Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO2, malate, abscisic acid or interruption of water supply

Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 levels close them. This CO2 response is maintained in the dark. To elucidate mechanisms underlying the dark CO2 response we introduced pH- and potential-sensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiration were simultaneously monitored. Upon an increase in CO2 concentration transient changes in apoplastic pH (occasionally brief acidification, but always followed by alkalinization) and in membrane potential (brief hyperpolarization followed by depolarization) accompanied stomatal closure. Alkalinization and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening in response to CO2-free air the apoplastic pH increased while the membrane potential initially depolarized before it transiently hyperpolarized. To examine whether changes in apoplastic malate concentrations represent a closing signal for stomata, malate was fed into the transpiration stream. Although malate caused apoplastic alkalinization and membrane depolarization reminiscent of the effects observed with CO2 and abscisic acid, this dicarboxylate closed the stomata only partially and less effectively than CO2. Apoplastic alkalinization was also observed and stomata closed partially when KCl was fed to the leaves. Respiration increased on feeding of malate or KCl, or while abscisic acid closed the stomate. From these results we conclude that CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H+-ATPases during changes in stomatal aperture. Responses to potassium malate and KCl are not restricted to guard cells and neighbouring cells.     

Does ascorbate in the mesophyll cell walls form the first line of defence against ozone? Testing the concept using broad bean (Vicia faba L.)

Broad bean (Vicia faba L.) plants were exposed, in duplicate controlled environment chambers, to charcoal/Purafil-filtered air (CFA-grown plants) or to 75 nmol mol(-1) ozone (O(3)) for 7 h d(-1) (O(3)-grown plants) for 28 d, and then exposed to 150 nmol mol(-1) O(3 )for 8 h. The concentration of ascorbate (ASC) was determined in leaf extracellular washing fluid (apoplast) and in the residual leaf tissue (symplast) after 0, 4 and 8 h acute fumigation, and after a 16 h "recovery" period in CFA. Changes in stomatal conductance were measured in vivo in order to model pollutant uptake, while the light-saturated rate of CO(2) assimilation (A:(sat)) was recorded as an indicator of O(3)-induced intracellular damage. Measurements of A:(sat) revealed enhanced tolerance to 150 nmol mol(-1) O(3) in plants pre-exposed to the pollutant compared with equivalent plants grown in CFA, consistent with the observed reduction in pollutant uptake due to lower stomatal conductance. The concentration of ASC in the leaf apoplast (ASC(apo)) declined upon O(3)-treatment in both CFA- and O(3)-grown plants, consistent with the oxidation of ASC(apo) under O(3)-stress. Furthermore, the decline in ASC(apo) was reversible in O(3)-grown plants after a 16 h "recovery" period, but not in plants grown in CFA. No significant change in the level and/or redox state of ASC in the symplast (ASC(symp)) was observed in plants exposed to 150 nmol mol(-1) O(3), and there was no difference in the constitutive level of ASC(symp) between CFA- and O(3)-grown plants. Model calculations indicated that the reaction of O(3) with ASC(apo) in the leaves of Vicia faba is potentially sufficient to intercept a substantial proportion (30-40%) of the O(3)entering the plant under environmentally-relevant conditions. The potential role of apoplastic ASC in mediating the tolerance of leaves to O(3) is discussed.     

Other factors than apoplastic ascorbate contribute to the differential ozone tolerance of two clones of Trifolium repens L.

Apoplastic reactive oxygen intermediates, which are formed during the exposure of a higher plant to ozone (O₃), have been proposed to be detoxified by apoplastic ascorbate (ASC). An investigation to determine whether the differential sensitivity of two white clover clones (Trifolium repens L. cv Regal) to O₃ was related with their levels of ASC, glutathione derivatives or with the total antioxidative capacity. In contrast to what might be expected, the sensitive clone of white clover (NC-S) constitutively showed a 72% higher concentration of apoplastic ASC compared to the O₃-tolerant clone (NC-R). Furthermore, NC-S also showed a higher redox status of apoplastic ASC. These results indicate that higher ASC levels in the apoplast of NC-S are not sufficient to induce a higher O₃ tolerance. The redox status, but not the absolute concentration of homoglutathione in the symplast was found to be constitutively higher in NC-R than in NC-S. It is not clear, however, whether homoglutathione is a direct cause of the differential O₃ detoxification capacity of both clones. Total antioxidative capacity measurements ruled out the contribution of other low-molecular antioxidants to the relative tolerance of NC-R. It was concluded that elevated apoplastic ASC levels can not always be sufficient to render a plant O₃ tolerant.     

Mechanisms underlying the amelioration of O3-induced damage by elevated atmospheric concentrations of CO2

There is growing evidence that rising atmospheric CO2 concentrations will reduce or prevent reductions in the growth and productivity of C3 crops attributable to ozone (O3) pollution. In this study, the role of pollutant exclusion in mediating this response was investigated through growth chamber-based investigations on leaves 4 and 7 of spring wheat (Triticum aestivum cv. Hanno). In the core experiments, plants were raised at two atmospheric CO2 concentrations (ambient [350 micro l l(-1)] or elevated CO2 [700 micro l l(-1)] under two O3 regimes (charcoal/Purafil-filtered air [<5 nl l(-1) O3] or ozone-enriched air [75 nl l(-1) 7 h d(-1)]). A subsequent experiment used an additional O3 treatment where the goal was to achieve equivalent daily O3 uptake over the life-span of leaves 4 and 7 under ambient and CO2-enriched conditions, through daily adjustment of exposures based on measured shifts in stomatal conductance. Plant growth and net CO2 assimilation were stimulated by CO2-enrichment and reduced by exposure to O3. However, the impacts of O3 decreased with plant age (i.e. leaf 7 was more resistant to O3 injury than leaf 4); a finding consistent with ontogenic shifts in the tolerance of plant tissue and/or acclimation to O3-induced oxidative stress. In the combined treatment, elevated CO2 protected against the adverse effects of O3 and reduced cumulative O3 uptake (calculated from measurements of stomatal conductance) by c. 10% and 35% over the life-span of leaves 4 and 7, respectively. Analysis of the relationship between O3 uptake and the decline in the maximum in vivo rate of Rubisco carboxylation (Vcmax) revealed the protection afforded by CO2-enrichment to be due, to a large extent, to the exclusion of the pollutant from the leaf interior (as a consequence of the decline in stomatal conductance triggered by CO2-enrichment), but there was evidence (especially from flux-response relationships constructed for leaf 4) that CO2-enrichment resulted in additional effects that alleviated the impacts of ozone-induced oxidative stress on photosynthesis.     

Antioxidant Systems and O2.−/H2O2 Production in the Apoplast of Pea Leaves. Its Relation with Salt-Induced Necrotic Lesions in Minor Veins

The present work describes, for the first time, the changes that take place in the leaf apoplastic antioxidant defenses in response to NaCl stress in two pea (Pisum sativum) cultivars (cv Lincoln and cv Puget) showing different degrees of sensitivity to high NaCl concentrations. The results showed that only superoxide dismutase, and probably dehydroascorbate reductase (DHAR), were present in the leaf apoplastic space, whereas ascorbate (ASC) peroxidase, monodehydroascorbate reductase (MDHAR), and glutathione (GSH) reductase (GR) seemed to be absent. Both ASC and GSH were detected in the leaf apoplastic space and although their absolute levels did not change in response to salt stress, the ASC/dehydroascorbate and GSH to GSH oxidized form ratios decreased progressively with the severity of the stress. Apoplastic superoxide dismutase activity was induced in NaCl-treated pea cv Puget but decreased in NaCl-treated pea cv Lincoln. An increase in DHAR and GR and a decrease in ASC peroxidase, MDHAR, ASC, and GSH levels was observed in the symplast from NaCl-treated pea cv Lincoln, whereas in pea cv Puget an increase in DHAR, GR, and MDHAR occurred. The results suggest a strong interaction between both cell compartments in the control of the apoplastic ASC content in pea leaves. However, this anti-oxidative response does not seem to be sufficient to remove the harmful effects of high salinity. This finding is more evident in pea cv Lincoln, which is characterized by a greater inhibition of the growth response and by a higher rise in the apoplastic hydrogen peroxide content, O(2)(.-) production and thiobarbituric acid-reactive substances, and CO protein levels. This NaCl-induced oxidative stress in the apoplasts might be related to the appearance of highly localized O(2)(.-)/H(2)O(2)-induced necrotic lesions in the minor veins in NaCl-treated pea plants. It is possible that both the different anti-oxidative capacity and the NaCl-induced response in the apoplast and in the symplast from pea cv Puget in comparison with pea cv Lincoln contributes to a better protection of pea cv Puget against salt stress.     

Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate

 This paper presents a mathematical model which enables the semi-quantification of ozone (O₃) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments – based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O₃ reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O₃ taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O₃ in the cell wall. Received: 13 May 1999 / Accepted: 5 August 1999     

Elevated CO2 induces physiological, biochemical and structural changes in leaves of Arabidopsis thaliana

Leaves of Arabidopsis thaliana grown under elevated or ambient CO2 (700 or 370 micromol mol(-1), respectively) were examined for physiological, biochemical and structural changes. Stomatal characters, carbohydrate and mineral nutrient concentrations, leaf ultrastructure and plant hormone content were investigated using atomic absorption spectrophotometry, transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA). Elevated CO2 reduced the stomatal density and stomatal index of leaves, and also reduced stomatal conductance and transpiration rate. Elevated CO2 increased chloroplast number, width and profile area, and starch grain size and number, but reduced the number of grana thylakoid membranes. Under elevated CO2, the concentrations of carbohydrates and plant hormones, with the exception of abscisic acid, increased whereas mineral nutrient concentrations declined. These results suggest that the changes in chloroplast ultrastructure may primarily be a consequence of increased starch accumulation. Accelerated A. thaliana growth and development in elevated CO2 could in part be attributed to increased foliar concentrations of plant hormones. The reductions in mineral nutrient concentrations may be a result of dilution by increased concentrations of carbohydrates and also of decreases in stomatal conductance and transpiration rate.     

The role of ozone flux and antioxidants in the suppression of ozone injury by elevated CO2 in soybean

The projected rise in atmospheric CO2 concentration is expected to increase growth and yield of many agricultural crops. The magnitude of this stimulus will partly depend on interactions with other components of the atmosphere such as tropospheric O3. Elevated CO2 concentrations often lessen the deleterious effects of O3, but the mechanisms responsible for this response have received little direct examination. Previous studies have indicated that protection against O3 injury by elevated CO2 can be attributed to reduced O3 uptake, while other studies suggest that CO2 effects on anti-oxidant metabolism might also be involved. The aim of this experiment was to test further the roles of O3 flux and antioxidant metabolism in the suppression of O3 injury by elevated CO2. In a two-year experiment, soybean [Glycine max (L.) Merr.] was exposed from emergence to maturity to charcoal-filtered air or charcoal-filtered air plus a range of O3 concentrations in combination with ambient or approximately twice-ambient CO2 concentrations in open-top field chambers. Experimental manipulation of O3 concentrations and estimates of plant O3 uptake indicated that equivalent O3 fluxes that suppressed net photosynthesis, growth, and yield at ambient concentrations of CO2 were generally much less detrimental to plants treated concurrently with elevated CO2. These responses appeared unrelated to treatment effects on superoxide dismutase, glutathione reductase, and peroxidase activities and glutathione concentration. Total ascorbic acid concentration increased by 28-72% in lower canopy leaves in response to elevated CO2 and O3 but not in upper canopy leaves. Increasing concentrations of atmospheric CO2 will likely ameliorate O3 damage to many crops due to reduced O3 uptake, increased carbon assimilation, and possibly as yet undetermined additional factors. The results of this study further suggest that elevated CO2 may increase the threshold O3 flux for biomass and yield loss in soybean.     

The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees

The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H₂O₂ levels in lignifying tissues, whose capacity to produce and to accumulate H₂O₂ is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H₂O₂ in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O₂ to remove ASC, it could regulate O₂ availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H₂O₂ production in lignifying tissues) at substrate level, by controlling the tension of O₂. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.     

Relationship of Plasmalemma Redox Activity to K+ Uptake by Dunaliella salina Cells

The plasmalemma-bond redox system localized within the plasmalemma of unicellular green alga Dunaliella salina was studied. This system oxidized exogenous NADH, increased O2 consumption to 165 % and increased the pH of the external medium, while K+ influx was inhibited. With no NADH added, ferricyanide stimulated K+ uptake about 3 folds. In the presence of exogenous NADH, ferricyanide was rapidly reduced and the external medium was acidified, generating a greater electrochemical proton gradient across the plasmalemma, thus resulting an 6-fold increase of K+ influx. Typical inhibitors of plasmalemma H+-ATPase and redox system inhibited K+ uptake to different extent. That the inhibition of K+ uptake by vanadate could be resumed partly by addition of NADH and ferricyanide indicated that plasmalemma redox system operated in association with the H+-ATPase to exert an influence on K+ transportation. A model was presented in which the implication of two possible redox chains and H+-ATPase in generating an electrochemical potential gradient for protons (△uH+) was discussed.     

小麦根质膜原位膜微囊与翻转膜微囊的氧化还原特性比较

用二相法和不连续蔗糖梯度离心分别制得小麦根质膜的原位膜微囊和翻转膜微囊。两者比较可知：质膜内外两侧均表现出较高的氧化还原活性;膜内侧的ＮＡＤ（Ｐ）Ｈ氧化和Ｆｅ（ＣＮ）还原速率高于外侧。质膜内外两侧都能还原ＥＤＴＡ－Ｆｅ３＋,但外侧的还原活性高于内侧。质膜内外两侧均有Ｏ２吸收,同时都可被ＳＨＡＭ刺激,被ＫＣＮ抑制。质膜内侧和外侧都可产生,最适ＰＨ值为６．０;既可被ＳＨＡＭ刺激,也可被ＳＯＤ、过氧化氢酶和ＫＣＮ抑制。     

Stomatal uptake and stomatal deposition of ozone in isoprene and monoterpene emitting plants

Volatile isoprenoids were reported to protect plants against ozone. To understand whether this could be the result of a direct scavenging of ozone by these molecules, the stomatal and non-stomatal uptake of ozone was estimated in plants emitting isoprene or monoterpenes. Ozone uptake by holm oak (Quercus ilex, a monoterpene emitter) and black poplar (Populus nigra, an isoprene emitter) was studied in whole plant enclosures (continuously stirred tank reactors, CSTR). The ozone uptake by plants was estimated measuring ozone concentration at the inlet and outlet of the reactors, after correcting for the uptake of the enclosure materials. Destruction of ozone at the cuticle or at the plant stems was found to be negligible compared to the ozone uptake through the stomata. For both plant species, a relationship between stomatal conductance and ozone uptake was found. For the poplar, the measured ozone losses were explained by the uptake of ozone through the stomata only, and ozone destruction by gas phase reactions with isoprene was negligible. For the oak, gas phase reactions of ozone with the monoterpenes emitted by the plants contributed significantly to ozone destruction. This was confirmed by two different experiments showing a) that in cases of high stomatal conductance but under low CO(2) concentration, a reduction of monoterpene emission was still associated with reduced O(3) uptake; and b) that ozone losses due to the gas phase reactions only can be measured when using the exhaust from a plant chamber to determine the gas phase reactivity in an empty reaction chamber. Monoterpenes can therefore relevantly scavenge ozone at leaf level contributing to protection against ozone.     

The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors.

The apoplastic pH of the substomatal cavity is an essential determinant of stomatal movement. In detached leaves of Vicia faba substomatal apoplastic pH and its dependence on external (stress) factors was investigated using a non-invasive approach: pH-microsensors were inserted into open stomata, and upon contact with the apoplastic fluid, pH was measured continuously, as apoplastic pH was challenged by changed conditions of light, atmosphere (NH(3), CO(2)), and xylem sap (abscisic acid, cyanide, fusicoccin, pH, inorganic salts). Apoplastic pH proved extremely sensitive to infiltration and local flooding, which rapidly increased the apoplastic pH by more than 1.5 pH units. Recovery from infiltration took several hours, during which light effects on the apoplastic pH were strongly impeded. This indicates that pH tests carried out under such conditions may not be representative of the undisturbed leaf. NH(3), flushed across the stomata, yielded a rapid apoplastic alkalinization from which an apoplastic buffer capacity of 2-3 mM per pH unit was calculated. Fusicoccin, fed into the xylem sap acidified the apoplast, whereas cyanide alkalized it, thus underscoring the importance of the plasma membrane H(+) pump for apoplastic pH regulation. To address the question to what extent pH was a drought signal, the effect of iso-osmotic pH changes, fed into the xylem through the petiole were tested. It is demonstrated that the apoplastic response remained below 0.1 pH per pH unit imposed, regardless of the buffer capacity. An increase in the osmolarity of the bath solution (harbouring the cut petiole) using KCl, NaCl, CaCl(2) or sorbitol alkalized the substomatal apoplast. It is suggested that pH may only act as drought signal when accompanied by elevated osmolarity.     

Apoplastic pH and FeIII reduction in young sunflower (Helianthus annuus) roots

The relationship between the apoplastic pH in young sunflower roots ( Helianthus annuus L.) and the plasmalemma ferric chelate reductase (FC-R; EC 1.16.1.7) activity in roots was investigated. The hypothesis was tested that a high apoplastic pH depresses FC-R activity, thereby restricting the uptake of Fe²⁺ into the cytosol. Until recently, little has been known about this relationship, because pH and redox reaction measurements are difficult to perform within the confines of the root apoplast. We recorded the apoplastic pH by means of the fluorescence ratio in conjunction with video microscopy by covalently tagging fluorescein boronic acid to OH groups of the root cell wall. Fe^III reduction was measured using a similar approach by tagging ferrozine diboronic acid with OH groups of the cell wall. Ferrozine forms an Fe²⁺ complex, thus indicating the reduction of ferric iron. In roots bathing in buffered outer solutions of different pH, a high pH sensitivity of apoplastic Fe^III reduction was found, with the highest ferric iron reduction rates at an apoplastic pH of 4.9; above an apoplastic pH of 5.3, no reduction was observed. Nitrate in the bathing solution increased the apoplastic pH and hence depressed the Fe^III reduction; ammonium had the reverse effect. Nitrate together with HCO₃^–, a combination which is typical of calcareous soils, had the strongest depressing effect. From the results, it can be concluded that the main reason for the frequently occurring iron deficiency chlorosis of plants grown on calcareous soils is the inhibition of Fe^III reduction in the apoplast, and hence Fe²⁺ uptake into the cytosol.     

Reactive oxygen species signaling and stomatal movement in plant responses to drought stress and pathogen attack

Stomata, the pores formed by a pair of guard cells, are the main gateways for water transpiration and photosynthetic CO_2 exchange, as well as pathogen invasion in land plants. Guard cell movement is regulated by a combination of environmental factors, including water status, light, CO_2 levels and pathogen attack, as well as endogenous signals, such as abscisic acid and apoplastic reactive oxygen species(ROS). Under abiotic and bioticstress conditions, extracellular ROS are mainly produced by plasma membrane-localized NADPH oxidases, whereas intracellular ROS are produced in multiple organelles. These ROS form a sophisticated cellular signaling network, with the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here, we review recent progress in understanding the molecular mechanisms of the ROS signaling network,primarily during drought stress and pathogen attack. We summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and CO_2 signaling, and immunity responses.Finally, we discuss ROS accumulation and communication between organelles and cells. This information provides a conceptual framework for understanding how ROS signaling is integrated with various signaling pathways during plant responses to abiotic and biotic stress stimuli.     

The influence of elevated rhizosphere dissolved inorganic carbon concentrations on respiratory O2 and CO2 flux in tomato roots

Respiratory CO2 and O2 flux were measured in hydroponically grown Lycopersicon esculentum (L.) Mill. cv. F144 plants at either low (O mol mol^-1) or elevated CO2 concentrations (>2000 mol mol^-1) supplied to the roots. In NO3^- fed plants the consumption of O2 and the engagement of the alternative pathway were increased by elevated dissolved inorganic carbon (DIC = CO2 + HCO3^-) concentrations. This was ascribed to the influence of organic acids on the TCA cycle and electron transport pathways. Inhibition of O2 consumption by elevated DIC in NH4^--fed plants may be due to the reduction requirements of anaplerotic carbon entering the TCA cycle or the removal of carbon from the TCA cycle for amino acid synthesis. In both NO3^- and NH4⁺-fed plants elevated DIC inhibited CO2 release due to high rates of DIC incorporation by phosphoenolpyruvate carboxylase (PEPc) activity. Transient net CO2 consumption due to the inhibition of respiration by salicylhydroxamic acid and KCN, together with high respiratory quotients after the addition of inhibitors of carbonic anhydrase (CA) activity, were also ascribed to high rates of DIC incorporation at elevated DIC concentrations. Ethoxyzolamide, an inhibitor of CA activity, inhibited both DI¹⁴C incorporation into organic products and NO3^- uptake by 81% and 40%, respectively. This, together with a 32% increase in DI¹⁴C accumulation and inhibition of NO3^- uptake upon inhibition of anion transport by diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) may indicate the exchange of HCO3^- for NO3^- across the root plasmalemma. It was concluded that dark incorporation of HCO3^- by PEPc increased at elevated rhizosphere DIC concentrations and that the products of DIC incorporation may stimulate respiratory electron transport. Additional reducing energy and carbon skeletons from the tricarboxylic acid (TCA) cycle would therefore be available for respiration and the reduction and incorporation of NO3^- into amino acids.Key words: Tomato, PEPc, respiration, carbon dioxide nitrate.      

Specification of adaxial and abaxial stomata, epidermal structure and photosynthesis to CO2 enrichment in maize leaves

Acclimation to CO2 enrichment was studied in maize plants grown to maturity in either 350 or 700 microl l-1 CO2. Plants grown with CO2 enrichment were significantly taller than those grown at 350 microl l-1 CO2 but they had the same number of leaves. High CO2 concentration led to a marked decrease in whole leaf chlorophyll and protein. The ratio of stomata on the adaxial and abaxial leaf surfaces was similar in all growth conditions, but the stomatal index was considerably increased in plants grown at 700 microl l-1 CO2. Doubling the atmospheric CO2 content altered epidermal cell size leading to fewer, much larger cells on both leaf surfaces. The photosynthesis and transpiration rates were always higher on the abaxial surface than the adaxial surface. CO2 uptake rates increased as atmospheric CO2 was increased up to the growth concentrations on both leaf surfaces. Above these values, CO2 uptake on the abaxial surface was either stable or increased as CO2 concentration increased. In marked contrast, CO2 uptake rates on the adaxial surface were progressively inhibited at concentrations above the growth CO2 value, whether light was supplied directly to this or the abaxial surface. These results show that maize leaves adjust their stomatal densities through changes in epidermal cell numbers rather than stomatal numbers. Moreover, the CO2-response curve of photosynthesis on the adaxial surface is specifically determined by growth CO2 abundance and tracks transpiration. Conversely, photosynthesis on the abaxial surface is largely independent of CO2 concentration and rather independent of stomatal function.     

Soil and plant water relations determine photosynthetic responses of C3 and C4 grasses in a semi-arid ecosystem under elevated CO2

To model the effect of increasing atmospheric CO2 on semi-arid grasslands, the gas exchange responses of leaves to seasonal changes in soil water, and how they are modified by CO2, must be understood for C3 and C4 species that grow in the same area. In this study, open-top chambers were used to investigate the photosynthetic and stomatal responses of Pascopyrum smithii (C3) and Bouteloua gracilis (C4) grown at 360 (ambient CO2) and 720 micro mol mol-1 CO2 (elevated CO2) in a semi-arid shortgrass steppe. Assimilation rate (A) and stomatal conductance (gs) at the treatment CO2 concentrations and at a range of intercellular CO2 concentrations and leaf water potentials (psileaf) were measured over 4 years with variable soil water content caused by season and CO2 treatment. Carboxylation efficiency of ribulose bisphosphate carboxylase/oxygenase (Vc,max), and ribulose bisphosphate regeneration capacity (Jmax) were reduced in P. smithii grown in elevated CO2, to the degree that A was similar in elevated and ambient CO2 (when soil moisture was adequate). Photosynthetic capacity was not reduced in B. gracilis under elevated CO2, but A was nearly saturated at ambient CO2. There were no stomatal adaptations independent of photosynthetic acclimation. Although photosynthetic capacity was reduced in P. smithii growing in elevated CO2, reduced gs and transpiration improved soil water content and psileaf in the elevated CO2 chambers, thereby improving A of both species during dry periods. These results suggest that photosynthetic responses of C3 and C4 grasses in this semi-arid ecosystem will be driven primarily by the effect of elevated CO2 on plant and soil water relations.     

Interactive Effects of Elevated CO2 and Ozone on Leaf Thermotolerance in Field-grown Glycine max

Humans are increasing atmospheric CO2, ground-level ozone (O3), and mean and acute high temperatures. Laboratory studies show that elevated CO2 can increase thermotolerance of photosynthesis in C3 plants. O3-related oxidative stress may offset benefits of elevated CO2 during heat-waves. We determined effects of elevated CO2 and O3 on leaf thermotolerance of field-grown Glycine max (soybean, C3). Photosynthetic electron transport (φet) was measured in attached leaves heated in situ and detached leaves heated under ambient CO2 and O3. Heating decreased φet, which O3 exacerbated. Elevated CO2 prevented O3-related decreases during heating, but only increased φet under ambient O3 in the field. Heating decreased chlorophyll and carotenoids, especially under elevated CO2. Neither CO2 nor O3 affected heat-shock proteins. Heating increased catalase (except in high O3) and CulZn-superoxide dismutase (SOD), but not MnSOD; CO2 and O3 decreased catalase but neither SOD. Soluble carbohydrates were unaffected by heating, but increased in elevated CO2. Thus, protection of photosynthesis during heat stress by elevated CO2 occurs in field-grown soybean under ambient O3, as in the lab, and high CO2 limits heat damage under elevated O3, but this protection is likely from decreased photorespiration and stomatal conductance rather than production of heat-stress adaptations.