| Popis: |
Metabolism of glycolate via the photorespiratory pathway in C3 plants consumes not only ATP and reducing equivalents but results also in approximately 25% loss of the carbon from glycolate. In the present study, a novel biochemical pathway for the metabolism of glycolate was established in the chloroplast of Arabidopsis thaliana plants. The new pathway aims 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 tartronic semialdehyde reductase (TSR). The minimal E.coli glycolate dehydrogenase enzyme is formed from three different polypeptides. As an alternative, a glycolate dehydrogenase (AtGDH) derived from A. thaliana was used. Transgenic A. thaliana plants containing the necessary genes for the novel pathway were generated. Variable amounts of foreign proteins as well as RNA were detected by Western blot and RT-PCR, respectively. Enzymatic assays showed that the proteins are active in planta. Biochemical, physiological and biophysical analyses were performed under ambient and enhanced photorespiratory conditions using different transgenic lines for evaluating the impact of the novel pathway in planta. By measuring the Gly/Ser ratio, a clear reduction in photorespiration was observed in transgenic plants expressing the novel pathway genes compared to wild type plants. A clear decrease in the amount of CO2 released in the plant mitochondria during photorespiration was also obvious in transgenic lines. The ammonia release bioassay provides an additional evidence for the partial suppression of photorespiration in some of the transgenic lines. Furthermore, establishment of the glycolate pathway in the plant chloroplasts results in a decrease in the CO2 compensation point (Gamma*). The CO2 assimilation rates in transgenic plants were also enhanced under photorespiratory conditions. Finally, plant growth measurements revealed that the transgenic plants expressing the glycolate pathway in their chloroplasts have bigger leaf area as well as bigger rosette diameter compared to the control plants. Moreover, the total fresh and dry weight measurements showed that the total plant productivity was enhanced. Interestingly, most of the described effects were also observed in plants that only overexpressed a functional GDH. However, these effects were stronger in plants overexpressing all necessary elements of the glycolate pathway. Moreover, the phenotypical effects were much stronger when the bacterial GDH was compared to the plant GDH. Taken together, it can be concluded that expression of the novel pathway in C3 plant chloroplast does not only result in a reduction of photorespiration but it also enhances plant growth. |
