Differential expression of CuZn-superoxide dismutases in Pinus sylvestris needles exposed to SO2 and NO2

Mature Pinus sylvestris trees were exposed to air-pollution strees in an open field fumigation experiment. The trees were exposed to low-levels of sulphur dioxide and nitrogen oxides throughout the whole growing season (June to October 1991). Three age-classes (current, one- and two-year-old) of needles were collected from exposed and control trees and the expression of chloroplastic and cytosolic CuZn-superoxide dismutase (SOD; EC 1.15.1.1) genes was studied. The levels of chloroplastic and cytosolic CuZn-SOD mRNA were significantly (up to 2.3-fold) higher in exposed trees for all age-classes of needles as compared to control trees. No significant differences were observed between different needle age-classes in CuZn-SOD mRNA induction. The chloroplastic and cytosolic CuZn-SOD mRNA were induced to the same extent, however, the level of chloroplastic CuZn-SOD mRNA was higher than the cytosolic one. Small differences were observed in CuZn-SOD mRNA levels for individual trees both within exposed and control areas. Protein activity measurements of the CuZn-SOD isozymes did not reveal any significant differences between control and exposed trees. The results are discussed in relation to protection against oxidant stress from air pollutants.     

Diagnostic parameters for selecting against novel spruce (Picea abies) decline: IV. Response of photosynthesis and transpiration to SO2+NO2 exposures

The dose- and time-response effects of 3 days of 6 h day-time sequential exposures to NO₂, SO₂ and SO₂+NO₂ of 0.45–1.81 μl l⁻¹ (ppm) SO₂ and 1.50–7.65 μl l⁻¹ NO₂ on photosynthesis, transpiration and dark respiration were examined for nine Carpatho-Ukrainian half-sib families and a population from the GFR ('Westerhof') of Norway spruce [ Piecea abies (L.) Karst.], all in their 5th growing season.
SO₂+NO₂ inhibited photosynthesis and transpiration and stimulated dark respiration more than SO₂ alone. SO₂ and SO₂+NO₂ at the lowest concentrations inhibited night transpiration, but increased it at the highest concentration, the strongest effects being obtained with combined exposures. Photosynthesis of the different half-sib families was affected significantly differently by SO₂+NO₂ exposures. NO₂ alone had no effects.
Sensitivity to transpiration decline correlated negatively with branch density. Height of trees correlated postitively with decline sensitivity in the seed orchard. The distribution of photosynthesis and transpiration sensitivities over all tested half-sib families correlated negatively with the distribution of decline sensitivity of their parents in a rural Danish seed orchard. The relative photosynthesis and transpiration sensitivities may thus serve as diagnostic parameters for selecting against novel spruce decline.     

Persistent effects of low concentrations of SO2 and NO2 on photosynthesis in Scots pine (Pinus sylvestris) needles

In an open-field experiment, 50-year-old trees of Scots pine (Pinus sylvestris L.) were fumigated with low concentrations of SO₂ and NO₂ (10–15 nl I^?1) during the growing season in four consecutive years (1988 to 1991). Results from the autumn and early winter of 1991 and 1992 are presented. The maximum photochemical efficiency of photosystem II (PSII), as indicated by the ratio of variable to maximum fluorescence (F_v/F_M) was assessed in current and one-year-old needles from the top and the bottom of the canopy. Furthermore, simultaneous measurements of photosynthetic O₂ evolution and chlorophyll fluorescence were made in current-year needles at 20°C. In general, the F_v/F_M ratio as well as the gross rate of O₂ evolution in needles of fumigated trees was not significantly different from that in needles of control trees during the fumigation period. However, both current and one-year-old needles sampled in November and December 1991 from the top of the canopy of fumigated trees had significantly lower F_v/F_M values than corresponding needles of control trees. Similar differences in F_v/F_M correlated with the treatments were observed in needles from the bottom of the canopy, indicating that the depression of F_v/F_M in needles of fumigated trees was not due to an increased susceptibility to photoinhibition. In 1992, when no fumigation occurred, differences in F_v/F_M between the treatments were not significant during autumn and early winter. The gross rate of O₂ evolution at high irradiances was significantly lower in current-year needles of fumigated trees sampled in November and December 1991 than in those of control trees. Furthermore, a nearly identical linear relationship between the quantum yield of PSII electron transport determined from chlorophyll fluorescence and the quantum yield of O₂ evolution (gross rate of O₂ evolution/PPFD) was found during autumn and early winter. This appeared to be largely a result of changes in the thermal energy dissipation within PSII. The observed differences in photosynthetic characteristics correlated with the different treatments after the fumigation period is suggested to be mainly caused by increased sensitivity of the needles of fumigated trees to low and subfreezing temperatures. However, current-year needles of fumigated trees tended to have a lower N content than those of control trees, which may partly explain the differences in gross photosynthesis between fumigated and control trees.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Physiological effects of a long term exposure to low concentrations of NH3, NO2 and SO2 on Douglas fir (Pseudotsuga menziesii)

The above-ground parts of two years old seedlings of Douglas fir (Pseudotsuga menziesii) were exposed to filtered air, NH₃, NO₂₊, SO₂ (66, 96 and 95 μg m^?3, respectively), to a mixture of NO₂+NH₃ (55 + 82 μg m^?3) or SO₂+NO₂ (128 + 129 μg m^?3), for 8 months in fumigation chambers. Both chlorophyll fluorescence and gas exchange measurements were carried out on shoots which had sprouted at the beginning of the exposure period. The chlorophyll fluorescence measurements were performed after 3 and 5 months of exposure (average shoot age 70 and 140 days, respectively). Light response curves of electron transport rate (J) were determined, in which J was deduced from chlorophyll fluorescence. In addition, light response curves of net CO₂ assimilation were determined after 5 months of exposure. After 3 months of exposure (average shoot age 70 days) all exposure treatments showed a lower maximum electron transport rate (J_max) as compared to the control shoots (filtered air). A large reduction (45%) was observed for shoots exposed to SO₂+NO₂. During the exposure period between 3 and 5 months (average shoot age 70 and 140 days, respectively) a decrease of J_max was observed for all treatments. J_max had further declined some time after termination of the exposure, when average shoot age was 310 days. Shoots exposed to SO₂ and SO₂+NO₂ also showed a reduction in maximum net CO₂ assimilation (P_max) as compared to the control shoots. However, shoots exposed to NO₂ showed no reduction and even a higher P_max was observed for shoots exposed to NH₃ or NO₂+NH₃. Needles of these treatments also showed a higher chlorophyll content which might explain the contradictory results obtained for these treatments: the increased amount of photosynthetic units counteracts the reduction in J_max and consequently no reduction in P_max is measured. Shoots exposed to SO₂ and SO₂+NO₂ also showed a reduction in maximum stomatal conductance (g_s). However, the stomatal opening was larger than could be expected on basis of their (maximum) CO₂ assimilation rate. Consequently, water use efficiency of these shoots was lower than that of the control shoots. Also shoots exposed to NO₂ had a lower water use efficiency due to a significantly higher maximum g_s. Shoots exposed to NH₃ showed a high transpiration rate in the dark, indicating imperfect stomatal closure.     

Blue-light-induced pH changes associated with NO3−, NO2− and Cl− uptake by the green alga Monoraphidium braunii

In M. braunii, the uptake of NO₃⁻ and NO₂⁻ is blue-light-dependent and is associated with alkalinization of the medium. In unbuffered cell suspensions irradiated with red light under a CO₂-free atmosphere, the pH started to rise 10s after the exposure to blue light. When the cellular NO₃⁻ and NO₂⁻ reductases were active, the pH increased to values of around 10, since the NH₄⁺ generated was released to the medium. When the blue light was switched off, the pH stopped increasing within 60 to 90s and remained unchanged under background red illumination. Titration with H₂SO₄ of NO₃⁻ or NO₂⁻ uptake and reduction showed that two protons were consumed for every one NH₄⁺ released. The uptake of Cl⁻ was also triggered by blue light with a similar 10 s time response. However, the Cl⁻ -dependent alkalinization ceased after about 3 min of blue light irradiation. When the blue light was turned off, the pH immediately (15 to 30 s) started to decline to the pre-adjusted value, indicating that the protons (and presumably the Cl⁻) taken up by the cells were released to the medium. When the cells lacked NO₃⁻ and NO₂⁻ reductases, the shape of the alkalinization traces in the presence of NO₃⁻ and NO₂⁻ was similar to that in the presence of Cl⁻, suggesting that NO₃⁻ or NO₂⁻ was also released to the medium. Both the NO₃⁻ and Cl⁻-dependent rates of alkalinization were independent of mono- and divalent cations.     

Enriched rhizosphere CO2 concentrations can ameliorate the influence of salinity on hydroponically grown tomato plants

Our previous work indicated that salinity caused a shift in the predominant site of nitrate reduction and assimilation from the shoot to the root in tomato plants. In the present work we tested whether an enhanced supply of dissolved inorganic carbon (DIC, CO₂+ HCO₃) to the root solution could increase anaplerotic provision of carbon compounds for the increased nitrogen assimilation in the root of salinity-stressed Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were grown in hydroponic culture with 0 or 100mM NaCl and aeration of the root solution with either ambient or CO₂-enriched air (5000 μmol mol^?1). The salinity-treated plants accumulated more dry weight and higher total N when the roots were supplied with CO₂-enriched aeration than when aerated with ambient air. Plants grown with salinity and enriched DIC also had higher rates of NO^?₃ uptake and translocated more NO^?₃ and reduced N in the xylem sap than did equivalent plants grown with ambient DIC. Incorporation of DIC was measured by supplying a 1 -h pulse of H¹⁴CO^?₃ to the roots followed by extraction with 80% ethanol. Enriched DIC increased root incorporation of DIC 10-fold in both salinized and non-salinized plants. In salinity-stressed plants, the products of dissolved inorganic ¹⁴C were preferentially diverted into amino acid synthesis to a greater extent than in non-salinized plants in which label was accumulated in organic acids. It was concluded that enriched DIC can increase the supply of N and anaplerotic carbon for amino acid synthesis in roots of salinized plants. Thus enriched DIC could relieve the limitation of carbon supply for ammonium assimilation and thus ameliorate the influence of salinity on NO^?₃ uptake and assimilation as well as on plant growth.     

Influence of NaCl, Cd(NO3)2 and air humidity on transpiration of Tamarix aphylla

Transpiration rates of young Tamarix aphylla (L.) Karst, plants grown in hydroponics were measured under NaCl- and Cd(NO₃)₂-stress. Transpiration rates were negatively correlated with the relative humidity of the ambient air at all NaCl concentrations investigated. Low and intermediate concentrations of Cd²⁺ (45 and 90 μ M , respectively) in the medium caused an increase in transpiration rates. This was particularly pronounced at low levels of relative humidity. At 180 μ M Cd²⁺, transpiration rates dropped, probably as a result of root damage due to Cd²⁺ toxicity. Since the transpiration rates differed by a factor of ca 3 between day and night, it is concluded that the stomata did not lose their ability to regulate transpiration under the influence of NaCl or of Cd(NO₃)₂. The transpiration behaviour of T. aphylla indicates that the effect of water vapour pressure (presented as relative humidity) on the degree of stomatal opening is small. Under conditions of ample water supply transpiration follows the evaporative demand of the ambient air and is influenced by the water uptake capacity of the root system as well as by other environmental factors, e.g. light.     

The effects of reduced and elevated CO2 and O2 on the seaweed Lomentaria articulata

We grew a non-bicarbonate using red seaweed, Lomentaria articulata (Huds.) Lyngb., in media aerated with four O₂ concentrations between 10 and 200% of current ambient [O₂] and four CO₂ concentrations between 67 and 500% of current ambient [CO₂], in a factorial design, to determine the effects of gas composition on growth and physiology. The relative growth rate of L. articulata increased with increasing [CO₂] up to 200% of current ambient [CO₂] but was unaffected by [O₂]. The relative growth enhancement, on a carbon basis, was 52% with a doubling of [CO₂] but fell to 23% under 5× ambient [CO₂]. Plants collected in winter responded more extremely to [CO₂] than did plants collected in the summer, although the overall pattern was the same. Discrimination between stable carbon isotopes (Δ¹³C) increased with increasing [CO₂] as would be expected for diffusive CO₂ acquisition. Tissue C and N were inversely related to [CO₂]. Growth in terms of biomass appeared to be limited by conversion of photosynthate to new biomass rather than simply by diffusion of CO₂, suggesting that non-bicarbonate-using macroalgae, such as L. articulata, may not be directly analogous to C3 higher plants in terms of their responses to changing gas composition.     

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.