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.

Chloroplast development requires the coordinated action of various proteins, many of which remain to be identified. Here, we report two novel genes, Mesophyll-cell RNAi Library line 7 (MRL7) and MRL7-Like (MRL7-L), that are involved in this process. An Arabidopsis knock-down transgenic plant (MRL7-RNAi) with delayed-greening phenotype was isolated from an RNA interference (RNAi) transformant library. Cotyledons and young leaves of MRL7-RNAi were pale in seedlings and gradually greened as the plant matured, while a knock-out in the MRL7 gene was seedling lethal. The MRL7 protein was shown to co-localize with a marker protein for nucleoids in chloroplasts, indicative of a role for the protein in chloroplast nucleic acid metabolism. Accordingly, chloroplast development was arrested upon loss of MRL7 function and the expression of plastid-encoded genes transcribed by plastid-encoded RNA polymerase (PEP) was significantly reduced in MRL7 knock-down and knock-out plants. A paralog of MRL7 (MRL7-L) was identified in the Arabidopsis genome. Both MRL7 and MRL7-L are only found in land plants and encode previously uncharacterized proteins without any known conserved domain. Like MRL7, knock-down of MRL7-L also resulted in a virescent phenotype, and a similar effect on plastid gene expression. However, the MRL7-L protein was localized to the chloroplast stroma. Taken together, our data indicate that the two paralogous proteins MRL7 and MRL7-L have essential but distinct roles during early chloroplast development and are involved in regulation of plastid gene expression.

Redox modulation of protein activity by thioredoxins (TRXs) plays a key role in cellular regulation. Thioredoxin z (TRX z) and its interaction partner fructokinase-like protein 1 (FLN1) represent subunits of the plastid-encoded RNA polymerase (PEP), suggesting a role of both proteins in redox regulation of chloroplast gene expression. Loss of TRX z or FLN1 expression generates a PEP-deficient phenotype and renders the plants incapable to grow autotrophically. This study shows that PEP function in trx z and fln1 plants can be restored by complementation with redox-inactive TRX z C106S and FLN1 C105/106A protein variants, respectively. The complemented plants showed wild-type levels of chloroplast gene expression and were restored in photosynthetic capacity, indicating that redox regulation of PEP through TRX z/FLN1 per se is not essential for autotrophic growth. Promoter–reporter gene studies indicate that TRX z and FLN1 are expressed during early phases of leaf development while expression ceases at maturation. Taken together, our data support a model in which TRX z and FLN1 are essential structural components of the PEP complex and their redox activity might only play a role in the fine tuning of PEP function.