Here, we characterize a plastidial thioredoxin (TRX) isoform from Arabidopsis thaliana that defines a previously unknown branch of plastidial TRXs lying between x-and y-type TRXs and thus was named TRX z. An Arabidopsis knockout mutant of TRX z had a severe albino phenotype and was inhibited in chloroplast development. Quantitative real-time RT-PCR analysis of the mutant suggested that the expressions of genes that depend on a plastid-encoded RNA polymerase (PEP) were specifically decreased. Similar results were obtained upon virus-induced gene silencing (VIGS) of the TRX z ortholog in Nicotiana benthamiana. We found that two fructokinase-like proteins (FLN1 and FLN2), members of the pfkB-carbohydrate kinase family, were potential TRX z target proteins and identified conserved Cys residues mediating the FLN-TRX z interaction. VIGS in N. benthamiana and inducible RNA interference in Arabidopsis of FLNs also led to a repression of PEPdependent gene transcription. Remarkably, recombinant FLNs displayed no detectable sugar-phosphorylating activity, and amino acid substitutions within the predicted active site imply that the FLNs have acquired a new function, which might be regulatory rather than metabolic. We were able to show that the FLN2 redox state changes in vivo during light/dark transitions and that this change is mediated by TRX z. Taken together, our data strongly suggest an important role for TRX z and both FLNs in the regulation of PEP-dependent transcription in chloroplasts.

Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b 6 f complex to the metabolic demand for ATP and NADPH. While the cytochrome b 6 f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclearencoded g-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastidencoded atpB gene (encoding the essential b-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b 6 f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO 2 fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed. INTRODUCTIONThe capacity of the photosynthetic light reactions to provide ATP and NADPH must be closely adjusted to their metabolic consumption by the Calvin cycle, the subsequent reactions of dark metabolism such as starch synthesis, and other anabolic pathways within the chloroplast. Upon hyperactivity of the light reactions, the metabolic regeneration of NADP + , ADP, and P i will limit photosynthetic electron transport, resulting in detrimental side reactions. NADP + limitation would result in electron transfer to alternative acceptors, such as O 2 , generating reactive oxygen species. These can damage the photosynthetic apparatus itself and also initiate cell death responses (Kim et al., 2008).Reduced ADP and P i regeneration results in substrate limitation of the thylakoid ATP synthase, reducing proton efflux from the lumen and resulting in an increase of the proton motive force (pmf) across the thylakoid membrane (Takizawa et al., 2008;Kiirats et al., 2009). Under standard growth conditions, the pmf is partitioned into an electrochemical component (DC) and a proton gradient (DpH) in such a way that the pH value of the thylakoid lumen is usually kept between 7.0 and 6.5 (Takizawa et al., 2007). However, in response to short-term imbalances between proton translocation into the lumen by photosynthetic electron transport and use of the pmf for ATP synthesis, the pH of the thylakoid lumen can drop below 6.5. This initiates photoprotective feedback responses such as nonphotochemical quenching (qN), which is the thermal dissipation of excess excitation energy in the photosystem II (PSII) antenna bed in the...

The intricate assembly of photosystem I (PSI), a large multiprotein complex in the thylakoid membrane, depends on auxiliary protein factors. One of the essential assembly factors for PSI is encoded by ycf3 (hypothetical chloroplast reading frame number 3) in the chloroplast genome of algae and higher plants. To identify novel factors involved in PSI assembly, we constructed an epitope-tagged version of ycf3 from tobacco (Nicotiana tabacum) and introduced it into the tobacco chloroplast genome by genetic transformation. Immunoaffinity purification of Ycf3 complexes from the transplastomic plants identified a novel nucleus-encoded thylakoid protein, Y3IP1 (for Ycf3-interacting protein 1), that specifically interacts with the Ycf3 protein. Subsequent reverse genetics analysis of Y3IP1 function in tobacco and Arabidopsis thaliana revealed that knockdown of Y3IP1 leads to a specific deficiency in PSI but does not result in loss of Ycf3. Our data indicate that Y3IP1 represents a novel factor for PSI biogenesis that cooperates with the plastid genome-encoded Ycf3 in the assembly of stable PSI units in the thylakoid membrane.

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