A novel approach for the suppression of photorespiration in C 3 plants by gene transfer

In the present study, a novel principle is proposed to increase the CO2 concentration in the vicinity of Rubisco thereby 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 Tratronic semialdehyde reductase (TSR). In order to establish the pathway in the chloroplast of tobacco (Nicotiana tabacum), all necessary genes were cloned in both prokaryotic and plant expression vectors in N-terminal translational fusion to a His-tag. The genes were first expressed in bacteria and the enzymatic activity of the constructs was shown. Transgenic tobacco plants were created containing all genes necessary for the proposed pathway by plastdial as well as nuclear transformation. Plastidial transformation was done by constructing a single polycistronic operon with five open reading frames. Nuclear transformation was performed by Agrobacterium-mediated transformation of single or double constructs. Plastid transformants were checked by southern blot analysis. Many transgenic plants with a variable size of shortened transgenic sequences were detected. Moreover, many of the transgenic plants also displayed drastic chlorosis and stunted growth. However, few plants showed integration of the complete operon and the next generation of those plants is currently analysed. Initially, transgenic plants containing TSR and GCL were created by nuclear transformation due to the lack of a suitable glycolate oxidising enzyme. Variable amounts of foreign proteins were detected in Western blots. Enzymatic assays showed that the proteins are active in planta. However transgenic plants containing GCL protein again showed a chlorotic phenotype. A putative open reading frame was identified in the Arabidopsis genome sequence with homology to glycolate-oxidizing enzymes. The open reading frame was cloned and expressed in bacteria. Enzymatic assays and complementation tests showed that the protein is indeed a glycolate dehydrogenase. The gene is preferentially expressed in illuminated leaves and the enzyme is located inside the mitochondria. This protein forms an optimised starting point for the completion of the proposed pathway