Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the first reaction in the C 4 photosynthetic pathway, the conversion of atmospheric CO 2 to bicarbonate in the mesophyll cytosol. To examine the importance of the enzyme to the functioning of the C 4 photosynthetic pathway, Flaveria bidentis (L.) Kuntze, a C 4 dicot, was genetically transformed with an antisense construct in which the cDNA encoding a putative cytosolic CA (CA3) was placed under the control of a constitutive promoter. Some of the primary transformants had impaired CO 2 assimilation rates and required high CO 2 for growth. The T 1 progeny of four primary transformants were used to examine the quantitative relationship between leaf CA activity and CO 2 assimilation rate. CA activity was determined in leaf extracts with a mass spectrometric technique that measured the rate of 18 O exchange from doubly labelled 13 C 18 O 2 . Steady-state CO 2 assimilation rates were unaffected by a decrease in CA activity until CA activity was less than 20% of wild type when they decreased steeply. Transformants with less than 10% of wild-type CA activity had very low CO 2 assimilation rates and grew poorly at ambient CO 2 partial pressure. Reduction in CA activity also increased the CO 2 partial pressure required to saturate CO 2 assimilation rates. The present data show that CA activity is essential for the functioning of the C 4 photosynthetic pathway.

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C 3 species, Rubisco operates in a low CO 2 environment, which is suboptimal for both catalysis and carbamylation. In C 4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO 2 atmosphere close to saturation. To explore the role of Rubisco activase in C 4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C 4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T 1 progeny required high CO 2 (.1 kPa) for growth. They had very low CO 2 assimilation rates at high light and ambient CO 2 , and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO 2 . The amount of Rubisco was similar to that of wild-type plants. Experiments with the T 1 progeny of these four primary transformants showed that CO 2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C 4 photosynthetic pathway.The C 4 photosynthetic pathway is a biochemical CO 2 -concentrating mechanism that provides elevated CO 2 partial pressure (pCO 2 ) at the site of Rubisco carboxylation in the bundle sheath. This suppresses photorespiration and allows Rubisco (EC 4.1.1.39) to operate close to its maximal rate, such that CO 2 assimilation in C 4 plants is effectively CO 2 saturated in air (Hatch, 1987). Similar to photosynthesis in C 3 species, there is a strong correlation between Rubisco content of leaves and CO 2 assimilation rate at high irradiance, demonstrating that Rubisco carboxylation is among the rate-limiting steps of C 4 photosynthesis (Usuda et al., 1984;Hunt et al., 1985;Sage et al., 1987;Furbank et al., 1996;von Caemmerer et al., 1997). For CO 2 fixation to take place, Rubisco's catalytic sites must first be activated. This requires the carbamylation of a Lys residue within Rubisco's catalytic sites to allow the binding of a Mg 21 (for review, see Andrews and Lorimer, 1987). Sugar phosphates can also bind to Rubisco catalytic sites, and this interferes with both the process of carbamylation and full activity of the carbamylated enzyme (Badger and Lorimer, 1981;Edmondson et al., 1990a;Zhu and Jensen, 1991). In vivo, the activation and maintenance of Rubisco activity are facilitated by a second protein called Rubisco activase. Activase was first identified by analysis of the rca mutant of Arabidopsis (Arabidopsis thaliana), which required high CO 2 fo...