Leaf Air Spaces Research Articles

Mesophyll airspace unsaturation drives C4 plant success under vapor pressure deficit stress

A fundamental assumption in plant science posits that leaf air spaces remain vapor saturated, leading to the predominant view that stomata alone control leaf water loss. This concept has been pivotal in photosynthesis and water-use efficiency research. However, recent evidence has refuted this longstanding assumption by providing evidence of unsaturation in the leaf air space of C3 plants under relatively mild vapor pressure deficit (VPD) stress. This phenomenon represents a nonstomatal mechanism restricting water loss from the mesophyll. The potential ubiquity and physiological implications of this phenomenon, its driving mechanisms in different plant species and habitats, and its interaction with other ecological adaptations have. In this context, C4 plants spark particular interest for their importance as crops, bundle sheath cells' unique anatomical characteristics and specialized functions, and notably higher water-use efficiency relative to C3 plants. Here, we confirm reduced relative humidities in the substomatal cavity of the C4 plants maize, sorghum, and proso millet down to 80% under mild VPD stress. We demonstrate the critical role of nonstomatal control in these plants, indicating that the role of the CO2 concentration mechanism in CO2 management at a high VPD may have been overestimated. Our findings offer a mechanistic reconciliation between discrepancies in CO2 and VPD responses reported in C4 species. They also reveal that nonstomatal control is integral to maintaining an advantageous microclimate of relatively higher CO2 concentrations in the mesophyll air space of C4 plants for carbon fixation, proving vital when these plants face VPD stress.

Extreme undersaturation in the intercellular airspace of leaves: a failure of Gaastra or Ohm?

Recent reports of extreme levels of undersaturation in internal leaf air spaces have called into question one of the foundational assumptions of leaf gas exchange analysis, that leaf air spaces are effectively saturated with water vapour at leaf surface temperature. Historically, inferring the biophysical states controlling assimilation and transpiration from the fluxes directly measured by gas exchange systems has presented a number of challenges, including: (1) a mismatch in scales between the area of flux measurement, the biochemical cellular scale and the meso-scale introduced by the localization of the fluxes to stomatal pores; (2) the inaccessibility of the internal states of CO2 and water vapour required to define conductances; and (3) uncertainties about the pathways these internal fluxes travel. In response, plant physiologists have adopted a set of simplifying assumptions that define phenomenological concepts such as stomatal and mesophyll conductances. Investigators have long been concerned that a failure of basic assumptions could be distorting our understanding of these phenomenological conductances, and the biophysical states inside leaves. Here we review these assumptions and historical efforts to test them. We then explore whether artefacts in analysis arising from the averaging of fluxes over macroscopic leaf areas could provide alternative explanations for some part, if not all, of reported extreme states of undersaturation. Spatial heterogeneities can, in some cases, create the appearance of undersaturation in the internal air spaces of leaves. Further refinement of experimental approaches will be required to separate undersaturation from the effects of spatial variations in fluxes or conductances. Novel combinations of current and emerging technologies hold promise for meeting this challenge.

Understanding airspace in leaves: 3D anatomy and directional tortuosity.

The leaf intercellular airspace is a tortuous environment consisting of cells of different shapes, packing densities, and orientation, all of which have an effect on the travelling distance of molecules from the stomata to the mesophyll cell surfaces. Tortuosity, the increase in displacement over the actual distance between two points, is typically defined as encompassing the whole leaf airspace, but heterogeneity in pore dimensions and orientation between the spongy and palisade mesophyll likely result in heterogeneity in tortuosity along different axes and would predict longer traveling distance along the path of least tortuosity, such as vertically within the columnar cell matrix of the palisade layer. Here, we compare a previously established geometric method to a random walk approach, novel for this analysis in plant leaves, in four different Eucalyptus species. The random walk method allowed us to quantify directional tortuosity across the whole leaf profile, and separately for the spongy and palisade mesophyll. For all species tortuosity was higher in the palisade mesophyll than the spongy mesophyll and horizontal (parallel to the epidermis) tortuosity was consistently higher than vertical (from epidermis to epidermis) tortuosity. We demonstrate that a random walk approach improves on previous geometric approaches and is valuable for investigating CO2 and H2 O transport within leaves.

Quantification of plant morphology and leaf thickness with optical coherence tomography

Optical coherence tomography (OCT) can be a valuable imaging tool for in vivo and label-free digital plant phenotyping. However, for imaging leaves, air-filled cavities limit the penetration depth and reduce the image quality. Moreover, up to now quantification of leaf morphology with OCT has been done in one-dimensional or two-dimensional images only, and has often been limited to relative measurements. In this paper, we demonstrate a significant increase in OCT imaging depth and image quality by infiltrating the leaf air spaces with water. In the obtained high-quality OCT images the top and bottom surface of the leaf are digitally segmented. Moreover, high-quality en face images of the leaf are obtained from numerically flattened leaves. Segmentation in three-dimensional OCT images is used to quantify the spatially resolved leaf thickness. Based on a segmented leaf image, the refractive index of an infiltrated leaf is measured to be 1.345±0.004, deviating only 1.2% from that of pure water. Using the refractive index and a correction for refraction effects at the air-leaf interface, we quantitatively mapped the leaf thickness. The results show that OCT is an efficient and promising technique for quantitative phenotyping on leaf and tissue level.

Determining the Subcellular Localization of Fluorescently Tagged Proteins Using Protoplasts Extracted from Transiently Transformed Nicotiana benthamiana Leaves.

In plants, stable expression is arguably the method of choice to test transgene function but it is a slow and labor-intensive process. This bottleneck generally limits the number of transgenes that can be tested, and as such hinders construct optimization. In the face of this challenge, transient expression in Nicotiana benthamiana leaves has emerged as a powerful screening platform to test gene expression, as well as subcellular distribution and function of many proteins within a week. This system relies on the infiltration of Agrobacterium tumefaciens (Agrobacterium) carrying DNA of interest into the leaf air spaces of N. benthamiana plants. Agrobacterium rapidly transforms the plant cells and the leaves can be analyzed within a few days. Investigating the subcellular localization of a protein of interest often relies on its fusion to a fluorescent tag. While the amount of accumulation of such fusion proteins can often be gauged by observing the fluorescence of the tag at the whole-leaf level, subcellular protein distribution is best determined in protoplasts extracted from transformed leaves. Here I present a simple and effective method to transform N. benthamiana leaves with Agrobacterium and to prepare protoplasts from these leaves to characterize the subcellular localization of proteins of interest.

Concerted anatomical change associated with crassulacean acid metabolism in the Bromeliaceae.

Crassulacean acid metabolism (CAM) is a celebrated example of convergent evolution in plant ecophysiology. However, many unanswered questions surround the relationships among CAM, anatomy and morphology during evolutionary transitions in photosynthetic pathway. An excellent group in which to explore these issues is the Bromeliaceae, a diverse monocot family from the Neotropics in which CAM has evolved multiple times. Progress in the resolution of phylogenetic relationships among the bromeliads is opening new and exciting opportunities to investigate how evolutionary changes in leaf structure has tracked, or perhaps preceded, photosynthetic innovation. This paper presents an analysis of variation in leaf anatomical parameters across 163C3 and CAM bromeliad species, demonstrating a clear divergence in the fundamental aspects of leaf structure in association with the photosynthetic pathway. Most strikingly, the mean volume of chlorenchyma cells of CAM species is 22 times higher than that of C3 species. In two bromeliad subfamilies (Pitcairnioideae and Tillandsioideae), independent transitions from C3 to CAM are associated with increased cell succulence, whereas evolutionary trends in tissue thickness and leaf air space content differ between CAM origins. Overall, leaf anatomy is clearly and strongly coupled with the photosynthetic pathway in the Bromeliaceae, where the independent origins of CAM have involved significant anatomical restructuring.

The rapid A-Ci response: photosynthesis in the phenomic era.

Phenotyping for photosynthetic gas exchange parameters is limiting our ability to select plants for enhanced photosynthetic carbon gain and to assess plant function in current and future natural environments. This is due, in part, to the time required to generate estimates of the maximum rate of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) carboxylation (Vc,max ) and the maximal rate of electron transport (Jmax ) from the response of photosynthesis (A) to the CO2 concentration inside leaf air spaces (Ci ). To relieve this bottleneck, we developed a method for rapid photosynthetic carbon assimilation CO2 responses [rapid A-Ci response (RACiR)] utilizing non-steady-state measurements of gas exchange. Using high temporal resolution measurements under rapidly changing CO2 concentrations, we show that RACiR techniques can obtain measures of Vc,max and Jmax in ~5 min, and possibly even faster. This is a small fraction of the time required for even the most advanced gas exchange instrumentation. The RACiR technique, owing to its increased throughput, will allow for more rapid screening of crops, mutants and populations of plants in natural environments, bringing gas exchange into the phenomic era.

Rethinking Guard Cell Metabolism.

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere and, therefore, play a pivotal role in regulating CO2 uptake for photosynthesis as well as water loss through transpiration. Guard cells, which flank the stomata, undergo adjustments in volume, resulting in changes in pore aperture. Stomatal opening is mediated by the complex regulation of ion transport and solute biosynthesis. Ion transport is exceptionally well understood, whereas our knowledge of guard cell metabolism remains limited, despite several decades of research. In this review, we evaluate the current literature on metabolism in guard cells, particularly the roles of starch, sucrose, and malate. We explore the possible origins of sucrose, including guard cell photosynthesis, and discuss new evidence that points to multiple processes and plasticity in guard cell metabolism that enable these cells to function effectively to maintain optimal stomatal aperture. We also discuss the new tools, techniques, and approaches available for further exploring and potentially manipulating guard cell metabolism to improve plant water use and productivity.

Mesophyll photosynthesis and guard cell metabolism impacts on stomatal behaviour.

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere. Guard cells determine stomatal aperture and must operate to ensure an appropriate balance between CO2 uptake for photosynthesis (A) and water loss, and ultimately plant water use efficiency (WUE). A strong correlation between A and stomatal conductance (gs ) is well documented and often observed, but the underlying mechanisms, possible signals and metabolites that promote this relationship are currently unknown. In this review we evaluate the current literature on mesophyll-driven signals that may coordinate stomatal behaviour with mesophyll carbon assimilation. We explore a possible role of various metabolites including sucrose and malate (from several potential sources; including guard cell photosynthesis) and new evidence that improvements in WUE have been made by manipulating sucrose metabolism within the guard cells. Finally we discuss the new tools and techniques available for potentially manipulating cell-specific metabolism, including guard and mesophyll cells, in order to elucidate mesophyll-derived signals that coordinate mesophyll CO2 demands with stomatal behaviour, in order to provide a mechanistic understanding of these processes as this may identify potential targets for manipulations in order to improve plant WUE and crop yield.

Hydraulic limitation not declining nitrogen availability causes the age‐related photosynthetic decline in loblolly pine (Pinus taeda L.)

Declining net primary production (NPP) with forest age is often attributed to a corresponding decline in gross primary production (GPP). We tested two hypotheses explaining the decline of GPP in ageing stands (14-115 years old) of Pinus taeda L.: (1) increasing N limitation limits photosynthetic capacity and thus decreases GPP with increasing age; and (2) hydraulic limitations increasingly induce stomatal closure, reducing GPP with increasing age. We tested these hypotheses using measurements of foliar nitrogen, photosynthesis, sap-flow and dendroclimatological techniques. Hypothesis (1) was not supported; foliar N retranslocation did not increase and declines were not observed in foliar N, leaf area per tree or photosynthetic capacity. Hypothesis (2) was supported; declines were observed in light-saturated photosynthesis, leaf- and canopy-level stomatal conductance, concentration of CO(2) inside leaf air-spaces (corroborated by an increase in wood δ(13) C) and specific leaf area (SLA), while stomatal limitation and the ratio of sapwood area (SA) to leaf area increased. The sensitivity of radial growth to inter-annual variation in temperature and drought decreased with age, suggesting that tree water use becomes increasingly conservative with age. We conclude that hydraulic limitation increasingly limits the photosynthetic rates of ageing loblolly pine trees, possibly explaining the observed reduction of NPP.

Predicting the effects of gas diffusivity on photosynthesis and transpiration of plants grown under hypobaria

As part of a Bio-regenerative Life Support System (BLSS) for long-term space missions, plants will likely be grown at reduced pressure. This low pressure will minimize structural requirements for growth chambers on missions to the Moon or Mars. However, at reduced pressures the diffusivity of gases increases. This will affect the rates at which CO 2 is assimilated and water is transpired through stomata. To understand quantitatively the possible effects of reduced pressure on plant growth, CO 2 and H 2O transport were calculated for atmospheres of various total pressures (101, 66, 33, 22, 11 kPa) and CO 2 concentrations (0.04, 0.1 and 0.18 kPa). The diffusivity of a gas is inversely proportional to total pressure and shows dramatic increases at pressures below 33 kPa (1/3 atm). A mathematical relationship based on the principle of thermodynamics was applied to low pressure conditions and can be used for calculating the transpiration and photosynthesis of plants grown in hypobaria. At 33 kPa total pressure, the stomatal conductance increases by a factor of three with the boundary layer conductance increasing by a factor of ∼1.7, since the leaf conductance is a function of both stomatal and the boundary layer conductance, the overall conductance will increase resulting in significantly higher levels of transpiration as the pressure drops. The conductance of gases is also regulated by stomatal aperture in an inverse relationship. The higher CO 2 concentration inside the leaf air space during low pressure treatments may result in higher CO 2 assimilation and partial stomata closure, resulting in a decrease in transpiration rate. The results of this analysis offer guidelines for experiments in pressure and high CO 2 environments to establish ideal conditions for minimizing transpiration and maximizing the plant biomass yield in BLSS.

Determination of leaf fresh mass after storage between moist paper towels: constraints and reliability of the method

To ensure comparability among leaf fresh mass measurements it is important to handle the leaves in a standardized manner. In the present work constraints of a commonly used method to achieve full turgor, storage between damp paper towels, were investigated. After overnight rehydration in a saturated atmosphere, the fresh mass of leaves of 14 species was measured, and the leaves were stored between paper towels (two treatments: moist and wet) at 4 °C. Their mass was measured after 24, 48, and 72 h. Leaf fresh mass increased during the first 24 h of storage between moist paper towels by an average of 1.8%, between wet towels by 3.3%. Among the species, the increase of leaf fresh mass between moist towels correlated with the species' desiccation propensity, indicating that it was rehydration from water loss during initial handling. On the other hand, between wet towels the fresh mass increase was associated with the species' leaf tissue structure, and it continued to increase beyond 24 h, indicating that the increase was a result of water penetration into the leaf air spaces. It is concluded that storage between moist paper towels results in reliable values of leaf fresh mass, and that desiccated leaves rehydrate well between moist towels. However, care has to be taken to avoid too wet conditions as they may lead to erroneously high fresh mass values, especially in species with large air spaces. Furthermore, exposure to unsaturated atmospheric conditions during handling has to be minimized.

Effects of photon flux density on the morphology, photosynthesis and growth of a CAM orchid, Doritaenopsis during post-micropropagation acclimatization

Micropropagated plantlets are fragile and often lack sufficient vigour to survive the acclimatization shock during transplantation to the soil. Effects of photosynthetic photon flux densities (PPFDs) on growth, photosynthesis and anatomy of micropropagated Doritaenopsis were studied after 4 months of acclimatization in a greenhouse at 25 °C. The plantlets were transferred to three different PPFDs for four months, i.e. ‘low light’ (175), ‘intermediate light’ (270) and ‘high light’ (450 μmol m−2 s−1). For most of the growth parameters measured i.e. leaf length, leaf area, leaf width, fresh weight, dry weight, chlorophyll (Chl) a/b ratio, were greater for the intermediate light levels after 4 months of acclimatization. The only exception was leaf thickness, which was increased more under high light levels. Results showed that the survival of Doritaenopsis plantlets was greatest (90%) in low light and intermediate light (89%) compared with only (73%) at high light. However, at low light levels, pigment concentrations (chlorophyll a, b and total chlorophyll) were higher. Net CO2 assimilation (A), stomatal conductance (g) and transpiration (E) were higher in plantlets grown at high level PPFD than at low after 4 months of acclimatization. Photosynthetic efficiency (Fv/Fm) decreased insignificantly; only at mid day for the high light treatment whereas leaf temperature and stomatal closure increased compared to low light. Scanning electron microscopic (SEM) images of leaves from acclimatized plantlets showed an increase in wax formation for the high light grown plantlets compared to those at low light. Microscopic analysis of acclimatized root sections showed highly developed multiseriate-velamen layers and higher root cell activity; while shoots had larger leaf air spaces than those of in vitro grown plantlets. These results suggest that physiological acclimation occurs at the intermediate PPFD (270 μmol m−2 s−1) in Doritaenopsis compared to treatment at the high light level.

The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: any role for stomatal response?

This paper examines the dependence of whole leaf hydraulic conductance to liquid water (K(L)) on irradiance when measured with a high pressure flowmeter (HPFM). During HPFM measurements, water is perfused into leaves faster than it evaporates hence water infiltrates leaf air spaces and must pass through stomates in the liquid state. Since stomates open and close under high versus low irradiance, respectively, the possibility exists that K(L) might change with irradiance if stomates close tightly enough to restrict water movement. However, the dependence of K(L) on irradiance could be due to a direct effect of irradiance on the hydraulic properties of other tissues in the leaf. In the present study, K(L) increased with irradiance for 6 of the 11 species tested. Whole leaf conductance to water vapour, g(L), was used as a proxy for stomatal aperture and the time-course of changes in K(L) and g(L) was studied during the transition from low to high irradiance and from high to low irradiance. Experiments showed that in some species K(L) changes were not paralleled by g(L) changes. Measurements were also done after perfusion of leaves with ABA which inhibited the g(L) response to irradiance. These leaves showed the same K(L) response to irradiance as control leaves. These experimental results and theoretical calculations suggest that the irradiance dependence of K(L) is more consistent with an effect on extravascular (and/or vascular) tissues rather than stomatal aperture. Irradiance-mediated stimulation of aquaporins or hydrogel effects in leaf tracheids may be involved.

CO(2)-concentrating: consequences in crassulacean acid metabolism.

The consequences of CO(2)-concentrating in leaf air-spaces of CAM plants during daytime organic acid decarboxylation in Phase III of CAM (crassulacean acid metabolism) are explored. There are mechanistic consequences of internal CO(2) partial pressures, p(i)(CO(2)). These are (i) effects on stomata, i.e. high p(i)(CO(2)) eliciting stomatal closure in Phase III, (ii) regulation of malic acid remobilization from the vacuole, malate decarboxylation and refixation of CO(2) via Rubisco (ribulose bisphosphate carboxylase/oxygenase), and (iii) internal signalling functions during the transitions between Phases II and III and III and IV, respectively, in the natural day/night cycle and in synchronizing the circadian clocks of individual leaf cells or leaf patches in the free-running endogenous rhythmicity of CAM. There are ecophysiological consequences. Obvious beneficial ecophysiological consequences are (i) CO(2)-acquisition, (ii) increased water-use- efficiency, (iii) suppressed photorespiration, and (iv) reduced oxidative stress by over-energization of the photosynthetic apparatus. However, the general potency of these beneficial effects may be questioned. There are also adverse ecophysiological consequences. These are (i) energetics, (ii) pH effects and (iii) Phase III oxidative stress. A major consequence of CO(2)-concentrating in Phase III is O(2)-concentrating, increased p(i)(CO(2)) is accompanied by increased p(i)(O(2)). Do reversible shifts of C(3)/CAM-intermediate plants between the C(3)-CAM-C(3) modes of photosynthesis indicate that C(3)-photosynthesis provides better protection from irradiance stress? There are many open questions and CAM remains a curiosity.

Long-distance signaling within Coleus x hybridus leaves; mediated by changes in intra-leaf CO2?

Rapid long-distance signaling in plants can occur via several mechanisms, including symplastic electric coupling and pressure waves. We show here in variegated Coleus leaves a rapid propagation of electrical signals that appears to be caused by changes in intra-leaf CO2 concentrations. Green leaf cells, when illuminated, undergo a rapid depolarization of their membrane potential (Vm) and an increase in their apoplastic pH (pHa) by a process that requires photosynthesis. This is followed by a slower hyperpolarization of Vm and apoplastic acidification, which do not require photosynthesis. White (chlorophyll-lacking) leaf cells, when in isolated white leaf segments, show only the slow response, but when in mixed (i.e. green and white) segments, the rapid Vm depolarization and increase in pHa propagate over more than 10 mm from the green to the white cells. Similarly, these responses propagate 12-20 mm from illuminated to unilluminated green cells. The fact that the propagation of these responses is eliminated when the leaf air spaces are infiltrated with solution indicates that the signal moves in the apoplast rather than the symplast. A depolarization of the mesophyll cells is induced in the dark by a decrease in apoplastic CO2 but not by an increase in pHa. These results support the hypothesis that the propagating signal for the depolarization of the white mesophyll cells is a photosynthetically induced decrease in the CO2 level of the air spaces throughout the leaf.

Erratum

Erratum

46] Assay for singlet oxygen generation by plant leaves exposed to ozone

Ozone is a well-established atmospheric toxin for plants. It is known to diffuse through the open stomata of leaves into their inner air spaces. Chameides has carried out a mathematical analysis of the interaction of ozone with plants and concluded that most of the ozone entering a leaf will react with the ascorbic acid present within the cell walls of the plant cells lining the leaf air spaces. Ascorbic acid concentrations on the order of 300 μM within the cell wall, combined with the large rate constant for the reaction of ozone with ascorbic acid, ensure that no ozone will reach the plasmalemmas of the plant cells. Because the reaction of ascorbic acid with ozone produces singlet oxygen in high yield, plants exposed to an ozone-containing atmosphere should generate singlet oxygen. The singlet oxygen generated within the cell wall should also not reach the plant cell plasmalemma, as singlet oxygen reacts rapidly with ascorbic acid and, in addition, is quenched efficiently by water. However, because most of the singlet oxygen is generated close to the outer surface of the cell wall, some singlet oxygen should diffuse back into the air spaces of the leaf.

Subcellular localization and stress responses of superoxide dismutase isoforms from leaves in the C3‐CAM intermediate halophyte Mesembryanthemum crystallinum L.

BSA, bovine serum albumin CAM, Crassulacean acid metabolism DTT, dithiothreitol EDTA, ethylenediaminetetraacetic acid FPLCfast protein liquid chromatography HEPES, N‐(2‐hydroxyethyl)piperazine‐Ń‐(ethanesulphonic acid) ME, β‐mercaptoethanol NBT, nitro blue tetrazolium PAGE, polyacrylamide gel electrophoresis SDS, sodium dodecyl sulphate SDS‐PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis Rubisco, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (EC 4.1.1.39) SOD, superoxide dismutase (EC 1.15.1.1) TEMED, N,N,Ń,Ń‐tetramethylethylenediamine Tris, Tris (hydroxymethyl) aminomethane Tricine, N‐Tris(hydroxymethyl)methylglycine Treatment of Mesembryanthemum crystallinum for several days with 0·4 kmol m–3 NaCl in the root medium, in parallel to an increase of the cell sap osmolarity enhances activity of important antioxidative enzymes, such as superoxide dismutases (SODs). M. crystallinum is equipped with three SOD isoforms. These isoforms were identified as Mn‐, Fe‐, and Cu/Zn‐SODs, respectively. Mn‐SOD was found in the mitochondrial fraction, Fe‐SOD in the chloroplast fraction, and Cu/Zn‐SOD is probably localized in the cytosol. The Fe‐SOD found in M. crystallinum is the first iron‐containing SOD enzyme to be characterized in the plant family Aizoaceae. Salt treatment increases the activity of this isoform earlier than the other SODs. Molecular masses of SOD isoforms were determined as 82, 48 and 34 kDa for Mn‐, Fe‐, Cu/Zn‐SODs, respectively. Native Mn‐SOD seems to be a tetramer, while Fe‐SOD and Cu/Zn‐SOD are dimers. All SOD isoforms show high thermal stability. Mn‐SOD is active even after short heating at 90 °C and Fe‐SOD at 70 °C. Moreover, high concentrations of β‐mercaptoethanol used as a reducing agent did not destroy the function of all isoforms. With the salinity treatment in M. crystallinum, Crassulacean acid metabolism (CAM) is induced. Build‐up of large stationary O2 concentrations in the leaf air spaces is associated with the photosynthetic CO2 reduction behind closed stomata in phase III of CAM. This illustrates why M. crystallinum may require higher antioxidative activities under NaCl stress and also explains earlier findings that CAM plants are more resistant than C3 plants to environmental stress as imposed by, for example, SO2 and O3.

The influence of ozone and nutrition on δ13C in Betula pendula.

In the cellulose of stems and leaves, δ13C was investigated in a birch clone (Betula pendula), which was exposed throughout the growing season to either <3 (control) or 90/40 nl O3 1-1 (day/night). Each regime was split into plants under high or low nutrient supply. δ13C was increased (becoming less negative), in stems rather than leaves, by both high nutrition (+2‰) and O3 stress (+1‰). Whereas high nutrition raised the wateruse efficiency (WUE) while lowering the CO2 concentration in the inner leaf air space (c i), WUE decreased and c i increased under O3 stress. Therefore, only the nutritional effect on the carbon isotope fractionation was reproduced by the model of Farquhar et al. (1982) which estimates WUE by means of δ13C based on c i. c i was not biased by 'patchiness' in respect to stomatal opening. The latter was verified by microscopical analysis and the complete water infiltration of the birch leaves through the stomata, independent of the diurnal course of the leaf conductance for water vapour. Under low nutrient supply, the activity of phosphoenol pyruvate carboxylase (PEPC) was roughly doubled by ozone to about 1.3% of the total carboxylation capacity (by PEPC + rubisco), and was increased to 1.7% under high nutrition. The fractionation model, extended to account for varying activities of the carboxylating enzymes, indicated that stimulated PEPC was the cause of elevated δ13C, although c i was increased under O3 stress. The stimulation of PEPC and, as a consequence, elevated δ13C are discussed as part of a whole-plant acclimation to O3 stress.