Molecular and biochemical analyses of transgenic nicotiana tabacum plants metabolizing glycolate in the chloroplasts

The photorespiratory pathway in C3 plants consumes not only ATP and reducing equivalents but also results in loss of ~ 25% carbon that has been fixed during the process of photosynthesis. In the present study, an alternative biochemical pathway for the metabolism of glycolate was established in the chloroplasts of tobacco (Nicotiana tabacum) plants. The new pathway aims at increasing the refixation of CO2 inside the chloroplasts and thereby at suppressing photorespiration in C3 plants. The pathway is derived from E. coli and converts the glycolate formed during photorespiration into glycerate. Three enzymatic activities are required: glycolate dehydrogenase (GDH), glyoxylate carboligase (GCL), and tartronic semialdehyde reductase (TSR). Instead of E. coli GDH, the glycolate dehydrogenase from Arabidopsis thaliana (AtGDH) was used. Transgenic N. tabacum plants containing the necessary genes were generated. The expression level of the transgenes was analyzed by RT-PCR and the respective enzymatic activity assays showed that the proteins are active in planta. Various physiological, biochemical and photosynthetic measurements were performed under ambient and enhanced photorespiratory conditions to evaluate the impact of the established pathway in planta. By measuring the postillumination burst (PIB), a clear reduction in photorespiration was determined in plants transgenic for AtGDH. A further reduction of the photorespiratory flow was observed when all the transgenes were expressed in one plant (GTA). Additionally, the establishment of the bacterial glycolate pathway in plant chloroplasts results in a decrease of the CO2 compensation point (Gamma). The CO2 assimilation rates in transgenic plants were enhanced under photorespiratory conditions. Moreover, leaves of transgenic plants expressing the glycolate pathway showed higher glucose and fructose, end products of photosynthesis. Leaf fresh and dry weight measurements revealed that total plant productivity might be enhanced. Most of the above described effects were also observed in plants that overexpressed glycolate dehydrogenase alone. It can be concluded that expression of the bacterial glycolate pathway in C3 plant chloroplasts results in a reduction of photorespiration and an enhancement of plant growth