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.     

Temperature acclimation of photosynthesis: mechanisms involved in the changes in temperature dependence of photosynthetic rate

Growth temperature alters temperature dependence of the photosynthetic rate (temperature acclimation). In many species, the optimal temperature that maximizes the photosynthetic rate increases with increasing growth temperature. In this minireview, mechanisms involved in changes in the photosynthesis-temperature curve are discussed. Based on the biochemical model of photosynthesis, change in the photosynthesis-temperature curve is attributable to four factors: intercellular CO2 concentration, activation energy of the maximum rate of RuBP (ribulose-1,5-bisphosphate) carboxylation (Vc max), activation energy of the rate of RuBP regeneration (Jmax), and the ratio of Jmax to Vc max. In the survey, every species increased the activation energy of Vc max with increasing growth temperature. Other factors changed with growth temperature, but their responses were different among species. Among these factors, activation energy of Vc max may be the most important for the shift of optimal temperature of photosynthesis at ambient CO2 concentrations. Physiological and biochemical causes for the change in these parameters are discussed.     

Growth temperature can alter the temperature dependent stimulation of photosynthesis by elevated carbon dioxide in Albutilon theophrasti

Stimulation of photosynthesis in response to elevated carbon dioxide concentration [CO2] in the short-term (min) should be highly temperature dependent at high photon flux. However, it is unclear if long-term (days, weeks) adaptation to a given growth temperature alters the temperature-dependent stimulation of photosynthesis to [CO2]. In velveltleaf (Albutilon theophrasti), the response of photosynthesis, determined as CO2 assimilation, was measured over a range of internal CO2 concentrations at 7 short-term measurement (12, 16, 20, 24, 28, 32, 36 degrees C) temperatures for each of 4 long-term growth (16, 20, 28 and 32 degrees C) temperatures. In vivo estimates of VCmax, the maximum RuBP saturated rate of carboxylation, and Jmax, the light-saturated rate of potential electron transport, were determined from gas exchange measurements for each temperature combination. Overall, previous exposure to a given growth temperature adjusted the optimal temperatures of Jmax and VCmax with subsequently greater enhancement of photosynthesis at elevated [CO2] (i.e., a greater enhancement of photosynthesis at elevated [CO2] was observed at low measurement temperatures for A. theophrasti grown at low growth temperatures compared with higher growth temperatures, and vice versa for plants grown and measured at high temperatures). Previous biochemical based models used to predict the interaction between rising [CO2] and temperature on photosynthesis have generally assumed no growth temperature effect on carboxylation kinetics or no limitation by Jmax. In the current study, these models over predicted the temperature dependence of the photosynthetic response to elevated [CO2] at temperatures above 24 degrees C. If these models are modified to include long-term adjustments of Jmax and VCmax to growth temperature, then greater agreement between observed and predicted values was obtained.     

Anatomical basis of variation in mesophyll resistance in eastern Australian sclerophylls: news of a long and winding path

In sclerophylls, photosynthesis is particularly strongly limited by mesophyll diffusion resistance from substomatal cavities to chloroplasts (r (m)), but the controls on diffusion limits by integral leaf variables such as leaf thickness, density, and dry mass per unit area and by the individual steps along the diffusion pathway are imperfectly understood. To gain insight into the determinants of r (m) in leaves with varying structure, the full CO(2) physical diffusion pathway was analysed in 32 Australian species sampled from sites contrasting in soil nutrients and rainfall, and having leaf structures from mesophytic to strongly sclerophyllous. r (m) was estimated based on combined measurements of gas exchange and chlorophyll fluorescence. In addition, r (m) was modelled on the basis of detailed anatomical measurements to separate the importance of different serial resistances affecting CO(2) diffusion into chloroplasts. The strongest sources of variation in r (m) were S (c)/S, the exposed surface area of chloroplasts per unit leaf area, and mesophyll cell wall thickness, t (cw). The strong correlation of r (m) with t (cw) could not be explained by cell wall thickness alone, and most likely arose from a further effect of cell wall porosity. The CO(2) drawdown from intercellular spaces to chloroplasts was positively correlated with t (cw), suggesting enhanced diffusional limitations in leaves with thicker cell walls. Leaf thickness and density were poorly correlated with S (c)/S, indicating that widely varying combinations of leaf anatomical traits occur at given values of leaf integrated traits, and suggesting that detailed anatomical studies are needed to predict r (m) for any given species.     

叶肉细胞导度研究进展

叶肉细胞导度指叶片叶肉细胞内部的CO2扩散能力,在植物生理生态及全球气候变化和陆地生态系统相互关系的研究中具有重要作用。系统介绍了叶肉细胞导度的发现、发展过程及其研究进展、几种目前国际上常用的叶肉细胞导度测度方法的原理、计算过程;强调了叶肉细胞导度作为光合作用扩散过程一部分的重要意义,明确了叶肉细胞导度的定义及分布范围。并探讨了不同方法的优缺点及注意事项。总结分析了叶肉细胞导度对不同环境因子(温度、水分及环境中CO2和O3浓度等)的响应,从不同角度对叶肉细胞导度的生态学意义进行了简单的概括。对叶肉细胞导度的未来研究进行了展望。     

Effects of HgCl(2) on CO(2) dependence of leaf photosynthesis: evidence indicating involvement of aquaporins in CO(2) diffusion across the plasma membrane

Experiments were conducted to examine whether mercury-sensitive aquaporins facilitate photosynthetic CO(2) diffusion across the plasma membrane of leaf mesophyll cells. Discs without abaxial epidermes from Vicia faba leaflets were treated with HgCl(2), an inhibitor of aquaporins. Hydraulic conductivity of the plasma membrane of these discs, measured as the weight loss of the discs in the 1 M sorbitol solution, was inhibited by sub-mM concentrations of HgCl(2) by 70 to 80%. Photosynthetic CO(2) fixation was also inhibited by the HgCl(2) treatment in a similar concentration range. When 0.3 mM HgCl(2) solution was fed to the V. faba leaflets with intact epidermes via the transpiration stream, the rate of photosynthesis on leaf area basis (A) measured at photosynthetically active photon flux density of 700 micromol m(-2) s(-1) and at leaf temperature of 25 degrees C, decreased by about 20 to 30% at any CO(2) concentration in the intercellular spaces (C(i)). However, when CO(2) concentration in the chloroplast stroma (C(c)) was calculated from fluorescence and gas exchange data and A was plotted against C(c), A at low C(c) concentrations did not differ before and after the treatment. The conductance for CO(2) diffusion from the intercellular spaces to the chloroplast stroma (g(i)) decreased to 40 and 30% of the control value, when the leaflets were fed with 0.3 mM and 1.2 mM HgCl(2), respectively. Similar results were obtained with leaves of Phaseolus vulgaris. Although effects of HgCl(2) were not specific, the present results showed that HgCl(2) consistently lowered g(i). It is, thus, probable that the photosynthetic CO(2) uptake across the plasma membrane of the mesophyll cells is facilitated by mercury-sensitive aquaporins.     

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.     

Determining Photosynthetic Parameters from Leaf CO2 Exchange and Chlorophyll Fluorescence (Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Specificity Factor,Dark Respiration in the Light,Excitation Distribution between Photosystems,Alternative Electron Transport Rate,and Mesophyll Diffusion Resistance

Using simultaneous measurements of leaf gas exchange and chlorophyll fluorescence, we determined the excitation partitioning to photosystem II (PSII), the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase, the dark respiration in the light, and the alternative electron transport rate to acceptors other than bisphosphoglycerate, and the transport resistance for CO2 in the mesophyll cells for individual leaves of herbaceous and tree species. The specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase for CO2 was determined from the slope of the O2 dependence of the CO2 compensation point between 1.5 and 21% O2. Its value, on the basis of dissolved CO2 and O2 concentrations at 25.5[deg]C, varied between 86 and 89. Dark respiration in the light, estimated from the difference between the CO2 compensation point and the CO2 photocompensation point, was about 20 to 50% of the respiration rate in the dark. The excitation distribution to PSII was estimated from the extrapolation of the dependence of the PSII quantum yield on F/Fm to F = 0, where F is steady-state and Fm is pulse-satuarated fluorescence, and varied between 0.45 and 0.6. The alternative electron transport rate was found as the difference between the electron transport rates calculated from fluorescence and from gas exchange, and at low CO2 concentrations and 10 to 21% O2, it was 25 to 30% of the maximum electron transport. The calculated mesophyll diffusion resistance accounted for about 20 to 30% of the total mesophyll resistance, which also includes carboxylation resistance. Whole-leaf photosynthesis is limited by gas phase, mesophyll diffusion, and carboxylation resistances in nearly the same proportion in both herbaceous species and trees.     

植物光合作用模型参数的温度依存性研究进展

综述了植物光合作用与温度响应模型研究的进展,围绕光合作用生化模型的4个主要参数：胞间CO₂浓度、RuBP最大碳同化速率(V_{c max})的活化能、RuBP最大再生速率(J_max)的活化能和J_max/V_{c max},讨论了影响光合作用 温度响应曲线的内在机理.随着生长温度的升高,所有物种的V_{c max}活化能均呈增加趋势,而其他参数的变化因物种不同而存在明显差异,说明V_{c max}的活化能可能是决定光合作用温度依存性的首要参数.最后分析了研究中存在的问题并提出研究展望,认为应整合叶片与群落水平的光合作用模型,从叶面积、太阳辐射、冠层结构、冠层小气候和光合能力等方面研究植物群落对全球变化的响应机理.这对于人们理解和准确估算植物生长、群落碳收支和生态系统初级生产力具有重要意义.     

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.     

Seasonal change in the balance between capacities of RuBP carboxylation and RuBP regeneration affects CO2 response of photosynthesis in Polygonum cuspidatum

The balance between the capacities of RuBP (ribulose-1,5-bisphosphate) carboxylation (V(cmax)) and RuBP regeneration (expressed as the maximum electron transport rate, J(max)) determines the CO(2) dependence of the photosynthetic rate. As it has been suggested that this balance changes depending on the growth temperature, the hypothesis that the seasonal change in air temperature affects the balance and modulates the CO(2) response of photosynthesis was tested. V(cmax) and J(max) were determined in summer and autumn for young and old leaves of Polygonum cuspidatum grown at two CO(2) concentrations (370 and 700 micromol mol(-1)). Elevated CO(2) concentration tended to reduce both V(cmax) and J(max) without changing the J(max):V(cmax) ratio. The seasonal environment, on the other hand, altered the ratio such that the J(max):V(cmax) ratio was higher in autumn leaves than summer leaves. This alternation made the photosynthetic rate more dependent on CO(2) concentration in autumn. Therefore, when photosynthetic rates were compared at growth CO(2) concentration, the stimulation in photosynthetic rate was higher in young-autumn than in young-summer leaves. In old-autumn leaves, the stimulation of photosynthesis brought by a change in the J(max):V(cmax) ratio was partly offset by accelerated leaf senescence under elevated CO(2). Across the two seasons and the two CO(2) concentrations, V(cmax) was strongly correlated with Rubisco and J(max) with cytochrome f content. These results suggest that seasonal change in climate affects the relative amounts of photosynthetic proteins, which in turn affect the CO(2) response of photosynthesis.     

Responses to Doubled CO2 in Medicago sativa: Studies on Ecophysiology and Simulation Modelling

A world-wide spread forage grass, Medicago sativa, was grown in two open-top chambers maintained at either normal (350 μmol · mol-1) or doubled (700 μmol · mol-1) CO2 concentration, from seedling to maturity. During the whole growth season, ecophysiological measurements and observations were conducted over different phenological stages and the main results were as follows: (1) With similar environmental factors, in terms of RH (relative humidity), irradiance, and watering, a slight shift in temperature (about 0.77℃, averaged over the whole growth season) within the chamber maintained at doubled CO2 did not affect instantaneous physiological processes at leaf level, but had some impacts on the effect of doubled CO2 over a long period. (2) Over the whole growth season, physiological variables showed differences between two chambers. The net photosynthesis of plant grown under higher CO2 increased by 18.7 %, while stomatal conductance fell slightly as compared with that of the control. So was the water use efficiency which was also 30.1% higher than the control. Based on the above results from field studies, we concluded that photosynthetically active radiation (PAR) and RH were the main factors affecting photosynthesis and stomatal behavior. Then we combined a widely accepted model of C3 leaf photosynthesis with an empirical model of stomatal conductance and made some modifications according to our experiments. This model was parameterized using field data sets of net CO2 assimilation, stomatal conductance, intercellular CO2 concentration of plants grown at both doubled and control CO2 levels. Variances of main parameters between two treatments reflected some biochemical changes in leaf cell. The maximum efficiency of light energy conversion (α) increased by 22 % and light-saturated rate of electron transport (Jmax) rose by 15 %. The maximum stomatal conductance was slightly reduced by 8 %. The increases in parameters (α and Jmax) indicate accelerated biochemical processes in leaf cell, which means that the photosynthetic capacity of M. sativa may increase at elevated CO2. These results agree well with biochemical measurements at cell level.     

The effect of temperature on C(4)-type leaf photosynthesis parameters

C(4)-type photosynthesis is known to vary with growth and measurement temperatures. In an attempt to quantify its variability with measurement temperature, the photosynthetic parameters - the maximum catalytic rate of the enzyme ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) (V(cmax)), the maximum catalytic rate of the enzyme phosphoenolpyruvate carboxylase (PEPC) (V(pmax)) and the maximum electron transport rate (J(max)) - were examined. Maize plants were grown in climatic-controlled phytotrons, and the curves of net photosynthesis (A(n)) versus intercellular air space CO(2) concentrations (C(i)), and A(n) versus photosynthetic photon flux density (PPFD) were determined over a temperature range of 15-40 degrees C. Values of V(cmax), V(pmax) and J(max) were computed by inversion of the von Caemmerer & Furbank photosynthesis model. Values of V(pmax) and J(max) obtained at 25 degrees C conform to values found in the literature. Parameters for an Arrhenius equation that best fits the calculated values of V(cmax), V(pmax) and J(max) are then proposed. These parameters should be further tested with C(4) plants for validation. Other model key parameters such as the mesophyll cell conductance to CO(2) (g(i)), the bundle sheath cells conductance to CO(2) (g(bs)) and Michaelis-Menten constants for CO(2) and O(2) (K(c), K(p) and K(o)) also vary with temperature and should be better parameterized.     

Determination of mesophyll diffusion resistance in Chamaerion angustifolium by the method of three-dimensional reconstruction of the leaf cell packing

A detailed quantitative analysis of the three-dimensional organization of the mesophyll was performed, and mesophyll diffusion resistance to CO₂ in the leaves of Chamaerion angustifolium formed under different irradiance was calculated using an original method of stereometric cellular packing. For each type of leaves (sun and shade), we determined structural components of gas exchange: the volume of mesophyll per unit leaf area (V _mes), the volume of the intercellular space in the mesophyll (V _is), the area of the total mesophyll surface (S), the area of the free mesophyll surface facing the intercellular spaces (S _mes), and the ratios of the total and the free mesophyll surfaces to its volume (S/V and S _mes/V). As compared with sun leaves, in the shade leaves of Ch. angustifolium, S and V _mes decreased twofold, tissue density was reduced twofold, and the share of the intercellular space in the mesophyll rose from 49 to 72%. In shade, the diffusion resistance of the mesophyll increased by 1.8 times because of changes in the leaf structure. At the same time, the ratio S _mes/V was found to increase by 1.4 times, which facilitated the diffusion of CO₂. In the shade leaves of Ch. angustifolium, the diffusion resistance of the intercellular air spaces was reduced twofold as a result of an increase in their share in the leaf mesophyll and simplification of their geometry. Thus, the method of three-dimensional reconstruction of sun and shade leaves of Ch. angustifolium showed a comprehensive rearrangement of the mesophyll spatial organization in shade and revealed the structural mechanisms of changes in the resistance to CO₂ diffusion within the leaf.     

Seasonal changes in temperature dependence of photosynthetic rate in rice under a free-air CO(2) enrichment

BACKGROUND AND AIMS: Influences of rising global CO(2) concentration and temperature on plant growth and ecosystem function have become major concerns, but how photosynthesis changes with CO(2) and temperature in the field is poorly understood. Therefore, studies were made of the effect of elevated CO(2) on temperature dependence of photosynthetic rates in rice (Oryza sativa) grown in a paddy field, in relation to seasons in two years. METHODS: Photosynthetic rates were determined monthly for rice grown under free-air CO(2) enrichment (FACE) compared to the normal atmosphere (570 vs 370 micromol mol(-1)). Temperature dependence of the maximum rate of RuBP (ribulose-1,5-bisphosphate) carboxylation (V(cmax)) and the maximum rate of electron transport (J(max)) were analysed with the Arrhenius equation. The photosynthesis-temperature response was reconstructed to determine the optimal temperature (T(opt)) that maximizes the photosynthetic rate. KEY RESULTS AND CONCLUSIONS: There was both an increase in the absolute value of the light-saturated photosynthetic rate at growth CO(2) (P(growth)) and an increase in T(opt) for P(growth) caused by elevated CO(2) in FACE conditions. Seasonal decrease in P(growth) was associated with a decrease in nitrogen content per unit leaf area (N(area)) and thus in the maximum rate of electron transport (J(max)) and the maximum rate of RuBP carboxylation (V(cmax)). At ambient CO(2), T(opt) increased with increasing growth temperature due mainly to increasing activation energy of V(cmax). At elevated CO(2), T(opt) did not show a clear seasonal trend. Temperature dependence of photosynthesis was changed by seasonal climate and plant nitrogen status, which differed between ambient and elevated CO(2).     

Relationships between leaf conductance to CO2 diffusion and photosynthesis in micropropagated grapevine plants, before and after ex vitro acclimatization

In vitro-cultured plants typically show a low photosynthetic activity, which is considered detrimental to subsequent ex vitro acclimatization. Studies conducted so far have approached this problem by analysing the biochemical and photochemical aspects of photosynthesis, while very little attention has been paid to the role of leaf conductance to CO(2) diffusion, which often represents an important constraint to CO(2) assimilation in naturally grown plants. Mesophyll conductance, in particular, has never been determined in in vitro plants, and no information exists as to whether it represents a limitation to carbon assimilation during in vitro growth and subsequent ex vitro acclimatization. In this study, by means of simultaneous gas exchange and chlorophyll fluorescence measurements, the stomatal and mesophyll conductance to CO(2) diffusion were assessed in in vitro-cultured plants of the grapevine rootstock '41B' (Vitis vinifera 'Chasselas'xVitis berlandieri), prior to and after ex vitro acclimatization. Their impact on electron transport rate partitioning and on limitation of potential net assimilation rate was analysed. In vitro plants had a high stomatal conductance, 155 versus 50 mmol m(-2) s(-1) in acclimatized plants, which ensured a higher CO(2) concentration in the chloroplasts, and a 7% higher electron flow to the carbon reduction pathway. The high stomatal conductance was counterbalanced by a low mesophyll conductance, 43 versus 285 mmol m(-2) s(-1), which accounted for a 14.5% estimated relative limitation to photosynthesis against 2.1% estimated in acclimatized plants. It was concluded that mesophyll conductance represents an important limitation for in vitro plant photosynthesis, and that in acclimatization studies the correct comparison of photosynthetic activity between in vitro and acclimatized plants must take into account the contribution of both stomatal and mesophyll conductance.     

Estimation of Mesophyll Conductance to CO(2) Flux by Three Different Methods

The resistance to diffusion of CO₂ from the intercellular airspaces within the leaf through the mesophyll to the sites of carboxylation during photosynthesis was measured using three different techniques. The three techniques include a method based on discrimination against the heavy stable isotope of carbon, ¹³C, and two modeling methods. The methods rely upon different assumptions, but the estimates of mesophyll conductance were similar with all three methods. The mesophyll conductance of leaves from a number of species was about 1.4 times the stomatal conductance for CO₂ diffusion determined in unstressed plants at high light. The relatively low CO₂ partial pressure inside chloroplasts of plants with a low mesophyll conductance did not lead to enhanced O₂ sensitivity of photosynthesis because the low conductance caused a significant drop in the chloroplast CO₂ partial pressure upon switching to low O₂. We found no correlation between mesophyll conductance and the ratio of internal leaf area to leaf surface area and only a weak correlation between mesophyll conductance and the proportion of leaf volume occupied by air. Mesophyll conductance was independent of CO₂ and O₂ partial pressure during the measurement, indicating that a true physical parameter, independent of biochemical effects, was being measured. No evidence for CO₂-accumulating mechanisms was found. Some plants, notably Citrus aurantium and Simmondsia chinensis, had very low conductances that limit the rate of photosynthesis these plants can attain at atmospheric CO₂ level.     

Temperature response of mesophyll conductance. Implications for the determination of Rubisco enzyme kinetics and for limitations to photosynthesis in vivo

CO(2) transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g(m)), is finite. Therefore, it will limit photosynthesis when CO(2) is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude of g(m). The process dominating g(m) is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO(2) in water have all been suggested. The response of g(m) to temperature (10 degrees C-40 degrees C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q(10)) of approximately 2.2 for g(m), suggesting control by a protein-facilitated process because the Q(10) for diffusion of CO(2) in water is about 1.25. Further, g(m) values are maximal at 35 degrees C to 37.5 degrees C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g(m) to calculate CO(2) at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10 degrees C to 40 degrees C. Using these parameters, we determined the limitation imposed on photosynthesis by g(m). Despite an exponential rise with temperature, g(m) does not keep pace with increased capacity for CO(2) uptake at the site of Rubisco. The fraction of the total limitations to CO(2) uptake within the leaf attributable to g(m) rose from 0.10 at 10 degrees C to 0.22 at 40 degrees C. This shows that transfer of CO(2) from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature.     

Ternary effects on the gas exchange of isotopologues of carbon dioxide

The ternary effects of transpiration rate on the rate of assimilation of carbon dioxide through stomata, and on the calculation of the intercellular concentration of carbon dioxide, are now included in standard gas exchange studies. However, the equations for carbon isotope discrimination and for the exchange of oxygen isotopologues of carbon dioxide ignore ternary effects. Here we introduce equations to take them into account. The ternary effect is greatest when the leaf-to-air vapour mole fraction difference is greatest, and its impact is greatest on parameters derived by difference, such as the mesophyll resistance to CO(2) assimilation, r(m) . We show that the mesophyll resistance to CO(2) assimilation has been underestimated in the past. The impact is also large when there is a large difference in isotopic composition between the CO(2) inside the leaf and that in the air. We show that this partially reconciles estimates of the oxygen isotopic composition of CO(2) in the chloroplast and mitochondria in the light and in the dark, with values close to equilibrium with the estimated oxygen isotopic composition of water at the sites of evaporation within the leaf.     

Small decreases in SBPase cause a linear decline in the apparent RuBP regeneration rate, but do not affect Rubisco carboxylation capacity

The response of net photosynthetic CO(2) uptake (A) to increasing leaf intercellular CO(2) concentration (c(i)) was determined in antisense Nicotiana tabacum plants, derived from six independent transformation lines, displaying a range of sedoheptulose-1, 7-bisphosphatase (SBPase) activities. The maximum in vivo ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation (V(c,max)) and RuBP regeneration (J(max)) rates were calculated from the steady-state measurements of the A to c(i) response curves. In plants with reductions in SBPase activity of between 9% and 60%, maximum RuBP regeneration capacity declined linearly (r(2)=0.79) and no significant change in apparent in vivo Rubisco activity (V(c,max)) was observed in these plants. No correlation between V(c,max) and a decrease in capacity for RuBP regeneration was observed (r(2)=0.14) in the SBPase antisense plants. These data demonstrate that small decreases in SBPase activity limit photosynthetic carbon assimilation by reducing the capacity for RuBP regeneration.