrations. For the FQR pathway, inhibition was found to be classically competitive with Fd, and the NDH pathway showing partial competition with Fd. We propose a straightforward model for regulation of CEF in plants in which CEF is activated under conditions when stromal ATP low, but is downregulated as ATP levels build up, allowing for effective ATP homeostasis. The differences in K_i values suggest a two-tiered regulatory system, where the highly efficient proton pumping NDH is activated with moderate decreases in ATP, with the less energetically-efficient FQR pathway being activated under more severe ATP depletion.     

Changes in the activity of proteases and trypsin inhibitors in wheat leaves in the initial period of cold hardening and subsequent dehardening

The dynamics of amidase, cysteine protease, and trypsin inhibitor activities were studied in the leaves of wheat (Triticum aestivum L.) seedlings grown under controlled conditions (25°C, illuminance 10 kLx, 14-h photoperiod) and subjected to cold hardening (5°C, 10 kLx, 14-h photoperiod). Changes in the activity of amidases and cysteine proteases proved to precede an increase in cold resistance during cold hardening and a decrease in cold resistance after the end of cold hardening. The activity of trypsin inhibitors changed only during cold hardening. It is suggested that amidases, cysteine proteases, and trypsin inhibitors are involved in the cold adaptation of plants.     

Heat shock increases thermotolerance of photosynthetic electron transport and the content of chloroplast membranes and lipids in wheat leaves

Preliminary heating of 15-16-day-old wheat (Triticum aestivum L.) plants for 3 h at 37–38°C (heat shock, HS) increased the tolerance of photosynthetic electron transport (determined as the reduction of 2,6-dichlorophenol indophenol by isolated chloroplasts) toward heating of leaves at 42–48°C in high light (100 klx). At the same time, HS did not affect the activity of the xanthophyll cycle reactions in the 30–48°C temperature range. HS exposure induced an increase in the thylakoid length, the number of grana, and the average number of thylakoids per granum. The volume of the thylakoid system increased 1.4-fold. Such indices as the total content of chlorophylls (a + b), the chlorophyll a/b ratio, as well as the contents of individual carotenoids, chloroplast membrane proteins, and the soluble leaf proteins remained unchanged. The de novo photosynthetic membrane formation was accompanied by the 1.5-fold increase in major chloroplast lipids. It was concluded that, in mature wheat chloroplasts, HS induced the formation of thylakoids characterized by a changed molecular structure and by increased lipid/protein and lipid/chlorophyll ratios.     

Source of glycolate and cyclic changes in photosynthetic and photorespiratory activity during the development of barley leaves

¹⁴CO₂ assimilation, RuBP earboxylase and PEP carboxylase activities show cyclic changes during the development of barley leaves. Cyclic changes, but in phase opposition with respect to carboxylating enzymes, are shown by RuBP oxygenase, phosphoglycolate phosphatase, glycolate oxidase and nitrate reductase activities. The oxygenase function of RuBP carboxylase appears to be the primary source of glycolate in young leaves, whereas in old ones glycolate could be supplied from some source in addition to RuBP oxygenase activity.     

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.     

Effect of nitrogen application and elevated CO2 on photosynthetic gas exchange and electron transport in wheat leaves

Nitrogen (N) availability is a critical factor affecting photosynthetic acclimation of C₃ plants under elevated atmospheric CO₂ concentration ([CO₂]_e). However, current understanding of N effects on photosynthetic electron transport rate and partitioning, as well as its impact on photosynthesis under [CO₂]_e, is inadequate. Using controlled environment open-top chambers, wheat (Triticum aestivum L.) was grown at two N levels (0 and 200 mg(N) kg^?1 soil) and two atmospheric CO₂ concentrations of 400 ([CO₂]_a) and 760 μmol mol^?1([CO₂]_e) during 2009 and 2010. Under [CO₂]_e high N availability increased stomatal conductance and transpiration rate, reduced limitations on the activity of triose phosphate isomerase, a Calvin cycle enzyme, and increased the rate of net photosynthesis (P _N). Considering photosynthetic electron transport rate and partitioning aspects, we suggest that greater N availability increased P _N under [CO₂]_e due to four following reasons: (1) higher N availability enhanced foliar N and chlorophyll concentrations, and the actual photochemical efficiency of photosystem (PS) II reaction centers under irradiance increased, (2) increase of total electron transport rate and proportion of open PSII reaction centers, (3) enhancement of the electron transport rate of the photochemical and carboxylation processes, and (4) reduced limitations of the Calvin cycle enzymes on the photosynthetic electron transport rate. Consequently, sufficient N improved light energy utilization in wheat flag leaves under [CO₂]_e, thus benefiting to photosynthetic assimilation.     

Interaction between starch breakdown, acetate assimilation, and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii

Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700(+), the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700(+) directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700(+) reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration.