Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.     

Photosynthetic net O<Subscript>2</Subscript> evolution enhancement as a sign of acclimation to phosphorus deficiency in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) leaves

Primary leaves of bean (Phaseolus vulgaris L.) seedlings cultivated for 14 days in a growth chamber on complete (control) and phosphate deficient (−P) Knop liquid medium were used for measurements. The −P leaves were smaller and showed an increased specific leaf area (SLA). Their inorganic phosphate (P_i) concentration was considerably lowered. They did not show any significant changes in chlorophyll (Chl) (a + b) concentration and in their net CO₂ assimilation rate when it was estimated under the conditions close to those of the seedlings growth. Light response curves of photosynthetic net O₂ evolution (P _NO₂) of the leaves for the irradiation range up to 500 μmol(photon) m⁻² s⁻¹ were determined, using the leaf-disc Clark oxygen electrode. The measurements were taken under high CO₂ concentration of about 1 % and O₂ concentrations of 21 % or lowered to about 3 % at the beginning of measurement. The results obtained at 21 % O₂ and the irradiations close to or higher than those used during the seedlings growth revealed the phosphorus stress suppressive effect on the leaf net O₂ evolution, however, no such effect was observed at lower irradiations. Other estimated parameters of P _NO₂ such as: apparent quantum requirement (QR_A) and light compensation point (LCP) for the control and −P leaves were similar. However, with a high irradiation and lowered O₂ concentration the rate of P _NO₂ for the −P leaves was markedly higher than that for the control, in relation to both the leaf area and leaf fresh mass. This difference also disappeared at low irradiations, but the estimated reduced QR_A values indicate, under those conditions, the increased yield of photosynthetic light reaction, especially in the −P leaves. The presented results confirm the suggestion that during the initial phase of insufficient phosphate feeding the acclimations in the light phase of photosynthesis, both structural and functional appear. They correspond, probably, to the increased energy costs of carbon assimilation under phosphorus stress, e.g. connected with raised difficulties in phosphate uptake and turnover and enhanced photorespiration. Under the experimental conditions especially advantageous for the dark phase of photosynthesis (saturating CO₂ and PAR, low O₂ concentration), those acclimations may be manifested as an enhancement of photosynthetic net O₂ evolution.     

Brassinosteroids Alleviate Heat-Induced Inhibition of Photosynthesis by Increasing Carboxylation Efficiency and Enhancing Antioxidant Systems in <Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>

To investigate the effects of exogenously applied brassinosteroids on the thermotolerance of plants, leaf CO₂ assimilation, chlorophyll fluorescence parameters, and antioxidant enzyme metabolism were examined in tomato (Lycopersicon esculentum Mill. cv. 9021) plants with or without 24-epibrassinolide (EBR) application. Tomato plants were exposed to 40/30°C for 8 days and then returned to optimal conditions for 4 days. High temperature significantly decreased the net photosynthetic rate (P _n), stomatal conductance (G _s), and maximum carboxylation rate of Rubisco (V _cmax), the maximum potential rate of electron transport contributed to ribulose-1,5-bisphosphate (RuBP), as well as the relative quantum efficiency of PSII photochemistry (Ф_PSII), photochemical quenching (q _P), and increased nonphotochemical quenching (NPQ). However, only slight reversible photoinhibition occurred during heat stress. Interestingly, EBR pretreatment significantly alleviated high-temperature-induced inhibition of photosynthesis. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPOD), and catalase (CAT) increased during heat treatments, and these increases proved to be more significant in EBR-treated plants. EBR application also reduced total hydrogen peroxide (H₂O₂) and malonaldehyde (MDA) contents, while significantly increasing shoot weight following heat stress. It was concluded that EBR could alleviate the detrimental effects of high temperatures on plant growth by increasing carboxylation efficiency and enhancing antioxidant enzyme systems in leaves.     

The activity of antioxidative enzymes,contents of H<Subscript>2</Subscript>O<Subscript>2</Subscript> and of ascorbate in tomato leaves of cultivars more or less sensitive to infection with <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

The aim of the present studies was to compare H₂O₂ and ascorbate contents as well as peroxidase (PO) and catalase (CAT) activities in leaves of less susceptible cultivar Perkoz and more susceptible Corindo after B. cinerea infection. Increase in H₂O₂ contents in both Perkoz and Corindo cytosol was observed, however, it appeared earlier in the less susceptible cultivar. The increase in PO activity in the cytosol fraction was observed 48 hours after infection in both cultivars but it was greater in the less susceptible Perkoz. No significant differences between the tested cultivars were observed in ascorbate peroxidase (APX) activity and in reduced and oxidated ascorbate contents. PO activity was thoroughly analyzed in the apoplast fraction. It was measured with syringaldazine (S), tetramethylbenzidine (TMB) and ferulic acid (FA)—substrates characteristic of isoenzymes involved in lignification and stiffening of a cell wall. Increase in PO activity with these substrates was observed earlier in cultivar Perkoz than in cultivar Corindo. Similarly, increase in PO activity with NADH appeared significantly earlier in cultivar Perkoz. Apoplastic PO was separated with DEAE Sepharose and two fractions binding and non-binding were obtained. Binding PO fraction was significantly more active especially with S, TMB and NADH after B. cinerea infection. The increase in the enzyme activity was mostly observed in cultivar Perkoz. Binding PO was separated by electrophoresis on acrylamide gel and revealed six enzymatic forms from which three were much more active after infection in cultivar Perkoz. The obtained results suggest that cell wall strengthening mediated by apoplast PO is a key factor responsible for different resistance of tomato cultivars Perkoz and Corindo to B. cinerea infection.     

Elevated CO<Subscript>2</Subscript> ameliorated oxidative stress induced by elevated O<Subscript>3</Subscript> in <Emphasis Type="Italic">Quercus mongolica</Emphasis>

Using open top chambers, the effects of elevated O₃ (80 nmol mol⁻¹) and elevated CO₂ (700 μmol mol⁻¹), alone and in combination, were studied on young trees of Quercus mongolica. The results showed that elevated O₃ increased malondialdehyde content and decreased photosynthetic rate after 45 days of exposure, and prolonged exposure (105 days) induced significant increase in electrolyte leakage and reduction of chlorophyll content. All these changes were alleviated by elevated CO₂, indicating that oxidative stress on cell membrane and photosynthesis was ameliorated. After 45 days of exposure, elevated O₃ stimulated activities of superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11), but the stimulation was dampened under elevated CO₂ exposure. Furthermore, ascorbate (AsA) and total phenolics contents were not higher in the combined gas treatment than those in elevated O₃ treatment. It indicates that the protective effect of elevated CO₂ against O₃ stress was achieved hardly by enhancing ROS scavenging ability after 45 days of exposure. After 105 days of exposure, elevated O₃ significantly decreased activities of SOD, catalase (CAT, EC 1.11.1.6) and APX and AsA content. Elevated CO₂ suppressed the O₃-induced decrease, which could ameliorate the oxidative stress in some extent. In addition, elevated CO₂ increased total phenolics content in the leaves both under ambient O₃ and elevated O₃ exposure, which might contribute to the protection against O₃-induced oxidative stress as well.     

Effects of salinity on chlorophyll fluorescence and CO<Subscript>2</Subscript> fixation in C<Subscript>4</Subscript> estuarine grasses

The effects of salinity (sea water at 0 ‰ versus 30 ‰) on gross rates of O₂ evolution (J _O2) and net rates of CO₂ uptake (P _N) were measured in the halotolerant estuarine C₄ grasses Spartina patens, S. alterniflora, S. densiflora, and Distichlis spicata in controlled growth environments. Under high irradiance, salinity had no significant effect on the intercellular to ambient CO₂ concentration ratio (C _i/C _a). However, during photosynthesis under limiting irradiance, the maximum quantum efficiency of CO₂ fixation decreased under salinity across species, suggesting there is increased leakage of the CO₂ delivered to the bundle sheath cells by the C₄ pump. Growth under salinity did not affect the maximum intrinsic efficiency of photosystem 2, PS2 (F_V/F_M) in these species, suggesting salinity had no effect on photosynthesis by inactivation of PS2 reaction centers. Under saline conditions and high irradiance, P _N was reduced by 75 % in Spartina patens and S. alterniflora, whereas salinity had no effect on P _N in S. densiflora or D. spicata. This inhibition of P _N in S. patens and S. alterniflora was not due to an effect on stomatal conductance since the ratio of C _i/C _a did not decrease under saline conditions. In growth with and without salt, P _N was saturated at ∼500 μmol(quantum) m⁻² s⁻¹ while J _O2 continued to increase up to full sunlight, indicating that carbon assimilation was not tightly coupled to photochemistry in these halophytic species. This increase in alternative electron flow under high irradiance might be an inherent function in these halophytes for dissipating excess energy.     

Plasma membrane-generated ROS and their possible contribution to leaf cell growth of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) MSC16 mitochondrial mutant

Reactive oxygen species (ROS) generally regarded as harmful products of oxygenic metabolism causing oxidative stress and cell damage are also important for control and regulation of biological processes. ROS can be generated by various enzymatic activities and removed by an array of ROS-scavenging molecules in the cell. In plants, the generation of ROS initiated by the plasma membrane NADPH oxidase can be used for controlled polymer breakdown leading to cell wall loosening during extension growth. The mosaic (MSC16) mitochondrial mutant of cucumber (Cucumis sativus L.) has marked phenotypic changes, including a slower growth rate which partially may result from disturbed leaf carbon and energy metabolism and ROS/antioxidants equilibrium. Cytochemical localization of H₂O₂ in leaf cells showed lower total level of H₂O₂ particularly in the apoplast of MSC16 leaf cells as compared to WT. The activity of plasma membrane NADPH oxidase (EC 1.6.3.1) was about 30% lower in plasmalemma vesicles isolated from MSC16 leaf tissue as compared to WT. The total foliar ascorbate pool (reduced and oxidized) was about 35% higher in MSC16 compared to WT leaves due to an increased content of the oxidized form. About 3% of the whole-leaf ascorbate was localized in the apoplast but in MSC16 it was considerably more reduced. We conclude that the lower apoplastic ROS content caused by decreased activity of plasma membrane NADPH oxidase and lower amounts of H₂O₂ in the apoplast may also contribute to altered growth of the MSC16 cucumber mutant.     

Seasonal physiological responses and biomass growth in a bioenergy crop (<Emphasis Type="Italic">Phalaris arundinacea</Emphasis> L.) under elevated temperature and CO<Subscript>2</Subscript>, subjected to different water regimes in boreal conditions

We investigated the seasonal variability of effects of elevated temperature (+3.5°C), CO₂ elevation (700 μmol mol⁻¹) and varying water regimes (high to low water content) on physiological responses and biomass growth of reed canary grass (Phalaris arundinacea L., local field-grown cultivar) grown in a boreal environment. In controlled environment greenhouses, various physiological and growth parameters of grass, i.e., light-saturated net photosynthetic rates (P _nmax), water use efficiency (WUE) and optimal photochemical efficiency of photosystem II (F _v/F _m), and leaf area development and biomass of plant organs (leaf, stem, coarse, and fine root) were measured. During the early measurement periods, elevated temperature enhanced leaf photosynthesis and above-ground biomass of reed canary grass; however, this resulted in earlier senescence and lower biomass at the end of measurement period, compared to ambient temperature. This effect was more pronounced under water limitation. Elevated CO₂ enhanced P _nmax, WUE, and leaf area and total plant biomass (above- and below-ground) over growing season. The explanation for imbalance between stimulated photosynthesis and increase in above-ground biomass was that CO₂ enrichment causes a greater increase in the plant’s root system. The combination of elevated temperature and CO₂ slightly increases the growth of plant. Adequate water availability favored photosynthesis and biomass growth of reed canary grass. The temperature- and drought-induced stresses were partially mitigated by elevated CO₂. Other cultivars should be tested in order to identify those that are better adapted to elevated temperatures and CO₂ and variable water levels.     

Development of gypsy moth larvae feeding on red maple saplings at elevated CO<Subscript>2</Subscript> and temperature

Predicted increases in atmospheric CO₂ and global mean temperature may alter important plant-insect associations due to the direct effects of temperature on insect development and the indirect effects of elevated temperature and CO₂ enrichment on phytochemicals important for insect success. We investigated the effects of CO₂ and temperature on the interaction between gypsy moth (Lymantria dispar L.) larvae and red maple (Acer rubrum L.) saplings by bagging first instar larvae within open-top chambers at four CO₂/temperature treatments: (1) ambient temperature, ambient CO₂, (2) ambient temperature, elevated CO₂ (+300 l l^-1 CO₂), (3) elevated temperature (+3.5°C), ambient CO₂, and (4) elevated temperature, elevated CO₂. Larvae were reared to pupation and leaf samples taken biweekly to determine levels of total N, water, non-structural carbohydrates, and an estimate of defensive phenolic compounds in three age classes of foliage: (1) immature, (2) mid-mature and (3) mature. Elevated growth temperature marginally reduced (P <0.1) leaf N and significantly reduced (P <0.05) leaf water across CO₂ treatments in mature leaves, whereas leaves grown at elevated CO₂ concentration had a significant decrease in leaf N and a significant increase in the ratio of starch:N and total non-structural carbohydrates:N. Leaf N and water decreased and starch:N and total non-structural carbohydrates:N ratios increased as leaves aged. Phenolics were unaffected by CO₂ or temperature treatment. There were no interactive effects of CO₂ and temperature on any phytochemical measure. Gypsy moth larvae reached pupation earlier at the elevated temperature (female =8 days, P <0.07; male =7.5 days, P <0.03), whereas mortality and pupal fresh weight of insects were unrelated to either CO₂, temperature or their interaction. Our data show that CO₂ or temperature-induced alterations in leaf constituents had no effect on insect performance; instead, the long-term exposure to a 3.5°C increase in temperature shortened insect development but had no effect on pupal weight. It appears that in some tree-herbivorous insect systems the direct effects of an increased global mean temperature may have greater consequences for altering plant-insect interactions than the indirect effects of an increased temperature or CO₂ concentration on leaf constituents.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> production and antioxidant responses in seeds and early seedlings of two different rice varieties exposed to aluminum

The effect of Al stress on H₂O₂ production of rice (Oryza sativa L.) seedlings and difference in responses of antioxidant enzymes between Al-tolerant variety (Azucena) and Al-sensitive rice one (IR 64) were investigated. Aluminum-induced H₂O₂ production and malondialdehyde (MDA) content were more pronounced for IR 64 than for Azucena. In the presence of 2 mM Al, addition of 10 mM imidazole (inhibitor of NADPH oxidase) and 1 mM azide (inhibitor of peroxidase) significantly decreased H₂O₂ production by 16% and 43% for Azucena, and 21% and 68% for IR 64, respectively. Under Al treatment, the Al-tolerant variety Azucena had significantly higher activities of catalase, ascorbate peroxidase, dehydroascorbate reducase, glutathione peroxidase and glutathione reductase, and higher concentrations of reduced glutathione than the Al-sensitive one IR 64. Treatment with buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased H₂O₂ production in both varieties in the presence and absence of Al. In contrast, the treatment with GSH significantly decreased the production of H₂O₂ induced by Al stress. Results suggest that GSH may play an important role in scavenging H₂O₂ caused by Al stress.     

Partitioning of photosynthetic electron flow between CO<Subscript>2</Subscript> assimilation and O<Subscript>2</Subscript> reduction in sunflower plants under water deficit

In sunflower (Helianthus annuus L.) grown under controlled conditions and subjected to drought by withholding watering, net photosynthetic rate (P _N) and stomatal conductance (g _s) of attached leaves decreased as leaf water potential (Ψ_w) declined from −0.3 to −2.9 MPa. Although g _s decreased over the whole range of Ψ_w, nearly constant values in the intercellular CO₂ concentrations (C _i) were observed as Ψ_w decreased to −1.8 MPa, but C _i increased as Ψ_w decreased further. Relative quantum yield, photochemical quenching, and the apparent quantum yield of photosynthesis decreased with water deficit, whereas non-photochemical quenching (q_NP) increased progressively. A highly significant negative relationship between q_NP and ATP content was observed. Water deficit did not alter the pyridine nucleotide concentration but decreased ATP content suggesting metabolic impairment. At a photon flux density of 550 μmol m⁻² s⁻¹, the allocation of electrons from photosystem (PS) 2 to O₂ reduction was increased by 51 %, while the allocation to CO₂ assimilation was diminished by 32 %, as Ψ_w declined from −0.3 to −2.9 MPa. A significant linear relationship between mean P _N and the rate of total linear electron transport was observed in well watered plants, the correlation becoming curvilinear when water deficit increased. The maximum quantum yield of PS2 was not affected by water deficit, whereas q_P declined only at very severe stress and the excess photon energy was dissipated by increasing q_NP indicating that a greater proportion of the energy was thermally dissipated. This accounted for the apparent down-regulation of PS2 and supported the protective role of q_NP against photoinhibition in sunflower.     

Foliar quality influences tree-herbivore-parasitoid interactions: effects of elevated CO<Subscript>2</Subscript>, O<Subscript>3</Subscript>, and plant genotype

This study examined the effects of carbon dioxide (CO₂)-, ozone (O₃)-, and genotype-mediated changes in quaking aspen (Populus tremuloides) chemistry on performance of the forest tent caterpillar (Malacosoma disstria) and its dipteran parasitoid (Compsilura concinnata) at the Aspen Free-Air CO₂ Enrichment (FACE) site. Parasitized and non-parasitized forest tent caterpillars were reared on two aspen genotypes under elevated levels of CO₂ and O₃, alone and in combination. Foliage was collected for determination of the chemical composition of leaves fed upon by forest tent caterpillars during the period of endoparasitoid larval development. Elevated CO₂ decreased nitrogen levels but had no effect on concentrations of carbon-based compounds. In contrast, elevated O₃ decreased nitrogen and phenolic glycoside levels, but increased concentrations of starch and condensed tannins. Foliar chemistry also differed between aspen genotypes. CO₂, O₃, genotype, and their interactions altered forest tent caterpillar performance, and differentially so between sexes. In general, enriched CO₂ had little effect on forest tent caterpillar performance under ambient O₃, but reduced performance (for insects on one aspen genotype) under elevated O₃. Conversely, elevated O₃ improved forest tent caterpillar performance under ambient, but not elevated, CO₂. Parasitoid larval survivorship decreased under elevated O₃, depending upon levels of CO₂ and aspen genotype. Additionally, larval performance and masses of mature female parasitoids differed between aspen genotypes. These results suggest that host-parasitoid interactions in forest systems may be altered by atmospheric conditions anticipated for the future, and that the degree of change may be influenced by plant genotype.     

Growth and photosynthetic responses of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings to O<Subscript>3 </Subscript>under different nitrogen loads

To obtain the basic data for evaluating the critical level of ozone (O₃) to protect Japanese deciduous broad-leaved forest tree species, the growth and photosynthetic responses of Fagus crenata seedlings to O₃ under different nitrogen (N) loads were investigated. The seedlings were grown in potted andisol supplied with N as NH₄NO₃ solution at 0, 20 or 50 kg ha⁻¹ year⁻¹ and were exposed to charcoal-filtered air or O₃ at 1.0, 1.5 and 2.0 times the ambient concentration for two growing seasons. The interactive effect of O₃ and N load on the whole-plant dry mass of the seedlings at the end of the second growing season was significant. The O₃-induced reduction in the whole-plant dry mass of the seedlings was greater in the relatively high N treatment than that in the low N treatment. This interactive effect was mainly due to the difference in the degree of O₃-induced reduction in net photosynthesis among the N treatments. The degree of O₃-induced reduction in N availability to photosynthesis was greater in the relatively high N treatment than that in the low N treatment. In conclusion, the sensitivity of growth and photosynthetic parameters of F. crenata seedlings to O₃ become high with increasing amounts of N added to the soil. Therefore, N deposition from the atmosphere should be taken into account to evaluate the critical level of O₃ to protect Japanese deciduous broad-leaved forest tree species.     

Carbon balance in a monospecific stand of an annual herb <Emphasis Type="Italic">Chenopodium album</Emphasis> at an elevated CO<Subscript>2</Subscript> concentration

Elevated CO₂ enhances carbon uptake of a plant stand, but the magnitude of the increase varies among growth stages. We studied the relative contribution of structural and physiological factors to the CO₂ effect on the carbon balance during stand development. Stands of an annual herb Chenopodium album were established in open-top chambers at ambient and elevated CO₂ concentrations (370 and 700 μmol mol⁻¹). Plant biomass growth, canopy structural traits (leaf area, leaf nitrogen distribution, and light gradient in the canopy), and physiological characteristics (leaf photosynthesis and respiration of organs) were studied through the growing season. CO₂ exchange of the stand was estimated with a canopy photosynthesis model. Rates of light-saturated photosynthesis and dark respiration of leaves as related with nitrogen content per unit leaf area and time-dependent reduction in specific respiration rates of stems and roots were incorporated into the model. Daily canopy carbon balance, calculated as an integration of leaf photosynthesis minus stem and root respiration, well explained biomass growth determined by harvests (r ² = 0.98). The increase of canopy photosynthesis with elevated CO₂ was 80% at an early stage and decreased to 55% at flowering. Sensitivity analyses suggested that an alteration in leaf photosynthetic traits enhanced canopy photosynthesis by 40–60% throughout the experiment period, whereas altered canopy structure contributed to the increase at the early stage only. Thus, both physiological and structural factors are involved in the increase of carbon balance and growth rate of C. album stands at elevated CO₂. However, their contributions were not constant, but changed with stand development.     

Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of <Emphasis Type="Italic">Brassica napus</Emphasis> leaf protoplasts

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H₂O₂ and enzymes involved in H₂O₂ generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H₂O₂ and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H₂O₂ was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H₂O₂ in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H₂O₂ generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.     

Leaf senescence in response to elevated atmospheric CO<Subscript>2</Subscript> concentration and low nitrogen supply

This review reports the physiological and metabolic changes in plants during development under elevated atmospheric carbon dioxide concentration and/or limited-nitrogen supply in order to establish their effects on leaf senescence induction. Elevated CO₂ concentration and nitrogen supply modify gene expression, protein content and composition, various aspects of photosynthesis, sugar metabolism, nitrogen metabolism, and redox state in plants. Elevated CO₂ usually causes sugar accumulation and decreased nitrogen content in plant leaves, leading to imbalanced C/N ratio in mature leaves, which is one of the main factors behind premature senescence in leaves. Elevated CO₂ and low nitrogen decrease activities of some antioxidant enzymes and thus increase H₂O₂ production. These changes lead to oxidative stress that results in the degradation of photosynthetic pigments and eventually induce senescence. However, this accelerated leaf senescence under conditions of elevated CO₂ and limited nitrogen can mobilize nutrients to growing organs and thus ensure their functionality.     

Kinetics of the electron transfer reaction of Cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>552</Subscript> adsorbed on biomimetic electrode studied by time-resolved surface-enhanced resonance Raman spectroscopy and electrochemistry

Cytochrome c ₅₅₂ (Cyt-c ₅₅₂) and its redox partner ba ₃ -oxidase from Thermus thermophilus possess structural differences compared with Horse heart cytochrome c (cyt-c)/cytochrome c oxidase (CcO) system, where the recognition between partners and the electron transfer (ET) process is initiated via electrostatic interactions. We demonstrated in a previous study by surface-enhanced resonance Raman (SERR) spectroscopy that roughened silver electrodes coated with uncharged mixed self-assembled monolayers HS–(CH₂) _n –CH₃/HS–(CH₂) _{n + 1}–OH 50/50, n = 5, 10 or 15, was a good model to mimic the Cyt-c ₅₅₂ redox partner. All the adsorbed molecules are well oriented on such biomimetic electrodes and transfer one electron during the redox process. The present work focuses on the kinetic part of the heterogeneous ET process of Cyt-c ₅₅₂ adsorbed onto electrodes coated with such mixed SAMs of different alkyl chain length. For that purpose, two complementary methods were combined. Firstly cyclic voltammetry shows that the ET between the adsorbed Cyt-c ₅₅₂ and the biomimetic electrode is direct and reversible. Furthermore, it allows the estimation of both the density surface coverage of adsorbed Cyt-c ₅₅₂ and the kinetic constants values. Secondly, time-resolved SERR (TR-SERR) spectroscopy showed that the ET process occurs without conformational change of the Cyt-c ₅₅₂ heme group and allows the determination of kinetic constants. Results show that the kinetic constant values obtained by TR-SERR spectroscopy could be compared to those obtained from cyclic voltammetry. They are estimated at 200, 150 and 40 s⁻¹ for the ET of Cyt-c ₅₅₂ adsorbed onto electrodes coated with mixed SAMs HS–(CH₂) _n –CH₃/HS–(CH₂) _{n + 1}–OH 50/50, n = 5, 10 or 15, respectively. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

不同生境下濒危植物膝柄木幼树的生态适应性

膝柄木是我国极度濒危植物,也是广西滨海过渡带天然植被的重要组成树种.为了解光因子对膝柄木天然更新的限制影响,该文对林缘、林窗、林下三种不同光照生境下膝柄木幼树的生理和生长指标的年际变化特征进行了研究.结果表明:(1)光合有效辐射不足影响了膝柄木幼树的生长.林下幼树的地径、株高和叶面积增长量显著降低,而生长于光照充足林缘...