Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures

The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5° C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO₂. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO₂ assimilation measured at 8° C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO₂-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose, (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO₂-assimilation rates at low temperature, but nevertheless remained limited by phosphate, (iii) Maximum rates of CO₂ assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis. This work was supported by the Agricultural and Food Research Council, UK (Research grant PG50/67) and by the Science and Engineering Reserach Council, UK. C.A.L. was supported by the British Council, by an Overseas Research Student Award and by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil.     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

Limitation of photosynthesis by changes in temperature

The aim of this work was to examine the effect of abrupt changes in temperature in the range 5 to 30°C upon the rate of photosynthetic carbon assimilation in leaves of barley (Hordeum vulgare L.). Measurement of the CO₂-assimilation rate in relation to the intercellular partial pressure of CO₂ at different temperatures and O₂ concentrations and at saturating irradiance showed that as the temperature was decreased photosynthesis was saturated at progressively lower CO₂ partial pressures and that the transition between the CO₂-limited and ribulose-1,5-bisphosphate-regeneration-limited rate became more abrupt. Feeding of orthophosphate to leaves resulted in an increased rate of CO₂ assimilation at lower temperatures at around ambient or higher CO₂ partial pressures both in 20% O₂ and in 2% O₂ and it removed the abruptness in the transition between the CO₂-limited and ribulose-1,5-bisphosphate-regeneration-limited rates. Phosphate feeding tended to inhibit carbon assimilation at higher temperatures. The response of carbon assimilation to temperature was altered by feeding orthophosphate, by changing the concentrations of CO₂ or of O₂ or by leaving plants in the dark at 4°C for several hours. Similarly, the response of carbon assimilation to phosphate feeding or to changes in 2% O₂ was altered by leaving the plants in the dark at 4°C. The mechanism of limitation of photosynthesis by an abrupt lowering of temperature is discussed in the light of the results.Abbreviations A rate of CO₂ assimilation - P _i intercellular partial pressure of CO₂ - RuBP ribulose-1,5-bisphosphate     

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO₂. After determination of the rate of CO₂ assimilation at known intercellular CO₂ pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO₂, and fell rapidly as the CO₂-assimilation rate increased with increasing intercellular partial pressures of CO₂, indicating that bundle-sheath CO₂ concentrations fell at low intercellular partial pressures of CO₂. In contrast, the amount of phosphoenolpyruvate and of C₄-cycle intermediates declined at low intercellular partial pressures of CO₂. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C₄ cycles.Abbreviations PEP phosphoenolpyruvate - PGA glycerate-3-phosphate - p _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - triose-P triose phosphates     

Secondary fluorescence kinetics of spinach leaves in relation to the onset of photosynthetic carbon assimilation

When spinach leaves are re-illuminated, after dark periods of 90 s or less, an initial fluorescence peak is observed which rapidly gives way to a much lower terminal value. After 2 min or more in the dark, however, there is a secondary rise, at about 50–70 s, which then gives way, more slowly, to approximately the same low terminal value as before. The secondary rise is eliminated or disguised by feeding D,L-glyceraldehyde (a specific inhibitor of photosynthetic carbon assimilation) and by manose, 2-deoxyglucose and glucosamine, all of which are believed to sequester cytoplasmic orthophosphate. This secondary rise in fluorescence is discussed in relation to photosynthetic induction and the manner in which these compounds may modulate fluorescence by their effect on the availability of orthophosphate and their consequent impact on the adenylate status of the stroma.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - CCCP carbonylcyanidchlorophenylhydrazon     

Simultaneous and independent effects of abscisic acid on stomata and the photosynthetic apparatus in whole leaves

(±)-Abscisic acid (ABA) at 10^-5 M was added to the transpiration stream of leaves of 16 species (C₃ and C₄, monocotyledons and dicotyledons). Stomatal responses followed one of three patterns: i) stomata that were wide and insensitive to CO₂ initially, closed partially and became sensitive to CO₂; ii) for stomata that were sensitive to CO₂ before the application of ABA, the range of highest sensitivity to CO₂ shifted from high to low intercellular partial pressures of CO₂, for instance in leaves of Zea mays from 170–350 to 70–140 bar; iii) when stomata responded strongly to ABA, their conductance was reduced to a small fraction of the initial conductance, and sensitivity to CO₂ was lost. The photosynthetic apparatus was affected by applications of ABA to various degrees, from no response at all (in agreement with several previous reports on the absence of effects of ABA on photosynthesis) through a temporary decrease of its activity to a lasting reduction. Saturation curves of photosynthesis with respect to the partial pressure of CO₂ in the intercellular spaces indicated that application of ABA could produce three phenomena: i) a reduction of the initial slope of the saturation curve (which indicates a diminished carboxylation efficiency); ii) a reduction of the level of the CO₂-saturated rate of assimilation (which indicates a reduction of the ribulose-1,5-bisphosphate regeneration capacity); and iii) an increase of the CO₂ compensation point. Photosynthesis of isolated mesophyll cells was not affected by ABA treatments. Responses of the stomatal and photosynthetic apparatus were usually synchronous and often proportional to each other, with the result that the partial pressure of CO₂ in the intercellular spaces frequently remained constant in spite of large changes in conductance and assimilation rate. Guard cells and the photosynthetic apparatus were able to recover from effects of ABA applications while the ABA supply continued. Recovery was usually partial, in the case of the photosynthetic apparatus occasionally complete. Abscisic acid did not cause stomatal closure or decreases in the rate of photosynthesis when it was applied during a phase of stomatal opening and induction of photosynthesis that followed a transition from darkness to light.Abbreviations and symbols A rate of CO₂ assimilation - ABA (±)-abscisic acid - c _a partial pressure of CO₂ in the ambient air or in the gas supplied to the leaf chambers - c _i partial pressure of CO₂ in the intercellular spaces of a leaf - e _a partial pressure of H₂O in the air - g conductance for water vapor - J quantum flux - T ₁ leaf temperature     

Some relationships between contents of photosynthetic intermediates and the rate of photosynthetic carbon assimilation in leaves of Zea mays L.

The relationship between the gas-exchange characteristics of attached leaves of Zea mays L. and the contents of photosynthetic intermediates was examined at different intercellular partial pressure of CO₂ and at different irradiances at a constant intercellular partial pressure of CO₂. (i) The behaviour of the pools of the C₄-cycle intermediates, phosphoenolpyruvate and pyruvate, provides evidence for light regulation of their consumption. However, light regulation of phosphoenolpyruvate carboxylase does not influence the assimilation rate at limiting intercellular partial pressures of CO₂. (ii) A close correlation between the pools of phosphoenolpyruvate and glycerate-3-phosphate exists under many different flux conditions, consistent with the notion that the pools of C₄ and C₃ cycles are connected via the interconversion of glycerate-3-phosphate and phosphoenolpyruvate. (iii) The ratio of triose-phosphate to glycerate-3-phosphate is used as an indicator of the availability of ATP and NADPH. Changes of this ratio with CO₂ and with irradiance are compared with results obtained in C₃ leaves and indicate that the mechanism of regulation of carbon assimilation by light in leaves of C₄ plants may differ from that in C₃ plants. (iv) The behaviour of the ribulose-1,5-bisphosphate pool with CO₂ and irradiance is contrasted with the behaviour of these pools measured in leaves of C₃ plants.Abbreviations P _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - PEP phosphoenolpyruvate - triose-P triose phosphates - PGA glycerate-3-phosphate     

The relationship between carbon dioxide fixation and chlorophyll a fluorescence during induction of photosynthesis in maize leaves at different temperatures and carbon dioxide concentrations

The rate of CO₂ fixation (F_c) and 680 nm chlorophyll fluorescence emission (F680) were measured simultaneously during induction of photosynthesis in Zea mays L. leaves under varying experimental conditions in order to assess the validity of fluorescence as an indicator of in vivo photosynthetic carbon assimilation. Z. mays leaves showed typical Kautsky fluorescence induction curves consisting of a fast rise in emission (O to P) followed by a slow quenching via a major transient (S-M) to a steady-state (T). After an initial lag, net CO₂ assimilation commenced at a point corresponding to the onset of the S-M transient on the F680 induction curve. Subsequently, F_c and F680 always arrived at a steady-state simultaneously. Decreasing the dark-adaption period increased the rate of induction of both parameters. Alteration of leaf temperature produced anti-parallel changes in induction characteristics of F_c and F680. Reducing the CO₂ level to below that required for saturation of photosynthesis also produced anti-parallel changes during induction, however, at CO₂ concentrations tenfold greater than the atmospheric level the rate of F680 quenching from P to T was appreciably reduced without a similar change in the induction of F_c. Removal of CO₂ at steady-state produced only a small increase in F680 and a correspondingly small decrease in F680 occurred when CO₂ was re-introduced. The complex relationship between chlorophyll fluorescence and carbon assimilation in vivo is discussed and the applicability of fluorescence as an indicator of carbon assimilation is considered.Abbreviations F_c rate of CO₂ fixation - F680 fluorescence emission at 680 nm     

Short-term effects of nitrate,nitrite and ammonium assimilation on photosynthesis,carbon partitioning and protein phosphorylation in maize

Maize (Zea mays L. cv. Contessa) was grown with a nitrogen supply that was just sufficient to support maximal biomass production. The third leaves from 14-to 21-d-old plants were harvested and net photosynthesis allowed to attain steady-state rates at an irradiance of either 250 or 700 mol·m^–2·s^–1. Nitrogen in the form of either KNO₃, KNO₂ or NH₄Cl was then supplied to the leaves through the transpiration stream. In all cases the addition of the nitrogen source resulted in an approximate doubling of the total amino-acid content of the leaves within 1 h. The glutamine pool increased to ten times the level found in control leaves in the light in the absence of added nitrogen. Glutamine accounted for about 21–24% of the total amino-acid content in leaves fed with 40 mM NH₄Cl. Nitrate caused a rapid, but transient inhibition of the rate of net CO₂ assimilation, accompanied by an increase in the activity of phosphoenolpyruvate carboxylase and a decrease in the maximum extractable activity of sucrose-phosphate synthase. This demonstrates that the activities of phospho-enolpyruvate carboxylase and sucrose-phosphate synthase are modulated by NO ₃ ^– in the C₄ plant maize, in a similar manner to that observed in C₃ plants. Nitrite or ammonium feeding resulted in decreased rates of CO₂ assimilation for as long as the nitrogen source was supplied. In all cases the degree of inhibition was greatest at high irradiance and least at low irradiance, even though the total amino-acid contents of the leaves were comparable at the time when maximum inhibition of CO₂ assimilation occurred. Measurements of chlorophyll-a fluorescence showed that the quantum efficiency of PSII decreased and non-radiative dissipation of excitation energy increased as CO₂ assimilation was inhibited by nitrate or nitrite. These metabolites had no direct effect on thylakoid PSII-based electron transport. Ammonium ions weakly inhibited O₂ evolution at high concentrations. The addition of nitrogen (KNO ₃ ^– , KNO₂ or NH₄Cl) caused a significant decrease in the phosphorylation state of the light-harvesting chlorophyll-a/b-binding protein of the thylakoid membranes. We conclude that the response of photosynthetic carbon assimilation and electron transport in maize is essentially similar whether nitrogen is supplied in the form of nitrate, nitrite or ammonium, with the noteworthy exception that the nitrogen-induced inhibition of photosynthesis is transient when leaves are supplied with NO ₃ ^– but sustained when NO ₂ ^– or NH ₄ ⁺ is provided. We suggest that the observed modulation of phosphoenolpyruvate carboxylase and sucrose-phosphate synthase is mediated by the increase in the endogenous level of glutamine. Furthermore, the transient nature of the inhibition of CO₂ assimilation in the case of NO ₃ ^– , but not NO ₂ ^– or NH ₄ ⁺ , may be due to regulation of nitrate reductase.Abbreviations and Symbol Chl chlorophyll - FB-Pase fructose-1,6-bisphosphatase - Gln glutamine - Glu glutamic acid - K_D index of the rate of thermal energy dissipation within the PSII antenna - LHCII light-harvesting chlorophyll-a/b-binding protein - PEPCase phosphoenolpyruvate carboxylase - PFD photon flux density - SPS sucrose-phosphate synthase - _PSII relative quantum efficiency for electron transport by PSII We wish to thank Gabriel Cornic (Structure et métabolisme des plantes, Université de Paris-Sud, Orsay, France) for useful discussion. We are grateful to Sylvie Ferrario (Laboratoire du Métabolisme, I.N.R.A., Versailles) for optimising the conditions of assay and extraction of SPS and PEPCase from maize leaves.     

The relationship between phosphate status and photosynthesis in leaves

Spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) were grown in hydroponic culture with varying levels of orthophosphate (Pi). When leaves were fed with 20 mmol·l^–1 Pi at low CO₂ concentrations, a temporary increase of CO₂ uptake was observed in Pi-deficient leaves but not in those from plants grown at 1 mmol·l^–1 Pi. At high concentrations of CO₂ (at 21% or 2% O₂) the Pi-induced stimulation of CO₂ uptake was pronounced in the Pi-deficient leaves. The contents of phosphorylated metabolites in the leaves decreased as a result of Pi deficiency but were restored by Pi feeding. These results demonstrate that there is an appreciable capacity for rapid Pi uptake by leaf mesophyll cells and show that the effects of long-term phosphate deficiency on photosynthesis may be reversed (at least temporarily) within minutes by feeding with Pi.Abbreviation Pi orthophosphate     

Distribution of reducing power between photosynthetic carbon and nitrogen assimilation in Scenedesmus

Fixation of CO₂ and N assimilation were studied in synchronous cultures of Scenedesmus obtusiusculus Chod. under saturating and limiting light. Within the photon-flux range studied, the cells maintained C to N assimilation ratios of 7–10 with either NO ₃ ^- , NO ₂ ⁺ or NH ₄ ⁺ as the N source. Competitive interactions between C and N assimilation were pronounced under light limitation and were proportional to the oxidation status of the N source. Fixation of CO₂ at saturating light was also slightly reduced by NO ₂ ^- and NH ₄ ⁺ . In the absence of CO₂, NO ₃ ^- uptake and reduction was light-saturated at a comparatively low photon flux, whereas NO ₂ ^- uptake and reduction was considerably faster in the absence of CO₂ than in its presence. The pools of reduced pyridine nucleotides (NADPH and NADH) were largely unaffected by the presence or absence of the different N sources. The regulatory influences of CO₂ fixation on N assimilation are discussed in terms of coupling between the rates of CO₂ fixation and NH ₄ ⁺ assimilation, as well as the existance of control mechanisms for NO ₃ ^- uptake and reduction.Abbreviations Chl chlorophyll - PF photon flux     

Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Purification and characterization of D-glycerate-3-kinase from maize leaves

Glycerate kinase (GK; EC 2.7.1.31) from maize (Zea mays L.) leaves was purified by a sequence of ammonium-sulfate precipitations and chromatography on diethylaminoethyl-cellulose, hydroxyapatite, Sephadex G-75SF and dye ligand (Green A) columns. The purest preparation was almost 1300-fold enriched and had a specific activity of 68 mol · min^-1 · (mg protein) ^-1. The enzyme was a monomer of a relative molecular mass (M_r) of 44 kDa (kdalton) as determined by gel filtration, electrophoresis in dissociating conditions and by immunoblots. The enzyme was only weakly recognized by polyclonal antibodies against purified spinach GK, indicating substantial differences in molecular structure of the two proteins. Highly reducing conditions stabilized GK activity and were required for activation of crude leaf enzyme. The enzyme had a broad pH optimum of 6.8–8.5, and formed 3-phosphoglycerate and ADP as reaction products. Apparent K _ms for D-glycerate and Mg-ATP were 0.11 and 0.25 mM, respectively. The enzyme was strongly affected by a number of phosphoesters, especially by 3-phosphoglycerate (K _i= 0.36 mM), fructose bisphosphates and nucleoside bisphosphates. Inhibition by 3-phosphoglycerate was competitive to Mg-ATP and noncompetitive to D-glycerate. Pyruvate was found noncompetitive to D-glycerate (K _is=4 mM). The ratio of stromal concentration of Mg-ATP to phosphoesters, particularly to 3-phosphoglycerate, may be of importance in the regulation of GK during C₄-photosynthesis.Abbreviations DEAE diethylaminoethyl - kDa kdalton - GAP-DH glyceraldehyde phosphate dehydrogenase - GK glycerate kinase - LDH lactate dehydrogenase - 2-ME 2-mercaptoethanol - M_r relative molecular mass - PEP phosphoenolpyruvate - PGA(PK) phosphoglycerate (phosphokinase) - PK pyruvate kinase - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis     

Effects of high temperature on the germination of maize (Zea mays L.)

Poor emergence of maize seedlings, due to high soil temperatures, is a major limitation of crop potential in the lowland tropics. Ability to germinate at high temperature (>c. 37° C) is related to the temperature sensitivity of the embryo, and there is considerable genotypic variation for this character.Respiration and mitochondrial phosphorylation proceed normally in seeds imbibing at 41° C, and ATP levels are adequate for germination. However, the specific activities of several important enzymes are lower, and the rate of protein synthesis is severely reduced compared with seeds imbibing at 28° C. The depression of the rate of protein synthesis in the embryos of several tropical hybrids imbibing at high temperature correlated with their known temperature sensitivity. It is concluded that protein synthesis is an especially temperature sensitive process in germinating maize embryos, and that this is the principal reason for the sensitivity of germinating maize seeds to high temperature.Abbreviations ADP adenosine-5-diphosphate - ATP adenosine-5-triphosphate - BSA bovine serum albumin - EDTA ethylenediaminetetra-acetic acid - HEPES N-2-hydroxyethylpiperazinc-N-2-ethanesulphonic acid - NADH nicotinamide-adenine dinucleotide, reduced form - PPO 2, 5-diphenyloxazole - PVP polyvinylpyrrolidone - SEM standard error of the mean - tris tris (hydroxymethyl)-methylamine     

The midday depression of CO2 assimilation in leaves of Arbutus unedo L.: diurnal changes in photosynthetic capacity related to changes in temperature and humidity

Parts of attached leaves of the sclerophyllous shrub Arbutus unedo were subjected to simulated mediterranean days. Gas exchange was recorded in order to recognize the causes of the midday depression in CO₂ assimilation. Depressions could be induced in part of a leaf: they were local responses. The CO₂-saturation curves of photosynthesis, determined during the morning and afternoon maxima of CO₂ assimilation and during the minimum at midday, established that depressions in CO₂ assimilation were in one-half of the investigated cases totally caused by reversible reductions in the photosynthetic capacity of the leaves, and in the other half almost totally caused by such reductions. An analysis of 37 daily courses showed that morning reductions and afternoon recoveries of stomatal conductance and rate of photosynthesis occurred simultaneously and in proportion to each other, with the result that the partial pressure of CO₂ in the intercellular spaces remained more or less constant. Midday depressions occurred also in detached leaves standing in water. The initiation of a midday depression was not caused by a circadian rhythm, nor was high quantum flux or high temperature a requirement. There was no correlation between the rate of water loss from the leaves, or the amount of water lost, with the degree of reduction of the photosynthetic capacity. However, depressions occurred if an apparent threshold in the water-vapor pressure difference between leaf and air was exceeded. This critical value varied between about 20 and 30 mbar, depending on the leaf investigated. The dominating role of humidity in the induction of the midday depression was further demonstrated when leaf temperature was held constant and the vapor-pressure difference was made to follow the pattern of the mediterranean day: depressions occurred. Depressions however were hardly noticeable when the water-vapor pressure difference was held constant and leaf temperature was allowed to vary. In another set of experiments, leaves were subjected to variations in temperature and humidity independent of the time of the day, under otherwise constant conditions. Photosynthetic capacity and stomatal conductance proved to be almost insensitive to changes in temperature (in a range extending from 20 to 37° C) as long as the water vapor-pressure difference was held constant. If it was not, the rate of photosynthesis began to decline with increasing temperature after a threshold water-vapor pressure difference was exceeded. The position of the resulting apparent temperature optimum of photosynthesis depended on the humidity of the air. We suggest that the ability of A. unedo to respond to a dry atmosphere with a reversible reduction of its photosynthetic capacity (by a still unknown mechanism) is the result of a co-evolution with the development of a strong stomatal sensitivity to changes in humidity.     

Effect of carbon dioxide and temperature on photosynthetic CO2 uptake and photorespiratory CO2 evolution in sunflower leaves

Using an open gas-exchange system, apparent photosynthesis, true photosynthesis (TPS), photorespiration (PR) and dark respiration of sunflower (Helianthus annuus L.) leaves were determined at three temperatures and between 50 and 400 l/l external CO₂. The ratio of PR/TPS and the solubility ratio of O₂/CO₂ in the intercellular spaces both decreased with increasing CO₂. The rate of PR was not affected by the CO₂ concentration in the leaves and was independent of the solubility ratio of oxygen and CO₂ in the leaf cell. At photosynthesis-limiting concentrations of CO₂, the ratio of PR/TPS significantly increased from 18 to 30°C and the rate of PR increased from 4.3 mg CO₂ dm^-2 h^-1 at 18°C to 8.6 mg CO₂ dm^-2 h^-1 at 30°C. The specific activity of photorespired CO₂ was CO₂-dependent but temperature-independent, and the carbon traversing the glycolate pathway appeared to be derived both from recently fixed assimilate and from older reserve materials. It is concluded that PR as a percentage of TPS is affected by the concentrations of O₂ and CO₂ around the photosynthesizing cells, but the rate of PR may also be controlled by other factors.Abbreviations APS apparent photosynthesis (net CO₂ uptake) - PR photorespiration (CO₂ evolution in light) - RuBP ribulose-1,5-bisphosphate - TPS true photosynthesis (true CO₂ uptake)     

In-situ localization of nitrate reductase in maize roots

The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues ofZea mays L. (W64A W182L) was determined using post-embedding immunogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cytoplasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchymal cells of the pith lying next to the xylem. A positive signal for NR protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when affinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant NR protein when adequate controls and signal-enhancement procedures were used.Abbreviations IgG immunoglobulin G - NR nitrate reductase - PEPCase phosphoenolpyruvate carboxylase This research was funded by Natural Sciences and Engineering Research Council (NSERC) of Canada grants ISE0125461 (AO), OGP0106265 (JSG) and an NSERC Visiting Scientist Award to E.F.     

Subcellular and immunocytochemical localization of the enzymes involved in ammonia assimilation in mesophyll and bundle-sheath cells of maize leaves

The cellular localization of the enzymes involved in primary nitrogen assimilation was investigated following separation of mesophyll protoplasts and bundle-sheath cells of maize (Zea mays L.) leaves. Determination of the enzymatic activities in the two types of cell revealed that nitrate and nitrite reductase are principally located in the mesophyll cells whereas glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) are present in both tissues with a preferential location in the bundle-sheath strands. In order to confirm the results obtained by this conventional biochemical method we have used an in-situ immunofluorescence technique to unambiguously localize GS and Fd-GOGAT at the cellular level. Thin-sectioned maize leaves treated with specific GS and Fd-GOGAT antisera followed by conjugation with fluorescein-isothiocyanate-labelled sheep anti-rabbit immunoglobulins clearly show that GS is equally distributed within the leaf whereas Fd-GOGAT is mostly present in the chloroplasts of the bundle-sheath cells. The cellular localization of nitrate reductase, nitrite reductase, GS-2 and Fd-GOGAT in maize leaf cell types strongly indicates that primary nitrogen assimilation functions in the mesophyll cells while photorespiratory nitrogen recycling is restricted to the bundle-sheath cells.     

Control of photosynthesis in barley leaves with reduced activities of glutamine synthetase or glutamate synthase

Wild-type and mutant plants of barley (Hordeum vulgare L. cv. Maris Mink) lacking activities of chloroplastic glutamine synthetase (GS) and of ferredox-in-dependent glutamate synthase (Fd-GOGAT) were crossed to generate heterozygous plants. Crosses of the F² generation containing GS activities between 47 and 97 of the wild-type and Fd-GOGAT activities down to 63 of the wild-type have been selected to study the control of both enzymes on photorespiratory carbon and nitrogen metabolism. There were no major pleiotropic effects. Decreased GS had a small impact on leaf protein and the total activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). The activation state of Rubisco was unaffected in air, but a decrease in GS influenced the activation state of Rubisco in low CO₂. In illuminated leaves, the amino-acid content decreased with decreasing GS, while the content of ammonium rose, showing that even small reductions in GS limit ammonium re-assimilation and may bring about a loss of nitrogen from the plants, and hence a reduction in protein and Rubisco. Leaf amino-acid contents were restored, and ammonium and nitrate contents decreased, by leaving plants in the dark for 24 h. The ratios of serine to glycine decreased with a decrease in GS when plants were kept at moderate photon flux densities in air, suggesting a possible feedback on glycine decarboxylation. This effect was absent in high light and low CO₂. Under these conditions ammonium contents exhibited an optimum and amino-acid contents a minimum at a GS activity of 65 of the wild-type, suggesting an inhibition of ammonium release in mutants with less than 65 GS. The leaf contents of glutamate, glutamine, aspartate, asparagine, and alanine largely followed changes in the total amino-acid contents determined under different environmental conditions. Decreased Fd-GOGAT resulted in a decrease in leaf protein, chlorophyll, Rubisco and nitrate contents. Chlorophyll a/b ratios and specific leaf fresh weight were lower than in the wild-type. Leaf ammonium contents were similar to the wild-type and total leaf amino-acid contents were only affected in low CO₂ at high photon flux densities, but mutants with decreased Fd-GOGAT accumulated glutamine and contained less glutamate.Abbreviations Chl chlorophyll - FBPase fructose-1,6-bisphosphatase - Fd-GOGAT ferredoxin-dependent glutamine: 2-oxoglutarate aminotransferase - GS glutamine synthetase - PEP phosphoenolpyruvate - PFD photon flux density - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase This research was jointly supported by the Agricultural and Food Research Council and the Science and Engineering Research Council, U.K. in the programme on Biochemistry of Metabolic Regulation in Plants (PG50/555).