Lateral diffusion of CO2 in leaves is not sufficient to support photosynthesis

Lateral diffusion of CO(2) was investigated in photosynthesizing leaves with different anatomy by gas exchange and chlorophyll a fluorescence imaging using grease to block stomata. When one-half of the leaf surface of the heterobaric species Helianthus annuus was covered by 4-mm-diameter patches of grease, the response of net CO(2) assimilation rate (A) to intercellular CO(2) concentration (C(i)) indicated that higher ambient CO(2) concentrations (C(a)) caused only limited lateral diffusion into the greased areas. When single 4-mm patches were applied to leaves of heterobaric Phaseolus vulgaris and homobaric Commelina communis, chlorophyll a fluorescence images showed dramatic declines in the quantum efficiency of photosystem II electron transport (measured as F(q)'/F(m)') across the patch, demonstrating that lateral CO(2) diffusion could not support A. The F(q)'/F(m)' values were used to compute images of C(i) across patches, and their dependence on C(a) was assessed. At high C(a), the patch effect was less in C. communis than P. vulgaris. A finite-volume porous-medium model for assimilation rate and lateral CO(2) diffusion was developed to analyze the patch images. The model estimated that the effective lateral CO(2) diffusion coefficients inside C. communis and P. vulgaris leaves were 22% and 12% of that for free air, respectively. We conclude that, in the light, lateral CO(2) diffusion cannot support appreciable photosynthesis over distances of more than approximately 0.3 mm in normal leaves, irrespective of the presence or absence of bundle sheath extensions, because of the CO(2) assimilation by cells along the diffusion pathway.     

Lateral diffusion of CO2 from shaded to illuminated leaf parts affects photosynthesis inside homobaric leaves

Gas exchange is generally regarded to occur between the leaf interior and ambient air, i.e. in vertical (anticlinal) directions of leaf blades. However, inside homobaric leaves, gas movement occurs also in lateral directions. The aim of the present study was to ascertain whether lateral CO2 diffusion affects leaf photosynthesis when illuminated leaves are partially shaded. Measurements using gas exchange and chlorophyll fluorescence imaging techniques were performed on homobaric leaves of Vicia faba and Nicotiana tabacum or on heterobaric leaves of Glycine max and Phaseolus vulgaris. For homobaric leaves, gas exchange inside a clamp-on leaf chamber was affected by shading the leaf outside the chamber. The quantum yield of photosystem II (Phi(PSII)) was highest directly adjacent to a light/shade border (LSB). Phi(PSII) decreased in the illuminated leaf parts with distance from the LSB, while the opposite was observed for nonphotochemical quenching. These effects became most pronounced at low stomatal conductance. They were not observed in heterobaric leaves. The results suggest that plants with homobaric leaves can benefit from lateral CO2 flux, in particular when stomata are closed (e.g. under drought stress). This may enhance photosynthetic, instead of nonphotochemical, processes near LSBs in such leaves and reduce the photoinhibitory effects of excess light.     

Lateral CO2 diffusion inside dicotyledonous leaves can be substantial: quantification in different light intensities

Substantial lateral CO(2) diffusion rates into leaf areas where stomata were blocked by grease patches were quantified by gas exchange and chlorophyll a fluorescence imaging in different species across the full range of photosynthetic photon flux densities (PPFD). The lateral CO(2) flux rate over short distances was substantial and very similar in five dicotyledonous species with different vascular anatomies (two species with bundle sheath extensions, sunflower [Helianthus annuus] and dwarf bean [Phaseolus vulgaris]; and three species without bundle sheath extensions, faba bean [Vicia faba], petunia [Petunia hybrida], and tobacco [Nicotiana tabacum]). Only in the monocot maize (Zea mays) was there little or no evident lateral CO(2) flux. Lateral diffusion rates were low when PPFD <300 micromol m(-2) s(-1) but approached saturation in moderate PPFD (300 micromol m(-2) s(-1)) when lateral CO(2) diffusion represented 15% to 24% of the normal CO(2) assimilation rate. Smaller patches and higher ambient CO(2) concentration increased lateral CO(2) diffusion rates. Calculations with a two-dimensional diffusion model supported these observations that lateral CO(2) diffusion over short distances inside dicotyledonous leaves can be important to photosynthesis. The results emphasize that supply of CO(2) from nearby stomata usually dominates assimilation, but that lateral supply over distances up to approximately 1 mm can be important if stomata are blocked, particularly when assimilation rate is low.     

Air pressure in clamp-on leaf chambers: a neglected issue in gas exchange measurements

Air pressure in leaf chambers is thought to affect gas exchange measurements through changes in partial pressure of the air components. However, other effects may come into play when homobaric leaves are measured in which internal lateral gas flow may occur. When there was no pressure difference between the leaf chamber and ambient air (DeltaP=0), it was found in previous work that lateral CO(2) diffusion could affect measurements performed with clamp-on leaf chambers. On the other hand, overpressure (DeltaP>0) in leaf chambers has been reported to minimize artefacts possibly caused by leaks in chamber sealing. In the present work, net CO(2) exchange rates (NCER) were measured under different DeltaP values (0.0-3.0 kPa) on heterobaric and homobaric leaves. In heterobaric leaves which have internal barriers for lateral gas movement, changes in DeltaP had no significant effect on NCER. For homobaric leaves, effects of DeltaP>0 on measured NCER were significant, obviously due to lateral gas flux inside the leaf mesophyll. The magnitude of the effect was largely defined by stomatal conductance; when stomata were widely open, the impact of DeltaP on measured NCER was up to 7 mumol CO(2) m(-2) s(-1) kPa(-1). Since many other factors are also involved, neither DeltaP=0 nor DeltaP>0 was found to be the 'one-size fits all' solution to avoid erroneous effects of lateral gas transport on measurements with clamp-on leaf chambers.     

Lateral gas diffusion inside leaves

Diffusion of CO2 inside leaves is generally regarded to be from the substomatal cavities to the assimilating tissues, i.e. in the vertical direction of the leaf blades. However, lateral gas diffusion within intercellular air spaces may be much more effective than hitherto considered. In a previous work it was demonstrated that, when 'clamp-on' leaf chambers are used, leaf internal 'CO2 leakage' beyond the leaf chamber gaskets may seriously affect gas exchange measurement. This effect has been used in the present paper to quantify gas conductance (g(leaf,l), mmol m(-2) s(-1)) in the lateral directions within leaves and significant differences between homo- and heterobaric leaves were observed. For the homobaric leaves, lateral gas conductance measured over a distance of 6 or 8 mm (the widths of the chamber gaskets) was 2-20% of vertical conductance taken from published data measured over much smaller distances of 108-280 microm (the thickness of the leaves). The specific internal gas diffusion properties of the leaves have been characterized by gas conductivities (g*(leaf), micromol m(-1) s(-1)). Gas conductivities in the lateral directions of heterobaric leaves were found to be small but not zero. In homobaric leaves, they were between 67 and 209 micromol m(-1) s(-1) and thus even larger than those in the vertical direction of the leaf blades (between 15 and 78 micromol m(-1) s(-1)). The potential implications for experimentalists performing gas exchange measurements are discussed.     

Does lateral gas diffusion in leaves matter?

Photosynthesis depends on the diffusion of gaseous CO(2) inside the leaf spaces from the stomatal entry point to the mesophyll cell walls. Although most research considers only the vertical diffusion from stomata on upper and/or leaf lower surfaces, some of the gas will diffuse in the lateral (paradermal) direction. The importance of lateral CO(2) diffusion is reviewed, and the anatomical characteristics of leaves, including the variation of air space volume between species and conditions are discussed. The contribution of the air space conductance to the limitation of photosynthesis by the overall CO(2) diffusion pathway is usually ignored. However, the need to consider three-dimensional diffusion at the small scale of a few stomata is emphasized because stomata are discrete, and separated by 20-300 microm. At the large scale of 100s of micrometres, there may be barriers to CO(2) caused by the vascular tissue, particularly if there are bundle sheath extensions. The possible extent and controls on CO(2) lateral and vertical diffusion in different species and conditions are illustrated using chlorophyll a fluorescence imaging techniques. It is clear that there is a range of effective lateral permeabilities depending on the particular vascular patterns and cell arrangements, and that species cannot be simply divided into homobaric and heterobaric anatomies. Lateral diffusion in more permeable leaves can be sufficient to affect measurements of leaf gas exchange, particularly when fluxes are low, although its contribution to leaf photosynthesis in natural conditions needs clarification.     

Should structure-function relations be considered separately for homobaric vs. heterobaric leaves?

Tree and shrub species can be differentiated into two major groups based on their substantially different leaf anatomy: heterobaric and homobaric. In contrast to homobaric leaves, heterobaric leaves have bundle sheath extensions (BSEs) that create transparent regions on their lamina. Recent studies have shown that BSEs transfer visible light to internal mesophyll layers, thus affecting the photosynthetic performance of heterobaric leaves. Whether the two leaf types also differ in other functional and structural traits has not been addressed, nor have any structure-function relations. Here, we measured key anatomical and physiological parameters and tested their relationships in 30 species with different leaf types. Heterobaric leaves were thinner with lower leaf mass per area, had higher nitrogen concentration per mass, were (13)C-enriched, and achieved comparable photosynthetic capacity per area but had higher photosynthetic capacity per mass compared to homobaric leaves. Relations between leaf construction cost, nitrogen concentration, and photosynthesis followed the general pattern of the "leaf economic spectrum," but differed between homobaric and heterobaric leaves. We suggest that the mechanisms controlling these relations differ between the two leaf types, presumably due to their distinct anatomy.     

Carbon dioxide signalling in plant leaves

The role of carbon dioxide (CO(2)) as a signal in biochemical regulation networks of plants is fathomed. Transport mechanisms of CO(2) and HCO3- are surveyed, which are the prerequisite for signalling. A CO(2) sensor is not known to date, but any reaction where CO(2)/HCO3- is a substrate can be a candidate. Carbon concentrating mechanisms, e.g., in higher plants C(4)-photosynthesis and crassulacean acid metabolism (CAM), generate high internal CO(2) concentrations, important for photosynthesis, but also as a basis for signalling via diffusion of CO(2). Spatiotemporal dynamics of desynchronization/synchronization of photosynthetic activity over leaves can be followed by chlorophyll fluorescence imaging. One example of desynchronization is based on patchiness of stomatal opening/closing in heterobaric leaves due to anatomic constraints of lateral CO(2) diffusion. During CAM, largely different internal CO(2) concentrations prevail in the leaves, offering opportunities to study the effect of lateral diffusion of CO(2) in synchronizing photosynthetic activity over the entire leaves.     

沙地柏叶型变化的生态意义

沙地柏（Sabina vulgaris）的叶包括刺叶和鳞叶两种类型。本文探讨沙地柏刺叶与鳞叶的结构和功能差异的生态意义。结果表明：（1）刺叶的角质层厚度、表皮细胞大小、叶肉细胞表面积和维管束大小都显著小于鳞叶;（2）刺叶的组织密度和失水系数都明显大于鳞叶;（3）刺叶的净光合速度、蒸腾速率和胞间CO2／大气CO2比都显著大于鳞叶,而前者的水分利用效率显著低于后者。这些结果指示：沙地柏刺叶的光合产物积累和水分丧失量大于鳞叶,但前者的抗旱保水性、耐辐射能力和水分利用效率都低于后者。刺叶和鳞叶的结构和功能差异表明：沙地柏叶型变化在一定程度上具有适应意义。     

Overestimation of respiration rates in commercially available clamp-on leaf chambers. Complications with measurement of net photosynthesis

The possible interference when measuring gas exchange with respiratory CO₂ produced under the gasket of commercially available clamp‐on leaf chambers was investigated. Two of these chambers were compared with a leaf chamber that accommodated an entire leaf without clamping it under a gasket. An overestimation of dark respiration rate (R_D) by 55% was found with Plantago major leaves, a species with homobaric leaves that have high resistance for lateral gaseous transport. The percentage was similar in the heterobaric Ficus benjamina, but was 32% in the highly porous homobaric Nicotiana tabacum. Net photosynthetic rate at low photon flux density was underestimated by 35% in the clamp‐on chamber. However, the gasket effect was not detectable at light saturation because the error was small in comparison with the high photosynthetic rates. Estimation of respiration in the light (R_L) in Nicotiana as derived from CO₂ exchange at low CO₂ concentrations was complicated by three factors. The inward diffusion of respiratory CO₂ from under the gasket was added to a diffusion of CO₂ from outside through the gasket material and through the leaf, which produced an even larger error in R_L in comparison with R_D at ambient CO₂. These errors are significant for estimations of carbon gain at whole plant and canopy level and also at the leaf level when photosynthetic rates are low. Possible improvements in gasket design and corrections of CO₂ exchange measurements for the gasket effect are discussed.     

Water supply and demand remain balanced during leaf acclimation of Nothofagus cunninghamii trees

Higher leaf vein density (D(vein) ) enables higher rates of photosynthesis because enhanced water transport allows higher leaf conductances to CO(2) and water. If the total cost of leaf venation rises in proportion to the density of minor veins, the most efficient investment in leaf xylem relative to photosynthetic gain should occur when the water transport capacity of the leaf (determined by D(vein) ) matches potential transpirational demand (determined by stomatal size and density). We tested whether environmental plasticity in stomatal density (D(stomata) ) and D(vein) were linked in the evergreen tree Nothofagus cunninghamii to achieve a balance between liquid and gas phase water conductances. Two sources of variation were examined; within-tree light acclimation, and differences in sun leaves among plants from ecologically diverse populations. Strong, linear correlations between D(vein) and D(stomata) were found at all levels of comparison. The correlations between liquid- and vapour-phase conductances implied by these patterns of leaf anatomy were confirmed by direct measurement of leaf conductance in sun and shade foliage of an individual tree. ? Our results provide strong evidence that the development of veins and stomata are coordinated so that photosynthetic yield is optimized relative to carbon investment in leaf venation.     

The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light. We investigated this light response of Adiantum stomata and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration nor closed under high intercellular CO2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO2. In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K+ accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The stomatal opening was completely inhibited by CsCl, a K+ channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K+ uptake.     

Cadmium effects on leaf transpiration of sugar beet (Beta vulgaris)

Seedlings of sugar beet ( Beta vulgaris L. cv Monohill) were cultivated for 4 weeks in nutrient solution containing different concentrations of CdCl₂ (0 to 10 μ M ). The effects of Cd on appearance and function of stomata and leaf cuticle were investigated by water loss measurements and microscopy. The leaf transpiration rate increased with increasing Cd concentrations while the sum total of stomatal aperture area per unit leaf area decreased. Already at low Cd levels. an increase of defective and undeveloped stomata was found in Cd treated plants. These stomata are closed or have small apertures and probably lack a functional closing mechanism. The number of intact stomata per unit leaf area was lower in leaves of Cd treated plants than in controls, and Cd induced closure of intact stomata. The total number of stomata per leaf area slightly increases with increasing Cd concentration. as does the percentage of small stomata. Furthermore. specific leaf area increased, while the density of leaf structure was decreased by Cd. From this observation we conclude that the increase in transpiration rate caused by Cd is primarily due to effects on the permeability of the leaf cuticle to water.     

Increase of photosynthesis and starch in potato under elevated CO2 is dependent on leaf age

Potato plants (Solanum tuberosum cv. Bintje) were grown in open top chambers under ambient (400 microL L(-1)) and elevated CO2 (720 microL L(-1)). After 50 days one half of each group was transferred to the other CO2 concentration and the effects were studied in relation to leaf age (old, middle-aged and young leaves) in each of the four groups. Under long-term exposure to elevated CO2, photosynthesis increased between 10% and 40% compared to ambient CO2. A subsequent shift of the same plants to ambient CO2 caused a 20-40% decline in photosynthetic rate, which was most pronounced in young leaves. After shifting from long-term ambient to elevated CO2, photosynthesis also increased most strongly in young leaves (90%); these experiments show that photosynthesis was downregulated in the upper young fully expanded leaves of potato growing long-term under elevated CO2. Soluble sugar content in all leaf classes under long-term exposure was stable irrespective of the CO2 treatment, however under elevated CO2 young leaves showed a strongly increased starch accumulation (up to 400%). In all leaf classes starch levels dropped in response to the shift from 720 to 400 microL L(-1) approaching ambient CO2 levels. After the shift to 720 microL L(-1), sucrose and starch levels increased, principally in young Leaves. There is clear evidence that leaves of different age vary in their responses to changes in atmospheric CO2 concentration.     

Characterization of juvenile and adult leaves of Eucalyptus globulus showing distinct heteroblastic development: photosynthesis and volatile isoprenoids

Heteroblastic Eucalyptus (Eucalyptus globulus L.) leaves were characterized for their functional diversity examining photosynthesis and photosynthesis limitations, transpiration, and the emission of isoprene and monoterpenes. In vivo and combined analyses of gas-exchange, chlorophyll fluorescence, and light absorbance at 830 nm were made on the adaxial and abaxial sides of juvenile and adult leaves. When adult leaves were reversed to illuminate the abaxial side, photosynthesis and isoprene emission were significantly lower than when the adaxial side was illuminated. Monoterpene emission, however, was independent on the side illuminated and similarly partitioned between the two leaf sides. The abaxial side of adult leaves showed less diffusive resistance to CO(2) acquisition by chloroplasts, but also lower ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, than the adaxial leaf side. In juvenile leaves, photosynthesis, isoprene, and monoterpene emissions were similar when the adaxial or abaxial side was directly illuminated. In the abaxial side of juvenile leaves, photosynthesis did not match the rates attained by the other leaf types when exposed to elevated CO(2), which suggests the occurrence of a limitation of photosynthesis by ribulose bisphosphate (RuBP) regeneration. Accordingly, a reduced efficiency of both photosystems and a high non-radiative dissipation of energy was observed in the abaxial side of juvenile leaves. During light induction, the adaxial side of juvenile leaves also showed a reduced efficiency of photosystem II and a large non-radiative energy dissipation. Our report reveals distinct functional properties in Eucalyptus leaves. Juvenile leaves invest more carbon in isoprene, but not in monoterpenes, and have a lower water use efficiency than adult leaves. Under steady-state conditions, in adult leaves the isobilateral anatomy does not correspond to an equal functionality of the two sides, while in juvenile leaves the dorsiventral anatomy does not result in functional differences in primary or secondary metabolism in the two sides. However, photochemical limitations may reduce the efficiency of carbon fixation in the light, especially in the abaxial side of juvenile leaves.     

The role of bundle sheath extensions and life form in stomatal responses to leaf water status

Bundle sheath extensions (BSEs) are key features of leaf structure with currently little-understood functions. To test the hypothesis that BSEs reduce the hydraulic resistance from the bundle sheath to the epidermis (r(be)) and thereby accelerate hydropassive stomatal movements, we compared stomatal responses with reduced humidity and leaf excision among 20 species with heterobaric or homobaric leaves and herbaceous or woody life forms. We hypothesized that low r(be) due to the presence of BSEs would increase the rate of stomatal opening (V) during transient wrong-way responses, but more so during wrong-way responses to excision (V(e)) than humidity (V(h)), thus increasing the ratio of V(e) to V(h). We predicted the same trends for herbaceous relative to woody species given greater hydraulic resistance in woody species. We found that V(e), V(h), and their ratio were 2.3 to 4.4 times greater in heterobaric than homobaric leaves and 2.0 to 3.1 times greater in herbaceous than woody species. To assess possible causes for these differences, we simulated these experiments in a dynamic compartment/resistance model, which predicted larger V(e) and V(e)/V(h) in leaves with smaller r(be). These results support the hypothesis that BSEs reduce r(be). Comparison of our data and simulations suggested that r(be) is approximately 4 to 16 times larger in homobaric than heterobaric leaves. Our study provides new evidence that variations in the distribution of hydraulic resistance within the leaf and plant are central to understanding dynamic stomatal responses to water status and their ecological correlates and that BSEs play several key roles in the functional ecology of heterobaric leaves.     

Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging

High resolution imaging of chlorophyll a fluorescence was used to identify the sites at which ozone initially induces perturbations of photosynthesis in leaves of Phaseolus vulgaris. Leaves were exposed to 250 and 500 nmol mol(-1) ozone at a photosynthetically active photon flux density of 300 micromol m(-2) s(-1) for 3 h. Images of fluorescence parameters indicated that large decreases in both the maximum and operating quantum efficiencies of photosystem II had occurred in cells adjacent to stomata in the upper, but not lower, leaf surfaces. However, this treatment did not produce any significant changes in the maximum or operating quantum efficiencies of photosystem II in the leaves when estimated from fluorescence parameters measured with a conventional, integrating fluorometer. The localized decreases in photosystem II photochemical efficiencies were accompanied by an increase in the minimal fluorescence level, which is indicative of photoinactivation of photosystem II complexes and a decrease in stomatal conductance. Perturbations of photochemical efficiencies were not observed in cells associated with all of the stomata on the upper leaf surface or within cells distant from the upper leaf surface. It is concluded that ozone penetrates the leaf through stomata and initially damages only cells close to stomatal pores.     

Senescence and hyperspectral reflectance of cotton leaves exposed to ultraviolet-B radiation and carbon dioxide

The objectives of this study were to determine the effects of UV-B radiation and atmospheric carbon dioxide concentrations ([CO(2)]) on leaf senescence of cotton by measuring leaf photosynthesis and chlorophyll content and to identify changes in leaf hyperspectral reflectance occurring due to senescence and UV-B radiation. Plants were grown in controlled-environment growth chambers at two [CO(2)] (360 and 720 micro mol mol(-1)) and three levels of UV-B radiation (0, 7.7 and 15.1 kJ m(-2) day(-1)). Photosynthesis, chlorophyll, carotenoids and phenolic compounds along with leaf hyperspectral reflectance were measured on three leaves aged 12, 21 and 30 days in each of the treatments. No interaction was detected between [CO(2)] and UV-B for any of the measured parameters. Significant interactions were observed between UV-B and leaf age for photosynthesis and stomatal conductance. Elevated [CO(2)] enhanced leaf photosynthesis by 32%. On exposure to 0, 7.7 and 15.1 kJ of UV-B, the photosynthetic rates of 30-day-old leaves compared with 12-day-old leaves were reduced by 52, 76 and 86%, respectively. Chlorophyll pigments were not affected by leaf age at UV-B radiation of 0 and 7.7 kJ, but UV-B of 15.1 kJ reduced the chlorophylls by 20, 60 and 80% in 12, 21 and 30-day-old leaves, respectively. The hyperspectral reflectance between 726 and 1142 nm showed interaction for UV-B radiation and leaf age. In cotton, leaf photosynthesis can be used as an indicator of leaf senescence, as it is more sensitive than photosynthetic pigments on exposure to UV-B radiation. This study revealed that, cotton leaves senesced early on exposure to UV-B radiation as indicated by leaf photosynthesis, and leaf hyperspectral reflectance can be used to detect changes caused by UV-B and leaf ageing.     

How does elevated CO2 or ozone affect the leaf-area index of soybean when applied independently?

Changes in leaf-area index (LAI) may alter ecosystem productivity in elevated [CO2] or [O3]. By increasing the apparent quantum yield of photosynthesis (phi(c,max)), elevated [CO2] may increase maximum LAI. However, [O3] when elevated independently accelerates senescence and may reduce LAI. Large plots (20 m diameter) of soybean (Glycine max) were exposed to ambient (approx. 370 micromol mol(-1)) or elevated (approx. 550 micromol mol(-1)) CO2 or 1.2 times ambient [O3] using soybean free-air concentration enrichment (SoyFACE). In 2001 elevated CO2 had no detectable effect on maximum LAI, but in 2002 maximum LAI increased by 10% relative to ambient air. Elevated [CO2] also increased the phi(c,max) of shade leaves in both years. Elevated [CO2] delayed LAI loss to senescence by approx. 54% and also increased leaf-area duration. Elevated [O3] accelerated senescence, reducing LAI by 40% near the end of the growing season. No effect of elevated [O3] on photosynthesis was detected. Elevated [CO2] or [O3] affected LAI primarily by altering the rate of senescence; knowledge of this may aid in optimizing future soybean productivity.     

Gas exchange measurements, what can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error

The principles, equipment and procedures for measuring leaf and canopy gas exchange have been described previously as has chlorophyll fluorescence. Simultaneous measurement of the responses of leaf gas exchange and modulated chlorophyll fluorescence to light and CO2 concentration now provide a means to determine a wide range of key biochemical and biophysical limitations on photo synthesis in vivo. Here the mathematical frameworks and practical procedures for determining these parameters in vivo are consolidated. Leaf CO2 uptake (A) versus intercellular CO2 concentration (Ci) curves may now be routinely obtained from commercial gas exchange systems. The potential pitfalls, and means to avoid these, are examined. Calculation of in vivo maximum rates of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) carboxylation (Vc,max), electron transport driving regeneration of RuBP (Jmax), and triose-phosphate utilization (VTPU) are explained; these three parameters are now widely assumed to represent the major limitations to light-saturated photosynthesis. Precision in determining these in intact leaves is improved by the simultaneous measurement of electron transport via modulated chlorophyll fluorescence. The A/Ci response also provides a simple practical method for quantifying the limitation that stomata impose on CO2 assimilation. Determining the rate of photorespiratory release of oxygen (Rl) has previously only been possible by isotopic methods, now, by combining gas exchange and fluorescence measurements, Rl may be determined simply and routinely in the field. The physical diffusion of CO2 from the intercellular air space to the site of Rubisco in C3 leaves has long been suspected of being a limitation on photosynthesis, but it has commonly been ignored because of the lack of a practical method for its determination. Again combining gas exchange and fluorescence provides a means to determine mesophyll conductance. This method is described and provides insights into the magnitude and basis of this limitation.