Effects of Elevated [CO2] and Low Soil Moisture on the Physiological Responses of Mountain Maple (Acer spicatum L.) Seedlings to Light

Global climate change is expected to affect how plants respond to their physical and biological environments. In this study, we examined the effects of elevated CO₂ ([CO₂]) and low soil moisture on the physiological responses of mountain maple (Acer spicatum L.) seedlings to light availability. The seedlings were grown at ambient (392 µmol mol⁻¹) and elevated (784 µmol mol⁻¹) [CO₂], low and high soil moisture (M) regimes, at high light (100%) and low light (30%) in the greenhouse for one growing season. We measured net photosynthesis (A), stomatal conductance (g _s), instantaneous water use efficiency (IWUE), maximum rate of carboxylation (V _cmax), rate of photosynthetic electron transport (J), triose phosphate utilization (TPU)), leaf respiration (R _d), light compensation point (LCP) and mid-day shoot water potential (Ψ_x). A and g _s did not show significant responses to light treatment in seedlings grown at low soil moisture treatment, but the high light significantly decreased the C _i/C _a in those seedlings. IWUE was significantly higher in the elevated compared with the ambient [CO₂], and the effect was greater at high than the low light treatment. LCP did not respond to the soil moisture treatments when seedlings were grown in high light under both [CO₂]. The low soil moisture significantly reduced Ψ_x but had no significant effect on the responses of other physiological traits to light or [CO₂]. These results suggest that as the atmospheric [CO₂] rises, the physiological performance of mountain maple seedlings in high light environments may be enhanced, particularly when soil moisture conditions are favourable.     

The Stoichiometry between CO(2) and H Fluxes Involved in the Transport of Inorganic Carbon in Cyanobacteria

The pH of the medium during CO₂ uptake into the intracellular inorganic carbon (C_i) pool of a high CO₂-requiring mutant (E₁) and wild type of Anacystis nidulans R2 was measured. Experiments were performed under conditions where photosynthetic CO₂ fixation is inhibited. There was an acidification of the medium during CO₂ uptake in the light and an alkalization during CO₂ efflux after darkening. A one to one stoichiometry existed between the amounts of H⁺ appearing in the medium and CO₂ taken up into the intracellular C_i pool, regardless of the carbon species transported. The results indicate that (a) CO₂ is taken up simultaneously with an efflux of equimolar H⁺, probably produced as a result of CO₂ hydration during transport and (b) HCO₃⁻ produced by hydration of CO₂ in the medium was transported into the cells without accompanying net flux of H⁺ or OH⁻. The influx and efflux of C_i during C_i transport produced nonequilibrium between CO₂ and HCO₃⁻ in the medium, with the concentration of HCO₃⁻ being higher than that expected under equilibrium conditions. The nonequilibrium was present even under the conditions where the influx of C_i is compensated by its efflux. The direction of this nonequilibrium suggested that efflux of HCO₃⁻ occurs during uptake of C_i.     

Limitation to Photosynthesis in Water-stressed Leaves: Stomata vs. Metabolism and the Role of ATP

Decreasing relative water content (RWC) of leaves progressivelydecreases stomatal conductance (g_s), slowing CO₂ assimilation(A) which eventually stops, after which CO₂ is evolved. In somestudies, photosynthetic potential (A_pot), measured under saturatingCO₂, is unaffected by a small loss of RWC but becomes progressivelymore inhibited, and less stimulated by elevated CO₂, below athreshold RWC (Type 1 response). In other studies, A_pot andthe stimulation of A by elevated CO₂ decreases progressivelyas RWC falls (Type 2 response). Decreased A_pot is caused byimpaired metabolism. Consequently, as RWC declines, the relativelimitation of A by g_s decreases, and metabolic limitation increases.Causes of decreased A_pot are considered. Limitation of ribulosebisphosphate (RuBP) synthesis is the likely cause of decreasedA_pot at low RWC, not inhibition or loss of photosynthetic carbonreduction cycle enzymes, including RuBP carboxylase/oxygenase(Rubisco). Limitation of RuBP synthesis is probably caused byinhibition of ATP synthesis, due to progressive inactivationor loss of Coupling Factor resulting from increasing ionic (Mg²⁺)concentration, not to reduced capacity for electron or protontransport, or inadequate trans-thylakoid proton gradient (pH).Inhibition of A_pot by accumulation of assimilates or inadequateinorganic phosphate is not considered significant. DecreasedATP content and imbalance with reductant status affect cellmetabolism substantially: possible consequences are discussedwith reference to accumulation of amino acids and alterationsin protein complement under water stress.     

Correlation of Stomatal Conductance with Photosynthetic Capacity of Cotton Only in a CO2-Enriched Atmosphere: Mediation by Abscisic Acid?

Some evidence indicates that photosynthetic rate (A) and stomatal conductance (g) of leaves are correlated across diverse environments. The correlation between A and g has led to the postulation of a “messenger” from the mesophyll that directs stomatal behavior. Because A is a function of intercellular CO₂ concentration (c_i), which is in turn a function of g, such a correlation may be partially mediated by c_i if g is to some degree an independent variable. Among individual sunlit leaves in a cotton (Gossypium hirsutum L.) canopy in the field, A was significantly correlated with g (r² = 0.41, n = 63). The relative photosynthetic capacity of each leaf was calculated as a measure of mesophyll properties independent of c_i. This approach revealed that, in the absence of c_i effects, mesophyll photosynthetic capacity was unrelated to g (r² = 0.06). When plants were grown in an atmosphere enriched to about 650 microliters per liter of CO₂, however, photosynthetic capacity remained strongly correlated with g even though the procedure discounted any effect of variable c_i. This “residual” correlation implies the existence of a messenger in CO₂-enriched plants. Enriched CO₂ also greatly increased stomatal response to abscisic acid (ABA) injected into intact leaves. The data provide no evidence for a messenger to coordinate g with A at ambient levels of CO₂. In a CO₂-enriched atmosphere, though, ABA may function as such a messenger because the sensitivity of the system to ABA is enhanced.     

Mapping Intercellular CO2 Mole Fraction (Ci) in Rosa rubiginosa Leaves Fed with Abscisic Acid by Using Chlorophyll Fluorescence Imaging : Significance of Ci Estimated from Leaf Gas Exchange

Imaging of photochemical yield of photosystem II (PSII) computed from leaf chlorophyll fluorescence images and gas-exchange measurements were performed on Rosa rubiginosa leaflets during abscisic acid (ABA) addition. In air ABA induced a decrease of both the net CO₂ assimilation (A_n) and the stomatal water vapor conductance (g_s). After ABA treatment, imaging in transient nonphotorespiratory conditions (0.1% O₂) revealed a heterogeneous decrease of PSII photochemical yield. This decline was fully reversed by a transient high CO₂ concentration (7400 μmol mol⁻¹) in the leaf atmosphere. It was concluded that ABA primarily affected A_n by decreasing the CO₂ supply at ribulose-1,5-bisphosphate carboxylase/oxygenase. Therefore, the A_n versus intercellular mole fraction (C_i) relationship was assumed not to be affected by ABA, and images of C_i and g_s were constructed from images of PSII photochemical yield under nonphotorespiratory conditions. The distribution of g_s remained unimodal following ABA treatment. A comparison of calculations of C_i from images and gas exchange in ABA-treated leaves showed that the overestimation of C_i estimated from gas exchange was only partly due to heterogeneity. This overestimation was also attributed to the cuticular transpiration, which largely affects the calculation of the leaf conductance to CO₂, when leaf conductance to water is low.     

Mesophyll conductance to CO<Subscript>2</Subscript> in leaves of Siebold’s beech (<Emphasis Type="Italic">Fagus crenata</Emphasis>) seedlings under elevated ozone

Ozone is an air pollutant that negatively affects photosynthesis in woody plants. Previous studies suggested that ozone-induced reduction in photosynthetic rates is mainly attributable to a decrease of maximum carboxylation rate (V_cmax) and/or maximum electron transport rate (J_max) estimated from response of net photosynthetic rate (A) to intercellular CO₂ concentration (C_i) (A/C_i curve) assuming that mesophyll conductance for CO₂ diffusion (g_m) is infinite. Although it is known that C_i-based V_cmax and J_max are potentially influenced by g_m, its contribution to ozone responses in C_i-based V_cmax and J_max is still unclear. In the present study, therefore, we analysed photosynthetic processes including g_m in leaves of Siebold’s beech (Fagus crenata) seedlings grown under three levels of ozone (charcoal-filtered air or ozone at 1.0- or 1.5-times ambient concentration) for two growing seasons in 2016–2017. Leaf gas exchange and chlorophyll fluorescence were simultaneously measured in July and September of the second growing season. We determined the A, stomatal conductance to water vapor and g_m, and analysed A/C_i curve and A/C_c curve (C_c: chloroplast CO₂ concentration). We also determined the Rubisco and chlorophyll contents in leaves. In September, ozone significantly decreased C_i-based V_cmax. At the same time, ozone decreased g_m, whereas there was no significant effect of ozone on C_c-based V_cmax or the contents of Rubisco and chlorophyll in leaves. These results suggest that ozone-induced reduction in C_i-based V_cmax is a result of the decrease in g_m rather than in carboxylation capacity. The decrease in g_m by elevated ozone was offset by an increase in C_i, and C_c did not differ depending on ozone treatment. Since C_c-based V_cmax was also similar, A was not changed by elevated ozone. We conclude that g_m is an important factor for reduction in C_i-based V_cmax of Siebold’s beech under elevated ozone.     

Internal conductance to CO2 transfer of adult Fagus sylvatica: Variation between sun and shade leaves and due to free-air ozone fumigation

Two separate objectives were considered in this study. We examined (1) internal conductance to CO₂ (g_i) and photosynthetic limitations in sun and shade leaves of 60-year-old Fagus sylvatica, and (2) whether free-air ozone fumigation affects g_i and photosynthetic limitations. g_i and photosynthetic limitations were estimated in situ from simultaneous measurements of gas exchange and chlorophyll fluorescence on attached sun and shade leaves of F. sylvatica. Trees were exposed to ambient air (1× O₃) and air with twice the ambient ozone concentration (2× O₃) in a free-air ozone canopy fumigation system in southern Germany (Kranzberg Forest). g_i varied between 0.12 and 0.24 mol m⁻² s⁻¹ and decreased CO₂ concentrations from intercellular spaces (C_i) to chloroplastic (C_c) by approximately 55 μmol mol⁻¹. The maximum rate of carboxylation (V_cmax) was 22–39% lower when calculated on a C_i basis compared with a C_c basis. g_i was approximately twice as large in sun leaves compared to shade leaves. Relationships among net photosynthesis, stomatal conductance and g_i were very similar in sun and shade leaves. This proportional scaling meant that neither C_i nor C_c varied between sun and shade leaves. Rates of net photosynthesis and stomatal conductance were about 25% lower in the 2× O₃ treatment compared with 1× O₃, while V_cmax was unaffected. There was no evidence that g_i was affected by ozone.     

Interactions of Elevated CO2 Concentration and Drought Stress on Photosynthesis in Eucalyptus Cladocalyx F. Muell

Response of net photosynthetic rate (P _N), stomatal conductance (g _s), intercellular CO₂ concentration (c _i), and photosynthetic efficiency (F_v/F_m) of photosystem 2 (PS2) was assessed in Eucalyptus cladocalyx grown for long duration at 800 (C₈₀₀) or 380 (C₃₈₀) µmol mol^-1 CO₂ concentration under sufficient water supply or under water stress. The well-watered plants at C₈₀₀ showed a 2.2 fold enhancement of P _N without any change in g _s. Under both C₈₀₀ and C₃₈₀, water stress decreased P _N and g _s significantly without any substantial reduction of c _i, suggesting that both stomatal and non-stomatal factors regulated P _N. However, the photosynthetic efficiency of PS2 was not altered.     

Mechanism of Photosynthetic Carbon Dioxide Uptake by the Red Macroalga, Chondrus crispus

The aim of this study was to determine how Chondrus crispus, a marine red macroalga, acquires the inorganic carbon (C_i) it utilizes for photosynthetic carbon fixation. Analyses of C_i uptake were done using silicone oil centrifugation (using multicellular fragments of thallus), infrared gas analysis, and gas chromatography. Inhibitors of carbonic anhydrase (CA), the band 3 anion exchange protein and Na⁺/K⁺ exchange were used in the study. It was found that: (a) C. crispus does not accumulate C_i internally above the concentration attainable by diffusion; (b) the initial C_i fixtion rate of C. crispus fragments saturates at approximately 3 to 4 millimolar C_i; (c) CA is involved in carbon uptake; its involvement is greatest at high HCO₃⁻ and low CO₂ concentration, suggesting its participation in the dehydration of HCO₃⁻ to CO₂; (d) C. crispus has an intermediate C_i compensation point; and (e) no evidence of any active or facilitated mechanism for the transport of HCO₃⁻ was detected. These data support the view that photosynthetic C_i uptake does not involve active transport. Rather, CO₂, derived from HCO₃⁻ catalyzed by external CA, passively diffuses across the plasma membrane of C. crispus. Intracellular CA also enhances the fixation of carbon in C. crispus.     

Soil drying and its effect on leaf conductance and CO2 assimilation of Vigna unguiculata (L.) Walp

Summary Well watered plants of Vigna unguiculata (L.) Walp cv. California Blackeye No. 5 had maximum photosynthetic rates of 16 mol m^-2 s^-1 (at ambient CO₂ concentration and environmental parameters optimal for high CO₂ uptake). Leaf conductance declined with increasing water vapour concentration difference between leaf and air (w), but it increased with increasing leaf temperature at a constant small w. When light was varied, CO₂ assimilation and leaf conductance were correlated linearly. We tested the hypothesis that g was controlled by photosynthesis via intercellular CO₂ concentration (c _i). No unique relationship between (1) c _i, (2) the difference between ambient CO₂ concentration (c _a) and c _i, namely c _a-c _i, or (3) the c _i/c _a ratio and g was found. g and A appeared to respond to environmental factors fairly independently of each other. The effects of different rates of soil drying on leaf gas exchange were studied. At unchanged air humidity, different rates of soil drying were produced by using (a) different soils, (b) different irrigation schemes and (c) different soil volumes per plant. Although the soil dried to wilting point the relative leaf water content was little affected. Different soil drying rates always resulted in the same response of photosynthetic capacity (A _max) and corresponding leaf conductance (g(_Amax)) when plotted against percent relative plant-extractable soil water content (W _e %) but the relationship with relative soil water content (W _e ) was less clear. Above a range of W _e of 15%–25%, A _max and g(_Amax) were both high and responded little to decreasing W _e . As soon as W _e fell below this range, A _max and g(_Amax) declined. The data suggest root-to-leaf communication not mediated via relative leaf water content. However, g(_Amax) was initially more affected than A _max.List of abbreviations A CO₂ assimilation - A _max photosynthetic capacity at favourable ambient conditions - c _a CO₂ concentration of the air in the leaf chamber - c _i intercellular - CO₂ concentration - E transpiration - g leaf conductance - g(_Amax) leaf conductance corresponding to photosynthetic capacity - I photon flux rate - T _l leaf temperature - W _e relative plant-extractable soil water content - W _e absolute plant-extractable soil water content - W _l relative leaf water content - W _s relative soil water content - w difference in water vapour mole fraction between leaf and air - leaf water potential     

Limitation factors for photosynthesis in ‘Bluecrop’ highbush blueberry (Vaccinium corymbosum) leaves in response to moderate water stress

The levels of stomatal, mesophyll and biochemical limitations in CO₂ assimilation of ‘Bluecrop’ highbush blueberry leaves were compared at two different levels of leaf water potential. The leaf water potentials were ?1.49 and ?1.94 MPa in daily-irrigated (DI) and non-irrigated (NI) shrubs, respectively. The NI shrubs represented plants under moderate water stress. Mesophyll conductance (g _m) and chloroplastic CO₂ concentration (C _c) were estimated by combined measurements of gas exchange and chlorophyll fluorescence under various intercellular CO₂ concentrations (C _i). Net CO₂ assimilation rates (A _n) as a function of C _c were used for calculating maximum carboxylation efficiency (α _cmax) at the real sites of CO₂ assimilation. Maximum A _n (A _nmax) from the light response curves at 400 μmol mol^?1 air of ambient CO₂ concentration (C _a) were lower in the leaves of NI shrubs than in those of DI ones. However, electron transport rates were higher in the leaves of NI shrubs than in those of DI ones. The decrease in CO₂ assimilation following water stress may be caused by a decrease in g _m rather than a decrease in stomatal conductance (g _s) according to limitation analysis. Limitation rates by g _s, calculated at 400 μmol mol^?1 air of C _a in A _n-C _i curves, were not significantly different between the leaves of DI and NI shrubs. However, limitation rates by g _m from A _n-C _c curves were significantly higher in the leaves of NI shrubs than in those of DI ones. Maximum carboxylation efficiency (α _cmax) values calculated from the A _n-C _c curve, contrary to those calculated from the A _n-C _i curve, were higher in the leaves of NI shrubs than in those of DI ones. Consequently, mesophyll limitation than stomatal and biochemical limitations mainly down-regulated the photosynthesis in the leaves of ‘Bluecrop’ blueberry shrubs during moderate water stress.     

Stomatal response to blue light: water use efficiency in sugarcane and soybean*

Abstract. The significance of blue light-stimulated stomatal conductance for carbon assimilation (A), stomatal conductance (g), intercellular CO₂ (C_i), stomatal limitation of A (L), transpiration (E) and water use efficiency (W = A/E), was determined in a C₄ and a C₃ species. W and L were evaluated for steady-state gas exchange with constant, saturating red light (A_s, g_s, E_s), and for the integrated gas exchange above the steady state baseline induced by a single, brief pulse of blue light (A_p, g_p, E_p). Sugarcane (Saccharum spp. hybrid), a C₄ grass, and soybean (Glycine max) a C₃ dicot, were compared. Sugarcane exhibited typical C₄ behaviour, with A saturing at C_i of ca. 200 μmol mol^?1, compared to >500 μmol mol^?1 in soybean. Steady-state W was also considerably higher in sugarcane. The extent of stomatal opening in response to a blue light pulse, from baseline (g_s) to the maximum value of conductance during the opening response (g_m), was similar in the two species. More rapid opening and closing of stomata in sugarcane resulted in a smaller integrated magnitude of the conductance response (g_p) than in soybean. At the peak of the blue light response, both species exhibited similar levels of L. During the response to the pulse of blue light, A and C_i increased and L decreased to a greater extent in sugarcane than in soybean. As a result, the gas exchange attributed to the stomatal response to blue light exhibited a higher ratio of A_p to E_p (W_p) in sugarcane than in soybean. This W_p was lower in both species than was the W_s associated with the steady state gas exchange. The two species did not differ in the rate of induction of photosynthetic utilization of elevated C_i. The greater stimulation of A in sugarcane was attributed to its C₄ attributes of greater carboxylation efficiency (slope of the A versus C_i relationship), lower g_s and prevailing C_i,s, and greater L_s under steady-state red illumination. Despite saturation of A at low levels of C_i in C₄ species, the gas exchange attributed to the stomatal response to blue light decreased L and contributed considerably to carbon acquisition, while maintaining the high level of W associated with C₄ metabolism.     

Artefactual responses of mesophyll conductance to CO2 and irradiance estimated with the variable J and online isotope discrimination methods

Studies with the variable J method have reported that mesophyll conductance (g_m) rapidly decreases with increasing intercellular CO₂ partial pressures (C_i) or decreasing irradiance. Similar responses have been suggested with the online isotope discrimination method, although with less consistency. Here we show that even when the true g_m is constant, the variable J method can produce an artefactual dependence of g_m on C_i or irradiance similar to those reported in previous studies for any of the following factors: day respiration and chloroplastic CO₂ photocompensation point are estimated with Laisk method; C_i or electron transport rate is positively biased; net photosynthetic rate is negatively biased; insufficient NADPH is assumed while insufficient ATP limits RuBP regeneration. The isotopic method produces similar artefacts if fractionation of carboxylation or C_i is positively biased or Δ¹³ negatively biased. A non‐zero chloroplastic resistance to CO₂ movement results in a qualitatively different dependence of g_m on C_i or irradiance and this dependence is only sensitive at low C_i. We thus cannot rule out the possibility that previously reported dependence of g_m on C_i or irradiance is a methodological artefact. Recommendations are made to take advantage of sensitivities of the variable J and isotopic methods for estimating g_m.     

Photosynthesis Decrease and Stomatal Control of Gas Exchange in Abies alba Mill. in Response to Vapor Pressure Difference

The responses of steady state CO₂ assimilation rate (A), transpiration rate (E), and stomatal conductance (g_s) to changes in leaf-to-air vapor pressure difference (ΔW) were examined on different dates in shoots from Abies alba trees growing outside. In Ecouves, a provenance representative of wet oceanic conditions in Northern France, both A and g_s decreased when ΔW was increased from 4.6 to 14.5 Pa KPa⁻¹. In Nebias, which represented the dry end of the natural range of A. alba in southern France, A and g_s decreased only after reaching peak levels at 9.0 and 7.0 Pa KPa⁻¹, respectively. The representation of the data in assimilation rate (A) versus intercellular CO₂ partial pressure (C_i) graphs allowed us to determine how stomata and mesophyll photosynthesis interacted when ΔW was increased. Changes in A were primarily due to alterations in mesophyll photosynthesis. At high ΔW, and especially in Ecouves when soil water deficit prevailed, A declined, while C_i remained approximately constant, which may be interpreted as an adjustment of g_s to changes in mesophyll photosynthesis. Such a stomatal control of gas exchange appeared as an alternative to the classical feedforward interpretation of E versus ΔW responses with a peak rate of E. The gas exchange response to ΔW was also characterized by considerable deviations from the optimization theory of IR Cowan and GD Farquhar (1977 Symp Soc Exp Biol 31: 471-505).     

Carbonyl sulfide: an inhibitor of inorganic carbon transport in cyanobacteria

Cells of a high CO₂-requiring mutant (E₁) and wild type of Synechococcus PCC7942 were incubated with COS in the light, then suspended in COS-free medium and their CO₂ exchange was measured using an open gas-analysis system under the conditions where photosynthetic CO₂ fixation is inhibited. When the suspension of cells untreated with COS was illuminated, the rate of CO₂ uptake was high and addition of carbonic anhydrase during illumination released a large amount of CO₂ from the medium into the gas phase. The COS treatment in the light markedly reduced the rate of CO₂ uptake by the cells and the amount of CO₂ released by carbonic anhydrase. Incubation of cells with COS in the dark had no effect on the CO₂-exchange profile. The COS concentration required for 50% inhibition of CO₂ uptake was about 25 micromolar when the concentration of inorganic carbon (C_i) in the medium was 60 micromolar; higher C_i concentrations reduced the inhibitory effect of COS. Measurement of C_i uptake in E₁ cells by a silicone oil centrifugation method also indicated marked reduction of the activities of ¹⁴CO₂ and H¹⁴CO₃⁻ uptake in the cells treated with COS in the light. The results demonstrated that COS is a potent inhibitor of C_i transport.     

Photosynthetic characteristics of dwarf and fringe Rhizophora mangle L. in a Belizean mangrove

Twin Cays (Belize) is a highly oligotrophic mangrove archipelago dominated by Rhizophora mangle L. Ocean‐fringing trees are 3–7 m tall with a leaf area index (LAI) of 2.3, whereas in the interior, dwarf zone, trees are 1.5 m or less, and the LAI is 0.7. P‐fertilization of dwarf trees dramatically increases growth. As a partial explanation of these characteristics, it was hypothesized that differences in stature and growth rates would reflect differences in leaf photosynthetic capacity, as determined by the photochemical and biochemical characteristics at the chloroplast level. Gas exchange and chlorophyll fluorescence were used to compare photosynthesis of dwarf, fringe and fertilized trees. Regardless of zonation or treatment, net CO₂ exchange (A) and photosynthetic electron transport were light saturated at less than 500 µmol photons m^?2 s^?1, and low‐light quantum efficiencies were typical for healthy C₃ plants. On the other hand, light‐saturated A was linearly related to stomatal conductance (g_s), with seasonal, zonal and treatment differences in photosynthesis corresponding linearly to differences in the mean g_s. Overall, photosynthetic capacity appeared to be co‐regulated with stomatal conductance, minimizing the variability of C_i at ambient CO₂ (and hence, C_i/C_a). Based on the results of in vitro assays, regulation of photosynthesis in R. mangle appeared to be accomplished, at least in part, by regulation of Rubisco activity.     

Carbon Dioxide and Light Responses of Photosynthesis in Cowpea and Pigeonpea during Water Deficit and Recovery

Greenhouse-grown pigeonpea (Cajanus cajan, [L.] Millsp.; cultivar UW-10) and cowpea (Vigna unguiculata, [L.] Walp.; cultivar California No. 5) were well-watered (control) or subjected to low water potential by withholding water to compare their modes of adaptation to water-limited conditions. Leaf CO₂ exchange rate (CER), leaf diffusive conductance to CO₂ (g_l), and CO₂ concentration in the leaf intercellular air space (C_i) were determined at various CO₂ concentrations and photon flux densities (PFD) of photosynthetically active radiation (400 to 700 nanometer). In cowpea, g_l declined to less than 15% of controls and total water potential (ψ_w) at midafternoon declined to −0.8 megapascal after 5 days of withholding water, whereas g_l in pigeonpea was about 40% of controls even though midafternoon ψ_w was −1.9 megapascal. After 8 days of withholding water, ψ_w at midafternoon declined to −0.9 and −2.4 megapascals in cowpea and pigeonpea, respectively. The solute component of water potential (ψ_s) decreased substantially less in cowpea than pigeonpea. Photosynthetic CER at saturation photon flux density (PFD) and ambient external CO₂ concentration (360 microliters per liter) on day 5 of withholding decreased by 83 and 55% in cowpea and pigeonpea, respectively. When measured at external, CO₂ concentration in bulk air of 360 microliters per liter, the CER of cowpea had fully recovered to control levels 3 days after rewatering; however, at 970 microliters per liter the PFD-saturated CERs of both species were substantially lower than in controls, indicating residual impairment. In stressed plants of both species the CER responses to C_i from 250 to 600 microliters per liter indicated that a substantial nonstomatal inhibition of CER had occurred. Although the sensitivity of g_l to water limitation in cowpea suggested a dehydration avoidance response, parallel measurements of CER at various C_i and PFD indicated that photosynthetic activity of cowpea mesophyll was substantially inhibited by the water-limited treatment.     

The use of low [CO2] to estimate diffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive saplings

In this study it has been shown that increased diffusional resistances caused by salt stress may be fully overcome by exposing attached leaves to very low [CO₂] (～ 50 µmol mol^?1), and, thus a non‐destructive‐in vivo method to correctly estimate photosynthetic capacity in stressed plants is reported. Diffusional (i.e. stomatal conductance, g_s, and mesophyll conductance to CO₂, g_m) and biochemical limitations to photosynthesis (A) were measured in two 1‐year‐old Greek olive cultivars (Chalkidikis and Kerkiras) subjected to salt stress by adding 200 mm NaCl to the irrigation water. Two sets of A–C_i curves were measured. A first set of standard A–C_i curves (i.e. without pre‐conditioning plants at low [CO₂]), were generated for salt‐stressed plants. A second set of A–C_i curves were measured, on both control and salt‐stressed plants, after pre‐conditioning leaves at [CO₂] of ～ 50 µmol mol^?1 for about 1.5 h to force stomatal opening. This forced stomata to be wide open, and g_s increased to similar values in control and salt‐stressed plants of both cultivars. After g_s had approached the maximum value, the A–C_i response was again measured. The analysis of the photosynthetic capacity of the salt‐stressed plants based on the standard A–C_i curves, showed low values of the J_max (maximum rate of electron transport) to V_cmax (RuBP‐saturated rate of Rubisco) ratio (1.06), that would implicate a reduced rate of RuBP regeneration, and, thus, a metabolic impairment. However, the analysis of the A–C_i curves made on pre‐conditioned leaves, showed that the estimates of the photosynthetic capacity parameters were much higher than in the standard A–C_i responses. Moreover, these values were similar in magnitude to the average values reported by Wullschleger (Journal of Experimental Botany 44, 907–920, 1993) in a survey of 109 C₃ species. These findings clearly indicates that: (1) salt stress did affect g_s and g_m but not the biochemical capacity to assimilate CO₂ and therefore, in these conditions, the sum of the diffusional resistances set the limit to photosynthesis rates; (2) there was a linear relationship (r² = 0.68) between g_m and g_s, and, thus, changes of g_m can be as fast as those of g_s; (3) the estimates of photosynthetic capacity based on A–C_i curves made without removing diffusional limitations are artificially low and lead to incorrect interpretations of the actual limitations of photosynthesis; and (4) the analysis of the photosynthetic properties in terms of stomatal and non‐stomatal limitations should be replaced by the analysis of diffusional and non‐diffusional limitations of photosynthesis. Finally, the C₃ photosynthesis model parameterization using in vitro‐measured and in vivo‐measured kinetics parameters was compared. Applying the in vivo‐measured Rubisco kinetics parameters resulted in a better parameterization of the photosynthesis model.     

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (E_t; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m⁻². Mesophyll conductance (μ) was measured under 1.5% O₂, as well as postillumination CO₂ uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on E_t saturated at E_t = 30 μmol active sites m⁻² and μ = 11 mm s⁻¹ in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar E_t but reached a μ of only 6 to 8 mm s⁻¹. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm⁻¹ s⁻¹. Our data show that the saturation of the relationship between E_t and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.Rubisco (EC 4.1.1.39) catalyzes the irreversible carboxylation of RuBP to form two PGA molecules (in this work the oxygenase reaction was not active since a low O₂ concentration was used). RuBP carboxylation is the major rate-determining reaction in photosynthetic CO₂ assimilation. All factors that influence the photosynthetic rate do so by influencing the activity of Rubisco and the concentration of its substrates, CO₂ and RuBP. E_t in leaves may be as high as 75 μmol m⁻², and for the extracted enzyme K_m(CO₂) = 9.4 μm (Makino et al., 1985a) and K_m(RuBP) = 30 to 40 μm (Yeoh et al., 1981). In leaves photosynthesizing under atmospheric conditions, the concentration of RuBP may increase to 10 to 15 mm (Badger et al., 1984; Sharkey et al., 1986), but the concentration of CO₂ is usually about 4 to 8 μm in leaf intercellular spaces, depending on stomatal conductance. This CO₂ concentration is well below the K_m(CO₂) of the enzyme, and it is the initial slope of the kinetic curve V_M/K_m(CO₂), termed carboxylation conductance, that becomes important.r_c limits the CO₂-fixation rate in series with the other resistances, r_g and r_md. The carboxylation rates are usually expressed in relation to C_i or C_w. C_c is usually about 20% to 30% lower than C_w because of concentration decrease generated by the carboxylation flux on r_md. Considering the above, the carboxylation conductance in intact leaves in vivo may be found as the initial slope of the A versus C_c graph at low C_c values. If C_c cannot be calculated because r_md is unknown, the closest approximation is a plot of A versus C_w or A versus C_i. The true parameters of the carboxylase can be found only from experiments carried out in nonphotorespiratory conditions (1%–2% O₂); otherwise the competing oxygenase reaction consumes a part of RuBP and partially inhibits carboxylase activity.Because of technical problems with the measurement of A versus C_w relationships, in many studies only the net photosynthetic rate under atmospheric conditions (21% O₂) was related to Rubisco activity or content. Nevertheless, good correlation has been found (Makino et al., 1983; Hudson et al., 1992; Jacob and Lawlor, 1992; Jiang and Rodermel, 1995; Nakano et al., 1997). These results indicated that the level of Rubisco protein could be a limiting factor in photosynthesis throughout the life span of the leaf under natural environmental conditions. On the other hand, when Rubisco levels in leaves exceeded 4 g m⁻² (60 μmol m⁻²), the in vivo Rubisco activity (measured as photosynthesis under pC_i = 20 to 30 Pa and 21% O₂) became curvilinearly correlated with E_t (Makino et al., 1994, 1997). When measurements were made over the whole life span of wheat leaves, the measured rates of photosynthesis were lower in young leaves, which had high protein content, than would have been expected from the amount and activity of Rubisco (Lawlor et al., 1989).During senescence the decrease in Rubisco activity was initially greater than the decrease in net photosynthesis (Hall et al., 1978). In a willow canopy, Rubisco-specific activity was higher when the apparent E_t (N content in leaves) was smaller (Vapaavuori and Vuorinen, 1989). A similar nonlinearity was found in our previous experiments (Eichelmann and Laisk, 1990), in which we obtained a saturating relationship when E_t exceeded 30 μmol m⁻². In the latter work the initial slope of the A versus C_w curves under nonphotorespiratory conditions (1.5% O₂) was assumed to represent the Rubisco activity in vivo and was compared with the E_t. We discovered that growth light had the strongest influence on the saturation of the relationship between μ and E_t. In the present work we present insight into this relationship, using not only plants grown under different light intensities but also leaves adapted to different light intensities.     

Effect of nitrogen application and elevated CO2 on photosynthetic gas exchange and electron transport in wheat leaves

Nitrogen (N) availability is a critical factor affecting photosynthetic acclimation of C₃ plants under elevated atmospheric CO₂ concentration ([CO₂]_e). However, current understanding of N effects on photosynthetic electron transport rate and partitioning, as well as its impact on photosynthesis under [CO₂]_e, is inadequate. Using controlled environment open-top chambers, wheat (Triticum aestivum L.) was grown at two N levels (0 and 200 mg(N) kg^?1 soil) and two atmospheric CO₂ concentrations of 400 ([CO₂]_a) and 760 μmol mol^?1([CO₂]_e) during 2009 and 2010. Under [CO₂]_e high N availability increased stomatal conductance and transpiration rate, reduced limitations on the activity of triose phosphate isomerase, a Calvin cycle enzyme, and increased the rate of net photosynthesis (P _N). Considering photosynthetic electron transport rate and partitioning aspects, we suggest that greater N availability increased P _N under [CO₂]_e due to four following reasons: (1) higher N availability enhanced foliar N and chlorophyll concentrations, and the actual photochemical efficiency of photosystem (PS) II reaction centers under irradiance increased, (2) increase of total electron transport rate and proportion of open PSII reaction centers, (3) enhancement of the electron transport rate of the photochemical and carboxylation processes, and (4) reduced limitations of the Calvin cycle enzymes on the photosynthetic electron transport rate. Consequently, sufficient N improved light energy utilization in wheat flag leaves under [CO₂]_e, thus benefiting to photosynthetic assimilation.