Derivative-based time-adjusted analysis of diurnal and within-tree variation in the OJIP fluorescence transient of silver birch.

The JIP test, based on fast chlorophyll fluorescence (ChlF) kinetics and derived parameters, is a dependable tool for studying photosynthetic efficiency under varying environmental conditions. We extracted additional information from the whole OJIP and the normalized variable fluorescence (Vt) transient curve using first and second-order derivatives to visualize and localize points of landmark events. To account for light-induced variations in the fluorescence transient, we present a time-adjusted JIP test approach in which the derivatives of the transient curve are used to determine the exact timing of the J and I steps instead of fixed time points. We compared the traditional JIP test method with the time-adjusted method in analyzing fast ChlF measurements of silver birch (Betula pendula) in field conditions studying diurnal and within-crown variation. The time-adjusted JIP test method showed potential for studying ChlF dynamics, as it takes into account potential time shifts in the occurrence of J and I steps. The exact occurrence times of J and I steps and other landmark events coincided with the times of significant differences in fluorescence intensity. Chlorophyll fluorescence parameters were linearly related to photosynthetic photon flux density (PPFD) at different times of day, and the values obtained by the time-adjusted JIP test showed a stronger linear regression than the traditional JIP test. For fluorescence parameters having significant differences among different times of day and crown layers, the time-adjusted JIP test resulted in more clear differences than the traditional JIP test. Diurnal ChlF intensity data indicated that differences between the southern and northern provenance were only evident under low light conditions. Taken together, our results emphasize the potential relevance of considering the time domain in the analysis of the fast ChlF induction.

Immediate and carry-over effects of late-spring frost and growing season drought on forest gross primary productivity capacity in the Northern Hemisphere.

Forests are increasingly exposed to extreme global warming-induced climatic events. However, the immediate and carry-over effects of extreme events on forests are still poorly understood. Gross primary productivity (GPP) capacity is regarded as a good proxy of the ecosystem's functional stability, reflecting its physiological response to its surroundings. Using eddy covariance data from 34 forest sites in the Northern Hemisphere, we analyzed the immediate and carry-over effects of late-spring frost (LSF) and growing season drought on needle-leaf and broadleaf forests. Path analysis was applied to reveal the plausible reasons behind the varied responses of forests to extreme events. The results show that LSF had clear immediate effects on the GPP capacity of both needle-leaf and broadleaf forests. However, GPP capacity in needle-leaf forests was more sensitive to drought than in broadleaf forests. There was no interaction between LSF and drought in either needle-leaf or broadleaf forests. Drought effects were still visible when LSF and drought coexisted in needle-leaf forests. Path analysis further showed that the response of GPP capacity to drought differed between needle-leaf and broadleaf forests, mainly due to the difference in the sensitivity of canopy conductance. Moreover, LSF had a more severe and long-lasting carry-over effect on forests than drought. These results enrich our understanding of the mechanisms of forest response to extreme events across forest types.

Artificially decreased vapour pressure deficit in field conditions modifies foliar metabolite profiles in birch and aspen.

Relative air humidity (RH) is expected to increase in northern Europe due to climate change. Increasing RH reduces the difference of water vapour pressure deficit (VPD) between the leaf and the atmosphere, and affects the gas exchange of plants. Little is known about the effects of decreased VPD on plant metabolism, especially under field conditions. This study was conducted to determine the effects of artificially decreased VPD on silver birch (Betula pendula Roth.) and hybrid aspen (Populus tremula L.×P. tremuloides Michx.) foliar metabolite and nutrient profiles in a unique free air humidity manipulation (FAHM) field experiment during the fourth season of humidity manipulation, in 2011. Long-term exposure to decreased VPD modified nutrient homeostasis in tree leaves, as demonstrated by a lower N concentration and N:P ratio in aspen leaves, and higher Na concentration and lower K:Na ratio in the leaves of both species in decreased VPD than in ambient VPD. Decreased VPD caused a shift in foliar metabolite profiles of both species, affecting primary and secondary metabolites. Metabolic adjustment to decreased VPD included elevated levels of starch and heptulose sugars, sorbitol, hemiterpenoid and phenolic glycosides, and α-tocopherol. High levels of carbon reserves, phenolic compounds, and antioxidants under decreased VPD may modify plant resistance to environmental stresses emerging under changing climate.

Low vapour pressure deficit affects nitrogen nutrition and foliar metabolites in silver birch.

Air humidity indicated as vapour pressure deficit (VPD) is directly related to transpiration and stomatal function of plants. We studied the effects of VPD and nitrogen (N) supply on leaf metabolites, plant growth, and mineral nutrition with young micropropagated silver birches (Betula pendula Roth.) in a growth chamber experiment. Plants that were grown under low VPD for 26 d had higher biomass, larger stem diameter, more leaves, fewer fallen leaves, and larger total leaf area than plants that were grown under high VPD. Initially, low VPD increased height growth rate and stomatal conductance; however, the effect was transient and the differences between low and high VPD plants became smaller with time. Metabolic adjustment to low VPD reflected N deficiency. The concentrations of N, iron, chlorophyll, amino acids, and soluble carbohydrates were lower and the levels of starch, quercetin glycosides, and raffinose were higher in the leaves that had developed under low VPD compared with high VPD. Additional N supply did not fully overcome the negative effect of low VPD on nutrient status but it diminished the effects of low VPD on leaf metabolism. Thus, with high N supply, the glutamine to glutamate ratio and starch production under low VPD became comparable with the levels under high VPD. The present study demonstrates that low VPD affects carbon and nutrient homeostasis and modifies N allocation of plants.

Decreased anthocyanidin reductase expression strongly decreases silver birch (Betula pendula) growth and alters accumulation of phenolics.

Phenolics, formed via a complex phenylpropanoid pathway, are important defensive agents in plants and are strongly affected by nitrogen (N) fertilization. Proanthocyanidins (PAs) are one possible endpoint of the phenylpropanoid pathway, and anthocyanidin reductase (ANR) represents a key enzyme in PA biosynthesis. In this study, the expression of silver birch (Betula pendula) anthocyanidin reductase BpANR was inhibited using the RNA interference (RNAi) method, in three consequent BpANR RNAi (ANRi birches) lines. The growth, the metabolites of the phenylpropanoid pathway, and the number of resin glands of the ANRi birches were studied when grown at two N levels. ANRi birches showed decreased growth and reduction in PA content, while the accumulation of total phenolics in both stems and leaves increased. Moreover, ANRi birches produced more resin glands than did wild-type (WT) birches. The response of ANRi birches to N depletion varied compared with that of WT birches, and in particular, the concentrations of some phenolics in stems increased in WT birches and decreased in ANRi birches. Because the inhibition of PAs biosynthesis via ANR seriously affected birch growth and resulted in accumulation of the precursors, the native level of PAs in plant tissues is assumed to be the prerequisite for normal plant growth. This draws attention to the real plant developmental importance of PAs in plant tissues.

Polar day syndrome: differences in growth, photosynthetic traits and sink-size patterns between northern and southern Finnish silver birch (Betula pendula Roth) provenances in native and non-native photoperiods.

Continuous light (CL) is available throughout the polar day for plants in the Arctic during the growing season, whereas provenances of the same species experience a very different environment with non-CL (NCL) just a few latitudes to the south. Both provenances need to acclimate to climate warming, yet we lack comprehensive understanding of how their growth, photosynthesis and leaf traits differ. Further, the provenances presumably have morphological and physiological adaptations to their native environments and therefore differ in response to photoperiod. We tested the height growth, leaf longevity, biomass accumulation, biomass allocation and rates of gas exchange of northern (67°N) and southern (61°N) Finnish silver birch (Betula pendula Roth) origins in CL- and NCL-treatments in a 4-month chamber experiment. Irrespective of photoperiod, 67°N had higher area-based photosynthetic rate (Anet), stomatal conductance (gs) and relative height growth rate (RGR), but lower stomatal density and fewer branches and leaves than 61°N. Photoperiod affected height growth cessation, biomass and photosynthetic traits, whereas leaf longevity and many leaf functional traits remained unchanged. In CL, both provenances had lower gs, higher RGR, increased shoot:root ratio and increased sink sizes (more branching, more leaves, increased total plant dry weight) compared with NCL. In NCL, 67°N ceased height growth earlier than in CL, which altered biomass accumulation and distribution patterns. Northern conditions impose challenges for plant growth and physiology. Whether a provenance inhabits and is adapted to an area with or without CL can also affect its response to the changing climate. Northern birches may have adapted to CL and the short growing season with a 'polar day syndrome' of traits, including relatively high gas exchange rates with low leaf biomass and growth traits that are mainly limited by the environment and the earlier growth cessation (to avoid frost damage).

Strategy by latitude? Higher photosynthetic capacity and root mass fraction in northern than southern silver birch (Betula pendula Roth) in uniform growing conditions.

Growth of northern trees is limited by short growing seasons. In multi-year trials, northern trees usually grow less than southern ones but can have higher gas exchange, whereas differences in biomass allocation and its relation to photosynthesis are less known. We characterized silver birch (Betula pendula Roth) provenances from southern (latitude 61°) and northern (latitude 67°) Finland in uniform chamber conditions. In a time-series experiment, we measured traits related to growth, biomass allocation and photosynthesis, and determined gas exchange responses to temperature and light. We found provenance differences in photosynthetic capacity and growth. The northern provenance allocated relatively more to roots, having a higher root mass fraction and lower shoot:root ratio than the southern provenance. On the other hand, the northern provenance had fewer leaves and lower total leaf dry weight (DW) than the southern provenance. The northern provenance attained higher rates of net photosynthesis (Anet) and higher stomatal conductance (gs) in all measured temperatures and higher photosynthesis at the optimum temperature (Aopt) than the southern provenance, but there was no difference in the optimum temperature of photosynthesis (Topt, 18.3 °C for the southern provenance vs 18.9 °C for the northern one). Photosynthetic light response curves showed no between-provenance differences. In a time-series, the northern provenance had higher Anet than the southern provenance, but gs was similar. The northern provenance had higher maximum quantum yield of photosystem II photochemistry (Fv/Fm) than the southern provenance. There were no differences between provenances in height, total plant DW, shoot DW, root DW or shoot mass fraction. Our results suggest that the provenances occupy a common thermal niche, or can at least relatively quickly acclimate to a common growth temperature. Thus, carbon assimilation of these northern trees may not be significantly affected by rising temperatures alone. In an equal photoperiod and optimal conditions, we found different one-season biomass accumulation strategies: southern trees grow with more leaves, while northern trees reach similar total assimilation (total DW, height) with more efficient photosynthetic capacity per leaf area (higher gas exchange, higher Fv/Fm) and relatively more investment in the below-ground fraction of the plant.

Climate and Competitive Status Modulate the Variation in Secondary Metabolites More in Leaves Than in Fine Roots of Betula pendula.

Plant secondary metabolites have many important functions; they also determine the productivity and resilience of trees under climate change. The effects of environmental factors on secondary metabolites are much better understood in above-ground than in below-ground part of the tree. Competition is a crucial biotic stress factor, but little is known about the interaction effect of climate and competition on the secondary chemistry of trees. Moreover, competition effect is usually overlooked when analyzing the sources of variation in the secondary chemistry. Our aim was to clarify the effects of competitive status, within-crown light environment, and climate on the secondary chemistry of silver birch (Betula pendula Roth). We sampled leaves (from upper and lower crown) and fine roots from competitively dominant and suppressed B. pendula trees in plantations along a latitudinal gradient (56-67° N) in Fennoscandia, with mean annual temperature (MAT) range: -1 to 8°C. Secondary metabolites in leaves (SML) and fine roots (SMFR) were determined with an HPLC-qTOF mass spectrometer. We found that SML content increased significantly with MAT. The effect of competitive stress on SML strengthened in colder climates (MAT<4°C). Competition and shade initiated a few similar responses in SML. SMFR varied less with MAT. Suppressed trees allocated relatively more resources to SML in warmer climates and to SMFR in colder ones. Our study revealed that the content and profile of secondary metabolites (mostly phenolic defense compounds and growth regulators) in leaves of B. pendula varied with climate and reflected the trees' defense requirements against herbivory, exposure to irradiance, and competitive status (resource supply). The metabolic profile of fine roots reflected, besides defense requirements, also different below-ground competition strategies in warmer and colder climates. An increase in carbon assimilation to secondary compounds can be expected at northern latitudes due to climate change.

Genetic modification of the flavonoid pathway alters growth and reveals flexible responses to enhanced UVB - Role of foliar condensed tannins.

Accumulation of certain phenolics is a well-known response of plants to enhanced UVB radiation (280-315 nm), but few experiments have compared the relative importance of different phenolic groups for UVB resilience. To study how an altered phenolic profile affects the responses and resilience of silver birch (Betula pendula) to enhanced UVB, we used RNA interference (RNAi) targeting dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), or anthocyanidin reductase (ANR) to change the accumulation of phenolics. The unmodified control line and RNAi-modified plants were grown for 51 days under ambient or +32% enhanced UVB dose in a greenhouse. RNAi greatly affected phenolic profile and plant growth. There were no interactive effects of RNAi and UVB on growth or photosynthesis, which indicates that the RNAi and unmodified control plants were equally resilient. UVB enhancement led to an accumulation of foliar flavonoids and condensed tannins, and an increase in the density of stem glands and glandular trichomes on upper leaf surfaces in both the control and RNAi-modified plants. Our results do not indicate a photoprotective role for condensed tannins. However, decreased growth of high-flavonoid low-tannin DFRi and ANRi plants implies that the balance of flavonoids and condensed tannins might be important for normal plant growth.

Differential gene expression in senescing leaves of two silver birch genotypes in response to elevated CO2 and tropospheric ozone.

Long-term effects of elevated CO(2) and O(3) concentrations on gene expression in silver birch (Betula pendula Roth) leaves were studied during the end of the growing season. Two birch genotypes, clones 4 and 80, with different ozone growth responses, were exposed to 2x ambient CO(2) and/or O(3) in open-top chambers (OTCs). Microarray analyses were performed after 2 years of exposure, and the transcriptional profiles were compared to key physiological characteristics during leaf senescence. There were genotypic differences in the responses to CO(2) and O(3). Clone 80 exhibited greater transcriptional response and capacity to alter metabolism, resulting in better stress tolerance. The gene expression patterns of birch leaves indicated contrasting responses of senescence-related genes to elevated CO(2) and O(3). Elevated CO(2) delayed leaf senescence and reduced associated transcriptional changes, whereas elevated O(3) advanced leaf senescence because of increased oxidative stress. The combined treatment demonstrated that elevated CO(2) only temporarily alleviated the negative effects of O(3). Gene expression data alone were insufficient to explain the O(3) response in birch, and additional physiological and biochemical data were required to understand the true O(3) sensitivity of these clones.

Elevation of night-time temperature increases terpenoid emissions from Betula pendula and Populus tremula.

Volatile organic compounds (VOCs) are expected to have an important role in plant adaptation to high temperatures. The impacts of increasing night-time temperature on daytime terpenoid emissions and related gene expression in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The plants were grown under five different night-time temperatures (6, 10, 14, 18, and 22 degrees C) while daytime temperature was kept at a constant 22 degrees C. VOC emissions were collected during the daytime and analysed by gas chromatography-mass spectrometry (GC-MS). In birch, emissions per leaf area of the C11 homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT) and several sesquiterpenes were consistently increased with increasing night-time temperature. Total sesquiterpene (SQT) emissions showed an increase at higher temperatures. In aspen, emissions of DMNT and beta-ocimene increased from 6 degrees C to 14 degrees C, while several other monoterpenes and the SQTs (Z,E)-alpha-farnesene and (E,E)-alpha-farnesene increased up to 18 degrees C. Total monoterpene and sesquiterpene emission peaked at 18 degrees C, whereas isoprene emissions decreased at 22 degrees C. Leaf area increased across the temperature range of 6-22 degrees C by 32% in birch and by 59% in aspen. Specific leaf area (SLA) was also increased in both species. The genetic regulation of VOC emissions seems to be very complex, as indicated by several inverse relationships between emission profiles and expression of several regulatory genes (DXR, DXS, and IPP). The study indicates that increasing night temperature may strongly affect the quantity and quality of daytime VOC emissions of northern deciduous trees.

Differences in growth and gas exchange between southern and northern provenances of silver birch (Betula pendula Roth) in northern Europe.

Due to its ubiquity across northern latitudes, silver birch (Betula pendula Roth) is an attractive model species for studying geographical trait variation and acclimation capacity. Six birch provenances from 60 to 67°N across Finland were grown in a common garden and studied for provenance and genotype variation. We looked for differences in height growth, photosynthetic gas exchange and chlorophyll content index (CCI) and compared the gas exchange of early and late leaves on short and long shoots, respectively. The provenances stratified into southern and northern groups. Northern provenances attained less height growth increment and had higher stomatal conductance (gs) and lower intrinsic water-use efficiency (WUE, Anet/gs) than southern provenances, whereas net photosynthesis (Anet) or CCI did not show clear grouping. Short shoot leaves had lower gs and higher WUE than long shoot leaves in all provenances, but there was no difference in Anet between shoot types. The separation of the provenances into two groups according to their physiological responses might reflect the evolutionary history of B. pendula. Latitudinal differences in gas exchange and water use traits can have plausible consequences for global carbon and water fluxes in a warming climate.

Emissions of volatile organic compounds and leaf structural characteristics of European aspen (Populus tremula) grown under elevated ozone and temperature.

Northern forest trees are challenged to adapt to changing climate, including global warming and increasing tropospheric ozone (O(3)) concentrations. Both elevated O(3) and temperature can cause significant changes in volatile organic compound (VOC) emissions as well as in leaf anatomy that can be related to adaptation or increased stress tolerance, or are signs of damage. Impacts of moderately elevated O(3) (1.3x ambient) and temperature (ambient + 1 degrees C), alone and in combination, on VOC emissions and leaf structure of two genotypes (2.2 and 5.2) of European aspen (Populus tremula L.) were studied in an open-field experiment in summer 2007. The impact of O(3) on measured variables was minor, but elevated temperature significantly increased emissions of total monoterpenes and green leaf volatiles. Genotypic differences in the responses to warming treatment were also observed. alpha-Pinene emission, which has been suggested to protect plants from elevated temperature, increased from genotype 5.2 only. Isoprene emission from genotype 2.2 decreased, whereas genotype 5.2 was able to retain high isoprene emission level also under elevated temperature. Elevated temperature also caused formation of thinner leaves, which was related to thinning of epidermis, palisade and spongy layers as well as reduced area of palisade cells. We consider aspen genotype 5.2 to have better potential for adaptation to increasing temperature because of thicker photosynthetic active palisade layer and higher isoprene and alpha-pinene emission levels compared to genotype 2.2. Our results show that even a moderate elevation in temperature is efficient enough to cause notable changes in VOC emissions and leaf structure of these aspen genotypes, possibly indicating the effort of the saplings to adapt to changing climate.

Rising atmospheric CO2 concentration partially masks the negative effects of elevated O3 in silver birch (Betula pendula Roth).

This review summarizes the main results from a 3-year open top chamber experiment, with two silver birch (Betula pendula Roth) clones (4 and 80) where impacts of 2x ambient [CO2] (EC) and [O3] (EO) and their combination (EC + EO) were examined. Growth, physiology of the foliage and root systems, crown structure, wood properties, and biological interactions were assessed to understand the effects of a future climate on the biology of silver birch. The clones displayed great differences in their reaction to EC and EO. Growth in clone 80 increased by 40% in EC and this clone also appeared O3-tolerant, showing no growth reduction. In contrast, growth in clone 4 was not enhanced by EC, and EO reduced growth with root growth being most affected. The physiological responses of the clones to EO were smaller than expected. We found no O3 effect on net photosynthesis in either of the clones, and many parameters indicated no change compared with chamber controls, suggesting active detoxification and defense in foliage. In EO, increased rhizospheric respiration over time and accelerated leaf senescence was common in both clones. We assumed that elevated O3 offsets the positive effects of elevated CO2 when plants were exposed to combined EC + EO treatment. In contrast, the responses to EC + EO mostly resembled the ones in EC, at least partly due to stomatal closure, which thus reduced O3 flux to the leaves. However, clear cellular level symptoms of oxidative stress were observed also in EC + EO treatment. Thus, we conclude that EC masked most of the negative O3 effects during long exposure of birch to EC + EO treatment. Biotic interactions were not heavily affected. Only some early season defoliators may suffer from faster maturation of leaves due to EO.

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Expression of senescence-associated genes in the leaves of silver birch (Betula pendula).

Development was monitored throughout the entire life span of silver birch (Betula pendula Roth.) leaves. The focus was on senescence-related changes in photosynthesis and gene expression. The youngest fully developed leaves were compared with older senescing leaves in two silver birch lines: the wild-type line R and a late-senescing line R3.1. Line R3.1 was found among transgenic lines produced with a plasmid containing sense-RbcS and nptII under the control of the 35S CaMV promoter. Compared with the wild type, line R3.1 showed no general change in the mRNA levels of RbcS or Rubisco protein; therefore, it can be considered a line whose phenotype is due to insertional mutagenesis. Leaf senescence started earlier in line R than in line R3.1. Senescence was characterized by declining photosynthesis as indicated by decreases in chlorophyll fluorescence, the amount and activity of Rubisco, and the level of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS1) mRNA. Some well-known senescence-associated genes (SAGs) encoding cysteine proteinases (Cyp1, Cyp2) and a pathogenesis-related gene (Pr1) were associated with leaf senescence. The expression pattern of Cyp1 indicated that it could serve as a molecular marker of leaf senescence in silver birch. Several genes related to energy metabolism, antioxidants and phenylpropanoid biosynthesis showed enhanced expression during leaf senescence. A distinct pattern in transcript abundance during leaf development was revealed for some of the identified SAGs.

Volatile organic compounds emitted from silver birch of different provenances across a latitudinal gradient in Finland.

Climate warming is having an impact on distribution, acclimation and defence capability of plants. We compared the emission rate and composition of volatile organic compounds (VOCs) from silver birch (Betula pendula (Roth)) provenances along a latitudinal gradient in a common garden experiment over the years 2012 and 2013. Micropropagated silver birch saplings from three provenances were acquired along a gradient of 7° latitude and planted at central (Joensuu 62°N) and northern (Kolari 67°N) sites. We collected VOCs emitted by shoots and assessed levels of herbivore damage of three genotypes of each provenance on three occasions at the central site and four occasions at the northern site. In 2012, trees of all provenances growing at the central site had higher total VOC emission rates than the same provenances growing at the northern site; in 2013 the reverse was true, thus indicating a variable effect of latitude. Trees of the southern provenance had lower VOC emission rates than trees of the central and northern provenances during both sampling years. However, northward or southward translocation itself had no significant effect on the total VOC emission rates, and no clear effect on insect herbivore damage. When VOC blend composition was studied, trees of all provenances usually emitted more green leaf volatiles at the northern site and more sesquiterpenes at the central site. The monoterpene composition of emissions from trees of the central provenance was distinct from that of the other provenances. In summary, provenance translocation did not have a clear effect in the short-term on VOC emissions and herbivory was not usually intense at the lower latitude. Our data did not support the hypothesis that trees growing at lower latitudes would experience more intense herbivory, and therefore allocate resources to chemical defence in the form of inducible VOC emissions.

Searching for common responsive parameters for ozone tolerance in 18 rice cultivars in India: Results from ethylenediurea studies.

Eighteen rice (Oryza sativa) cultivars were screened for ozone (O3) tolerance and for the most responsive parameters with ethylenediurea (EDU) treatments at two experimental sites experiencing high ambient O3 conditions in the Indo-Gangetic Plains (IGP) of India. EDU was applied at 15 day intervals until the final harvest phase as a foliar spray at 300 ppm in order to protect the plants from the adverse effects of O3. Antioxidant activity, malondialdehyde content (MDA), chlorophyll content, gas exchange, and chlorophyll fluorescence (Fv/Fm) at the vegetative and flowering phases and harvest-related parameters were studied, for a total of 24 parameters. Seven of the studied cultivars had higher than average grainweightplant(-1) in all site and treatment combinations and can be recommended for cultivation in areas suffering from high O3 concentrations. The most responsive parameters with EDU treatment in high O3 across all cultivars were superoxide dismutase (SOD) and catalase (CAT) activities, the contents of oxidised (GSSG) and reduced (GSH) glutathione and MDA, and shoot weight plant(-1). These results indicated that the O3 scavenging activity of EDU is mediated through an antioxidant defence system rather than a direct effect on physiological parameters, such as photosynthesis and stomatal conductance.

Ozone affects ascorbate and glutathione biosynthesis as well as amino acid contents in three Euramerican poplar genotypes.

Ozone is an air pollutant that causes oxidative stress by generation of reactive oxygen species (ROS) within the leaf. The capacity to detoxify ROS and repair ROS-induced damage may contribute to ozone tolerance. Ascorbate and glutathione are known to be key players in detoxification. Ozone effects on their biosynthesis and on amino acid metabolism were investigated in three Euramerican poplar genotypes (Populus deltoides Bartr. × Populus nigra L.) differing in ozone sensitivity. Total ascorbate and glutathione contents were increased in response to ozone in all genotypes, with the most resistant genotype (Carpaccio) showing an increase of up to 70%. Reduced ascorbate (ASA) concentration at least doubled in the two most resistant genotypes (Carpaccio and Cima), whereas the most sensitive genotype (Robusta) seemed unable to regenerate ASA from oxidized ascorbate (DHA), leading to an increase of 80% of the oxidized form. Increased ascorbate (ASA + DHA) content correlated with the increase in gene expression in its biosynthetic pathway, especially the putative gene of GDP-l-galactose phosphorylase VTC2. Increased cysteine availability combined with increased expression of γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) genes allows higher glutathione biosynthesis in response to ozone, particularly in Carpaccio. In addition, ozone caused a remobilization of amino acids with a decreased pool of total amino acids and an increase of Cys and putrescine, especially in Carpaccio. In addition, the expression of genes encoding threonine aldolase was strongly induced only in the most tolerant genotype, Carpaccio. Reduced ascorbate levels could partly explain the sensitivity to ozone for Robusta but not for Cima. Reduced ascorbate level alone is not sufficient to account for ozone tolerance in poplar, and it is necessary to consider several other factors including glutathione content.