Estimation of diffusive resistance of bundle sheath cells to CO2 from modeling of C4 photosynthesis

This account is focused upon the early part of my career in order to illuminate the logistics and the culture of our science in the period 1936 to 1949. A roundabout path took me from a farm in Pennsylvania to a PhD under George Burr at Minnesota in 1939. In studying the photosynthetic competence of chlorophyll formed by the green alga Chlorella in darkness, I stumbled upon the phenomenon of photoinhibition. In a two-year postdoctorate at the Smithsonian Institution, I worked under E.D. McAlister. Our major accomplishment was in making simultaneous recordings of fluorescence and CO₂ uptake during the induction period. Variations in photosynthetic behavior of Chlorella led to a study of culture conditions and a recognition of the changing conditions which occur in batch cultures. A continuous culture apparatus (turbidostat) was developed as a means of attaining steady-state growth and production of uniform experimental material. I exploited the device in work at my first (and only) position at The University of Texas in 1941 and subsequent years. Study of the CO₂/O₂ gas exchange ratio led to the recognition of the important role of nitrate in the photosynthetic metabolism of algae. The account ends with the 1949 American Association for the Advancement of Science symposium.Invited and edited by Govindjee     

Characterisation of seeds of a C4 phosphoenolpyruvate carboxylase-deficient mutant of Amaranthus edulis

Seeds from the C(4) plant Amaranthus edulis were studied as part of the characterisation of a mutant (designated LaC(4) 2.16), which contains reduced amounts (5% of wild type) of the photosynthetic leaf form of phosphoenolpyruvate carboxylase (PEPC). On a per seed basis, the amount of PEPC activity was not significantly altered, while the weight and protein content of the mutant seeds were 34% lower than that of the wild type. Western gel blot analysis detected two PEPC polypeptides with molecular masses of 105 kDa (minor) and 100 kDa (major). The determination of in vitro phosphorylation in reconstituted assays revealed the presence of both calcium-dependent and calcium-independent PEPC-kinase activities in protein extracts of wild-type and mutant seeds. However, PEPC proteins were phosphorylated in dry seeds, and PEPC phosphorylation did not occur in vivo during seed imbibition in the presence of (32) P-phosphate. In contrast, (32) P-phosphate was incorporated into a range of proteins in wild-type seeds, but not in mutant seeds. In addition, ATP content was much reduced in germinating mutant seeds and this did not increase following the supply of phosphate. Collectively, these data suggest that the deficiency in C(4) PEPC in mutant A. edulis leaves has no effect on C(3) -type PEPC content and phosphorylation state in seeds, but causes impairment of energy production, thereby accounting for the reduced germination of the mutant.     

C3 plants enhance rates of photosynthesis by reassimilating photorespired and respired CO2

Photosynthetic carbon gain in plants using the C₃ photosynthetic pathway is substantially inhibited by photorespiration in warm environments, particularly in atmospheres with low CO₂ concentrations. Unlike C₄ plants, C₃ plants are thought to lack any mechanism to compensate for the loss of photosynthetic productivity caused by photorespiration. Here, for the first time, we demonstrate that the C₃ plants rice and wheat employ a specific mechanism to trap and reassimilate photorespired CO₂. A continuous layer of chloroplasts covering the portion of the mesophyll cell periphery that is exposed to the intercellular air space creates a diffusion barrier for CO₂ exiting the cell. This facilitates the capture and reassimilation of photorespired CO₂ in the chloroplast stroma. In both species, 24–38% of photorespired and respired CO₂ were reassimilated within the cell, thereby boosting photosynthesis by 8–11% at ambient atmospheric CO₂ concentration and 17–33% at a CO₂ concentration of 200 µmol mol^?1. Widespread use of this mechanism in tropical and subtropical C₃ plants could explain why the diversity of the world's C₃ flora, and dominance of terrestrial net primary productivity, was maintained during the Pleistocene, when atmospheric CO₂ concentrations fell below 200 µmol mol^?1.     

Bundle sheath leakiness and light limitation during C4 leaf and canopy CO2 uptake

Perennial species with the C(4) pathway hold promise for biomass-based energy sources. We have explored the extent that CO(2) uptake of such species may be limited by light in a temperate climate. One energetic cost of the C(4) pathway is the leakiness () of bundle sheath tissues, whereby a variable proportion of the CO(2), concentrated in bundle sheath cells, retrodiffuses back to the mesophyll. In this study, we scale from leaf to canopy level of a Miscanthus crop (Miscanthus x giganteus hybrid) under field conditions and model the likely limitations to CO(2) fixation. At the leaf level, measurements of photosynthesis coupled to online carbon isotope discrimination showed that leaves within a 3.3-m canopy (leaf area index = 8.3) show a progressive increase in both carbon isotope discrimination and as light decreases. A similar increase was observed at the ecosystem scale when we used eddy covariance net ecosystem CO(2) fluxes, together with isotopic profiles, to partition photosynthetic and respiratory isotopic flux densities (isofluxes) and derive canopy carbon isotope discrimination as an integrated proxy for at the canopy level. Modeled values of canopy CO(2) fixation using leaf-level measurements of suggest that around 32% of potential photosynthetic carbon gain is lost due to light limitation, whereas using determined independently from isofluxes at the canopy level the reduction in canopy CO(2) uptake is estimated at 14%. Based on these results, we identify as an important limitation to CO(2) uptake of crops with the C(4) pathway.     

The isolation and characterisation of a mutant of the C4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C₄ leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F₂ plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO₂ assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C₄ enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C₄ photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME. Received: 2 April 1998 / Accepted: 7 May 1998     

Control of photosynthesis by the carbohydrate level in leaves of the C4 plant Amaranthus edulis L.

Photosynthesis was studied in relation to the carbohydrate status in intact leaves of the C₄ plant Amaranthus edulis. The rate of leaf net CO₂ assimilation, stomatal conductance and intercellular partial pressure of CO₂ remained constant or showed little decline towards the end of an 8-h period of illumination in ambient air (340 bar CO₂, 21% O₂). When sucrose export from the leaf was inhibited by applying a 4-h cold-block treatment (1°C) to the petiole, the rate of photosynthesis rapidly decreased with time. After the removal of the cold block from the petiole, further reduction in photosynthetic rate occurred, and there was no recovery in the subsequent light period. Although stomatal conductance declined with time, intercellular CO₂ partial pressure remained relatively constant, indicating that the inhibition of photosynthesis was not primarily caused by changes in stomatal aperture. Analysis of the leaf carbohydrate status showed a five- to sixfold increase in the soluble sugar fraction (mainly sucrose) in comparison with the untreated controls, whereas the starch content was the same. Leaf osmotic potential increased significantly with the accumulation of soluble sugars upon petiole chilling, and leaf water potential became slightly more negative. After 14 h recovery in the dark, photosynthesis returned to its initial maximum value within 1 h of illumination, and this was associated with a decline in leaf carbohydrate levels overnight. These data show that, in Amaranthus edulis, depression in photosynthesis when translocation is impaired is closely related to the accumulation of soluble sugars (sucrose) in source leaves, indicating feedback control of C₄ photosynthesis. Possible mechanisms by which sucrose accumulation in the leaf may affect the rate of photosynthesis are discussed with regard to the leaf anatomy of C₄ plants.Abbreviations and symbols A net CO₂ assimilation rate - Ci intercellular CO₂ partial pressure - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate - water potential - osmotic pressure     

CO(2) Concentrating Mechanism of C(4) Photosynthesis: Permeability of Isolated Bundle Sheath Cells to Inorganic Carbon

Diffusion of inorganic carbon into isolated bundle sheath cells from a variety of C₄ species was characterized by coupling inward diffusion of CO₂ to photosynthetic carbon assimilation. The average permeability coefficient for CO₂ (P_CO2) for five representatives from the three decarboxylation types was approximately 20 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. The average value for the NAD-ME species Panicum miliaceum (10 determinations) was 26 with a standard deviation of 6 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. A P_CO2 of at least 500 micromoles per minute per milligram chlorophyll per millimolar was determined for cells isolated from the C₃ plant Xanthium strumarium. It is concluded that bundle sheath cells are one to two orders of magnitude less permeable to CO₂ than C₃ photosynthetic cells. These data also suggest that CO₂ diffusion in bundle sheath cells may be made up of two components, one involving an apoplastic path and the other a symplastic (plasmodesmatal) path, each contributing approximately equally.     

C4 photosynthesis, atmospheric CO2, and climate

The objectives of this synthesis are (1) to review the factors that influence the ecological, geographical, and palaeoecological distributions of plants possessing C₄ photosynthesis and (2) to propose a hypothesis/model to explain both the distribution of C₄ plants with respect to temperature and CO₂ and why C₄ photosynthesis is relatively uncommon in dicotyledonous plants (hereafter dicots), especially in comparison with its widespread distribution in monocotyledonous species (hereafter monocots). Our goal is to stimulate discussion of the factors controlling distributions of C₄ plants today, historically, and under future elevated CO₂ environments. Understanding the distributions of C₃/C₄ plants impacts not only primary productivity, but also the distribution, evolution, and migration of both invertebrates and vertebrates that graze on these plants. Sixteen separate studies all indicate that the current distributions of C₄ monocots are tightly correlated with temperature: elevated temperatures during the growing season favor C₄ monocots. In contrast, the seven studies on C₄ dicot distributions suggest that a different environmental parameter, such as aridity (combination of temperature and evaporative potential), more closely describes their distributions. Differences in the temperature dependence of the quantum yield for CO₂ uptake (light-use efficiency) of C₃ and C₄ species relate well to observed plant distributions and light-use efficiency is the only mechanism that has been proposed to explain distributional differences in C₃/C₄ monocots. Modeling of C₃ and C₄ light-use efficiencies under different combinations of atmospheric CO₂ and temperature predicts that C₄-dominated ecosystems should not have expanded until atmospheric CO₂ concentrations reached the lower levels that are thought to have existed beginning near the end of the Miocene. At that time, palaeocarbonate and fossil data indicate a simultaneous, global expansion of C₄-dominated grasslands. The C₄ monocots generally have a higher quantum yield than C₄ dicots and it is proposed that leaf venation patterns play a role in increasing the light-use efficiency of most C₄ monocots. The reduced quantum yield of most C₄ dicots is consistent with their rarity, and it is suggested that C₄ dicots may not have been selected until CO₂ concentrations reached their lowest levels during glacial maxima in the Quaternary. Given the intrinsic light-use efficiency advantage of C₄ monocots, C₄ dicots may have been limited in their distributions to the warmest ecosystems, saline ecosystems, and/or to highly disturbed ecosystems. All C₄ plants have a significant advantage over C₃ plants under low atmospheric CO₂ conditions and are predicted to have expanded significantly on a global scale during full-glacial periods, especially in tropical regions. Bog and lake sediment cores as well as pedogenic carbonates support the hypothesis that C₄ ecosystems were more extensive during the last glacial maximum and then decreased in abundance following deglaciation as atmospheric CO₂ levels increased. Received: 12 February 1997 / Accepted: 20 June 1997     

Oligocene CO2 decline promoted C4 photosynthesis in grasses

C4 photosynthesis is an adaptation derived from the more common C3 photosynthetic pathway that confers a higher productivity under warm temperature and low atmospheric CO2 concentration [1, 2]. C4 evolution has been seen as a consequence of past atmospheric CO2 decline, such as the abrupt CO2 fall 32-25 million years ago (Mya) [3-6]. This relationship has never been tested rigorously, mainly because of a lack of accurate estimates of divergence times for the different C4 lineages [3]. In this study, we inferred a large phylogenetic tree for the grass family and estimated, through Bayesian molecular dating, the ages of the 17 to 18 independent grass C4 lineages. The first transition from C3 to C4 photosynthesis occurred in the Chloridoideae subfamily, 32.0-25.0 Mya. The link between CO2 decrease and transition to C4 photosynthesis was tested by a novel maximum likelihood approach. We showed that the model incorporating the atmospheric CO2 levels was significantly better than the null model, supporting the importance of CO2 decline on C4 photosynthesis evolvability. This finding is relevant for understanding the origin of C4 photosynthesis in grasses, which is one of the most successful ecological and evolutionary innovations in plant history.     

Oxygen and electron flow in C4 photosynthesis: Mehler reaction, photorespiration and CO2 concentration in the bundle sheath

The photosynthetic linear electron transport rate in excess of that used for CO₂ reduction was evaluated in Sorghum bicolor Moench. [NADP-malic enzyme (ME)-type C₄ plant], Amaranthus cruentus L. (NAD-ME-type C₄ plant) and Helianthus annuus L. (C₃ plant) leaves at different CO₂ and O₂ concentrations. The electron transport rate (J _F) was calculated from fluorescence using the light partitioning factor (relative PSII cross-section) determined under conditions where excess electron transport was assumed to be negligible: low light intensities, 500 μmol CO₂ · mol⁻¹ and 2% O₂. Under high light intensities there was a large excess of J _F/4 at 10–100% O₂ in the C₃ plant due to photorespiration, but very little in sorghum and somewhat more in amaranth, showing that photorespiration is suppressed, more in the NADP-ME- and less in the NAD-ME-type species. It is concluded that when C₄ photosynthesis is limited by supply of atmospheric CO₂ to the C₄ cycle, the C₃ cycle becomes limited by regeneration of ribulose 1,5-bisphosphate (RuBP) which in turn limits RuBP oxygenase activity and photorespiration. The rate of excess electron transport over that consumed for CO₂ fixation in C₄ plants was very sensitive to the presence of O₂ in the gas phase, rapidly increasing between 0.01 and 0.1% O₂, and at 2% O₂ it was about two-thirds of that at 21% O₂. This shows the importance of the Mehler O₂ reduction as an electron sink, compared with photorespiration in C₄ plants. However, the rate of the Mehler reaction is still too low to fully account for the extra ATP which is needed in C₄ photosynthesis. Received: 8 November 1997 / Accepted: 26 December 1997     

C(4) photosynthesis: principles of CO(2) concentration and prospects for its introduction into C(3) plants

C(4) photosynthesis has a number of distinct properties that enable the capture of CO(2) and its concentration in the vicinity of Rubisco, so as to reduce the oxygenase activity of Rubisco, and hence the rate of photorespiration. The aim of this review is to discuss the properties of this CO(2)-concentrating mechanism, and thus to indicate the minimum requirements of any genetically-engineered system. In particular, the Kranz leaf anatomy of C(4) photosynthesis and the division of the C(4)-cycle between two cell types involves intercellular co-operation that requires modifications in regulation and transport to make C(4) photosynthesis work. Some examples of these modifications are discussed. Comparisons are made with the C(4)-type photosynthesis found in single-celled C(4)-type CO(2)-concentrating mechanisms, such as that found in the aquatic plant, Hydrilla verticillata and the single-celled C(4) system found in the terrestrial chenopod Borszczowia aralocaspica. The outcome of recent attempts to engineer C(4) enzymes into C(3) plants is discussed.     

Development of C4 photosynthesis in sorghum leaves grown under free-air CO2 enrichment (FACE)

The developmental pattern of C4 expression has been well characterized in maize and other C4 plants. However, few reports have explored the possibility that the development of this pathway may be sensitive to changes in atmospheric CO2 concentrations. Therefore, both the structural and biochemical development of leaf tissue in the fifth leaf of Sorghum bicolor plants grown at elevated CO2 have been characterized. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) activities accumulate rapidly as the leaf tissue differentiates and emerges from the surrounding whorl. Rubisco was not expressed in a cell-specific manner in the youngest tissue at the base of the leaf, but did accumulate before PEPC was detected. This suggests that the youngest leaf tissue utilizes a C3-like pathway for carbon fixation. However, this tissue was in a region of the leaf receiving very low light and so significant rates of photosynthesis were not likely. Older leaf tissue that had emerged from the surrounding whorl into full sunlight showed the normal C4 syndrome. Elevated CO2 had no effect on the cell-specific localization of Rubisco or PEPC at any stage of leaf development, and the relative ratios of Rubisco to PEPC remained constant during leaf development. However, in the oldest tissue at the tip of the leaf, the total activities of Rubisco and PEPC were decreased under elevated CO2 implying that C4 photosynthetic tissue may acclimate to growth under elevated CO2.     

Inorganic Carbon Diffusion between C(4) Mesophyll and Bundle Sheath Cells: Direct Bundle Sheath CO(2) Assimilation in Intact Leaves in the Presence of an Inhibitor of the C(4) Pathway

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO₂ assimilation or oxygen evolution, over a wide range of CO₂ concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO₂ near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO₂ concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO₃⁻, indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of ¹⁴C-incorporation from ¹⁴CO₂ into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO₂ occurs predominantly by direct CO₂ fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO₂ concentrations, permeability coefficients for CO₂ flux into bundle sheath cells were determined for a range of C₄ species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C₃ plants. These results support the concept that a CO₂ permeability barrier exists to allow the development of high CO₂ concentrations in bundle sheath cells during C₄ photosynthesis.     

Mehler reaction plays a role in C3 and C4 photosynthesis under shade and low CO2

Photosynthesis Research - Alternative electron fluxes such as the cyclic electron flux (CEF) around photosystem I (PSI) and Mehler reaction (Me) are essential for efficient photosynthesis because...     

The efficiency of C(4) photosynthesis under low light conditions: assumptions and calculations with CO(2) isotope discrimination

Leakiness (Φ), the proportion of carbon fixed by phosphoenolpyruvate carboxylation that leaks out of the bundle-sheath cells, determines C(4) photosynthetic efficiency. Large increases in Φ have been described at low irradiance. The underlying mechanisms for this increase remain uncertain, but changes in photorespiration or the energy partitioning between the C(4) and C(3) cycles have been suggested. Additionally, values of Φ at low light could be magnified from assumptions made when comparing measured photosynthetic discrimination against (13)C (Δ) with the theoretical formulation for Δ. For example, several simplifications are often made when modelling Δ to predict Φ including: (i) negligible fractionation during photorespiration and dark respiration; (ii) infinite mesophyll conductance; and (iii) CO(2) inside bundle-sheath cells (C(s)) is much larger than values in mesophyll cells (C(m)). Theoretical models for C(4) photosynthesis and C(4) Δ were combined to evaluate how these simplifications affect calculations of Δ and Φ at different light intensities. It was demonstrated that the effects of photorespiratory fractionations and mesophyll conductance were negligible at low light. Respiratory fractionation was relevant only when the magnitude of the fractionation factor was artificially increased during measurements. The largest error in estimating Φ occurred when assuming C(s) was much larger than C(m) at low light levels, when bundle-sheath conductance was large (g(s)), or at low O(2) concentrations. Under these conditions, the simplified equation for Δ overestimated Φ, and compromised comparisons between species with different g(s), and comparisons across O(2) concentrations.     

The temperature response of C(3) and C(4) photosynthesis

We review the current understanding of the temperature responses of C(3) and C(4) photosynthesis across thermal ranges that do not harm the photosynthetic apparatus. In C(3) species, photosynthesis is classically considered to be limited by the capacities of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), ribulose bisphosphate (RuBP) regeneration or P(i) regeneration. Using both theoretical and empirical evidence, we describe the temperature response of instantaneous net CO(2) assimilation rate (A) in terms of these limitations, and evaluate possible limitations on A at elevated temperatures arising from heat-induced lability of Rubisco activase. In C(3) plants, Rubisco capacity is the predominant limitation on A across a wide range of temperatures at low CO(2) (<300 microbar), while at elevated CO(2), the limitation shifts to P(i) regeneration capacity at suboptimal temperatures, and either electron transport capacity or Rubisco activase capacity at supraoptimal temperatures. In C(4) plants, Rubisco capacity limits A below 20 degrees C in chilling-tolerant species, but the control over A at elevated temperature remains uncertain. Acclimation of C(3) photosynthesis to suboptimal growth temperature is commonly associated with a disproportional enhancement of the P(i) regeneration capacity. Above the thermal optimum, acclimation of A to increasing growth temperature is associated with increased electron transport capacity and/or greater heat stability of Rubisco activase. In many C(4) species from warm habitats, acclimation to cooler growth conditions increases levels of Rubisco and C(4) cycle enzymes which then enhance A below the thermal optimum. By contrast, few C(4) species adapted to cooler habitats increase Rubisco content during acclimation to reduced growth temperature; as a result, A changes little at suboptimal temperatures. Global change is likely to cause a widespread shift in patterns of photosynthetic limitation in higher plants. Limitations in electron transport and Rubisco activase capacity should be more common in the warmer, high CO(2) conditions expected by the end of the century.     

Synthesis and characterisation of the carboxylate linked osmium clusters [{Os3H(CO)10}2(CO2CH2CO2)], [{Os3H(CO)10}2(CO2C2H4CO2)], [{Os3H(CO)10}2(C4O4)] and [{Os3H(CO)10}2(C4O4){Co2(CO)6}]

Reactions between the activated cluster [Os₃(CO)₁₀(NCMe)₂] and malonic acid, succinic acid and dicarboxylic acetylene, respectively, lead to the formation of the linked cluster complexes [{Os₃H(CO)₁₀}₂(CO₂CH₂CO₂)] (1), [{Os₃H(CO)₁₀}₂(CO₂C₂H₄CO₂)] (2), and [{Os₃H(CO)₁₀}₂(C₄O₄)] (3) in good yield. Cluster 3 was subsequently treated with [Co₂(CO)₈] and this results in the addition of a “Co₂(CO)₆” group giving [{Os₃H(CO)₁₀}₂(C₂O₄){Co₂(CO)₆}] (4). The X-ray crystal structures are reported for 2–4. In each structure the two triangular triosmium units are linked by the carboxylate groups and within each complex the carboxylate groups are chelating and bridge two osmium atoms.     

CO2 efflux, CO2 concentration and photosynthetic refixation in stems of Eucalyptus globulus (Labill.)

In spite of the importance of respiration in forest carbon budgets,the mechanisms by which physiological factors control stem respirationare unclear. An experiment was set up in a Eucalyptus globulusplantation in central Portugal with monoculture stands of 5-year-oldand 10-year-old trees. CO₂ efflux from stems under shaded andunshaded conditions, as well as the concentration of CO₂ dissolvedin sap [CO₂^*], stem temperature, and sap flow were measuredwith the objective of improving our understanding of the factorscontrolling CO₂ release from stems of E. globulus. CO₂ effluxwas consistently higher in 5-year-old, compared with 10-year-old,stems, averaging 3.4 versus 1.3 µmol m^–2 s^–1,respectively. Temperature and [CO₂^*] both had important, andsimilar, influences on the rate of CO₂ efflux from the stems,but neither explained the difference in the magnitude of CO₂efflux between trees of different age and size. No relationshipwas found between efflux and sap flow, and efflux was independentof tree volume, suggesting the presence of substantial barriersto the diffusion of CO₂ from the xylem to the atmosphere inthis species. The rate of corticular photosynthesis was thesame in trees of both ages and only reduced CO₂ efflux by 7%,probably due to the low irradiance at the stem surface belowthe canopy. The younger trees were growing at a much fasterrate than the older trees. The difference between CO₂ effluxfrom the younger and older stems appears to have resulted froma difference in growth respiration rather than a differencein the rate of diffusion of xylem-transported CO₂. Key words: Eucalyptus globulus, refixation, stem respiration Received 19 May 2008; Revised 14 September 2008 Accepted 8 October 2008