Metabolic regulation mediated by thioredoxins and NADPH-dependent thioredoxin reductases in Arabidopsis thaliana L.

Abstract

Thioredoxins (TRXs) consist of a family of oxi-redox proteins capable of (de)activating enzymes, being an important post-translational mechanism for metabolic regulation. Plant TRXs are found in different cell compartments, such as chloroplast, mitochondria, and cytosol. TRXs are activated by specific TRX reductases (TRs), according to their subcellular location. For instance,

chloroplasts contain two TRs, named FTR (ferredoxin-dependent TR) and NTRC (NADPH- dependent TR C), which receives reducing power from ferredoxin and NADPH, respectivelly. On

the other hand, plant cells contain two other NTRs (NTRA and NTRB) that are mainly located in

cytosol and mitochondria. NTRA/B are thus the main TRs responsible for reducing the non- chloroplastic TRXs. Previous works showed that the single ntrc mutant and the double ntrab

mutant have reduced growth, but are still viable, i.e. they can complete the full life cycle and produce viable seeds. However, plants lacking all NTRs remained to be investigated, raising the question on whether the plant NTR system is essential for plant growth and development. In paralell, previous works suggested that the mitocondrial NTR/TRX system can regulate the interplay between carbon and nitrogen metabolisms, but how redox-mediated mechanisms regulate these metabolisms remained to be deeply investigated. Aiming to address these questions, this thesis was divided into three parts. We first reviewed how TRX-mediated mechanisms regulate the primary metabolism, especially the (photo)respiratory metabolism. The second and third chapters involve the characterization of Arabidopsis thaliana L mutants lacking different NTRs and TRXs. The second chapter describes the unprecedented characterization of the triple ntrabc mutant, which lacks all NTRs (NTR A, B and C), whilst the third involves experiments using mutants lacking TRX h2, TRX o1 or NTRA/B aiming to investigate how the NTR/TRX system regulates glutamine synthetase (GS) and the fluxes throughout the GS/GOGAT (glutamate synthase) cycle. Our results showed that the triple ntrabc mutant showed a leaf pale green phenotype with strong reduction in growth and substantial metabolic changes. Despite this, the ntrabc remained viable and was able to complete the full developmental cycle, including the production of viable seeds. These results suggest that the NTR system is highly important for plant growth, but not essential for plant development. Furthermore, our results indicate that the mitochondrial NTR/TRX system is key for the regulation of the redox status of GS and the metabolic fluxes thorughout the GS/GOGAT cycle, which is important for plant high light stress acclimation. Thus, our works provide important and unprecendented informations regarding the regulation of primary metabolism mediated by NTRs and TRXs.