Relationship between active oxygen species and cardenolide production in cell cultures of Digitalis thapsi: effect of calcium restriction

Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H₂O₂ accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H₂O₂, the superoxide anion was also formed before dismutating to H₂O₂. Additionally, exogenous H₂O₂ or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H₂O₂ and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl₃. All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.     

Flooding, gas exchange and hydraulic root conductivity of highbush blueberry

Highbush blueberry plants ( Vaccinium corymbosum L. cv. Bluecrop) growing in containers were flooded in the laboratory for various durations to determine the effect of flooding on carbon assimilation, photosynthetic response to varying CO₂ and O₂ concentrations and apparent quantum yield as measured in an open flow gas analysis system. Hydraulic conductivity of the root was also measured using a pressure chamber. Root conductivity was lower and the effect of increasing CO₂ levels on carbon assimilation less for flooded than unflooded plants after short-(i-2 days), intermediate-(10–14 days) and long-term (35–40 days) flooding. A reduction in O₂ levels surrounding the leaves from 21 to 2% for unflooded plants increased carbon assimilation by 33% and carboxylation efficiency from 0.012 to 0.021 mol CO₂ fixed (mol CO₂)⁻¹. Carboxylation efficiency of flooded plants, however, was unaffected by a decrease in percentage O₂, averaging 0.005 mol CO₂ fixed (mol CO₂)⁻¹. Apparent quantum yield decreased from 2.2 × 10⁻¹ mol of CO₂ fixed (mol light)⁻¹ for unflooded plants to 2.0 × 10⁻³ and 9.0 × 10⁻⁴ for intermediate- and long-term flooding durations, respectively. Shortterm flooding reduced carbon assimilation via a decrease in stomatal conductance, while longer flooding durations also decreased the carboxylation efficiency of the leaf.     

Effect of darkness and CO2 starvation on NH4+ and NO3− assimilation in the unicellular alga Cyanidium caldarium

Cyanidium caldarium (Tilden) Geitler, a non-vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO₂, assimilated NH₄⁺ at high rates both in the light and in the dark. The assimilation of NO₃⁻, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO₂-free air, NH₄⁺ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO₂ completely restored the capacity of the alga to assimilate NH₄⁺. NO₃⁻ assimilation, by contrast, was 33% higher in the absence of CO₂ and was linear with time. It is suggested that NO₃⁻ and NH₄⁺ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.     

The quenching of chlorophyll a fluorescence as a consequence of the transport of inorganic carbon by the cyanobacterium Synechococcus UTEX 625

The chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625 decreased upon the initiation of inorganic carbon transport. The fluorescence yield recovered upon the depletion of inorganic carbon from the medium or upon the addition of DCMU. The inhibition of photosynthetic CO₂ fixation by iodoacetamide did not prevent this reduction of fluorescence yield. Similar results were obtained for both Na⁺-stimulated HCO₃⁻ transport and for the transport (presumably of CO₂) that is stimulated by carbonic anhydrase. A transient lowering of the fluorescence yield was also observed when cell suspensions were pulsed with CO₂. In cells not inhibited with iodoacetamide, a very close quantitative relationship existed between the net rate of O₂ evolution and the maximum extent of fluorescence quenching seen as a function of the inorganic carbon concentration. The fluorescence quenching, however, was not due to CO₂ fixation but rather to the transport of inorganic carbon or the accumulation of the internal pool of inorganic carbon. If quenching is due to the latter it is not surprising that the extent of quenching corresponds to the maximum rate of photosynthesis as the rate of photosynthesis also depends on the size of the internal pool. The results with DCMU suggest that the quenching is Q quenching and transport must provide a mechanism for the oxidation of Q other than CO₂ fixation.     

Exchange of oxygen and its role in energy dissipation during drought stress in tomato plants

To elucidate how excess light energy is dissipated during water deficit, net photosynthesis (P_N), stomatal conductance (g_s), intercellular CO₂ concentration (c_i) and Chl a fluorescence were investigated in control and drought-stressed tomato plants ( Lycopersicon esculentum ). Gross O₂ evolution (E_o) and gross O₂ uptake (U_o) were determined by a mass spectrometric ¹⁶O/¹⁸O₂ isotope technique. Under drought stress P_N, g_s, c_i and U_o decline. While photochemical fluorescence quenching decreases under water deficit, non-photochemical quenching rises. The maximal efficiency of PSII measured in the dark is not affected by drought; however, in the light, E_o decreases under water deficit. The ratio P_N/E_o falls under stress while the ratio U_o/E_o increases. We conclude that tomato plants follow a double strategy to avoid photodamage under drought stress conditions: (1) a substantial portion of light energy is emitted as heat and PSII activity is downregulated. This results in a decrease in E_o as well as P_N and U_o. Despite reduced charge separation at PSII, the decline of CO₂ assimilation because of lowered stomatal conductance and metabolic changes results in the need of degrading excessive photosynthetic electrons. (2) Oxygen is used as an alternative electron acceptor in photorespiration or Mehler reaction and U_o rises relative to E_o.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

A precise potentiometric method for determination of algal activity in an open CO2 system

Abstract. The activity of the green alga Scenedesmus obliquus was studied in simplified nutrient solutions (20 mol m₋₃ NaNO₃, 20 mol m₋₃ NH₄C1, 20 mol m₋₃ NH₄NO₃, and 20 mol m₋₃ NaCl, respectively) at 25 °C. The experiments were performed under welldefined incident photon density fluxes ranging from 10 to 200 μmol m₂ s₋₁, Light-dependent changes in pH and alkalinity (A) were followed by means of a potentiometric method using a glass electrode. In the experiments, carbon dioxide with known partial pressure was bubbled through the algal suspension, and during dark periods ul intervals of 1 h, the solution was allowed to equilibrate with the gas phase. This technique was applied to calculate equilibrium values of pH and alkalinity at regular intervals during a 12-h period. Results obtained in NaNO₃, solution show a linear increase in A with time, at each level of illumination studied. After an initial drop, A also increases in NH₄NO₃, solution in a similar way to that in NaNO₃ solution. The change in A with time was also found to increase linearly with the photon density flux studied and no saturation level could be defined. In experiments in NaCl solution, no changes in A were registered while measurements in NH₄Cl solution showed a decrease in A with time.     

Physiological effects of long-term exposure to low and moderate concentrations of atmospheric NH3 on poplar leaves

Abstract. Poplar shoots ( Populus euramericana L.) obtained from cuttings were exposed for 6 or 8 weeks to NH₃ concentrations of 50 and 100 μgm⁻³ or filtered air in fumigation chambers. After this exposure the rates of NH₃ uptake, transpiration, CO₂ assimilation and respiration of leaves were measured using a leaf chamber. During the long-term exposure also modulated chlorophyll fluorescence measurements were carried out to obtain information about the photosynthetic performance of individual leaves. Both fluorescence and leaf chamber measurements showed a higher photosynthetic activity of leaves exposed to 100 μg NH₃ m⁻³. These leaves showed also a larger leaf conductance and a larger uptake rate of NH₃ than leaves exposed to 50 μg m⁻³ NH₃ or filtered air. The long-term NH₃ exposure did not induce an internal resistance against NH₃ transport in the leaf, nor did it affect the leaf cuticle. So, not only at a short time exposure, but also at a long-term exposure NH₃ uptake into leaves can be calculated from data on the boundary layer and stomatal resistance for H₂O and ambient NH₃-concentration. Furthermore, the NH₃ exposure had no effect on the relation between CO₂-assimilation and stomatal conductance, indicating that NH₃ in concentrations up to 100 μg m⁻³ has no direct effect on stomatal behaviour; for example, by affecting the guard or contiguous cells of the stomata.     

Effects of manganese toxicity on photosynthesis of white birch (Betula platyphylla var. japonica) seedlings

The effects of manganese (Mn) toxicity on photosynthesis in white birch ( Betula platyphylla var. japonica ) leaves were examined by the measurement of gas exchange and chlorophyll fluorescence in hydroponically cultured plants. The net photosynthetic rate at saturating light and ambient CO₂ (C_a) of 35 Pa decreased with increasing leaf Mn concentrations. The carboxylation efficiency, derived from the difference in CO₂ assimilation rate at intercellular CO₂ pressures attained at C_a of 13 Pa and O Pa, decreased with greater leaf Mn accumulation. Net photosynthetic rate at saturating light and saturating CO₂ (5%) also declined with leaf Mn accumulation while the maximum quantum yield of O₂ evolution at saturating CO₂ was not affected. The maximum efficiency of PSII photochemistry (F_v/F_m) was little affected by Mn accumulation in white birch leaves over a wide range of leaf Mn concentrations (2–17 mg g₋₁ dry weight). When measured in the steady state of photosynthesis under ambient air at 430 μmol quanta m₋₂ s₋₁, the levels of photochemical quenching (qP) and the excitation capture efficiency of open PSII (F'^v/F'^m) declined with Mn accumulation in leaves. The present results suggest that excess Mn in leaves affects the activities of the CO² reduction cycle rather than the potential efficiency of photochemistry, leading to increases in Q_A reduction state and thermal energy dissipation, and a decrease in quantum yield of PSII in the steady state.     

Chlorophyll fluorescence measured using the Fraunhofer line-depth principle and relationship to photosynthetic rate in the field

Abstract. A field study was conducted to determine the relationship of solar-excited chlorophyll a fluorescence to net CO₂ assimilation rate in attached leaves. The Fraunhofer line-depth principle was used to measure fluorescence at 656.3 nm wavelength while leaves remained exposed to full sunlight and normal atmospheric pressures of CO₂ and O₂. Fluorescence induction kinetics were observed when leaves were exposed to sunlight after 10 min in darkness. Subsequently, fluorescence varied inversely with assimilation rate. In the C₄ Zea mays , fluorescence decreased from 2.5 to 0.8 mW m^-2 nm^-1 as CO₂ assimilation rate increased from 1 to 8 μmol m^-2 s^-1 (r²= 0.520). In the C₃ Liquidambar styraciflua and Pinus taeda , fluorescence decreased from 6 to 2 mW m^-2 nm^-1 as assimilation rate increased from 2 to 5 or 0 to 2 μmol m^-2 s^-1 (r²= 0.44 and 0.45. respectively). The Fraunhofer line-depth principle enables the simultaneous measurement of solar-excited fluorescence and CO₂ assimilation rate in individual leaves, but also at larger scales. Thus, it may contribute significantly to field studies of the relationship of fluorescence to photosynthesis.     

Direct and indirect effects of light on stomata II. In Commelina communis L.

Abstract. Two experiments are described which test the normal correlations that arise between stomatal conductance, net CO₂ assimilation rate, and intercellular CO₂ concentration (C_i), using whole shoots of Commelina communis L. In the first, conductance increased with decreasing C_i, at four different quantum flux densities, such that there was no unique relationship between conductance and quantum flux density or C_i, In the second, conductance increased hyperbolically with increasing quantum flux density while C_i was held constant at 466, 302, and 46 μmiolmol⁻¹, and the response differed at each C_i. In neither experiment was conductance consistently related to net CO₂ assimilation rate in the mesophyll. In both experiments high C_i suppressed the response of conductance to light, while there was a large response of conductance to light at low C_i, indicating an interaction between the effects of light and CO₂ on stomata. The results show that the parallel responses of assimilation and conductance to light result in constant intercellular CO₂ concentrations, and not that stomata maintain a 'constant C_i'.     

Changes in photosystem II function during senescence of wheat leaves

Analyses of chlorophyll fluorescence were undertaken to investigate the alterations in photosystem II (PSII) function during senescence of wheat ( Triticum aestivum L. cv. Shannong 229) leaves. Senescence resulted in a decrease in the apparent quantum yield of photosynthesis and the maximal CO₂ assimilation capacity. Analyses of fluorescence quenching under steady‐state photosynthesis showed that senescence also resulted in a significant decrease in the efficiency of excitation energy capture by open PSII reaction centers (F'_v/F'_m) but only a slight decrease in the maximum efficiency of PSII photochemistry (F'_v/F'_m). At the same time, a significant increase in non‐photochemical quenching (q_N) and a considerable decrease in photochemical quenching (q_P) were observed in senescing leaves. Rapid fluorescence induction kinetics indicated a decrease in the rate of Q_A reduction and an increase in the proportion of Q_B‐non‐reducing PSII reaction during senescence. The decrease in both F'_v/F'_m and q_P explained the decrease in the actual quantum yield of PSII electron transport ((φ_PSII). We suggest that the modifications in PSII function, which led to the down‐regulation of photosynthetic electron transport, would be in concert with the lower demand for ATP and NADPH in the Calvin cycle which is often inhibited in senescing leaves.     

Photosynthesis of two poplar clones under long‐term exposure to ozone

The photosynthetic response was studied in two clones ( Populus deltoides × maximowiczii Eridano and Populus × euramericana I‐214), known for their differential response to ozone (O₃) in terms of visible symptoms, when exposed to O₃ (60 nl l⁻¹ 5 h day⁻¹, 7 and 15 days). The photosynthetic ability was tested using gas exchange and chlorophyll fluorescence analysis. O₃ caused a decrease in the CO₂ assimilation rate at light saturation level in mature leaves of both clones. Alterations of Chl fluorescence parameters, in particular the F_v/F_m ratio and non‐photochemical quenching were also observed. The effects were similar for both clones and it could not be concluded that differential effects on electron transport capacity were responsible for the observed reduction in photosynthesis. The reduction of photosynthetic rate in Eridano was due mainly to a reduced mesophyll activity, as evidenced by the increase in intercellular CO₂ concentration and the minimal changes in stomatal conductance. In contrast, in I‐214, stomatal effects were primarily responsible, although effects on the mesophyll cannot be excluded. Data obtained indicate that the effects observed at the mesophyll level may be attributed to indirect effects caused by membrane disorders.     

Depression of carbon flow to the glycogen pool induced by nitrogen assimilation in intact cells of Anacystis nidulans

The influence of nitrate and ammonium assimilation on glycogen metabolism has been determined in intact Anacystis nidulans cell actively fixing CO₂. Assimilation of nitrate or ammonium resulted in significant decreases in both the incorporation into glycogen of newly fixed carbon and the accumulation of glycogen by the cells, the magnitude of these effects depending on the light intensity. The depression in glycogen synthesis induced by nitrogen assimilation was more marked at low light intensity, and especially when ammonium was the nitrogen source. Under these conditions, specific radioactivity of the glycogen pool was particularly high, indicating enhanced turnover of glycogen. Thus, in addition to a more general depressing effect of nitrogen assimilation on the carbon flow to glycogen, degradation of glycogen appears to be stimulated by ammonium assimilation at low (but not at high) light intensity.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3. II. Effects of phosphorus limitation

The relationship between CO₂ assimilation rate, growth and partitioning of carbon among starch, sucrose, glucose and fructose were studied in phosphorus (P_i)-limited Lemna gibba L. G3. Two experimental models were used: 1) Cultures were grown at various stable, suboptimal rates regulated by the supply of P_i; 2) cultures growing at optimal rates were transferred to P_i-free medium. The response to a P_i deficiency can be divided into two phases. Phase I is characterized by hyperactivity of the sucrose synthesis pathway, leading to high levels of glucose and fructose. Phase II is characterized by starch accumulation associated with a decrease in the cytoplasmic pools of soluble sugars owing to inhibition of carbon export from the chloroplast. A strong negative correlation was found between the CO₂ assimilation rate and starch levels. No significant correlation was found between assimilation and ATP levels and decrease in relative growth rate did not significantly affect the adenylate energy charge (EC). The regulatory aspects of the partitioning of carbon among soluble sugars and starch as well as the negative correlation between carbohydrate levels and CO₂ assimilation at P_i-limited growth are discussed.     

The effects of cadmium on photosynthesis of Phaseolus vulgaris – a fluorescence analysis

Bean plants ( Phaseolus vulgaris L. cv. Scarlett), germinated in darkness for I week, were transferred to light (200 μmol m⁻² s⁻¹) and cultivated for I week in a complete nutrient solution. After this period, cadmium ions in the form of CdSO₄ were added at the concentrations of 0.10.20 and 50 μ M . The effects of this metal on the properties of photosystem II photochemistry were studied by means of modulated fluorescence analysis. Steady state photochemical quenching. non-photochemical quenching and terminal fluorescence were determined in control and cadmiumtreated plants. We postulate that, during short term exposure of plants to cadmium in the early stages of growth, the Calvin cycle reactions are more likely than photosystem II to be the primary target of the toxic influence of cadmium. The reduced demand for ATP and NADPH upon Calvin cycle inhibition causes a down-regulation of photosystem II photochemistry and of the yield of linear electron transport.     

Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77 K

Photoinhibition in Lemna gibba L. was studied by interpreting chlorophyll fluorescence characteristics at 77 K on the basis of the bipartite model of Butler and co-workers (Butler 1978). Application of this analysis to chloroplasts (isolated from plants before and after exposure to a photosynthetic photon flux density of 1 750 μmol m⁻² s⁻¹ at 3°C for 2 h) revealed that photoinhibition had the following effect on primary events in photosynthesis. Firstly, the fluorescence of PS II increased (44%) in the state of open traps (F_o) and decreased (32%) in the state of closed traps (F_m). It is suggested, that the F_o-decrease reflects increased quenching by radiationless decay, both effects occurring at PS II reaction centers. Secondly, the rate constant for transfer of excitation energy from PS II to PS I (k_T(μ→J)) increased by 34%. However, in the state of closed traps, the flux of excitation energy via this transfer process decreased, most likely because of increased quenching by radiationless decay at PS II reaction centers. Thirdly, the probability for fluorescence from PS I decreased (19%). This indicates increased quenching by radiationless decay.     

Effect of root zone carbon dioxide enrichment on ethylene inhibition of carbon assimilation in potato plants

Potato plants ( Solanum tuberosum L. var. Russet Burbank) treated with 1 μl ethylene 1⁻¹ of air showed an inhibition of CO₂ assimilation by 18%. The inhibition occurred after 3 h of exposure to ethylene and was not mediated through closure of the stomata. The enrichment of the root zone with CO₂ almost completely abolished the ethylene inhibition of CO₂ assimilation which was apparently due to an increase in the intercellular concentration of CO₂ in leaves following enrichment. The effect of application of CO₂ to the root zone on ethylene inhibition of CO₂ assimilation seemed to last for a few days. Potato plants treated with aminoethoxyvinlglycine (AVG) showed an increase in fresh and dry weight as compared to non-treated plants. Our results indicate that both CO₂ and AVG alter the effect of ethylene and promote growth in plants by inhibiting ethylene action and biosynthesis, respectively.     

Modification of the pII protein in response to carbon and nitrogen availability in filamentous heterocystous cyanobacteria

Abstract In the filamentous cyanobacterium Calothrix PCC 7504, which fixes N₂ aerobically, the modification state of the regulatory P_II protein (GlnB) was shown to depend on nitrogen and carbon availability, as observed in the unicellular non-fixing strain Synechococcus PCC 7942. However, the conditions for modifications, the time dependence of the process and the electrophoretic behavior of the native P_II isoforms differed somewhat between the two strains. In another strain, Calothrix PCC 7601, which has lost the capability to fix N₂, P_II was modified only if ammonia plus an inhibitor of glutamine synthetase were present. It is proposed that: (i) the behavior of the P_II proteins depends upon the physiological properties of the strains; and (ii) the modification system of P_II per se may differ between the two cyanobacterial genera.     

CO2 assimilation, respiration and chlorophyll fluorescence in peach leaves infected by Taphrina deformans

Photosynthesis, respiration and chlorophyll fluorescence parameters were determined in peach ( Prunus persica L. cv. Dixired) leaves naturally infected by Taphrina deformans (Berk.) Tul. and in healthy leaves (controls), in two successive springs. A drastic decrease in net photosynthesis and an evident increase in respiration in curled leaves were noted. The instantaneous PSII fluorescence yield, with no (F₀) and with (F₀) quenching component, and steady state fluorescence yield (under actinic light, F_s) were essentially unchanged. Maximal fluorescence in dark-adapted (F_m) and illuminated (F'_m) leaves and the corresponding variable fluorescence (F_v and F_v) clearly decreased. The indicators of PSII quantum yield (F_v/F_m) in dark-adapted leaves, and the potential PSII excitation capture efficiency (F'_v/F'_m) and the quantum yield of PSII (q_p [F'_v/F'_m]) in the light were also significantly lower in curled leaves. Decreasing tendencies were also noted for the PSII photochemical yield (photochemical quenching, q_p) and in the energy status of the chloroplast (non-photochemical quenching, q_N, and Stern-Vollmer value, NPQ) although the differences were not always significant. In curled leaves the main alteration documented is the imbalance between the drastic inhibition of CO₂ fixation and the moderate decrease in photochemical reactions (i.e. F_v/F_m and ΔF/F'_m), indicating changes in the energy flux.