Effect of heat wave on N2 fixation and N remobilisation of lentil (Lens culinaris MEDIK) grown under free air CO2 enrichment in a mediterranean-type environment.

The stimulatory effect of elevated [CO2 ] (e[CO2 ]) on crop production in future climates is likely to be cancelled out by predicted increases in average temperatures. This effect may become stronger through more frequent and severe heat waves, which are predicted to increase in most climate change scenarios. Whilst the growth and yield response of some legumes grown under the interactive effect of e[CO2 ] and heat waves has been studied, little is known about how N2 fixation and overall N metabolism is affected by this combination. To address these knowledge gaps, two lentil genotypes were grown under ambient [CO2 ] (a[CO2 ], ~400 µmol·mol-1 ) and e[CO2 ] (~550 µmol·mol-1 ) in the Australian Grains Free Air CO2 Enrichment facility and exposed to a simulated heat wave (3-day periods of high temperatures ~40 °C) at flat pod stage. Nodulation and concentrations of water-soluble carbohydrates (WSC), total free amino acids, N and N2 fixation were assessed following the imposition of the heat wave until crop maturity. Elevated [CO2 ] stimulated N2 fixation so that total N2 fixation in e[CO2 ]-grown plants was always higher than in a[CO2 ], non-stressed control plants. Heat wave triggered a significant decrease in active nodules and WSC concentrations, but e[CO2 ] had the opposite effect. Leaf N remobilization and grain N improved under interaction of e[CO2 ] and heat wave. These results suggested that larger WSC pools and nodulation under e[CO2 ] can support post-heat wave recovery of N2 fixation. Elevated [CO2 ]-induced accelerated leaf N remobilisation might contribute to restore grain N concentration following a heat wave.

Elevated [CO2 ] effects on crops: Advances in understanding acclimation, nitrogen dynamics and interactions with drought and other organisms.

Future rapid increases in atmospheric CO2 concentration [CO2 ] are expected, with values likely to reach ~550 ppm by mid-century. This implies that every terrestrial plant will be exposed to nearly 40% more of one of the key resources determining plant growth. In this review we highlight selected areas of plant interactions with elevated [CO2 ] (e[CO2 ]), where recently published experiments challenge long-held, simplified views. Focusing on crops, especially in more extreme and variable growing conditions, we highlight uncertainties associated with four specific areas. (1) While it is long known that photosynthesis can acclimate to e[CO2 ], such acclimation is not consistently observed in field experiments. The influence of sink-source relations and nitrogen (N) limitation on acclimation is investigated and current knowledge about whether stomatal function or mesophyll conductance (gm ) acclimate independently is summarised. (2) We show how the response of N uptake to e[CO2 ] is highly variable, even for one cultivar grown within the same field site, and how decreases in N concentrations ([N]) are observed consistently. Potential mechanisms contributing to [N] decreases under e[CO2 ] are discussed and proposed solutions are addressed. (3) Based on recent results from crop field experiments in highly variable, non-irrigated, water-limited environments, we challenge the previous opinion that the relative CO2 effect is larger under drier environmental conditions. (4) Finally, we summarise how changes in growth and nutrient concentrations due to e[CO2 ] will influence relationships between crops and weeds, herbivores and pathogens in agricultural systems.

Enhanced ozone strongly reduces carbon sink strength of adult beech (Fagus sylvatica)--resume from the free-air fumigation study at Kranzberg Forest.

Ground-level ozone (O(3)) has gained awareness as an agent of climate change. In this respect, key results are comprehended from a unique 8-year free-air O(3)-fumigation experiment, conducted on adult beech (Fagus sylvatica) at Kranzberg Forest (Germany). A novel canopy O(3) exposure methodology was employed that allowed whole-tree assessment in situ under twice-ambient O(3) levels. Elevated O(3) significantly weakened the C sink strength of the tree-soil system as evidenced by lowered photosynthesis and 44% reduction in whole-stem growth, but increased soil respiration. Associated effects in leaves and roots at the gene, cell and organ level varied from year to year, with drought being a crucial determinant of O(3) responsiveness. Regarding adult individuals of a late-successional tree species, empirical proof is provided first time in relation to recent modelling predictions that enhanced ground-level O(3) can substantially mitigate the C sequestration of forests in view of climate change.

Synopsis of the CASIROZ case study: carbon sink strength of Fagus sylvatica L. in a changing environment--experimental risk assessment of mitigation by chronic ozone impact.

Databases are needed for the ozone (O(3)) risk assessment on adult forest trees under stand conditions, as mostly juvenile trees have been studied in chamber experiments. A synopsis is presented here from an integrated case study which was conducted on adult FAGUS SYLVATICA trees at a Central-European forest site. Employed was a novel free-air canopy O(3) fumigation methodology which ensured a whole-plant assessment of O(3) sensitivity of the about 30 m tall and 60 years old trees, comparing responses to an experimental 2 x ambient O(3) regime (2 x O(3), max. 150 nl O(3) l (-1)) with those to the unchanged 1 x ambient O(3) regime (1 x O(3)=control) prevailing at the site. Additional experimentation on individual branches and juvenile beech trees exposed within the forest canopy allowed for evaluating the representativeness of young-tree and branch-bag approaches relative to the O(3) sensitivity of the adult trees. The 2 x O(3) regime did not substantially weaken the carbon sink strength of the adult beech trees, given the absence of a statistically significant decline in annual stem growth; a 3 % reduction across five years was demonstrated, however, through modelling upon parameterization with the elaborated database. 2 x O(3) did induce a number of statistically significant tree responses at the cell and leaf level, although the O(3) responsiveness varied between years. Shade leaves displayed an O(3) sensitivity similar to that of sun leaves, while indirect belowground O(3) effects, apparently mediated through hormonal relationships, were reflected by stimulated fine-root and ectomycorrhizal development. Juvenile trees were not reliable surrogates of adult ones in view of O(3) risk assessment. Branch sections enclosed in (climatized) cuvettes, however, turned out to represent the O(3) sensitivity of entire tree crowns. Drought-induced stomatal closure decoupled O(3) intake from O(3) exposure, as in addition, also the "physiologically effective O(3) dose" was subject to change. No evidence emerged for a need to lower the "Critical Level for Ozone" in risk assessment of forest trees, although sensitive tree parameters did not necessarily reflect a linear relationship to O(3) stress. Exposure-based concepts tended to overestimate O(3) risk under drought, which is in support of current efforts to establish flux-related concepts of O(3) intake in risk assessment.

Antioxidative defence of old growth beech (Fagus sylvatica) under double ambient O3 concentrations in a free-air exposure system.

In this study the influence of chronic free-air ozone exposure and of different meteorological conditions in the very dry year 2003 and the more humid year 2004 on the antioxidative system in sun and shade leaves of adult FAGUS SYLVATICA trees were investigated. Contents of ascorbate, glutathione, and alpha-tocopherol, as well as chloroplast pigments were determined under ambient (1 x O(3)) and double ambient (2 x O(3)) ozone concentrations. Ozone affected the antioxidative system in June and July, causing lower ascorbate contents in the apoplastic space, a more oxidized redox state of ascorbate and glutathione and an increase in pigment contents predominantly in the shade crown. For all measured parameters significant differences between the years were observed. In 2004 the redox state of ascorbate and glutathione was in a more reduced state and leaf contents of alpha-tocopherol, pigments of the xanthophyll cycle, beta-carotene, lutein, neoxanthin, and alpha-carotene were lower compared to 2003. Contents of total glutathione and chlorophyll a + b were increased in the second year. These results indicate a strong influence of the drought conditions in 2003 on the antioxidative system of beech overruling the ozone effects. Shade leaves showed lower contents of ascorbate in both years and the redox states of ascorbate and glutathione were more oxidized compared to sun leaves. Contents of photoprotective and accessory pigments generally were enhanced and the de-epoxidation state of the xanthophyll cycle was lower in the shade compared to the sun crown. Exhibiting less antioxidants shade leaves seem to be more sensitive against ozone than sun leaves.

Gas exchange and antioxidative compounds in young beech trees under free-air ozone exposure and comparisons to adult trees.

Three-year-old beech (Fagus sylvatica) seedlings growing in containers were placed into the sun and shade crown of a mature beech stand exposed to ambient (1 x O(3)) and double ambient (2 x O(3)) ozone concentrations at a free-air exposure system ("Kranzberg Forst", Germany). Pigments, alpha-tocopherol, glutathione, ascorbate, and gas exchange were measured in leaves during 2003 (a drought year) and 2004 (an average year). Sun-exposed seedlings showed higher contents of antioxidants, xanthophylls, and beta-carotene and lower contents of chlorophyll, alpha-carotene, and neoxanthin than shade-exposed seedlings. In 2003 sun-exposed seedlings showed higher contents of carotenoids and total glutathione and lower net photosynthesis rates (A(max)) compared to 2004. O(3) exposure generally affected the content of chlorophyll, the xanthophyll cycle, and the intercellular CO(2) concentration (c(i)). Seedlings differed from the adjacent adult trees in most biochemical and physiological parameters investigated: Sun exposed seedlings showed higher contents of alpha-tocopherol and xanthophylls and lower contents of ascorbate, chlorophyll, neoxanthin, and alpha-carotene compared to adult trees. Shade exposed seedlings had lower contents of xanthophylls, alpha-carotene, and alpha-tocopherol than shade leaves of old-growth trees. In 2003, seedlings had higher A(max), stomatal conductance (g(s)), and c(i) under 2 x O(3) than adult trees. The results showed that shade acclimated beech seedlings are more sensitive to O(3), possibly due to a lower antioxidative capacity per O(3) uptake. We conclude that beech seedlings are uncertain surrogates for adult beech trees.

Evidence that branch cuvettes are reasonable surrogates for estimating O3 effects in entire tree crowns.

Within the scope of quantifying ozone (O(3)) effects on forest tree crowns it is still an open question whether cuvette branches of adult trees are reasonable surrogates for O(3) responses of entire tree crowns and whether twigs exhibit autonomy in defense metabolism in addition to carbon autonomy. Therefore, cuvette-enclosed branches of mature beech (Fagus sylvatica) trees were compared with branches exposed to the same and different ozone regimes by a free-air fumigation system under natural stand conditions by means of a VICE VERSA experiment. For this purpose, cuvettes receiving 1 x O(3) air were mounted in trees exposed to 2 x O(3) and cuvettes receiving 2 x O(3) air were mounted in trees exposed to 1 x O (3) in the upper sun crown. At the end of the fumigation period in September 2004, leaves were examined for differences in gas exchange parameters, pigments, antioxidants, carbohydrates, and stable isotope ratios. No significant differences in foliar gas exchange, total carbohydrates, stable isotope ratios, pigment, and antioxidant contents were found as a consequence of cuvette enclosure (cuvette versus free-air branches) of the same O(3) concentrations besides increase of glucose inside the cuvettes and reduction of the de-epoxidation state of the xanthophyll cycle pigments. No significant ozone effect was found for the investigated gas exchange and most biochemical parameters. The total and oxidized glutathione level of the leaves was increased by the 2 x O(3) treatment in the cuvette and the free-air branches, but this effect was significant only for the free-air branches. From these results we conclude that cuvette branches are useful surrogates for examining the response of entire tree crowns to elevated O(3) and that the defence metabolism of twigs seems to be at least partially autonomous.

Evidence for potential impacts of ozone on Pinus cembra L. at mountain sites in Europe: an overview.

We summarize what is known about the impact of ozone (O(3)) on Pinus cembra in the timberline ecotone of the central European Alps and the Carpathian Mountains. In the central European Alps exposure to ambient and two-fold ambient O(3) throughout one growing season did neither cause any visible injury nor affect the photosynthetic machinery and biochemical parameters in current to 1-year-old needles. By contrast, in the southern French Alps and in the Carpathians 1-year-old needles of Pinus cembra trees showed visual symptoms similar to those observed in O(3) stressed pine stands in southern California. For the southern French Alps the observed symptoms could clearly be attributed O(3) and differences in O(3) uptake seems to be the likely key factor for explaining the observed decline. For the Carpathians however, other reasons such as drought may not be excluded in eliciting the observed symptoms. Thus, the action of O(3) has always to be evaluated in concert with other environmental impacts, determining the tree's sensitivity to stress.

Tree age dependence and within-canopy variation of leaf gas exchange and antioxidative defence in Fagus sylvatica under experimental free-air ozone exposure.

We characterized leaf gas exchange and antioxidative defence of two-year-old seedlings and 60-year-old trees of Fagus sylvatica exposed to ambient (1 x O3) or two-fold ambient (2 x O3) O3 concentrations (maximum of 150 ppb) in a free-air canopy exposure system throughout the growing season. Decline in photosynthesis from sun-exposed to shaded conditions was more pronounced in adult than juvenile trees. Seedling leaves and leaves in the sun-exposed canopy had higher stomatal conductance and higher internal CO2 concentrations relative to leaves of adult trees and leaves in shaded conditions. There was a weak overall depression of photosynthesis in the 2 x O3 variants across age classes and canopy positions. Pigment and tocopherol concentrations of leaves were significantly affected by canopy position and tree age, whereas differences between 1 x O3 and 2 x O3 regimes were not observed. Glutathione concentrations were significantly increased under 2 x O3 across both age classes and canopy levels. Seedlings differed from adult trees in relevant physiological and biochemical traits in ozone response. The water-soluble antioxidative systems responded most sensitively to 2 x O3 without regard of tree age or canopy position.

Effects of weak 16 3/2 Hz magnetic fields on growth parameters of young sunflower and wheat seedlings.

Previous studies on effects of magnetic fields on plants focussed on the power frequencies (50-60 Hz), but in Austria and Germany electric railways are powered by a frequency of 16 2/3) Hz. In the present study, sunflower and wheat seedlings were exposed to 16 2/3 Hz sinusoidal 20 muT (rms) vertical magnetic fields. Seeds were germinated in a germination roll and grown for 12 days under continued exposure. Seven series with sunflower and six series with wheat were done over 2 years. Sunflower seedlings exposed to experimental magnetic field showed small, but significant increases in total fresh weights, shoot fresh weights, and root fresh weights, whereas dry weights and germination rates remained unaffected. Experimentally treated wheat exhibited marginally (but significantly) higher root fresh and dry weights, total fresh weights, and higher germination rates.

Variation in morphological and biochemical O3 injury attributes of mature Jeffrey pine within canopies and between microsites.

Crown morphology and leaf tissue chemical and biochemical attributes associated with ozone (O3) injury were assessed in the lower, mid- and upper canopy of Jeffrey pine (Pinus jeffreyi Grev. & Balf.) growing in mesic and xeric microsites in Sequoia National Park, California. Microsites were designated mesic or xeric based on topography and bole growth in response to years of above-average precipitation. In mesic microsites, canopy response to O3 was characterized by thinner branches, earlier needle fall, less chlorotic leaf mottling, and lower foliar antioxidant capacity, especially of the aqueous fraction. In xeric microsites, canopy response to O3 was characterized by higher chlorotic leaf mottling, shorter needles, lower needle chlorophyll concentration, and greater foliar antioxidant capacity. Increased leaf chlorotic mottle in xeric microsites was related to drought stress and increased concurrent internal production of highly reactive oxygen species, and not necessarily to stomatal O3 uptake. Within-canopy position also influenced the expression of O3 injury in Jeffrey pine.

Tree- and needle-age-dependent variations in antioxidants and photoprotective pigments in Norway spruce needles at the alpine timberline.

To cope with environmental stress, plants are equipped with antioxidative (e.g., ascorbate, glutathione and alpha-tocopherol) and photoprotective (e.g., xanthophyll cycle pigments) defense systems. We investigated the defense capacities of three tree age classes (mature, sapling and seedling) of Norway spruce (Picea abies (L.) Karst.) at a field site near the timberline. Biochemical data were expressed on both a needle dry mass and a surface area basis. Compared with current-year needles, previous-year needles contained higher mass- and area-based concentrations of chlorophylls and alpha-tocopherol, and a larger xanthophyll cycle pool that was in a more epoxidized state. Total glutathione concentration was lower, the glutathione pool was more reduced and the ascorbate pool was more oxidized in previous-year needles than in current-year needles. Needle concentrations of glutathione and alpha-tocopherol increased and chlorophyll concentration decreased with increasing tree age when expressed on a surface area basis. On a dry mass basis, these trends were reversed or nonexistent. The ascorbate pool was more reduced and the glutathione pool was more oxidized in needles of mature trees than in needles of saplings and seedlings. The proportion of protective xanthophyll cycle pigments decreased and the de-epoxidation state increased with increasing tree age. We conclude that tree age and the basis of expression of antioxidant concentration--surface area or dry mass--are important in scaling from seedlings to large trees.

Multivariate patterns of biochemical responses of Pinus ponderosa trees at field plots in the San Bernardino Mountains, southern California.

Most environmental stress conditions promote the production of potentially toxic active oxygen species in plant cells. Plants respond with changes in their antioxidant and photoprotective systems. Antioxidants and pigments have been widely used to measure these responses. Because trees are exposed to multiple man-made and natural stresses, their responses are not reflected by changes in single stress markers, but by complex biochemical changes. To evaluate such response patterns, explorative multivariate statistics have been used. In the present study, 12 biochemical variables (chloroplast pigments, state of the xanthophyll cycle, alpha-tocopherol, ascorbate and dehydroascorbate, glutathione and oxidized glutathione) were measured in previous-year needles of field-grown Pinus ponderosa Dougl. ex Laws. The trees were sampled in two consecutive years in the San Bernardino Mountains in southern California, where a pollution gradient is overlaid by gradients in natural stresses (drought, altitude). To explore irradiance effects, needle samples were taken directly in the field (sun exposed) and from detached, dark-adapted branches. A principal component analysis on this data set (n = 80) resulted in four components (Components 1-4) that explained 67% of the variance in the original data. Component 1 was positively loaded by concentrations of alpha-tocopherol, total ascorbate and xanthophyll cycle pools, as well as by the proportion of de-epoxides in the xanthophyll cycle. It was negatively loaded by the proportion of dehydroascorbate in the ascorbate pool. Component 2 was negatively loaded by chlorophyll concentrations, and positively loaded by the ratios of lutein and beta-carotene to chlorophyll and by the de-epoxidation state of the xanthophyll cycle. Component 3 was negatively loaded by GSH concentrations and positively loaded by the proportions of GSSG and tocopherol concentrations. Component 4 was positively loaded by neoxanthin and negatively loaded by beta-carotene. The four components could be assigned to the concerted action of the biochemical protection system: high scores on Component 1 represent highly activated antioxidative defense, changes in pigment composition are represented in Components 2 and 4, and the glutathione system, which is important for antioxidant regeneration, is represented in Component 2. Although Component 1 scores were generally higher (indicating activation of antioxidant defense) in light-adapted needles relative to dark-adapted needles, they were also site dependent with increased scores at sites with less pollution, but higher natural stress impacts. High scores of Components 2 and 3 at the highest elevation site, which was only moderately polluted, indicated an increase in photoprotection by pigments and activation of the glutathione system. Significant differences between light- and dark-adapted needles in Components 2 and 3 were only found at the site with the highest pollution. Use of accumulated variables (components) instead of single biochemical variables enabled recognition of response patterns at particular sites and a better comparison with results of other studies is expected. Typical response patterns could be assigned to particular environmental stress combinations, providing a means of assessing potential biological risks within individual forest stands.

Antioxidants and protective pigments of Pinus ponderosa needles at gradients of natural stresses and ozone in the San Bernardino Mountains in California.

At the San Bernardino Mountains, California, a well documented gradient of ozone pollution overlays a natural stress gradient from mesic to dry and from lower elevation to higher elevation sites. In contrast to gradient studies in European regions, the highest ambient ozone levels are observed at low elevation and more mesic locations. In the present study, antioxidative and photoprotective systems in Pinus ponderosa needles were investigated at three plots--DW (1725 m, high ozone impact, mesic site), SW (1200 m, clean air, xeric site) and CO (above 2000 m, clean air and xeric site). Needles from the CO site contained significantly more total GSH (500 vs 300 nmol g(-1) dw in c needles), less alpha-carotene (6-10 vs 14-19 microg mg(-1) total chlorophyll) and chlorophyll (1.7-2 vs 2.5-2.6 mg g(-1) dw in c + 1 needles) than those at the DW site. Furthermore, their xanthophyll cycle pool was in a more de-epoxidized state at midday (up to 60% in c needles), and the carotenoid/chlorophylls ratios were generally higher. These patterns correspond to those observed at higher elevation plots in the Alps. On the other hand, needles from the high ozone site (DW) had a higher proportion of GSSG, indicating the onset of biochemical injury to needles. Needles from the SW site had intermediate proportions of GSSG. The results show the potential of environmental stressors to induce antioxidative and photoprotective responses in the absence of elevated ozone concentrations, but support the oxidative effects of ozone injury to ponderosa pine.

Effects of an oxidizing agent (hydrogen peroxide) on the glutathione system in epidermal cells of Allium cepa L. investigated by histochemical staining.

The glutathione system in epidermal cells of Allium cepa L. was measured by a quantitative image analysis method, using histochemical staining by monochlorobimane. Blockage of thiol groups with N-ethylmaleimide decreased fluorescence to a small rest fluorescence of maximum 5%. Pre-treatment with dithiothreitol increased fluorescence yield in all cells compared to the monochlorobimane treatment alone. These results correspond to the blockage or the reduction of the total pool of glutathione in the cells. After treatment with N-ethylmaleimide and subsequent incubation with dithiothreitol followed by labelling with bimane a low fluorescence yield was observed which correspond to the proportion of GSSG. Specimens that were incubated in H2O2 prior to the treatments described above exhibited a decrease in total glutathione and in reduced glutathione and an increase in the proportion of GSSG compared to the control.