Engineering Post-Rubisco Reactions

RuBP Regeneration
Flux control analysis indicated SBPase as the most likely rate-limiting step for regeneration of RuBP in the PCR cycle (Robinson and Walker, 1981; see Section 2.2). Furthermore, the two phosphatases FBPase and SBPase, as well as PRK, are light-regulated enzymes that avoid futile reactions in the dark. Regulation is exerted through the redox reaction of two SH-groups in these proteins (Buchanan, 1991). The SH-groups are also targets of hydrogen peroxide under oxidative stress that affects redox homeostasis (Shikanai et al., 1998).

In contrast to the plant PCR cycle, cyanobacterial and green algal PCR pathways are insensitive to oxidation by H₂O₂ and are not subject to light/dark regulation (Tamoi et al., 1998). This is because the enzyme involved in the rate-limiting step of these microorganisms lacks the functional redox-responding SH-groups (Tamoi et al., 1996a,b, 2001). While the plant and algal PCR cycles include FBPase and SBPase as separate entities, both metabolic steps are catalyzed by a single enzyme, FBP/SBPase, in the PCR cycle of Synchococcus (Tamoi et al., 1996b). The bifunctional enzyme lacks regulatory SH-groups. The gene for the cyanobacterial enzyme fused to a RuBisCO small subunit transit peptide has been introduced into tobacco (Miyagawa et al., 2001; Tamoi et al., 2005). The transformant created in this experiment revealed improved photosynthetic performance: transformed plants showed a 2.3-fold increase in chloroplast FBPase and SBP activities relative to wild type, accompanied by an increase in CO₂-fixation rate and dry matter to 125% and 150%, respectively, of the wild type (Fig. 4.3). The photosynthetic rates realized in these transformants may be the maximum attainable for C₃ photosynthesis because C₃ photosynthesis enters a Pi-limited state at such high CO₂-fixation rates (see section 2.1).

FIGURE 4.3 Phenotypes of the wild-type tobacco plant and the transformant expressing cyanobacterial FBPase/SBPase in chloroplasts. (A) Effect of increasing light irradiance on the net CO2 assimilation at 360 ppm of CO2, 25°C, and 60% relative humidity. The CO2 assimilation rate was measured using the fourth leaves down from the top of plant, after 12 weeks of culture. (B) Photographs of the wild plant and the transformant after 18 weeks of culture in 360-ppm CO2 at 400 mmol m-2 s-1.

With the exception of FBPase and SBPase, there were no detectable changes in these transformants in either total activities or amounts of enzymes involved in the PCR cycle. The only changes observed with the transformant were increases in RuBP levels and in the activation ratio of RuBisCO by a factor of 1.8–1.2 relative to the wild type (Miyagawa et al., 2001). These increases in photosynthetic rate are consistent with an increase in RuBisCO activation. Since RuBisCO activase requires a relatively high concentration of RuBP as judged from in vitro assays (Porits, 1990), the observed increase in activation seems to be due to the presence of the transgenic FBP/SBPase that appears to function by promoting regeneration of RuBP and, as a consequence, activating the activase. This study presents the first example of successful improvement of photosynthetic performance and productivity by the introduction of a single gene. In addition, it provides proof for the validity of the concept that single-gene transfers, based on precise knowledge of metabolic flux, its control, and enzyme activity regulation, can improve crop productivity. Similar, but smaller, effects have been reported in tobacco expressing FBPase and SBPase individually (Lefebvre et al., 2005; Tamoi et al., 2006).