Characterization of the β-hydroxyaspartate cycle and glycolate dehydrogenase as photorespiratory bypasses in Arabidopsis thaliana chloroplasts

Rubisco is the first enzyme of the Calvin-Benson-Bassham (CBB) cycle: it performs the carboxylation of ribulose-1,5-bisphosphate (RuBP) to 3-phosphoglycerate (3PGA). However, it is also possible for rubisco to oxygenate RuBP instead, which results in the production of toxic 2-phosphoglycolate (2PG). Photorespiration is a pathway that allows plants to detoxify 2PG, while recovering some carbon in the form of 3PGA. On the other hand, this process consumes energy and releases nitrogen and some of the previously fixated carbon. In C3 crops, this phenomenon translates to yearly losses between 20% and 50%: for this reason, researchers are focusing on improving photosynthesis by limiting photorespiration. The β-hydroxyaspartate cycle (BHAC), discovered in marine proteobacteria, allows these organisms to use glycolate, oxidized to glyoxylate, as a carbon and energy source, producing oxaloacetate with no carbon loss. Since glycolate is also an intermediate of photorespiration, the implementation of the BHAC in plants could be a valid photorespiratory bypass. This work focuses on the operation of BHAC in A. thaliana wild type plants and photorespiratory mutants. The four enzymes of this pathway are expressed in chloroplasts, along with glycolate dehydrogenase (GDH), necessary to generate glyoxylate. Analysis of the genotype through PCR led to the identification of five positive BHAC lines expressing all genes of the pathway. Western blotting allowed to verify the expression of four out of five exogenous proteins, while for one of them mRNA expression was verified through qRT-PCR. IC-MS analysis of positive individuals revealed a redirection of the flux of nitrogen and carbon, with an increase in intermediates of the gluconeogenesis and in the presence of acetylated and hydroxylated aminoacids. The substrates of the CBB cycle are also affected. Finally, A. thaliana plants were transformed with GDH alone to assess its contribution in the BHAC bypass and the possibility to be implemented as a photorespiratory bypass by itself.