Stomatal uptake of O3 in aspen and aspen-birch forests under free-air CO2 and O3 enrichment

Rising atmospheric carbon dioxide (CO₂) may alleviate the toxicological impacts of concurrently rising tropospheric ozone (O₃) during the present century if higher CO₂ is accompanied by lower stomatal conductance (g_s), as assumed by many models. We investigated how elevated concentrations of CO₂ and O₃, alone and in combination, affected the accumulated stomatal flux of O₃ (AFst) by canopies and sun leaves in closed aspen and aspen-birch forests in the free-air CO₂-O₃ enrichment experiment near Rhinelander, Wisconsin. Stomatal conductance for O₃ was derived from sap flux data and AFst was estimated either neglecting or accounting for the potential influence of non-stomatal leaf surface O₃ deposition. Leaf-level AFst (AFst_l) was not reduced by elevated CO₂. Instead, there was a significant CO₂ × O₃ interaction on AFst_l, as a consequence of lower values of g_s in control plots and the combination treatment than in the two single-gas treatments. In addition, aspen leaves had higher AFst_l than birch leaves, and estimates of AFst_l were not very sensitive to non-stomatal leaf surface O₃ deposition. Our results suggest that model projections of large CO₂-induced reductions in g_s alleviating the adverse effect of rising tropospheric O₃ may not be reasonable for northern hardwood forests.     

Will photosynthetic capacity of aspen trees acclimate after long-term exposure to elevated CO2 and O3?

Photosynthetic acclimation under elevated carbon dioxide (CO₂) and/or ozone (O₃) has been the topic of discussion in many papers recently. We examined whether or not aspen plants grown under elevated CO₂ and/or O₃ will acclimate after 11 years of exposure at the Aspen Face site in Rhinelander, WI, USA. We studied diurnal patterns of instantaneous photosynthetic measurements as well as A/C_i measurements monthly during the 2004-2008 growing seasons. Our results suggest that the responses of two aspen clones differing in O₃ sensitivity showed no evidence of photosynthetic and stomatal acclimation under either elevated CO₂, O₃ or CO₂ + O₃. Both clones 42E and 271 did not show photosynthetic nor stomatal acclimation under elevated CO₂ and O₃ after a decade of exposure. We found that the degree of increase or decrease in the photosynthesis and stomatal conductance varied significantly from day to day and from one season to another.     

Leaf size and surface characteristics of Betula papyrifera exposed to elevated CO2 and O3

Betula papyrifera trees were exposed to elevated concentrations of CO₂ (1.4 × ambient), O₃ (1.2 × ambient) or CO₂ + O₃ at the Aspen Free-air CO₂ Enrichment Experiment. The treatment effects on leaf surface characteristics were studied after nine years of tree exposure. CO₂ and O₃ increased epidermal cell size and reduced epidermal cell density but leaf size was not altered. Stomatal density remained unaffected, but stomatal index increased under elevated CO₂. Cuticular ridges and epicuticular wax crystallites were less evident under CO₂ and CO₂ + O₃. The increase in amorphous deposits, particularly under CO₂ + O_3, was associated with the appearance of elongated plate crystallites in stomatal chambers. Increased proportions of alkyl esters resulted from increased esterification of fatty acids and alcohols under elevated CO₂ + O₃. The combination of elevated CO₂ and O₃ resulted in different responses than expected under exposure to CO₂ or O₃ alone.     

Effects of elevated atmospheric CO2 and tropospheric O3 on tree branch growth and implications for hydrologic budgeting

The forest hydrologic budget may be impacted by increasing CO₂ and tropospheric O₃. Efficient means to quantify such effects are beneficial. We hypothesized that changes in the balance of canopy interception, stem flow, and through-fall in the presence of elevated CO₂ and O₃ could be discerned using image analysis of leafless branches. We compared annual stem flow to the results of a computerized analysis of all branches from the 2002, 2004, and 2006 annual growth whorls of 97 ten-year-old trees from the Aspen Free-Air CO₂ and O₃ Enrichment (Aspen FACE) experiment in Rhinelander, WI. We found significant effects of elevated CO₂ and O₃ on some branch metrics, and that the branch metrics were useful for predicting stem flow from birch, but not aspen. The results of this study should contribute to development of techniques for efficient characterization of effects on the forest hydrologic budget of increasing CO₂ and tropospheric O₃.     

Stomatal characteristics and infection biology of Pyrenopeziza betulicola in Betula pendula trees grown under elevated CO2 and O3

Two silver birch clones were exposed to ambient and elevated concentrations of CO₂ and O₃, and their combination for 3 years, using open-top chambers. We evaluated the effects of elevated CO₂ and O₃ on stomatal conductance (g_s), density (SD) and index (SI), length of the guard cells, and epidermal cell size and number, with respect to crown position and leaf type. The relationship between the infection biology of the fungus (Pyrenopeziza betulicola) causing leaf spot disease and stomatal characteristics was also studied. Leaf type was an important determinant of O₃ response in silver birch, while crown position and clone played only a minor role. Elevated CO₂ reduced the g_s, but had otherwise no significant effect on the parameters studied. No significant interactions between elevated CO₂ and O₃ were found. The infection biology of P. betulicola was not correlated with SD or g_s, but it did occasionally correlate positively with the length of the guard cells.     

DNA damage in Populus tremuloides clones exposed to elevated O3

The effects of elevated concentrations of atmospheric tropospheric ozone (O₃) on DNA damage in five trembling aspen (Populus tremuloides Michx.) clones growing in a free-air enrichment experiment in the presence and absence of elevated concentrations of carbon dioxide (CO₂) were examined. Growing season mean hourly O₃ concentrations were 36.3 and 47.3 ppb for ambient and elevated O₃ plots, respectively. The 4th highest daily maximum 8-h ambient and elevated O₃ concentrations were 79 and 89 ppb, respectively. Elevated CO₂ averaged 524 ppm (+150 ppm) over the growing season. Exposure to O₃ and CO₂ in combination with O₃ increased DNA damage levels above background as measured by the comet assay. Ozone-tolerant clones 271 and 8L showed the highest levels of DNA damage under elevated O₃ compared with ambient air; whereas less tolerant clone 216 and sensitive clones 42E and 259 had comparably lower levels of DNA damage with no significant differences between elevated O₃ and ambient air. Clone 8L was demonstrated to have the highest level of excision DNA repair. In addition, clone 271 had the highest level of oxidative damage as measured by lipid peroxidation. The results suggest that variation in cellular responses to DNA damage between aspen clones may contribute to O₃ tolerance or sensitivity.     

Spring leaf flush in aspen (Populus tremuloides) clones is altered by long-term growth at elevated carbon dioxide and elevated ozone concentration

Early spring leaf out is important to the success of deciduous trees competing for light and space in dense forest plantation canopies. In this study, we investigated spring leaf flush and how long-term growth at elevated carbon dioxide concentration ([CO₂]) and elevated ozone concentration ([O₃]) altered leaf area index development in a closed Populus tremuloides (aspen) canopy. This work was done at the Aspen FACE experiment where aspen clones have been grown since 1997 in conditions simulating the [CO₂] and [O₃] predicted for ∼2050. The responses of two clones were compared during the first month of spring leaf out when CO₂ fumigation had begun, but O₃ fumigation had not. Trees in elevated [CO₂] plots showed a stimulation of leaf area index (36%), while trees in elevated [O₃] plots had lower leaf area index (−20%). While individual leaf area was not significantly affected by elevated [CO₂], the photosynthetic operating efficiency of aspen leaves was significantly improved (51%). There were no significant differences in the way that the two aspen clones responded to elevated [CO₂]; however, the two clones responded differently to long-term growth at elevated [O₃]. The O₃-sensitive clone, 42E, had reduced individual leaf area when grown at elevated [O₃] (−32%), while the tolerant clone, 216, had larger mature leaf area at elevated [O₃] (46%). These results indicate a clear difference between the two clones in their long-term response to elevated [O₃], which could affect competition between the clones, and result in altered genotypic composition in future atmospheric conditions.     

Effects of chronic elevated ozone exposure on gas exchange responses of adult beech trees (Fagus sylvatica) as related to the within-canopy light gradient

The effects of elevated O₃ on photosynthetic properties in adult beech trees (Fagus sylvatica) were investigated in relation to leaf mass per area as a measure of the gradually changing, within-canopy light availability. Leaves under elevated O₃ showed decreased stomatal conductance at unchanged carboxylation capacity of Rubisco, which was consistent with enhanced δ¹³C of leaf organic matter, regardless of the light environment during growth. In parallel, increased energy demand for O₃ detoxification and repair was suggested under elevated O₃ owing to enhanced dark respiration. Only in shade-grown leaves, light-limited photosynthesis was reduced under elevated O₃, this effect being accompanied by lowered F_v/F_m. These results suggest that chronic O₃ exposure primarily caused stomatal closure to adult beech trees in the field regardless of the within-canopy light gradient. However, light limitation apparently raised the O₃ sensitivity of photosynthesis and accelerated senescence in shade leaves.     

Effects of high atmospheric CO2 concentration on root hydraulic conductivity of conifers depend on species identity and inorganic nitrogen source

We examined root hydraulic conductivity (L_p) responses of one-year-old seedlings of four conifers to the combined effects of elevated CO₂ and inorganic nitrogen (N) sources. We found marked interspecific differences in L_p responses to high CO₂ ranging from a 37% increase in P. abies to a 27% decrease in P. menziesii, but these effects depended on N source. The results indicate that CO₂ effects on root water transport may be coupled to leaf area responses under nitrate (NO₃⁻), but not ammonium (NH₄⁺) dominated soils. To our knowledge, this is the first study that highlights the role of inorganic N source and species identity as critical factors that determine plant hydraulic responses to rising atmospheric CO₂ levels. The results have important implications for understanding root biology in a changing climate and for models designed to predict feedbacks between rising atmospheric CO₂, N deposition, and ecohydrology.     

Temporal processes that contribute to nonlinearity in vegetation responses to ozone exposure and dose

Ozone interacts with plant tissue through distinct temporal processes. Sequentially, plants are exposed to ambient O₃ that (1) moves through the leaf boundary layer, (2) is taken up into plant tissue primarily through stomata, and (3) undergoes chemical interaction within plant tissue, first by initiating alterations and then as part of plant detoxification and repair. In this paper, we discuss the linkage of the temporal variability of apoplastic ascorbate with the diurnal variability of defense mechanisms in plants and compare this variability with daily maximum O₃ concentration and diurnal uptake and entry of O₃ into the plant through stomata. We describe the quantitative evidence on temporal variability in concentration and uptake and find that the time incidence for maximum defense does not necessarily match diurnal patterns for maximum O₃ concentration or maximum uptake. We suggest that the observed out-of-phase association of the diurnal patterns for the above three processes produces a nonlinear relationship that results in a greater response from the higher hourly average O₃ concentrations than from the lower or mid-level values. The fact that these out-of-phase processes affect the relationship between O₃ exposure/dose and vegetation effects ultimately impact the ability of flux-based indices to predict vegetation effects accurately for purposes of standard setting and critical levels. Based on the quantitative aspect of temporal variability identified in this paper, we suggest that the inclusion of a diurnal pattern for detoxification in effective flux-based models would improve the predictive characteristics of the models. While much of the current information has been obtained using high O₃ exposures, future research results derived from laboratory biochemical experiments that use short but elevated O₃ exposures should be combined with experimental results that use ambient-type exposures over longer periods of time. It is anticipated that improved understanding will come from future research focused on diurnal variability in plant defense mechanisms and their relationship to the diurnal variability in ambient O₃ concentration and stomatal conductance. This should result in more reliable O₃ exposure standards and critical levels.     

Responses to ozone pollution of alfalfa exposed to increasing salinity levels

Stomatal closure and biosynthesis of antioxidant molecules are two fundamental components of the physiological machinery that lead to stress adaptation during plant's exposure to salinity. Since high stomatal resistance may also contribute in counteracting O₃ damages, we hypothesized that soil salinization may increase O₃ tolerance of crops. An experiment was performed with alfalfa grown in filtered (AOT40 = 0 in both years) and non-filtered (AOT40 = 9.7 in 2005 and 6.9 ppm h in 2006) open-top chambers. Alfalfa yield was reduced by O₃ (−33%) only in plants irrigated with salt-free water, while the increasing levels of soil salinity until 1.06 dS m⁻¹ reduced both stomatal conductance and plant O₃ uptake, thus linearly reducing O₃ effects on yield. Therefore a reliable flux-based model for assessing the effects of O₃ on crop yield should take into account soil salinity.     

Gene expression responses of paper birch (Betula papyrifera) to elevated CO2 and O3 during leaf maturation and senescence

Gene expression responses of paper birch (Betula papyrifera) leaves to elevated concentrations of CO₂ and O₃ were studied with microarray analyses from three time points during the summer of 2004 at Aspen FACE. Microarray data were analyzed with clustering techniques, self-organizing maps, K-means clustering and Sammon's mappings, to detect similar gene expression patterns within sampling times and treatments. Most of the alterations in gene expression were caused by O₃, alone or in combination with CO₂. O₃ induced defensive reactions to oxidative stress and earlier leaf senescence, seen as decreased expression of photosynthesis- and carbon fixation-related genes, and increased expression of senescence-associated genes. The effects of elevated CO₂ reflected surplus of carbon that was directed to synthesis of secondary compounds. The combined CO₂ + O₃ treatment resulted in differential gene expression than with individual gas treatments or in changes similar to O₃ treatment, indicating that CO₂ cannot totally alleviate the harmful effects of O₃.     

Soil nitrogen transformations under elevated atmospheric CO₂ and O₃ during the soybean growing season

We investigated the influence of elevated CO₂ and O₃ on soil N cycling within the soybean growing season and across soil environments (i.e., rhizosphere and bulk soil) at the Soybean Free Air Concentration Enrichment (SoyFACE) experiment in Illinois, USA. Elevated O₃ decreased soil mineral N likely through a reduction in plant material input and increased denitrification, which was evidenced by the greater abundance of the denitrifier gene nosZ. Elevated CO₂ did not alter the parameters evaluated and both elevated CO₂ and O₃ showed no interactive effects on nitrifier and denitrifier abundance, nor on total and mineral N concentrations. These results indicate that elevated CO₂ may have limited effects on N transformations in soybean agroecosystems. However, elevated O₃ can lead to a decrease in soil N availability in both bulk and rhizosphere soils, and this likely also affects ecosystem productivity by reducing the mineralization rates of plant-derived residues.     

Diurnal changes in photosynthetic parameters of Populus tremuloides, modulated by elevated concentrations of CO2 and/or O3 and daily climatic variation

The diurnal changes in light-saturated photosynthesis (Pn) under elevated CO₂ and/or O₃ in relation to stomatal conductance (g_s), water potential, intercellular [CO₂], 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 [CO₂] resulted in more pronounced midday decline in Pn compared to ambient concentrations. Moreover, this decline was more pronounced under combined treatment compared to elevated CO₂ treatment.The positive impact of CO₂ 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.     

Belowground effects of enhanced tropospheric ozone and drought in a beech/spruce forest (Fagus sylvatica L./Picea abies [L.] Karst)

The effects of experimentally elevated O₃ on soil respiration rates, standing fine-root biomass, fine-root production and δ¹³C signature of newly produced fine roots were investigated in an adult European beech/Norway spruce forest in Germany during two subsequent years with contrasting rainfall patterns. During humid 2002, soil respiration rate was enhanced under elevated O₃ under beech and spruce, and was related to O₃-stimulated fine-root production only in beech. During dry 2003, the stimulating effect of O₃ on soil respiration rate vanished under spruce, which was correlated with decreased fine-root production in spruce under drought, irrespective of the O₃ regime. δ¹³C signature of newly formed fine-roots was consistent with the differing g_s of beech and spruce, and indicated stomatal limitation by O₃ in beech and by drought in spruce. Our study showed that drought can override the stimulating O₃ effects on fine-root dynamics and soil respiration in mature beech and spruce forests.     

Combined effects of elevated CO2 and natural climatic variation on leaf spot diseases of redbud and sweetgum trees

Atmospheric CO₂ concentrations are predicted to double within the next century and alter climate regimes, yet the extent that these changes will affect plant diseases remains unclear. In this study conducted over five years, we assessed how elevated CO₂ and interannual climatic variability affect Cercospora leaf spot diseases of two deciduous trees. Climatic data varied considerably between the five years and altered disease expression. Disease incidence and severity for both species were greater in years with above average rainfall. In years with above average temperatures, disease incidence for Liquidambar styraciflua was decreased significantly. When significant changes did occur, disease incidence and severity always increased under elevated CO₂. Chlorophyll fluorescence imaging of leaves revealed that any visible increase in disease severity induced by elevated CO₂ was mitigated by higher photosynthetic efficiency in the remaining undamaged leaf tissue and in a halo surrounding lesions.     

Ozone exposure and stomatal sluggishness in different plant physiognomic classes

Gas exchange responses to static and variable light were tested in three species: snap bean (Phaseolus vulgaris, two cultivars), California black oak (Quercus kelloggii), and blue oak (Q. douglasii). The effects of 1-month (snap beans) and 2-month (oaks) O₃ (ozone) exposure (70 ppb over 8 h per day in open-top chambers) were investigated. A delay in stomatal responses (i.e., ‘sluggish’ responses) to variable light was found to be both an effect of O₃ exposure and a reason for increased O₃ sensitivity in snap bean cultivars, as it implied higher O₃ uptake during times of disequilibrium. Sluggishness increased the time to open (thus limiting CO₂ uptake) and close stomata (thus increasing transpirational water loss) after abrupt changes in light level. Similar responses were shown by snap beans and oaks, suggesting that O₃-induced stomatal sluggishness is a common trait among different plant physiognomic classes.     

Assessment of growth and yield losses in two Zea mays L. cultivars (quality protein maize and nonquality protein maize) under projected levels of ozone

Rapid industrialization and economic developments have increased the tropospheric ozone (O₃) budget since preindustrial times, and presently, it is supposed to be a major threat to crop productivity. Maize (Zea mays L.), a C4 plant is the third most important staple crop at global level with a great deal of economic importance. The present study was conducted to evaluate the performance of two maize cultivars [HQPM1: quality protein maize (QPM)] and [DHM117: nonquality protein maize (NQPM)] to variable O₃ doses. Experimental setup included filtered chambers, nonfiltered chambers (NFC), and two elevated doses of O₃ viz. NFC+15 ppb O₃ (NFC+15) and NFC+30 ppb O₃ (NFC+30). During initial growth period, both QPM and NQPM plants showed hormetic effect that is beneficial due to exposure of low doses of a toxicant (NFC and NFC+15 ppb O₃), but at later stages, growth attributes were negatively affected by O₃. Growth indices showed the variable pattern of photosynthate translocation under O₃ stress. Foliar injury in the form of interveinal chlorosis and reddening of leaves due to increased production of anthocyanin pigments was observed at higher concentrations of O₃. One-dimensional gel electrophoresis of leaves taken from NFC+30 showed reductions of major photosynthetic proteins, and differential response was observed between the two test cultivars. Decline in the number of male flowers at elevated O₃ doses suggested damaging effect of O₃ on reproductive structures which might be a cause of productivity losses. Variable carbon allocation pattern particularly to husk leaves, foliar injury, and damage of photosynthetic proteins led to significant reductions in economic yield at higher O₃ doses. PCA showed that both the cultivars responded more or less similarly to O₃ stress in their respective groupings of growth and yield parameters, but magnitude of their response was variable. It is further supported by difference in the significance of correlations between variables of yield and AOT40. Cultivar response reflects that QPM performed better than NQPM against elevated O₃.     

Apoplastic ascorbate contributes to the differential ozone sensitivity in two varieties of winter wheat under fully open-air field conditions

We studied leaf apoplastic ascorbates in relation to ozone (O₃) sensitivity in two winter wheat (Triticum aestivum L.) varieties: Yangfumai 2 (Y2) and Yangmai 16 (Y16). The plants were exposed to elevated O₃ concentration 27% higher than the ambient O₃ concentration in a fully open-air field from tillering stage until final maturity. The less sensitive variety Y16 had higher concentration of reduced ascorbate in the apoplast and leaf tissue by 33.5% and 12.0%, respectively, than those in the more sensitive variety Y2, whereas no varietal difference was detected in the decline of reduced ascorbate concentration in response to elevated O₃. No effects of O₃ or variety were detected in either oxidized ascorbate or the redox state of ascorbate in the apoplast and leaf tissue. The lower ascorbate concentrations in both apoplast and leaf tissue should have contributed to the higher O₃ sensitivity in variety Y2.     

Elevated CO2 response of photosynthesis depends on ozone concentration in aspen

The effect of elevated CO₂ and O₃ on apparent quantum yield (?), maximum photosynthesis (P_max), carboxylation efficiency (V_cmax) and electron transport capacity (J_max) at different canopy locations was studied in two aspen (Populus tremuloides) clones of contrasting O₃ tolerance. Local light climate at every leaf was characterized as fraction of above-canopy photosynthetic photon flux density (%PPFD). Elevated CO₂ alone did not affect ? or P_max, and increased J_max in the O₃-sensitive, but not in the O₃-tolerant clone. Elevated O₃ decreased leaf chlorophyll content and all photosynthetic parameters, particularly in the lower canopy, and the negative impact of O₃ increased through time. Significant interaction effect, whereby the negative impact of elevated O₃ was exaggerated by elevated CO₂ was seen in Chl, N and J_max, and occurred in both O₃-tolerant and O₃-sensitive clones. The clonal differences in the level of CO₂ × O₃ interaction suggest a relationship between photosynthetic acclimation and background O₃ concentration.