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Hemiboreal forests form a transitional belt between boreal and temperate forests in Eurasia, and due to long-term climate warming they could potentially expand in a northerly direction. However, carbon (C) exchange studies in this transitional forest type are scarce. In 2014–2015 we analyzed CO2 exchange in a hemiboreal mixed forest at SMEAR Estonia (Station for Measuring Ecosystem-Atmosphere Relations) using eddy covariance (EC), soil chamber technique and biometric methods. Employing two plots that differ in forest stand composition (mixed (MP) and coniferous (CP)) located within the footprint of the EC tower, we evaluated heterotrophic and total soil respiration (Rh and Rs, respectively). Measurements showed that C, nitrogen (N), and soil organic matter (SOM) contents in topsoil were significantly higher in the coniferous plot, and the total C stock until 20 cm of mineral soil was higher by a factor of two (i.e., 170.6 and 94.8 t C ha−1 for the CP and MP, respectively). Only in 2015 there was a significant difference in Rs between the plots, while Rh exhibited no detectable difference. The cumulative Rs during the growing season was similar for both plots (CP and MP) and years: 532 and 548 g m−2, respectively, in 2014; 636 and 592 g m−2, respectively, in 2015. The same applied to Rh: 418 and 430 g m−2, respectively, in 2014; 406 and 387 g m−2, respectively, in 2015. The forest as a whole was a C sink. In 2015, net ecosystem exchange (NEE) was -585.62 ± 45.16 g C m−2 yr−1 (the average and SD of two gap-filling methods: MDS and nonlinear regressions). Gross primary production (GPP), and ecosystem respiration (RE) were -1280.62 ± 53.36, and 696.00 ± 98.51 g C m−2 yr−1, respectively (the average and SD of two flux partitioning methods: nighttime data-based and daytime data-based). NEE and GPP suggested a greater similarity to temperate forests, while RE was closer to more boreal forest stands. The mean value of net ecosystem production (NEP) of both plots, estimated using forest stand biometric, litter and chamber data was similar to the annual NEE value: 6.3 and −5.9 t C ha−1 yr−1, respectively.

The abandonment of agricultural areas is usually accompanied by the stochastic encroachment of shrubs and trees or deliberate afforestation, but this leads as a rule to the decline of grassland plant diversity. Future climate change may increase precipitation and air humidity in temperate and boreal forest zones, and thus change vegetation dynamics. We conducted a five-year experiment on the Free Air Humidity Manipulation experimental facility (FAHM) with planted deciduous trees to examine how both the grassland afforestation with deciduous trees and the increase of air humidity in interaction with forest soil supplementation and cover of herbaceous layer affect the species richness, composition and cover of bryophyte layer. The effects of the same factors on the cover of herbaceous layer were also analysed. The species of bryophytes and cover of herbaceous and bryophyte layer were surveyed in September of 2007–2011. During these early years of forest succession, herbaceous layer cover decreased and bryophyte cover increased, although the direct suppressing effect of tree canopy was observed on both layers. The input of forest soil as diaspore deposit of bryophytes had some positive effect on the cover of bryophytes, while the increased air humidity had quite limited effect. The density of tree canopy (defined in our study through Leaf Area Index) first promoted the species richness of bryophytes, particularly of species with grassland preference. However, together with the herbaceous layer it became suppressing in the last year. The species richness of bryophytes declined during the first years and then stabilised. The decline relied mainly on the disappearance of grassland species. The richness of bryophytes was supported by forest soil supplementation only in the initial year. The bryophyte species composition changed rapidly in first two years mainly due to decline of short-lived grassland species and perennial species started to dominate. Our results show that bryophyte succession during the afforestation is largely driven by natural loss of grassland specialists after tree canopy closure, while diaspore supplementation from forest and improved air moisture level had small effects.

Expected climate change in high latitudes includes increased air temperature, precipitation, and humidity in the coming decades. Simultaneously, climate extremes like heat waves and droughts become more frequent. In the Free-Air Humidity Manipulation (FAHM) experiment in silver birch ( Betula pendula Roth) and hybrid aspen ( Populus tremula L. × P. tremuloides Michx.) stands in Estonia, we focussed on two questions − how elevated humidity in high temperature and drought conditions influences soil greenhouse gas emissions, and what is the impact of tree species on greenhouse gas emissions. Soil carbon dioxide (CO 2 ), methane (CH 4 ), and nitrous oxide (N 2 O) fluxes were measured using the static chamber method in the 3rd and 4th years of humidification, in 2010 and 2011 respectively. Soil temperature, soil water potential (SWP), relative humidity, and precipitation were monitored; tree and understory growth, litter fluxes, substrate-induced respiration, and basal respiration were measured. During the severe drought in summer 2011 (SWP fell below −250 kPa), aspen stands had higher CO 2 emissions than birch stands and humidification increased CO 2 emission for both tree species. Generally, methane consumption was higher in control than in humidified conditions. Humidification reduced N 2 O emission in aspen stands in 2010. Hence elevated humidity, heat wave and drought, and tree species significantly affected soil greenhouse gas emissions, however CH 4 and N 2 O fluxes remained small in all cases. The results of this study can be used to infer the future greenhouse gas dynamics from mineral soils in northern forests and to forecast growth conditions for energy forestry in changing climate.

Increasing atmospheric humidity—a climate trend predicted for northern Europe—will reduce water flux through vegetation. Diminished transpirational water flux impacts various physiological processes, causing growth decline in deciduous trees. We propose, based on the results obtained from the long-term free air humidity manipulation experiment, concurrent mechanisms to explain the growth deceleration due to increases in relative air humidity. Reduced atmospheric evaporative demand diminishes nutrient uptake and leads to lower leaf nutritional status and to an unbalanced foliar phosphorus/nitrogen ratio (P:N), resulting in a decline in leaf photosynthetic capacity. Elevated relative humidity induces readjustment of foliar metabolism: disturbed N metabolism, accumulation of starch and changes in secondary metabolite contents probably impair both photosynthetic performance and growth. Increased carbohydrate content in the leaves suggests that sink strength of trees is reduced under elevated humidity. As a consequence of the stress, foliar development is hindered, observed at individual leaf or whole-tree foliage levels, lowering production potential of trees proportionally to their foliar area. Larger investments in stem xylem in relation to foliage cause an increase in the ratio of non-photosynthetic to photosynthetic tissues, leading to larger maintenance respiration costs determined by the volume of parenchymatous tissue. An increase in the proportion of living parenchyma cells in relation to dead xylem elements in sapwood additionally enhances respiration costs. Disproportionate changes in hydraulic versus stomatal conductance become a critical factor in the case of weather extremes, which limit canopy conductance and may induce dysfunction of the hydraulic system. Increasing environmental humidity creates favourable conditions for development of pathogens, increasing frequency of fungal damage.

Processes determining the carbon (C) balance of a forest ecosystem are influenced by a number of climatic and environmental factors. In Northern Europe, a rise in atmospheric humidity and precipitation is predicted. The study aims to ascertain the effect of elevated atmospheric humidity on the components of the C budget and on the C-sequestration capacity of a young birch forest. Biomass production, soil respiration, and other C fluxes were measured in young silver birch (Betula pendula Roth) stands growing on the Free Air Humidity Manipulation (FAHM) experimental site, located in South-East Estonia. The C input fluxes: C sequestration in trees and understory, litter input into soil, and methane oxidation, as well as C output fluxes: soil heterotrophic respiration and C leaching were estimated. Humidified birch stands stored C from the atmosphere, but control stands can be considered as C neutral. Two years of elevated air humidity increased C sequestration in the understory but decreased it in trees. Humidification treatment increased remarkably the C input to the soil. The main reason for such an increase was the higher root litter input into the soil, brought about by the more than two-fold increase of belowground biomass production of the understory in the humidification treatment. Elevated atmospheric humidity increased C sequestration in young silver birch stands, mitigating increasing CO2 concentration in the atmosphere. However, the effect of elevated atmospheric humidity is expected to decrease over time, as plants and soil organisms acclimate, and new communities emerge.

Elevated atmospheric humidity reduced bud size by restricting primordium growth and increased the frequency of bud break in fast-growing deciduous trees, but the responses are species-specific. The initiation, development, and growth potential of buds determine the structure of a tree crown. Climate change scenarios project increasing amounts of precipitation at high latitudes in the future, but the effects of a more humid climate on bud size and growth potential remain mostly unexplored. This study investigates the effects of atmospheric humidity on fast-growing deciduous trees, silver birch (Betula pendula Roth.) and hybrid aspen (Populus tremula L. × P. tremuloides Michx.), planted in a unique free-air experimental facility, designed for increasing air humidity during the growing season. Over four consecutive years (2009–2012), the size of overwintering buds was assessed in the upper crown of young trees, and additionally, annual height increments were assessed during the study period. Furthermore, bud contents, probabilities of bud death and break, and subsequent shoot growth were examined. In silver birch, misting effect on height growth depended on year, possibly due to acclimation or variable weather conditions, but bud size was consistently reduced. Nevertheless, misting restricted the growth rather than the initiation of leaf primordia in the bud, and decreased bud size did not translate into changes in the leaf area of future shoots. In contrast, the size of hybrid aspen buds was markedly reduced by misting only in 2009; however, increased humidity promoted bud break, reducing the proportion of dormant buds. The underlying mechanisms causing reduced bud size may involve interactions with shoot growth, but require further study. Although the effect of stimulated bud break is subtle in a given year, cumulative effects may modulate crown structure in the long term, facilitating the acclimation of tree growth to rising humidity in the future.

Climate change is predicted to bring about a rise in precipitation and atmospheric humidity at northern latitudes, which could have a considerable impact on forest ecology and management. This study investigates the effect of elevated relative air humidity (RH) on tree growth and biomass accumulation and allocation in six-year-old seedling-originated silver birch (Betula pendula Roth) and monoclonal hybrid aspen (Populus tremula L.×P. tremuloides Michx.) stands. The study was conducted in the unique Free Air Humidity Manipulation experimental facility in Estonia. Two understory vegetation types (forest and pasture) were used in the experimental plots. As a short-term response during the first manipulation years, a small rise (6–7%) in RH reduced the growth of both tree species. In the fifth humidification year, suppression of growth continued in aspen, but birch acclimated effectively to elevated RH – differences in biomass and stem diameter increments levelled out in humidified (H) and control (C) plots. The H birches ensured biomass accumulation by changing the biomass and morphology of the physiologically most active tree parts: increasing fine-root biomass, as well as specific areas of fine roots (SRA) and leaves (SLA). The effect of elevated RH on most leaf and fine root characteristics of aspens was in a similar direction as that of birches, but weaker, and the plasticity of the functional traits was also lower compared to birches, probably due to the lack of genetic variation in monoclonal aspens. The biomass of young aspens was 22% lower in H plots. The relative aboveground biomass allocation was not affected by elevated RH. The aboveground biomass of humidified birches was higher in plots with forest understory, where root competition was lower compared to the dense root systems of grasses in pasture understory plots. Aspen biomass was not affected by understory vegetation type. We conclude that young silver birches have sufficient phenotypic plasticity to acclimate to elevated RH and the forests retain biomass accumulation. However, the growth acclimation of planted forests of clonal plant material is more complicated and humidity-tolerance should be considered in the selection of new clones for areas where an increase in RH is anticipated.

The predicted increase in precipitation at northern latitudes is an important factor affecting the functioning of forest ecosystems, yet the influence of increased atmospheric humidity on the forest ecosystem carbon cycle is still largely unknown. Our objectives were to determine the effect of elevated atmospheric humidity on soil respiration in silver birch (Betula pendula Roth.) plantations in a Free Air Humidity Manipulation experimental facility (FAHM). Soil respiration was monitored monthly from May to October, in 2008 and 2009 in three control and in three misting plots; soil temperature and soil moisture were measured simultaneously. Fine-root and rhizome biomass of the understory was measured by sequential soil cores. Soil microbial biomass and basal respiration were measured using manometric respirometers. After the first two years of manipulation, humidification led to lower soil respiration. Soil temperature was the main factor influencing seasonal dynamics of soil respiration, describing up to 75% of the total variation of soil respiration. Soil moisture had a weak negative effect on soil respiration. Humidification caused a remarkable increase in below-ground biomass and the production of the understory, and a 28% increase in the basal respiration of microbes. However, above-ground biomass and root turnover rate of the understory were almost unchanged. Hence, elevated atmospheric humidity significantly affects carbon cycle of deciduous forest. However, further studies are necessary for a better understanding of soil respiration response to changes in atmospheric humidity, in order to predict carbon balance in changing climate conditions.

International audience; ContextAnthropogenic activity has increased the level of atmospheric CO2, which is driving an increase of global temperatures and associated changes in precipitation patterns. At Northern latitudes, one of the likely consequences of global warming is increased precipitation and air humidity.AimsIn this work, the effects of both elevated atmospheric CO2 and increased air humidity on trees commonly growing in northern European forests were assessed.MethodsThe work was carried out under field conditions by using Free Air Carbon dioxide Enrichment (FACE) and Free Air Humidity Manipulation (FAHM) systems. Leaf litter fall was measured over 4 years (FACE) or 5 years (FAHM) to determine the effects of FACE and FAHM on leaf phenology.ResultsIncreasing air humidity delayed leaf litter fall in Betula pendula, but not in Populus tremula × tremuloides. Similarly, under elevated atmospheric CO2, leaf litter fall was delayed in B. pendula, but not in Alnus glutinosa. Increased CO2 appeared to interact with periods of low precipitation in summer and high ozone levels during these periods to effect leaf fall.ConclusionsThis work shows that increased CO2 and humidity delay leaf fall, but this effect is species-specific.

Air moisture is one of the main factors causing the greenhouse effect, but humidity has not been thoroughly studied at the ecosystem level. In 2006 a free-air humidity manipulation (FAHM) facility was established in Estonia to investigate the effect of humidity on the performance of two tree species—silver birch (Betula pendula Roth.) and hybrid aspen (Populus tremula L. × P. tremuloides Michx.). The trial is located on an Endogleyic Planosol of former arable land and offers the opportunity to change relative air humidity through controlled artificial misting and drying. We measured the CO2, CH4 and N2O emission from the FAHM system using closed chamber and gas-chromatograph techniques from July 2009 to October 2010. Measurements were carried out once a month in three humidification (h) plots and in three control (c) plots. The vegetation period was rainy in 2009, but the next summer was relatively dry. The dry summer interacted better with the humidification. The CO2 flux decreased when the air moisture was higher than in the control plots. The soil acted as a sink for methane. Less CH4 was oxidized in the soil with increased humidity. Emission of N2O did not correlate with air humidity, although one could observe a clear tendency that there was a smaller N2O flux during the period with increased humidity. Expectedly, CO2 emission and CH4 consumption demonstrated strong positive correlations with soil temperature.

A facility for free air humidity manipulation (FAHM) was established to investigate the effect of increased air humidity on trees’ performance and their canopy functioning with respect to rising air humidity predicted for Northern Europe. The FAHM system enables air relative humidity (RH) to be increased up to 18 units (%) over the ambient level during mist fumigation, depending on the wind speed inside the experimental stand. Water was dispersed inside 14 × 14 m experimental plots in the form of mist with an average particle size of 50 μm from June to August in 2008, and from May to September in 2009. The average increase in RH was 7 units (%) over the whole period of humidification in 2008 ( P F ) in silver birch ( Betula pendula Roth.) trees was 24.8% ( P P P > 0.05) in silver birch on the days without misting. In hybrid aspen ( Populus tremula L. × P. tremuloides Michx.) the average difference in F between C and H plots was 61.1% ( P P F as a result of long-term acclimation to fumigation. The leaves of silver birch in a humidified plot demonstrated up to 2.4 °C lower ( P T L ) compared to the control plot in 2009. The decline in T L decreased the humidity gradient between leaf and air by about 1/3, whereas 2/3 of the effect was caused directly by changes in air humidity in the leaf boundary layer. Our preliminary data suggest that the FAHM experimental facility enables water fluxes through a deciduous tree canopy to be reduced and this effect is attributable both to the increased air humidity and decreased leaf temperature. Changes in these two basic factors may create considerable differences in the physiology, anatomy and nutrition of a whole tree, also affecting forest functioning in the light of global climate change.

The diurnal changes in light-saturated photosynthesis (Pn) under elevated CO(2) and/or O(3) in relation to stomatal conductance (g(s)), water potential, intercellular [CO(2)], leaf temperature and vapour-pressure difference between leaf and air (VPD(L)) were studied at the Aspen FACE site. Two aspen (Populus tremuloides Michx.) clones differing in their sensitivity to ozone were measured. The depression in Pn was found after 10:00 h. The midday decline in Pn corresponded with both decreased g(s) and decreased Rubisco carboxylation efficiency, Vc(max). As a result of increasing VPD(L), g(s) decreased. Elevated [CO(2)] resulted in more pronounced midday decline in Pn compared to ambient concentrations. Moreover, this decline was more pronounced under combined treatment compared to elevated CO(2) treatment. The positive impact of CO(2) on Pn was relatively more pronounced in days with environmental stress but relatively less pronounced during midday depression. The negative impact of ozone tended to decrease in both cases.

The aim of this study was to examine the effects of elevated carbon dioxide [CO2] and ozone [O3] and their interaction on wood chemistry and anatomy of five clones of 3-yearold trembling aspen (Populus tremuloides Michx.). Wood chemistry was studied also on paper birch (Betula papyrifera Marsh.) and sugar maple (Acer saccharum Marsh.) seedling-origin saplings of the same age. Material for the study was collected from the Aspen Free-Air CO2 Enrichment (FACE) experiment in Rhinelander, WI, USA, where the saplings had been exposed to four treatments: control (C; ambient CO2, ambient O3), elevated CO2 (560ppm during daylight hours), elevated O3 (1.5 � ambient during daylight hours) and their combination (CO2 1O3) for three growing seasons (1998‐2000). Wood chemistry responses to the elevated CO2 and O3 treatments differed between species. Aspen was most responsive, while maple was the least responsive of the three tree species. Aspen genotype affected the responses of wood chemistry and, to some extent, wood structure to the treatments. The lignin concentration increased under elevated O3 in four clones of aspen and in birch. However, elevated CO2 ameliorated the effect. In two aspen clones, nitrogen in wood samples decreased under combined exposure to CO2 and O3. Soluble sugar concentration in one aspen clone and starch concentration in two clones were increased by elevated CO2. In aspen wood, a-cellulose concentration changed under elevated CO2, decreasing under ambient O3 and slightly increasing under elevated O3. Hemicellulose concentration in birch was decreased by elevated CO2 and increased by elevated O3. In aspen, elevated O3 induced statistically significant reductions in distance from the pith to the bark and vessel lumen diameter, as well as increased wall thickness and wall percentage, and in one clone, decreased fibre lumen diameter. Our results show that juvenile wood properties of broadleaves, depending on species and genotype, were altered by atmospheric gas concentrations predicted for the year 2050 and that CO2 ameliorates some adverse effects of elevated O3 on wood chemistry.

Summary • Saplings of three aspen ( Populus tremuloides ) genotypes and seedlings of paper birch ( Betula papyrifera ) were exposed to elevated ozone (1.5 × ambient) and 560 p.p.m. CO 2 , singly and in combination, from 1998 at the Aspen-FACE (free-air CO 2 enrichment) site (Rhinelander, USA). • The plants were studied for H 2 O 2 accumulation within the leaf mesophyll, number of peroxisomes, level of gene expression for catalase ( Cat ), and changes in ultrastructure. • In tolerant clones, ozone-elicited excess H 2 O 2 production was restricted to the apoplast, without any ultrastructural injuries. This was associated with ozoneinduced proliferation of peroxisomes and increased transcript levels of Cat . In sensitive plants, ozone-induced H 2 O 2 accumulation continued from the cell wall to the plasma membrane, cytosol and chloroplasts, particularly in older leaves. However, chloroplastic precipitation was absent in the presence of elevated CO 2 . In the most sensitive aspen clone, H 2 O 2 accumulation was found in conjunction with chloroplast injuries, low number of peroxisomes and low cell wall volume, whereas in birch a simultaneous increase in cell wall thickness indicated defence activation. • Our results indicate that oxidative stress manifests as H 2 O 2 effects on leaf ultrastructure in sensitive trees exposed to elevated ozone. However, CO 2 enrichment appears to alleviate chloroplastic oxidative stress.

Summary 1. The impacts of elevated atmospheric CO 2 and/or O 3 have been examined over 4 years using an open-air exposure system in an aggrading northern temperate forest containing two different functional groups (the indeterminate, pioneer, O 3 -sensitive species Trembling Aspen, Populus tremuloides and Paper Birch, Betula papyrifera , and the determinate, late successional, O 3 -tolerant species Sugar Maple, Acer saccharum ). 2. The responses to these interacting greenhouse gases have been remarkably consistent in pure Aspen stands and in mixed Aspen/Birch and Aspen/Maple stands, from leaf to ecosystem level, for O 3 -tolerant as well as O 3 -sensitive genotypes and across various trophic levels. These two gases act in opposing ways, and even at low concentrations (1·5 × ambient, with ambient averaging 34‐36 nL L − 1 during the summer daylight hours), O 3 offsets or moderates the responses induced by elevated CO 2 . 3. After 3 years of exposure to 560 µ mol mol − 1 CO 2 , the above-ground volume of Aspen stands was 40% above those grown at ambient CO 2 , and there was no indication of a diminishing growth trend. In contrast, O 3 at 1·5 × ambient completely offset the growth enhancement by CO 2 , both for O 3 -sensitive and O 3 -tolerant clones. Implications of this finding for carbon sequestration, plantations to reduce excess CO 2 , and global models of forest productivity and climate change are presented.

Human activity causes increasing background concentrations of the greenhouse gases C02 and O3. Increased levels of C02 can be found in all terrestrial ecosystems. Damaging O3 concentrations currently occur over 29% of the world's temperate and subpolar forests but are predicted to affect fully 60% by 2100 (ref. 3). Although individual effects of C02 and O3, on vegetation have been widely investigated, very little is known about their interaction, and long-term studies on mature trees and higher trophic levels are extremely rare. Here we present evidence from the most widely distributed North American tree species, Populus tremuloides, showing that C02 and O3, singly and in combination, affected productivity, physical and chemical leaf defences and, because of changes in plant quality, insect and disease populations. Our data show that feedbacks to plant growth from changes induced by C02 and O3 in plant quality and pest performance are likely. Assessments of global change effects on forest ecosystems must therefore consider the interacting effects of C02 and O3 on plant performance, as well as the implications of increased pest activity.

We investigated the interaction of elevated CO2 and/or (Ozone) O3 on the occurrence and severity of aspen leaf rust (Malampsora medusae Thuem. f. sp. tremuloidae) on trembling aspen (Populus tremuloides MIchx.) Furthermore, we examined the role of changes in leaf surface properties induced by elevated CO2 and/or O3 in this host-pathogen interaction.

The Intergovernmental Panel of Climate Change (IPCC) has concluded that the greenhouse gases carbon dioxide (CO2) and tropospheric ozone (O3) are increasing concomitantly globally. Little is known about the effect of these interacting gases on growth, survival, and productivity of forest ecosystems. In this study we assess the effects of three successive years of exposure to combinations of elevated CO2 and O3 on growth responses in a five trembling aspen (Populus tremuloides) clonal mixture in a regenerating stand. The experiment is located in Rhinelander, Wisconsin, USA (45 N8 9W) and employs free air carbon dioxide and ozone enrichment (FACE) technology. The aspen stand was exposed to a factorial combination of four treatments consisting of elevated CO2 (560 ppm), elevated O3 (episodic exposure-90 m 11 1 hour 1 ), a combination of elevated CO2 and O3, and ambient control in 30 m treatment rings with three replications. Our overall results showed that our three growth parameters including height, diameter and volume were increased by elevated CO2, decreased by elevated O3, and were not significantly different from the ambient control under elevated CO2+O3. However, there were significant clonal differences in the responses; all five clones exhibited increased growth with elevated CO2, one clone showed an increase with elevated O3, and two clones showed an increase over the control with elevated CO2+O3, two clones showed a decrease, and one was not significantly different from the control. Notably, there was a significant increase in current terminal shoot dieback with elevated CO2 during the 1999–2000 dormant season. Dieback was especially prominent in two of the five clones, and was attributed to those clones growing longer into the autumnal season where they were subject to frost. Our results show that elevated O3 negates expected positive growth effects of elevated CO2 in Populus tremuloides in the field, and suggest that future climate model predictions should take into account the offsetting effects of elevated O3 on CO2 enrichment when estimating future growth of trembling aspen stands. # 2001 Published by Elsevier Science Ltd. All rights reserved.

To determine whether elevated CO 2 reduces or exacerbates the detrimental effects of O 3 on aspen ( Populus tremuloides Michx.), aspen clones 216 and 271 (O 3 tolerant), and 259 (O 3 sensitive) were exposed to ambient levels of CO 2 and O 3 or elevated levels of CO 2 , O 3 , or CO 2 +O 3 in the FACTS II (Aspen FACE) experiment, and physiological and molecular responses were measured and compared. Clone 259, the most O 3 -sensitive clone, showed the greatest amount of visible foliar symptoms as well as significant decreases in chlorophyll, carotenoid, starch, and ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) concentrations and transcription levels for the Rubisco small subunit. Generally, the constitutive (basic) transcript levels for phenylalanine ammonia-lyase ( PAL ) and chalcone synthase ( CHS ) and the average antioxidant activities were lower for the ozone sensitive clone 259 as compared to the more tolerant 216 and 271 clones. A significant decrease in chlorophyll a , b and total ( a + b ) concentrations in CO 2, O 3 , and CO 2+ O 3 plants was observed for all clones. Carotenoid concentrations were also significantly lower in all clones; however, CHS transcript levels were not significantly affected, suggesting a possible degradation of carotenoid pigments in O 3 -stressed plants. Antioxidant activities and PAL and 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase transcript levels showed a general increase in all O 3 treated clones, while remaining low in CO 2 and CO 2+ O 3 plants (although not all differences were significant). Our results suggest that the ascorbate-glutathione and phenylpropanoid pathways were activated under ozone stress and suppressed during exposure to elevated CO 2 . Although CO 2+ O 3 treatment resulted in a slight reduction of O 3 -induced leaf injury, it did not appear to ameliorate all of the harmful affects of O 3 and, in fact, may have contributed to an increase in chloroplast damage in all three aspen clones.

Rising atmospheric CO 2 may stimulate future forest productivity, possibly increasing carbon storage in terrestrial ecosystems, but how tropospheric ozone will modify this response is unknown. Because of the importance of fine roots to the belowground C cycle, we monitored fine-root biomass and associated C fluxes in regenerating stands of trembling aspen, and mixed stands of trembling aspen and paper birch at FACTS-II, the Aspen FACE project in Rhinelander, Wisconsin. Free-air CO 2 enrichment (FACE) was used to elevate concentrations of CO2 (average enrichment concentra- tion 535 µl l -1 ) and O 3 (53 nl l -1 ) in developing forest stands in 1998 and 1999. Soil respiration, soil pCO 2 , and dissolved organic carbon in soil solution (DOC) were monitored biweekly. Soil respiration was measured with a portable infrared gas analyzer. Soil pCO 2 and DOC samples were collected from soil gas wells and tension lysimeters, respectively, at depths of 15, 30, and 125 cm. Fine-root biomass averaged 263 g m-2 in con- trol plots and increased 96% under elevated CO 2. The increased root biomass was accompanied by a 39% in- crease in soil respiration and a 27% increase in soil pCO 2 . Both soil respiration and pCO 2 exhibited a strong seasonal signal, which was positively correlated with soil temperature. DOC concentrations in soil solution averaged ~12 mg l -1 in surface horizons, declined with depth, and were little affected by the treatments. A sim- plified belowground C budget for the site indicated that native soil organic matter still dominated the system, and that soil respiration was by far the largest flux. Ozone decreased the above responses to elevated CO2, but effects were rarely statistically significant. We con- clude that regenerating stands of northern hardwoods have the potential for substantially greater C input to soil due to greater fine-root production under elevated CO 2 . Greater fine-root biomass will be accompanied by greater soil C efflux as soil respiration, but leaching losses of C will probably be unaffected.

Leaf gas exchange parameters and the content of ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the leaves of two 2-year-old aspen (Populus tremuloides Michx.) clones (no. 216, ozone tolerant and no. 259, ozone sensitive) were determined to estimate the relative stomatal and mesophyll limitations to photosynthesis and to determine how these limitations were altered by exposure to elevated CO2 and/or O3. The plants were exposed either to ambient air (control), elevated CO2 (560 p.p.m.) elevated O3 (55 p.p.b.) or a mixture of elevated CO2 and O3 in a free air CO2 enrichment (FACE) facility located near Rhinelander, Wisconsin, USA. Light-saturated photosynthesis and stomatal conductance were measured in all leaves of the current terminal and of two lateral branches (one from the upper and one from the lower canopy) to detect possible age-related variation in relative stomatal limitation (leaf age is described as a function of leaf plastochron index). Photosynthesis was increased by elevated CO2 and decreased by O3 at both control and elevated CO2. The relative stomatal limitation to photosynthesis (ls) was in both clones about 10% under control and elevated O3. Exposure to elevated CO2 + O3 in both clones and to elevated CO2 in clone 259, decreased ls even further ‐ to about 5%. The corresponding changes in Rubisco content and the stability of Ci/Ca ratio suggest that the changes in photosynthesis in response to elevated CO2 and O3 were primarily triggered by altered mesophyll processes in the two aspen clones of contrasting O3 tolerance. The changes in stomatal conductance seem to be a secondary response, maintaining stable Ci under the given treatment, that indicates close coupling between stomatal and mesophyll processes.

Over the years, a series of trembling aspen (Populus tremuloides Michx.) clones differing in O3 sensitivity have been identified from OTC studies. Three clones (216 and 271[(O3 tolerant] and 259 [O3 sensitive]) have been characterized for O3 sensitivity by growth and biomass responses, foliar symptoms, gas exchange, chlorophyll content, epicuticular wax characteristics, and antioxidant production. In this study we compared the responses of these same clones exposed to O3 under field conditions along a natural O3 gradient and in a Free-Air CO2 and O3 Enrichment (FACE) facility. In addition, we examined how elevated CO2 affected O3 symptom development. Visible O3 symptoms were consistently seen (5 out of 6 years) at two of the three sites along the O3 gradient and where daily one-hour maximum concentrations were in the range of 96 to 125 ppb. Clonal differences in O3 sensitivity were consistent with our OTC rankings Elevated CO2 (200 ppm over ambient and applied during daylight hours during the growing season) reduced visible foliar symptoms for all three clones from 31 to 96% as determined by symptom development in elevated O3 versus elevated O3 + CO2 treatments. Degradation of the epicuticular wax surface of all three clones was found at the two elevated O3 gradient sites. This degradation was quantified by a coefficient of occlusion which was a measure of stomatal occlusion by epicuticular waxes. Statistically significant increases in stomatal occlusion compared to controls were found for all three clones and for all treatments including elevated CO2, elevated O3, and elevated CO2 + O3. Our results provide additional evidence that current ambient O3 levels in the Great Lakes region are causing adverse effects on trembling aspen. Whether or not elevated CO2 in the future will alleviate some of these adverse effects, as occurred with visible symptoms but not with epicuticular wax degradation, is unknown.

We developed free-air exposure regression-based models comprising annual growing season 4th highest daily maximum 8-h average ozone (O 3 ) concentration, growing degree days (GDD), and average wind speed (WS). The models include 95% confidence bands for determining uncertainty of prediction. The models are statistically significant, provide a high goodness of fit, and can be used within the ambient air context. Trembling aspen ( Populus tremuloides ) clones 216, 42E, 271, and 259 responded negatively to O 3 . Aspen clone 8L responded positively to growing season 4th highest daily maximum 8-h average O 3 concentration ≤90 ppb. White birch ( Betula papyrifera ) responded positively to growing season 4th highest daily maximum 8-h average O 3 concentration 3 concentration performed much better as a single O 3 exposure index for trembling aspen and white birch cross-sectional area growth than did W126, SUM06, AOT40, and maximum 1-h average O 3 concentration. Growing season 4th highest daily maximum 8-h average O 3 concentration is most closely associated with the actual measured response in the biological endpoint. The W126 index significantly overestimated the negative growth response of aspen and birch to O 3 . The growing season 4th highest daily maximum 8-h average O 3 concentration, cumulative GDD, and average WS-based model may provide an underutilized opportunity for scientifically defensible risk analysis within the North American air quality context.

Shoots of a soil- or sand-grown dwarf bean variety were exposed to O(3) concentrations in the range of 500 to 900 ppb for up to 5 h. The measured exchange rates of water vapor and CO(2) during exposures were used to calculate stomatal and mesophyll conductances averaged across all leaves. Changes in conductances were related to exposure duration and absorbed O(3) totals (AOT). Both conductances were more sensitive to AOT in sand-grown plants, which also had more visible injury under comparable AOT values. Measurements of the relationship between CO(2) exchange and internal CO(2) concentration of single leaflets of treated plants also showed greater sensitivity of CO(2)-saturated photosynthesis in sand-grown plants. Diffusional processes were not likely to have been the cause of dissimilar responses because the O(3) absorption rate was lower in sand-grown plants. A difference in the scaveninng capacities in cells is suggested to be the cause of the differences in sensitivity to acute O(3) exposure.

To determine whether elevated CO 2 reduces or exacerbates the detrimental effects of O 3 on aspen ( Populus tremuloides Michx.). Aspen clones 216 and 271 (O 3 tolerant), and 259 (O 3 sensitive) were exposed to ambient levels of CO 2 and O 3 or elevated levels of CO 2 , O 3 , or CO 2 + O 3 in the FACTS II (Aspen FACE) experiment, and physiological and molecular responses were measured and compared. Clone 259, the most O 3 -sensitive clone, showed the greatest amount of visible foliar symptoms as well as significant decreases in chlorophyll, carotenoid, starch, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) concentrations and transcription levels for the Rubisco small subunit. Generally, the constitutive (basic) transcript levels for phenylalanine ammonia-lyase (Pal) and chalcone synthase (Chs) and the average antioxidant activities were lower for the ozone sensitive clone 259 as compared to the more tolerant 216 and 271 clones. A significant decrease in chlorophyll a, b and total (a+b) concentrations in CO 2, O 3 , and CO 2 + O 3 plants was observed for all clones. Carotenoid concentrations were also significantly lower in all clones; however, Chs transcript levels were not significantly affected, suggesting a possible degradation of carotenoid pigments in O 3 -stressed plants. Antioxidant activities and Pal and 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase transcript levels showed a general increase in all O 3 treated clones, while remaining low in CO 2 and CO 2 + O 3 plants (although not all differences were significant). Our results suggest that the ascorbate-glutathione and phenylpropanoid pathways were activated under ozone stress and suppressed during exposure to elevated CO 2 . Although CO 2 + O 3 treatment resulted in a slight reduction of O 3 -induced leaf injury, it did not appear to ameliorate all of the harmful affects of O 3 and, in fact, may have contributed to an increase in chloroplast damage in all three aspen clones.

The International Panel of Climate Change (IPCC) has concluded that the greenhouse gases, carbon dioxide (CO2) and tropospheric ozone (O3), are increasing concomitantly globally. Little is known about the effect of these interacting gases on growth, survival, and productivity of forest ecosystems. In this study we assess the effects of three successive years of exposure to combinations of elevated CO2 and O3 on growth responses in a 5 trembling aspen (Populus tremuloides) clonal mixture in a regenerating stand. The experiment is located in Rhinelander, Wisconsin, USA (45° N 89° W) and employs free air carbon dioxide and ozone enrichment (FACE) technology. The aspen stand was exposed to a factorial combination of 4 treatments consisting of elevated CO2 (560 ppm), elevated O3 (episodic exposure—90 μl l −1 h −1 ), a combination of elevated CO2 and O3, and ambient control in 30 m treatment rings with 3 replications. Our overall results showed that our 3 growth parameters including height, diameter and volume were increased by elevated CO2, decreased by elevated O3, and were not significantly different from the ambient control under elevated CO2 + O3. However, there were significant clonal differences in the responses; all 5 clones exhibited increased growth with elevated CO2, one clone showed an increase with elevated O3, and two clones showed an increase over the control with elevated CO2 + O3, two clones showed a decrease, and one was not significantly different from the control. Notably, there was a significant increase in current terminal shoot dieback with elevated CO2 during the 1999–2000 dormant seasons. Dieback was especially prominent in two of the five clones, and was attributed to those clones growing longer into the autumnal season where they were subject to frost. In summary, our results show that elevated O3 negates expected positive growth effects of elevated CO2 in Populus tremuloides in the field. Our results suggest that future climate model predictions should take into account the offsetting effects of elevated O3 on CO2 enrichment when estimating future growth of trembling aspen stands.

Impacts of elevated atmospheric O3 and/or CO2 on three clones of aspen (Populus tremuloides Michx.) and birch (Betula papyrifera Marsh.) were studied to determine, whether or not elevated CO2 ameliorates O3-induced damage to leaf cells. The plants were exposed for 3 years at the Aspen FACE exposure site in Wisconsin (USA) prior to sampling for ultrastructural investigations on 19 June 1999. In the aspen clones, elevated CO2 increased chloroplast cover index, leaf and spongy mesophyll layer thickness, intercellular air space volume in mesophyll, amount of starch in chloroplasts and cytoplasmic lipids but decreased the number of plastoglobuli in chloroplasts. In contrast, elevated O3 decreased chloroplast cover index, starch content, and the proportion of cytoplasm and intercellular space in mesophyll, and increased the proportion of vacuoles, the amount of condensed vacuolar tannins and the number of plastoglobuli. Ozone also caused structural thylakoid injuries (dilation, distortion) and stromal condensation in chloroplasts, which was ameliorated by elevated CO2 by 5-66% in aspen clones and by 2-10% in birch. Birch ultrastructure was less affected by elevated CO2 or O3 stress compared to aspen. In the most O3-sensitive aspen clone, thinner leaves and cell walls, lower proportion of cell wall volume, and higher volume for vacuoles was found compared to more-tolerant clones.

At northern latitudes a rise in atmospheric humidity and precipitation is predicted as a consequence of global climate change. We studied several growth and functional traits of hybrid aspen (Populus tremula L.×P. tremuloides Michx.) in response to elevated atmospheric humidity (on average 7% over the ambient level) in a free air experimental facility during three growing seasons (2008-2010) in Estonia, which represents northern temperate climate (boreo-nemoral zone). Data were collected from three humidified (H) and three control (C) plots, and analysed using nested linear models. Elevated air humidity significantly reduced height, stem diameter and stem volume increments and transpiration of the trees whereas these effects remained highly significant also after considering the side effects from soil-related confounders within the 2.7 ha study area. Tree leaves were smaller, lighter and had lower leaf mass per area (LMA) in H plots. The magnitude and significance of the humidity treatment effect--inhibition of above-ground growth rate--was more pronounced in larger trees. The lower growth rate in the humidified plots can be partly explained by a decrease in transpiration-driven mass flow of NO(3) (-) in soil, resulting in a significant reduction in the measured uptake of N to foliage in the H plots. The results suggest that the potential growth improvement of fast-growing trees like aspens, due to increasing temperature and atmospheric CO(2) concentration, might be smaller than expected at high latitudes if a rise in atmospheric humidity simultaneously takes place.

Nature 420, 403–407 (2002). In this Letter, the conversion to SI units led to several errors. On page 404, left column, lines 16 and 17, the values should read 10–15 and 30–40 nanolitres per litre. On page 405, right column, lines 3 and 4, the values should read 360 microlitres per litre and 36.0–38.8 nanolitres per litre.

We investigated the interactive effects of elevated concentrations of carbon dioxide ([CO(2)]) and ozone ([O(3)]) on radial growth, wood chemistry and structure of five 5-year-old trembling aspen (Populus tremuloides Michx.) clones and the wood chemistry of paper birch (Betula papyrifera Marsh.). Material for the study was collected from the Aspen FACE (free-air CO(2) enrichment) experiment in Rhinelander, WI, where the saplings had been exposed to four treatments: control, elevated [CO(2)] (560 ppm), elevated [O(3)] (1.5 x ambient) and their combination for five growing seasons. Wood properties of both species were altered in response to exposure to the treatments. In aspen, elevated [CO(2)] decreased uronic acids (constituents of, e.g., hemicellulose) and tended to increase stem diameter. In response to elevated [O(3)] exposure, acid-soluble lignin concentration decreased and vessel lumen diameter tended to decrease. Elevated [O(3)] increased the concentration of acetone-soluble extractives in paper birch, but tended to decrease the concentration of these compounds in aspen. In paper birch, elevated [CO(2)] decreased and elevated [O(3)] increased starch concentration. The responses of wood properties to 5 years of fumigation differed from those previously reported after 3 years of fumigation.