Reductions of Rubisco activase by antisense RNA in the C4 plant Flaveria bidentis reduces Rubisco carbamylation and leaf photosynthesis

Abstract

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 C3 species, Rubisco operates in a low CO2 environment, which is suboptimal for both catalysis and carbamylation. In C4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO2 atmosphere close to saturation. To explore the role of Rubisco activase in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 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 T1 progeny required high CO2 (>1 kPa) for growth. They had very low CO2 assimilation rates at high light and ambient CO2, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO2. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T1 progeny of these four primary transformants showed that CO2 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 C4 photosynthetic pathway.

Figure 1.

CO₂ assimilation rates of wild type (black columns) and a number of kanamycin-resistant primary transformants (white columns). Gas exchange measurements were made at an ambient pCO₂ of 40 Pa, irradiance of 1,500 μmol m⁻² s⁻¹, and leaf temperature of 25°C. Activase amounts of primary transformants A, SS, Q, RR, and Aa are shown in Figure 2.

Figure 2.

Immunodetection of Rubisco activase in leaf extracts of F. bidentis wild type (Wt) and primary transformants A, SS, Q, RR, and Aa and of Arabidopsis separated by SDS-Page. Samples were loaded on gels on an equal-leaf area basis, and Rubisco activase polypeptides were detected with a reduced glutathione S-transferase/spinach activase fusion protein antiserum followed by a horseradish peroxidase conjugated secondary antibody and chemiluminescence.

Figure 3.

Rubisco carbamylation and Rubisco catalytic site content of leaves of selected primary transformants and wild-type plants. Leaf samples were collected in the growth cabinet under growth conditions of 2.5 kPa pCO₂ at an irradiance of 500 μmol quanta m⁻² s⁻¹ at 25°C.

Figure 4.

CO₂ assimilation rate as a function of intercellular pCO₂ measured on leaves of selected primary transformants (black symbols) and a wild-type plant (white symbols). Measurements were made at an irradiance of 1,500 μmol quanta m⁻² s⁻¹ and a leaf temperature of 25°C.

Figure 5.

CO₂ assimilation rate as a function of Rubisco activase content. Measurements were made on wild-type leaves (white symbols) and leaves of plants of the T₁ generation of primary transformants A, SS, Q, and RR (black symbols). Gas exchange measurements were made at an ambient pCO₂ of 38 Pa, irradiance of 1,500 μmol m⁻² s⁻¹, and leaf temperature 25°C. Activase content of leaves gas exchange measurements were made on was quantified by immunodetection as a percentage of wild-type levels. Further details are given in “Materials and Methods.”

Figure 6.

Rubisco carbamylation (A) and Rubisco content (B) as a function of Rubisco activase content in leaves of wild-type plants (white symbols) and leaves of plants of the T₁ generation of primary transformants A, SS, Q, and RR (black symbols). Triangles depict plants grown at 1 kPa pCO₂ and circles depict plants grown at 2.5 kPa pCO₂. For plants grown at 1 kPa pCO₂, leaf discs were sampled in the growth cabinet at approximately 40 pCO₂ and irradiance of 1,000 μmol m⁻² s⁻¹ and 30°C the day after gas exchange measurements. For plants grown at 2.5 kPa pCO₂, leaf discs were sampled immediately after gas exchange from the area had been measured at 38 Pa pCO₂, 1,500 μmol quanta m⁻² s⁻¹, and leaf temperature of 25°C.

Figure 7.

In vivo catalytic turnover rate of Rubisco, K_cat at 25°C, for F. bidentis leaves with different activase content. K_cat was calculated from gross CO₂ assimilation rates measured as described in Figure 5, and Rubisco carbamylated site content for leaves where leaf discs were sampled directly after gas exchange measurements (see Fig. 6 legend). Symbols are defined in Figure 6 legend. Average dark respiration rates measured were 2 μmol m⁻² s⁻¹, and this value was added to net CO₂ assimilation rate to obtain gross CO₂ assimilation rate.

Figure 8.

Modeled carbamylation status of Rubisco as a function of pCO₂ at RuBP saturation and different ratios of K_f/K_r′ using the mathematical model of activase function developed by Mate et al. (1996). The model assumes that activase activity increases K_f/K_r′, where K_r′ is the apparent Michaelis Menten constant for RuBP and K_f defines the ratio of uncarbamylated free Rubisco sites to uncarbamylated Rubisco site RuBP complexes in the steady state. The curves shown were calculated using equation (A14) from the appendix of Mate et al. (1996):where C denotes the CO₂ concentration, M the free magnesium concentration (5 mm), K_e and K_d are the dissociation constants of the Rubisco site CO₂ and Rubisco site CO₂ and magnesium complexes. K_d = 1,600 μm and K_eK_d = 160,000 μm² (Laing and Christeller, 1976; Lorimer et al., 1976).