SummaryDiatoms are widespread in aquatic ecosystems where they may be limited by the supply of inorganic carbon. Their carbon dioxide-concentrating mechanisms (CCMs) involving transporters and carbonic anhydrases (CAs) are well known, but the contribution of a biochemical CCM involving C 4 metabolism is contentious.The CCM(s) present in the marine-centric diatom, Thalassiosira pseudonana, were studied in cells exposed to high or low concentrations of CO 2 , using a range of approaches.At low CO 2 , cells possessed a CCM based on active uptake of CO 2 (70% contribution) and bicarbonate, while at high CO 2 , cells were restricted to CO 2 . CA was highly and rapidly activated on transfer to low CO 2 and played a key role because inhibition of external CA produced uptake kinetics similar to cells grown at high CO 2 . The activities of phosphoenolpyruvate (PEP) carboxylase (PEPC) and the PEP-regenerating enzyme, pyruvate phosphate dikinase (PPDK), were lower in cells grown at low than at high CO 2 . The ratios of PEPC and PPDK to ribulose bisphosphate carboxylase were substantially lower than 1, even at low CO 2 .Our data suggest that the kinetic properties of this species results from a biophysical CCM and not from C 4 type metabolism.

Communication between organelles and the nucleus is referred to as anterograde (nucleus to organelle) and retrograde (organelle to nucleus) signalling. In plants, the pentatricopeptide repeat (PPR) proteins represent a large family of nuclear-encoded proteins that are required for post-transcriptional control of chloroplast and mitochondria gene expression, and hence play a central role in the nuclear anterograde control of organelle genome expression. How PPR gene expression is controlled and regulated by retrograde signals is, however, still unknown. Here, we report a significant role for the general transcription factor TFIIF α-subunit (TFIIFα) in controlling PPR gene expression in Arabidopsis. First, we found that TFIIFα interacts with the BIN4 subunit of the Topoisomerase VI (Topo VI). Transcriptome analysis of TFIIF and Topo VI mutant lines then revealed that many PLS-type PPR genes involved in RNA editing are reciprocally controlled by TFIIF and Topo VI. The misexpression of CLB19 and DYW1 genes in two allelic tfIIfα mutants was associated with editing impairments in their plastid target RNAs rpoA and ndhD, respectively. Interestingly, we also detected a change in NDH activity in tfIIfα plants. We also show that TFIIFα and Topo VI coordinate the expression of NDH subunits encoded by the nuclear and plastid genomes. These results reveal the crucial role of the nuclear TFIIFα and Topo VI complexes in controlling plastid genome expression at multiple levels of regulation, including the particular regulation of PPR gene expression.

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically downregulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some TE loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine (SAM) synthase MAT3, which is required for H3K9me2 deposition. Topoisomerase VI promotes MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a mechanistic insight into the essential role of Topoisomerase VI in the delimitation of chromatin domains.

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains.