Structural and functional characterization of ferredoxin‐NADP⁺‐oxidoreductase using knock‐out mutants of Arabidopsis

Abstract

SummaryIn Arabidopsis thaliana, the chloroplast-targeted enzyme ferredoxin-NADP + -oxidoreductase (FNR) exists as two isoforms, AtLFNR1 and AtLFNR2, encoded by the genes At5g66190 and At1g20020, respectively. Both isoforms are evenly distributed between the thylakoids and soluble stroma, and they are separated by twodimensional electrophoresis in four distinct spots, suggesting post-translational modification of both isoforms. To reveal the functional specificity of AtLFNR1, we have characterized the T-DNA insertion mutants with an interrupted At5g66190 gene. Absence of AtLFNR1 resulted in a reduced size of the rosette with pale green leaves, which was accompanied by a low content of chlorophyll and light-harvesting complex proteins. Also the photosystem I/photosystem II (PSI/PSII) ratio was significantly lower in the mutant, but the PSII activity, measured as the F V /F M ratio, remained nearly unchanged and the excitation pressure of PSII was lower in the mutants than in the wild type. A slow re-reduction rate of P700 measured in the mutant plants suggested that AtLFNR1 is involved in PSI-dependent cyclic electron flow. Impaired function of FNR also resulted in decreased capacity for carbon fixation, whereas nitrogen metabolism was upregulated. In the absence of AtLFNR1, we found AtLFNR2 exclusively in the stroma, suggesting that AtLFNR1 is required for membrane attachment of FNR. Structural modeling supports the formation of a AtLFNR1-AtLFNR2 heterodimer that would mediate the membrane attachment of AtLFNR2. Dimer formation, in turn, might regulate the distribution of electrons between the cyclic and linear electron transfer pathways according to environmental cues.

Arabidopsis cryptochrome 1 functions in nitrogen regulation of flowering

“…FNR is a ubiquitous flavoenzyme that oxidizes the final reduced product of the photosynthetic electron transport chain, ferredoxin (Fd), to reduce NADP + , resulting in ATP production. Therefore, FNR levels (activity) regulate cellular NADPH/NADP + and ATP/AMP ratios (16,22). Consistent with LN induction of FNR1 expression, we detected higher NADPH/NADP + and lower AMP/ATP ratios in WT plants in LN conditions than in NN conditions (P < 0.05).…”

“…These results suggest that CRY1 may work downstream of FNR1 in the N-signaling pathway. Although previous studies reported that both the fnr2 mutant (16,22) and the cry2 mutant (19, 23) had a late-flowering phenotype, here these mutants exhibited normal responses to N changes (P < 0.05; SI Appendix, Table S3), suggesting that neither FNR2 nor CRY2 plays an essential role in N-regulated flowering time.…”

“…In this study, we used a modified method of suppressionsubtractive hybridization (SSH) to identify two key factors involved in N-regulated flowering: ferredoxin-NADP + -oxidoreductase (FNR1) (16) and the blue-light photoreceptor cryptochrome 1 (CRY1) (17). Their loss-of-function mutants are Significance Overapplication of nitrogen (N) fertilizer causes delayed flowering and negatively impacts the function and composition of natural ecosystems and climate.…”

De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits

“…Based on KEGG analysis of the four G. max-specific genes, Glyma01g37051 is involved in energy metabolism (ko00190 and ko00194), Glyma13g36351 and Glyma15g39541 in lipid metabolism (ko00590, ko00591 and ko00592), and Glyma15g39596 in cell growth and death (ko04210). Glyma01g37051 is a homolog of AtLFNR1 (ferredoxin-NADP + -oxidoreductase, AT5G66190), which in Arabidopsis is involved in photosystem Idependent cyclic electron now and affects rosette size 15 .…”

Nitrogen regulates CRY1 phosphorylation and circadian clock input pathways

“…Thus, C and N may regulate flowering through different pathways. Given that nitrogen levels regulate ferredoxin-NADP C -oxidoreductase and ATP synthesis rate (energy metabolism flow), 3,11 nitrogen metabolism and carbon metabolism may be tightly linked with each other. The crosstalk between C metabolite signals and N metabolite signals needs further investigations.…”