The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion.

The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.

The mitochondrial CMSII mutation of Nicotiana sylvestris impairs adjustment of photosynthetic carbon assimilation to higher growth irradiance.

The CMSII mutant of Nicotiana sylvestris, which lacks a functional mitochondrial complex I, was used to investigate chloroplast-mitochondria interactions in light acclimation of photosynthetic carbon assimilation. CMSII and wild-type (WT) plants were grown at 80 micromol m(-2) s(-1) photosynthetic active radiation (PAR; 80) and 350 micromol m(-2) s(-1) PAR (350). Carbon assimilation at saturating PFD was markedly higher in WT 350 leaves as compared with WT 80 leaves, but was similar in CMS 80 and CMS 350 leaves, suggesting that the mutant is unable to adjust photosynthesis to higher growth irradiance. WT 350 leaves showed several general characteristic light acclimation responses [increases in leaf specific area (LSA), total chlorophyll content, and chlorophyll a/b ratio, and a higher light compensation point]. In contrast, a similar chlorophyll content and chlorophyll a/b ratio were measured for both CMS 80 and CMS 350 leaves, while LSA and the light compensation point acclimated as in the WT. The failure of CMSII to adjust photosynthesis to growth PFD did not result from lower quantum efficiency of PSII, lower whole-chain electron transport rates (ETRs), or lower ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and sucrose phosphate synthase (SPS) capacities. Excess ETR not used for carbon assimilation was even higher in CMS 350 than in WT 350. Since photochemical fluorescence quenching and the initial activity of NADP malate dehydrogenase (NADP-MDH) were identical in WT 350 and CMS 350 leaves but the activation state of NADP-MDH was different, redox signals from primary ETR are not involved in the signal transduction of light acclimation, while a contribution of stromal redox state cannot be excluded. When mature plants were transferred between 350 and 80 conditions, the mutant showed acclimatory tendencies, although adjustments were not as rapid or as marked as in the WT, and the response of the initial activities of Rubisco and NADP-MDH was impaired or altered. Initial activities of Rubisco and SPS at limiting concentration were also affected in CMS 350 as compared with WT plants when compared at growth irradiance or after in situ activation at 1000 micromol m(-2) s(-1) PAR. The data demonstrate that chloroplast-mitochondria interactions are important in light acclimation, and modulation of the activation state of key photosynthetic enzymes could be an important mechanism in this cross-talk.

Diffusive and metabolic limitations to photosynthesis under drought and salinity in C(3) plants.

Drought and salinity are two widespread environmental conditions leading to low water availability for plants. Low water availability is considered the main environmental factor limiting photosynthesis and, consequently, plant growth and yield worldwide. There has been a long-standing controversy as to whether drought and salt stresses mainly limit photosynthesis through diffusive resistances or by metabolic impairment. Reviewing in vitro and in vivo measurements, it is concluded that salt and drought stress predominantly affect diffusion of CO(2) in the leaves through a decrease of stomatal and mesophyll conductances, but not the biochemical capacity to assimilate CO(2), at mild to rather severe stress levels. The general failure of metabolism observed at more severe stress suggests the occurrence of secondary oxidative stresses, particularly under high-light conditions. Estimates of photosynthetic limitations based on the photosynthetic response to intercellular CO(2) may lead to artefactual conclusions, even if patchy stomatal closure and the relative increase of cuticular conductance are taken into account, as decreasing mesophyll conductance can cause the CO(2) concentration in chloroplasts of stressed leaves to be considerably lower than the intercellular CO(2) concentration. Measurements based on the photosynthetic response to chloroplast CO(2) often confirm that the photosynthetic capacity is preserved but photosynthesis is limited by diffusive resistances in drought and salt-stressed leaves.

Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants.

Experimental studies on CO2 assimilation of mesophytic C3 plants in relation to relative water content (RWC) are discussed. Decreasing RWC slows the actual rate of photosynthetic CO2 assimilation (A) and decreases the potential rate (Apot). Generally, as RWC falls from c. 100 to c. 75%, the stomatal conductance (gs) decreases, and with it A. However, there are two general types of relation of Apot to RWC, which are called Type 1 and Type 2. Type 1 has two main phases. As RWC decreases from 100 to c. 75%, Apot is unaffected, but decreasing stomatal conductance (gs) results in smaller A, and lower CO2 concentration inside the leaf (Ci) and in the chloroplast (Cc), the latter falling possibly to the compensation point. Down-regulation of electron transport occurs by energy quenching mechanisms, and changes in carbohydrate and nitrogen metabolism are considered acclimatory, caused by low Ci and reversible by elevated CO2. Below 75% RWC, there is metabolic inhibition of Apot, inhibition of A then being partly (but progressively less) reversible by elevated CO2; gs regulates A progressively less, and Ci and CO2 compensation point, Gamma rise. It is suggested that this is the true stress phase, where the decrease in Apot is caused by decreased ATP synthesis and a consequent decreased synthesis of RuBP. In the Type 2 response, Apot decreases progressively at RWC 100 to 75%, with A being progressively less restored to the unstressed value by elevated CO2. Decreased gs leads to a lower Ci and Cc but they probably do not reach compensation point: gs becomes progressively less important and metabolic limitations more important as RWC falls. The primary effect of low RWC on Apot is most probably caused by limited RuBP synthesis, as a result of decreased ATP synthesis, either through inhibition of Coupling Factor activity or amount due to increased ion concentration. Carbohydrate synthesis and accumulation decrease. Type 2 response is considered equivalent to Type 1 at RWC below c. 75%, with Apot inhibited by limited ATP and RuBP synthesis, respiratory metabolism dominates and Ci and Gamma rise. The importance of inhibited ATP synthesis as a primary cause of decreasing Apot is discussed. Factors determining the Type 1 and Type 2 responses are unknown. Electron transport is maintained (but down-regulated) in Types 1 and 2 over a wide range of RWC, and a large reduced/oxidized adenylate ratio results. Metabolic imbalance results in amino acid accumulation and decreased and altered protein synthesis. These conditions profoundly affect cell functions and ultimately cause cell death. Type 1 and 2 responses may reflect differences in gs and in sensitivity of metabolism to decreasing RWC.

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping.

The role of cyclic electron transport has been re-examined in leaves of C3 plants because the bioenergetics of chloroplasts (H +/e = 3 in the presence of a Q-cycle; H+/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C3 photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by farred light and a large increase in the number of electrons available to reduce P700+ in the dark. At low CO2 and O2 molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, Fv/Fm) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a trans-thylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-redoxidised P700 and the decline in Fv/Fm brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO2-dependent O2 evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease Fv/Fm. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H+/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory.

Protection against photoinhibition in the alpine plant Geum montanum.

Geum montanum L. is an alpine plant usually found at altitudes between 1700 and 2600 m. Its wintergreen leaves can be subjected to very low temperatures and at the same time receive high photon flux densities at the beginning of the growth season when the snow melts. We report results of a study, performed with classical methods of biophysics, showing that leaves of G. montanum were remarkably tolerant to sunlight even at low temperatures. This tolerance results from the interplay of photorespiration and CO2 photosassimilation. When temperatures approach 0°C, responses include stomatal opening and CO2 uptake even under desiccation stress. This permits linear electron transport that is sufficient to avoid the excessive reduction of the electron transport chain which is known to lead to photodamage. In addition, excitation energy was shifted from photosystem (PS)II to PSI which is a very efficient energy quencher. Sensitivity of P700 in PSI to oxidation by far-red light was decreased and rates of dark reduction of photooxidized P700 were increased by actinic illumination, suggesting activation of cyclic electron transport. Consistent with this, far-red light was able to decrease the quantum yield of PSII (measured by the F v/F m ratio of chlorophyll fluorescence). We suggest that cyclic electron transport decreases the lumenal pH under strong light. In the presence of zeaxanthin, this increases energy dissipation at the PSII level. At low temperatures, P700 remained strongly oxidized under high irradiation while the primary electron acceptor of PSII, QA, was largely reduced. This shows efficient control of electron transport presumably at the level of the cytochrome b/f complex and suggests formation of a protective transthylakoid proton gradient even when linear electron transport is much reduced in the cold. Thus, several mechanisms cooperate to effectively protect the photosynthetic apparatus of G. montanum from photodamage. We see no indication of destructive "photostress" in this species during the growth season under alpine low-temperature and drought conditions.

Overexpression of iron superoxide dismutase in transformed poplar modifies the regulation of photosynthesis at low CO2 partial pressures or following exposure to the prooxidant herbicide methyl viologen.

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H2O2, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO2 assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O2) and at low (1%) O2. CO2-response curves for photosynthesis showed similar net CO2-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO2 (Ci) values below 200 microL L-1 but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O2 concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 micromol m-2 s-1) a progressive decrease in the ratio of variable (Fv) to maximal (Fm) fluorescence was observed with decreasing temperature. At 6(o)C the high-light-induced decrease in the Fv/Fm ratio was partially prevented by low O2 but values were comparable in all lines. Methyl viologen caused decreased Fv/Fm ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.

Effect of temperature on net CO2 assimilation and photosystem II quantum yield of electron transfer of French bean (Phaseolus vulgaris L.) leaves during drought stress.

The effect of leaf temperature on stomatal conductance and net CO2 uptake was studied on French bean (Phaseolus vulgaris L.) using either dehydrated attached leaves (25-40% water deficit) or cut leaves supplied with 10(-4) M abscisic acid (ABA) solution to the transpiration stream. Decreasing leaf temperature caused stomatal opening and increased net CO2 uptake (which was close to zero at around 25° C) to a level identical to that of control leaves (without water deficit) at around 15° C. (i) The ABA effect on stomatal closure was modulated by temperature and, presumably, ABA is at least partly responsible for stomatal closure of french bean submitted to a drought stress. (ii) For leaf temperatures lower than 15° C, net CO2 uptake was no longer limited by water deficit even on very dehydrated leaves. This shows that dehydrated leaves retain a substantial part of their photosynthetic capacity which can be revealed at normal CO2 concentrations when stomata open at low temperature. In contrast to leaves fed with ABA, decreasing the O2 concentration from 21% to 1% O2 did not increase either the rate of net CO2 uptake or the thermal optimum for photosynthesis of dehydrated leaves. The quantum yield of PSII electron flow (measured by ΔF/Fm) was lower in 1% O2 than in 21% O2 for each leaf pretreatment given (non-dehydrated leaves, dehydrated leaves, and leaves fed with ABA) even within a temperature range in which leaf photosynthesis at normal CO2 concentration was the same in these two O2 concentrations. It is concluded that this probably indicates an heterogeneity of photosynthesis, since this difference in quantum yield disappears when using high CO2 concentrations during measurements.

Partitioning of photosynthetic electron flow between CO2 and O 2 reduction in a C 3 leaf (Phaseolus vulgaris L.) at different CO 2 concentrations and during drought stress.

Photosystem II chlorophyll fluorescence and leaf net gas exchanges (CO2 and H2O) were measured simultaneously on bean leaves (Phaseolus vulgaris L.) submitted either to different ambient CO2 concentrations or to a drought stress. When leaves are under photorespiratory conditions, a simple fluorescence parameter ΔF/ Fm (B. Genty et al. 1989, Biochem. Biophys. Acta 990, 87-92; ΔF = difference between maximum, Fm, and steady-state fluorescence emissions) allows the calculation of the total rate of photosynthetic electron-transport and the rate of electron transport to O2. These rates are in agreement with the measurements of leaf O2 absorption using (18)O2 and the kinetic properties of ribulose-1,5bisphosphate carboxylase/oxygenase. The fluorescence parameter, ΔF/Fm, showed that the allocation of photosynthetic electrons to O2 was increased during the desiccation of a leaf. Decreasing leaf net CO2 uptake, either by decreasing the ambient CO2 concentration or by dehydrating a leaf, had the same effect on the partitioning of photosynthetic electrons between CO2 and O2 reduction. It is concluded that the decline of net CO2 uptake of a leaf under drought stress is only due, at least for a mild reversible stress (causing at most a leaf water deficit of 35%), to stomatal closure which leads to a decrease in leaf internal CO2 concentration. Since, during the dehydration of a leaf, the calculated internal CO2 concentration remained constant or even increased we conclude that this calculation is misleading under such conditions.

Chlorophyll fluorescence and photoinhibition in a tropical rainforest understory plant.

The data presented here deal with the effects of high-light exposure on the 77 K fluorescence characteristics of Elatostema repens. It is shown that the decrease of the variable fluorescence during the treatment is biphasic. The reactions responsible for the first phase of fluorescence quenching are saturated under 700 µmol photon m(-2) s(-1) and insensitive to streptomycin, whereas those responsible for the second phase are not yet saturated under 700 µ mol photon m(-2) s(-1) and sensitive to streptomycin. It is concluded that only the second phase of fluorescence quenching is associated with photoinhibitory processes. Rate and amplitude of recovery from photoinhibition are maximum under very low light (3.5 µ mol photon m(-2) s(-1)), and very small at a moderate light (160 µ mol photon m(-2) s(-1)) which does not cause photoinhibition. It is concluded that recovery processes are inhibited during photoinhibition. It is suggested that they could be associated with damage occuring on the oxidizing side of PSII.

Effect of dehydration and high light on photosynthesis of two C3 plants (Phaseolus vulgaris L. and Elatostema repens (Lour.) Hall f.).

The effect of drought on the photosynthetic functioning of two C3 plants, Phaseolus vulgaris and Elatostema repens, has been examined. Leaf net CO2 uptake measured in normal air was negligible at a leaf water deficit of about 30% while the calculated leaf intercellular CO2 concentration (Ci) was unchanged. However, both the maximal photosynthetic capacity (CO2-dependent O2 evolution) and apparent quantum yield, measured in the presence of saturating CO2 levels (5 to 14%), only started to decrease within the range of 25 to 30% leaf water deficit. This shows that the drought-induced inhibition seen in normal air is not caused by an inhibition of the photosynthetic mechanism, and that in this case Ci values can be misleading. Both 77 K and room-temperature fluorescence measurements indicate that the functioning of the photosystem-II reaction centre is hardly modified by water shortage. Furthermore, an analysis of photochemical chlorophyll fluorescence quenching shows, in the absence of CO2, that O2 can be an efficient acceptor of photosynthetic energy, even in severly dehydrated plants which do not show net CO2 uptake in normal air. In these plants, O2 is probably reduced mainly via Mehler-type reactions. High-light treatment given at low O2 increases photoinhibition as measured by the decrease of apparent quantum yield in dehydrated P. vulgaris, whereas, interestingly, 1% O2 protects dehydrated E. repens against high-light damage. The two plants could have different protective mechanisms depending upon the O2 level or different photoinhibitory sites or mechanisms.

Effect of high light intensities on oxygen evolution and the light activation of NADP-malate dehydrogenase in intact spinach chloroplasts.

The factors limiting the photosynthetic carbon metabolism of intact spinach (Spinacia oleracea L.) chloroplasts after a high-light pretreatment have been studied. Photosynthetic CO2 fixation was decreased and became more sensitive to the inhibitory effect of the cyclic-electron-flow inhibitor, antimycin A. Depending on the extent of photoinhibition, changing the balance of linear to cyclic electron flow by adding oxaloacetate and antimycin A either did not relieve, or partially relieved the photoinhibitory effect. The decrease in CO2 fixation appeared to be the consequence of either a limitation by photosystem-II activity (in the case of moderate inhibition) or, at least partially an unfavourable balance between the linear and cyclic electron flows (in the case of strong inhibition). The light activation of NADP-malate dehydrogenase (EC 1.1.1.82) was decreased only in the presence of CO2, i.e. when there was strong competition for reducing power; otherwise, it was unaffected by photoinhibitory treatments, in accordance with its low energy requirement.

Photoinhibition of Photosynthesis in Broken Chloroplasts as a Function of Electron Transfer Rates during Light Treatment.

Photoinhibition was studied in osmotically broken chloroplasts isolated from spinach leaves (Spinacia oleracea L.). Both whole chain electron transport (measured as ferricyanide-dependent O(2) evolution in the presence of NH(4)Cl) and photosystem II activity (measured as O(2) evolution in the presence of either silicomolybdate plus 3-(3,4-diphenyl)-1,1 dimethylurea or parabenzoquinone) showed similar decreases in activity in response to a photoinhibitory treatment (8 minutes of high light given in the absence of an electron acceptor other than O(2)). Photosystem I activity was less affected. Photoinhibition of silicomolybdate reduction was largely reversible by an 8 minute dark incubation following the light treatment. Decreasing the O(2) concentration during photoinhibition below 2% increased photoinhibition of whole chain electron transport. Addition of superoxide dismutase to the reaction medium did not affect photoinhibition. Photoinhibition of both photosystem I and photosystem II activity increased as the rate of electron transfer during the treatment increased, and was largely prevented when 3-(3,4-diphenyl)-1,1-dimethylurea was present during the photoinhibition period. Noncyclic photophosphorylation was decreased as a consequence of whole chain electron transfer photoinhibition. Since diphenyl carbazide added after light treatment did not relieve photoinhibition of dichlorophenol indophenol reduction, we conclude that the site of inhibition is located within or near the photosystem II reaction center.

Nonstomatal Inhibition of Net CO(2) Uptake by (+/-) Abscisic Acid in Pharbitis nil.

(+/-) Abscisic acid (ABA) injected into petioles of attached transpiring leaves of Pharbitis nil Chois. cv violet reduced the photosynthetic capacity of the mesophyll of these leaves as well as the stomatal conductance to CO(2) diffusion. Greater than 75% of the injected ABA was recovered as ABA, suggesting that ABA rather than some metabolite thereof was the active compound. The nonstomatal effect of ABA increased from 30% reduction in photosynthesis at 0.25 micromolar ABA in the leaf blade to 90% reduction at 18 micromolar. Despite the effect of ABA on the nonstomatal component of leaf net CO(2) uptake, it was calculated that a substantial part of the reduction in leaf net CO(2) uptake (50-80%) could be accounted for by the effect of ABA on stomatal conductance.

Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves.

Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO(2)-free buffer at 21% O(2), showed a decrease (30-70%) in CO(2)-dependent O(2) evolution and (14)CO(2) uptake. This photoinhibition was observed only when the O(2) concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO(2) concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO(2) concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O(2) evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO(2) concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O(2) evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O(2)-dependent photoinhibition of light saturated CO(2) fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O(2) for photoinhibition of CO(2) fixation capacity in isolated chloroplasts may be explained by an effect of O(2) in allowing metabolic depletion of Calvin cycle intermediates.