Optimierung der Photosynthese von C3-Pflanzen : C 4 -ähnlicher Zyklus und chloroplastidärer Bypass

Abstract

Most agronomic traits are polygenic in nature and rely on complex metabolic and regulatory pathways. Genetic modifications of these traits or introduction of new pathways require the transfer of multiple genes into the plant genome. Most of the existing methods like retransformation, cotransformation and crossing suffer from certain deficiencies like high expenditure of time or the integration of multiple copies (Dafny-Yelin und Tzfira, 2007; Naqvi et al., 2010). The transfer of the genes as a single multigene construct offers considerable advantages. Up to now there are only few efficient methods for assembly and subsequent transformation of multigene constructs into plants. In the context of this work a Gateway-based method could be established, which enables fusion of multiple fragments. The system consists of a Gateway-compatible destination vector and two special entry vectors with attR cassettes, which are flanked by incompatible attL sequences. By alternate use of the two entry vectors multiple transgenes can be recombined into the destination vector. Multigene constructs with 7 gene expression cassettes could be transferred successfully into tobacco by agrobacterium-mediated transformation. By biolistic transformation a multigene construct with 5 gene expression cassettes could be transferred into rice. Due to the bifunctionality of RUBISCO, the key enzyme of photosynthesis, besides carboxylation also oxygenation of ribulose-1,5-bisphosphat is catalyzed in the chloroplasts of higher plants (Bowes et al., 1971). Glycolate formed by this process is toxic and unusable for the plant (Zelitch et al., 2008) and have to be metabolized in the photorespiration. The conversion of glycolate not only consumes ATP and reduction equivalents, it also leads to loss of 25% carbon fixed in this metabolite. For C3 plants photorespiration means a significant reduction of photosynthesis efficiency. On the contrary C4 plants can considerably reduce the oxygenase activity of RUBISCO due to their CO2 concentration mechanism. By use of the MultiRound Gateway technology tobacco plants could be generated, which expressed the heterologous enzymes for a single cell C4 cycle following the example of Hydrilla verticillata. Besides a NADP-ME type C4 cylce, a NAD-ME type and a PCK type C4 cycle were constructed. However, there was no evidence for a CO2 concentration inside the chloroplasts. The heterologous expression of both malic enzymes (NADP-HvMe and NAD-EcMe) strongly affects plant growth. The higher the ME activities were, the slower was the plant growth. All types had in common a higher nitrogen content, which resulted in a lower C/N ratio. Also by use of the MultiRound Gateway technology rice plants with a chloroplastic photorespiratory bypass could be generated. The bypass relies on the catabolic glycolate pathway from E. coli and converts the glycolate inside the chloroplasts. This is intended to increase the CO2 concentration in the vicinity of RUBISCO and thereby suppress photorespiration. In Arabidopsis this pathway led to promising results (Kebeish et al., 2007). The expression of all genes required for this pathway (TSR, GCL, glcD, glcE, glcF) could be verified on the RNA level. To sum up, it can be said that the MultiRound Gateway technology is well suited for the assembly of multigene constructs and the constructed destination vector can be transferred into plants by agrobacterium-mediated transformation as well as biolistic transformation. The generated plants can serve as starting basis for further attempts to establish a single cell C4 cycle. A better balanced relation of enzymatic activities may avoid the negative effects on plant growth caused by high ME activities