Localization of root-type ferredoxin-NADP+ oxidoreductases in Arabidopsis thaliana

Abstract

Arabidopsis thaliana (Arabidopsis) genome encodes four plant-type ferredoxin-NADP+ oxidoreductases (FNRs) that mainly function in photosynthesis and anabolic metabolism. The two leaf-type FNRs present in chloroplasts are well characterized and operate in the last step of photosynthetic linear electron transfer by oxidizing ferredoxin and reducing NADP+ to NADPH. Two distinct root-type FNR isoforms, RFNR1 and RFNR2, function in the opposite direction producing reduced ferredoxin, which is further utilized in e.g. assimilation of nitrogen. Apart from a recent study showing that RFNR2 is required for nitrite detoxification, the functional specificities of the RFNR isoforms remain elusive. Determining the localization of the RFNR isoforms in distinct cell types and tissues could provide further insight into the biological roles of the isoforms. In this study, immunoblot analysis was used to determine tissue specific localization of the RFNR isoforms as well as to characterize mutants lacking functional RFNR1 or RFNR2. Additionally, tissue and cell specific localization of RFNR isoforms was studied by stably expressing YFP-tagged RFNRs in Arabidopsis. To achieve stable expression, binary vectors encoding YFP-tagged RFNRs were constructed and delivered into Arabidopsis by Agrobacterium tumefaciens mediated transformation. We have confirmed the presence of both RFNR isoforms in the roots and specific tissues of the shoot. Confocal imaging of Arabidopsis leaves expressing YFP-tagged RFNRs under a constitutively active 35S-promoter revealed chloroplastic localization of the fusion proteins suggesting that RFNRs are targeted to the plastids. Immunoblot analysis of samples from the rfnr mutant and wild type plants revealed no differences in the accumulation of proteins involved in nitrogen and carbon assimilation (GOGAT, NR, Rubisco, Rubisco activase)