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 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...

Photosynthetic Characteristics of Antisense Transgenic Rice Expressing Reduced Levels of Rubisco Activase

“…In rice, about 30% reduction in the Rubisco activase content led to 50% decrease in steadystate photosynthesis at high light in ambient air conditions (Jin et al, 2006), while the photosynthetic rates were unchanged until Rubisco activase contents decreased to 10% of that in control plants in tobacco (Mate et al, 1993) and Arabidopsis (Eckardt et al, 1997) and 30% of that in Flaveria bidentis (von Caemmerer et al, 2005). These results suggested that Rubisco activase in rice leaves has greater effects on photosynthesis compared with other plant species.…”

“…In order to clarify the effect of Rubisco activase on photosynthesis, transgenic plants with reduced Rubisco activase contents have been produced by the antisense RNA or RNAi technology in some plant species, tobacco (Mate et al, 1993), Arabidopsis thaliana (Eckardt et al, 1997), rice (Jin et al, 2004(Jin et al, , 2006 and Flaveria bidentis (von Caemmerer et al, 2005). In rice, about 30% reduction in the Rubisco activase content led to 50% decrease in steadystate photosynthesis at high light in ambient air conditions (Jin et al, 2006), while the photosynthetic rates were unchanged until Rubisco activase contents decreased to 10% of that in control plants in tobacco (Mate et al, 1993) and Arabidopsis (Eckardt et al, 1997) and 30% of that in Flaveria bidentis (von Caemmerer et al, 2005).…”

Characterization of Rubisco Activase Genes in Maize: An α-Isoform Gene Functions alongside a β-Isoform Gene

“…By transforming the C 4 plant Flaveria bidentis with an antisense RCA gene, von Caemmerer et al (2005) showed that RCA activity was essential for the proper functioning of the C 4 photosynthetic pathway. In maize, which is also a C 4 plant, the RCA protein content of leaves during grain filling was higher in high-yield populations than in low-yield populations (Martı'nez-Barajas et al, 1997; Morales et al, 1999).…”

Expression Quantitative Trait Loci Analysis of Two Genes Encoding Rubisco Activase in Soybean

“…The activities of RCA are thought to be key regulation points for photosynthesis under different environmental stress conditions (Crafts-Brandner and Salvucci, 2000;Pollock et al, 2003). Plants expressing reduced levels of RCA exhibit decreased levels of P N and/or growth (Mate et al, 1996;Eckardt et al, 1997;He et al, 1997), and those with very low or no RCA expression cannot survive in atmospheric CO 2 (Somerville et al, 1982;Salvucci et al, 1985Salvucci et al, , 1986Mate et al, 1993;von Caemmerer et al, 2005). These results make modulation of RCA an attractive experimental goal for the improvement of CO 2 fixation rates and, ultimately, crop productivity.…”