Rubisco is a large enzyme with a molecular mass of approximately 550 kD. The maximum rate of CO 2 fixation (i.e. ribulose-1,5-bisphosphate [RuBP] carboxylation) at CO 2 saturation is only 15 to 30 mol CO 2 mol 21 Rubisco protein s 21 at 25°C. Affinity to CO 2 is also low, and the K m , K c , at 25°C in the absence of oxygen is comparable to the CO 2 concentration in water equilibrated with air containing 39 Pa CO 2 (approximately 390 mL L 21 ), 13 mM. Moreover, RuBP carboxylation is competitively inhibited by RuBP oxygenation, which is the primary step of the energy-wasting process, photorespiration. If the CO 2 concentration in the chloroplast stroma is low, the carboxylation rate will decrease while the oxygenation rate will increase. Under such conditions, light energy and other resources, including nitrogen and water, are all wasted, eventually leading to a decrement of fitness of the plants. From these data, we may consider that structural features of the leaf contributing to the maintenance of the high CO 2 concentration in the chloroplast stroma may have been selected during evolution.In this Update, we focus on the key structural features that affect CO 2 concentration in the chloroplast stroma. First, we analyze the conductance for CO 2 diffusion from the substomatal cavity to the chloroplast stroma (mesophyll conductance [g m ], also called internal conductance). Because the low g m limits photosynthesis,

The CO2 concentration at the site of carboxylation inside the chloroplast stroma depends not only on the stomatal conductance, but also on the conductance of CO2 between substomatal cavities and the site of CO2 fixation. This conductance, commonly termed mesophyll conductance (gm), significantly constrains the rate of photosynthesis. Here we show that estimates of gm are influenced by the amount of respiratory and photorespiratory CO2 from the mitochondria diffusing towards the chloroplasts. This results in an apparent CO2 and oxygen sensitivity of gm that does not imply a change in intrinsic diffusion properties of the mesophyll, but depends on the ratio of mitochondrial CO2 release to chloroplast CO2 uptake. We show that this effect (1) can bias the estimation of the CO2 photocompensation point and non-photorespiratory respiration in the light; (2) can affect the estimates of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) kinetic constants in vivo; and (3) results in an apparent obligatory correlation between stomatal conductance and gm. We further show that the amount of photo(respiratory) CO2 that is refixed by Rubisco can be directly estimated through measurements of gm.

Photosynthesis is limited by the conductance of carbon dioxide (CO 2 ) from intercellular spaces to the sites of carboxylation. Although the concept of internal conductance (g i ) has been known for over 50 years, shortcomings in the theoretical description of this process may have resulted in a limited understanding of the underlying mechanisms. To tackle this issue, we developed a three-dimensional reaction-diffusion model of photosynthesis in a typical C 3 mesophyll cell that includes all major components of the CO 2 diffusion pathway and associated reactions. Using this novel systems model, we systematically and quantitatively examined the mechanisms underlying g i . Our results identify the resistances of the cell wall and chloroplast envelope as the most significant limitations to photosynthesis. In addition, the concentration of carbonic anhydrase in the stroma may also be limiting for the photosynthetic rate. Our analysis demonstrated that higher levels of photorespiration increase the apparent resistance to CO 2 diffusion, an effect that has thus far been ignored when determining g i . Finally, we show that outward bicarbonate leakage through the chloroplast envelope could contribute to the observed decrease in g i under elevated CO 2 . Our analysis suggests that physiological and anatomical features associated with g i have been evolutionarily fine-tuned to benefit CO 2 diffusion and photosynthesis. The model presented here provides a novel theoretical framework to further analyze the mechanisms underlying diffusion processes in the mesophyll.

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The relationship between chloroplast arrangement and diffusion of CO2 from substomatal cavities to the chloroplast stroma was investigated in Arabidopsis thaliana. Chloroplast position was manipulated by varying the amount of blue light and by cytochalasin D (CytD) treatment. We also investigated two chloroplast positioning mutants. Chloroplast arrangement was assessed by the surface area of chloroplasts adjacent to intercellular airspaces (Sc). Although it has been previously shown that long-term acclimation to high light is linked with a large Sc, we found that the shortterm chloroplast avoidance response reduces Sc. This effect was not apparent in the blue-light-insensitive phot2 mutant, which did not show the avoidance response. As expected, the smaller Sc induced by the avoidance response was coupled to a similar decrease in internal conductance. This reduction in internal conductance resulted in an increased limitation of the rate of photosynthesis. The limiting effect of Sc on internal conductance and photosynthesis was also shown in chup1, a mutant with a constant small Sc as the result of an unusual chloroplast arrangement. We conclude that chloroplast movements in A. thaliana can rapidly alter leaf morphological parameters, and this has significant consequences for the diffusion of CO2 through the mesophyll.

Maintaining high rates of photosynthesis in leaves requires efficient movement of CO 2 from the atmosphere to the mesophyll cells inside the leaf where CO 2 is converted into sugar. CO 2 diffusion inside the leaf depends directly on the structure of the mesophyll cells and their surrounding airspace, which have been difficult to characterize because of their inherently three-dimensional organization. Yet faster CO 2 diffusion inside the leaf was probably critical in elevating rates of photosynthesis that occurred among angiosperm lineages. Here we characterize the three-dimensional surface area of the leaf mesophyll across vascular plants. We show that genome size determines the sizes and packing densities of cells in all leaf tissues and that smaller cells enable more mesophyll surface area to be packed into the leaf volume, facilitating higher CO 2 diffusion. Measurements and modelling revealed that the spongy mesophyll layer better facilitates gaseous phase diffusion while the palisade mesophyll layer better facilitates liquid-phase diffusion. Our results demonstrate that genome downsizing among the angiosperms was critical to restructuring the entire pathway of CO 2 diffusion into and through the leaf, maintaining high rates of CO 2 supply to the leaf mesophyll despite declining atmospheric CO 2 levels during the Cretaceous.

Bypassing the photorespiratory pathway is regarded as a way to increase carbon assimilation and, correspondingly, biomass production in C 3 crops. Here, the benefits of three published photorespiratory bypass strategies are systemically explored using a systems-modeling approach. Our analysis shows that full decarboxylation of glycolate during photorespiration would decrease photosynthesis, because a large amount of the released CO 2 escapes back to the atmosphere. Furthermore, we show that photosynthesis can be enhanced by lowering the energy demands of photorespiration and by relocating photorespiratory CO 2 release into the chloroplasts. The conductance of the chloroplast membranes to CO 2 is a key feature determining the benefit of the relocation of photorespiratory CO 2 release. Although our results indicate that the benefit of photorespiratory bypasses can be improved by increasing sedoheptulose bisphosphatase activity and/or increasing the flux through the bypass, the effectiveness of such approaches depends on the complex regulation between photorespiration and other metabolic pathways.

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