Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in the carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control carotenoid biosynthesis and accumulation in plants. However, little is known about the mechanisms underlying their posttranscriptional regulation. Here we report that PSY and OR family proteins [Arabidopsis thaliana OR (AtOR) and AtOR-like] physically interacted with each other in plastids. We found that alteration of OR expression in Arabidopsis exerted minimal effect on PSY transcript abundance. However, overexpression of AtOR significantly increased the amount of enzymatically active PSY, whereas an ator ator-like double mutant exhibited a dramatically reduced PSY level. The results indicate that the OR proteins serve as the major posttranscriptional regulators of PSY. The ator or ator-like single mutant had little effect on PSY protein levels, which involves a compensatory mechanism and suggests partial functional redundancy. In addition, modification of PSY expression resulted in altered AtOR protein levels, corroborating a mutual regulation of PSY and OR. Carotenoid content showed a correlated change with OR-mediated PSY level, demonstrating the function of OR in controlling carotenoid biosynthesis by regulating PSY. Our findings reveal a novel mechanism by which carotenoid biosynthesis is controlled via posttranscriptional regulation of PSY in plants.arotenoids are a group of C40 isoprenoids synthesized in chloroplasts, chromoplasts, and other plastids in plants. Carotenoids serve as components of photosynthetic machinery, precursors for phytohormones, and important contributors to fruit nutritional quality and flower color (1, 2). The carotenoid biosynthetic pathway in higher plants has been well defined. However, identification of the regulatory mechanisms underlying carotenoid biosynthesis remains a challenge.Phytoene synthase (PSY) catalyzes the first committed step in carotenoid biosynthesis and controls carbon flux into the carotenoid biosynthetic pathway (1-5). Alteration of PSY expression exerts profound effects on carotenoid content (6-11). A number of factors are known to affect PSY gene expression (12)(13)(14)(15)(16)(17)(18). PSY is found to be repressed by phytochrome-interacting factors in etiolated Arabidopsis seedlings (16). PSY1 expression in tomato fruits is reported to be regulated by cis-carotenoids (14) and requires the MADS-Box transcription factor RIPENING INHIBITOR (18). Recently, it was discovered that PSY protein levels in carrot roots are modulated by a negative feedback emerging from carotenoids (19). The crucial role of PSY in carotenogenesis and the multiple factors affecting its expression suggest a complex regulatory system involved in controlling PSY. However, the factors involved in posttranscriptional regulation of PSY within plastids remain a mystery. No proteins have been re...

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using Fluorescent in situ Hybridisation to probe the subcellular location of mRNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which likely play roles in targeting photosynthetic proteins to the thylakoid membrane.

Summary Oxygenic photosynthesis requires the coordination of environmental stimuli with the regulation of transcription. The transcription factor RpaB is conserved from the simplest unicellular cyanobacteria to complex eukaryotic algae, representing more than 1 billion years of evolution. To predict the RpaB-controlled regulon in the cyanobacterium Synechocystis , we analyzed the positional distribution of binding sites together with high-resolution mapping data of transcriptional start sites (TSSs). We describe more than 150 target promoters whose activity responds to fluctuating light conditions. Binding sites close to the TSS mediate repression, whereas sites centered ∼50 nt upstream mediate activation. Using complementary experimental approaches, we found that RpaB controls genes involved in photoprotection, cyclic electron flow and state transitions, photorespiration, and nirA and isiA for which we suggest cross-regulation with the transcription factors NtcA or FurA. The deep integration of RpaB with diverse photosynthetic gene functions makes it one of the most important and versatile transcriptional regulators.

Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma–antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA–protein complexes and multisubunit protein complexes in a photosynthetic organism.

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.