Translocation of nuclear-encoded preproteins across the inner envelope of chloroplasts is catalyzed by the Tic translocon, consisting of Tic110, Tic40, Tic62, Tic55, Tic32, Tic20, and Tic22. Tic62 was proposed to act as a redox sensor of the complex because of its redox-dependent shuttling between envelope and stroma and its specific interaction with the photosynthetic protein ferredoxin-NADP(H) oxidoreductase (FNR). However, the nature of this close relationship so far remained enigmatic. A putative additional localization of Tic62 at the thylakoids mandated further studies examining how this feature might be involved in the respective redox sensing pathway and the interaction with its partner protein. Therefore, both the association with FNR and the physiological role of the third, thylakoid-bound pool of Tic62 were investigated in detail. Coexpression analysis indicates that Tic62 has similar expression patterns as genes involved in photosynthetic functions and protein turnover. At the thylakoids, Tic62 and FNR form high molecular weight complexes that are not involved in photosynthetic electron transfer but are dynamically regulated by light signals and the stromal pH. Structural analyses reveal that Tic62 binds to FNR in a novel binding mode for flavoproteins, with a major contribution from hydrophobic interactions. Moreover, in absence of Tic62, membrane binding and stability of FNR are drastically reduced. We conclude that Tic62 represents a major FNR interaction partner not only at the envelope and in the stroma, but also at the thylakoids of Arabidopsis thaliana and perhaps all flowering plants. Association with Tic62 stabilizes FNR and is involved in its dynamic and light-dependent membrane tethering.

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.

SummaryPhysiological roles of the two distinct chloroplast-targeted ferredoxin-NADP + oxidoreductase (FNR) isoforms in Arabidopsis thaliana were studied using T-DNA insertion line fnr1 and RNAi line fnr2. In fnr2 FNR1 was present both as a thylakoid membrane-bound form and as a soluble protein, whereas in fnr1 the FNR2 protein existed solely in soluble form in the stroma. The fnr2 plants resembled fnr1 in having downregulated photosynthetic properties, expressed as low chlorophyll content, low accumulation of photosynthetic thylakoid proteins and reduced carbon fixation rate when compared with wild type (WT). Under standard growth conditions the level of F 0 'rise' and the amplitude of the thermoluminescence afterglow (AG) band, shown to correlate with cyclic electron transfer (CET), were reduced in both fnr mutants. In contrast, when plants were grown under low temperatures, both fnr mutants showed an enhanced rate of CET when compared with the WT. These data exclude the possibility that distinct FNR isoforms feed electrons to specific CET pathways. Nevertheless, the fnr2 mutants had a distinct phenotype upon growth at low temperature. The fnr2 plants grown at low temperature were more tolerant against methyl viologen (MV)-induced cell death than fnr1 and WT. The unique tolerance of fnr2 plants grown at low temperature to oxidative stress correlated with an increased level of reduced ascorbate and reactive oxygen species (ROS) scavenging enzymes, as well as with a scarcity in the accumulation of thylakoid membrane protein complexes, as compared with fnr1 and WT. These results emphasize a critical role for FNR2 in the redistribution of electrons to various reducing pathways, upon conditions that modify the photosynthetic capacity of the plant.

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