Molekulare Mechanismen von Nitratreduktion, NO-Synthese und post-translationaler Regulation pflanzlicher Nitratreduktasen
Zusammenfassung der Projektergebnisse
The molybdenum-, heme- and FAD-containing enzyme nitrate reductase (NR) is essential for higher plants, as it catalyzes the first and rate-limiting step in the nitrogen assimilation pathway. In addition, NR is also considered to be a main source of nitric oxide in higher plants. Nitric oxide is a crucial signaling molecule in plants and is involved in many pathways throughout the plant life, from germination to senescence, but also in mediating the response to abiotic and biotic stress factors. Furthermore, plant nitrate reductase is known to be regulated on post-translational level by high-affinity binding of a 14-3-3 protein to a conserved phospho-serine residue and by low-affinity binding by a novel binding mechanism to the N-terminus of NR. Many plants including Arabidopsis thaliana express two isoforms of NR (AtNIA1 and AtNIA2), and multiple isoforms of 14-3-3 proteins (e. g. 13 in Arabidopsis thaliana). This project intended to shed light on the biochemical basis of the interplay of nitrate reduction versus nitrite reduction and the impact of 14-3-3 proteins on the NO synthetic activity of the different NR isoforms. In addition, we aimed to obtain structural data of NR potentially as part of the inhibited 14-3-3-phospho-NR complex in order to trap the dynamic and multidomain NR in a more stable complex. First, the planned biochemical characterization of Arabidopsis thaliana NR isoforms in terms of nitrate or nitrite reduction was successful, suggesting that AtNIA1 is the more efficient nitrite reductase while AtNIA2 is the preferred nitrate reductase. After starting this project, a new hypothesis was published based on data from the green alga Chlamydomonas reinhardtii, which showed that CrNR is not the primary physiological nitrite reductase, but that it rather forms a functional complex with another molybdenumdependent enzyme, named (mitochondrial) amidoxime reducing component ((m)ARC) in mammals. In this cytosolically localized NR:ARC complex, CrNR merely serves as electron transfer chain between the physiological electron donor NADH and the catalytically active nitrite reductase CrARC. As verification of this hypothesis for higher plants would change the entire concept of NR being the prime NO-forming nitrite reductase, we decided to prioritize testing this hypothesis in Arabidopsis thaliana. Consequently, we generated and characterized all proteins needed to test this hypothesis, including the two ARC isoforms AtARC1 and AtARC2, as well as the soluble domains of the cytochrome b5 (AtCb) and cytochrome b5 reductase (AtCbR), which were shown earlier to be the physiological electron suppliers for mammalian mARC. After confirming that AtARC1 and AtARC2 were active enyzmes, we performed kinetic experiments in order to test the suitability of nitrite as substrate, and to test the efficiency of electron supply from NADH via AtNR as found in Chlamydomonas or via AtCb and AtCbR as found in mammals. Furthermore, we analyzed whether AtARC1 or AtARC2 can form a stable complex with either AtNR or AtCb and AtCbR. In summary, we did not obtain any in vitro data that would support the results from Chlamydomonas reinhardtii. Therefore, we additionally performed in vivo studies and investigated arc1, arc2 and arc1arc2 mutant plants. Comparing their growth and development to wildtype plants did not reveal significant differences, and also nitric oxide synthase activity of the mutant plant extracts brought no significant differences to light. In aggregate, our study underlined the importance of NR in plant NO synthesis.
Projektbezogene Publikationen (Auswahl)
- (2019). Isoform-Specific NO Synthesis by Arabidopsis thaliana Nitrate Reductase. Plants 8, 67
Mohn, M.A., Thaqi, B., and Fischer-Schrader, K.
(Siehe online unter https://doi.org/10.3390/plants8030067) - (2022). Characterization of the amidoxime reducing components ARC1 and ARC2 from Arabidopsis thaliana. FEBS Journal, 2022 Sep;289(18):5656-5669
Maiber, L., Koprivova, A., Bender, D., Kopriva, S., Fischer-Schrader, K.
(Siehe online unter https://doi.org/10.1111/febs.16450)
