Targeted Proteomics in Characterizing Iron-Deprived Cyanobacteria : Insights Into The Regulation of Iron-Sulfur Cluster Biogenesis

Cyanobacteria comprise a diverse group of widely distributed gram‐negative bacteria, which have the unique capacity amongst prokaryotes to perform oxygenic photosynthesis. Acclimation to iron deprivation, a key theme in this Thesis, involves several metabolic changes, which are closely interlinked with the high demand for iron cofactors in the photosynthetic electron transfer chains of these organisms. In order to gain in‐depth information on the protein-level changes occurring under iron deprivation, a targeted MS‐based proteomics method, selected reaction monitoring (SRM), was developed for the cyanobacterial model species Synechocystis sp. PCC 6803. Altogether 106 proteins were selected as SRM‐target proteins, representing various key metabolic pathways and possible metabolic nodes linked with iron‐dependent enzymatic reactions. The SRM analysis resulted in a high‐quality dataset, which verified several responses to iron deprivation, such as those related to remodeling of photosynthetic apparatus and induction of several iron acquisition proteins. New information was obtained, for example, on the elevated levels of proteins acting as electron sinks to alleviate the overreduction of the photosynthetic electron transfer chain caused by the increase in Photosystem II to Photosystem I ratio under iron deprivation. As a demonstration of the sensitivity of the system, 64 of the quantified target proteins had not been detected in earlier discovery‐based proteomics assays. The validated SRM method was subsequently applied to study the regulation of iron‐sulfur biogenesis in Synechocystis sufR and isaR1 mutant strains. SufR has been characterized as a transcriptional repressor of the sufBCDS operon, which is responsible of the Fe‐S cluster biogenesis in cyanobacteria. Deletion of sufR caused drastic induction of the sufBCDS operon proteins under Fe‐sufficiency, while the proteins carrying Fe‐S cofactors were downregulated. Under extended Fe‐depletion, increased expression of the apo‐form Fe‐S proteins was observed in comparison to the wild type strain under the same conditions. IsaR1, a small regulatory RNA, was found to be responsible for the repression of several genes for Fe and Fe‐S proteins as well as tetrapyrrole biosynthesis genes under Fe‐deprivation. Hence, IsaR1 affects the photosynthetic apparatus both directly and indirectly upon acclimation to iron deprivation. Importantly, both SufR (transcriptional repressor protein) and IsaR1 (small regulatory RNA) were identified to repress the sufBCDS operon; SufR under Fe‐sufficiency and IsaR1 under Fe‐deprivation. Such complex mixed regulatory circuit highlights the importance of tight control over the biogenesis of Fe‐S clusters as part of the metabolic acclimation to varying iron conditions

SRM dataset of the proteome of inactivated iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803

This article contains SRM proteomics data related to the research article entitled ”Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses”[1]. The data described here provide comprehensive information on the applied SRM assays, together with the results of quantifying 94 Synechocystis sp. PCC 6803 proteins.  The data has been deposited in Panorama public  (https://panoramaweb.org/labkey/SufR) and from PASSEL under the PASS00765 identifier (http://www.peptideatlas.org/PASS/PASS00765).  </p

Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses

BackgroundIron-sulfur (Fe-S) clusters are protein-bound cofactors associated with cellular electron transport and redox sensing, with multiple specific functions in oxygen-evolving photosynthetic cyanobacteria. The aim here was to elucidate protein-level effects of the transcriptional repressor SufR involved in the regulation of Fe-S cluster biogenesis in the cyanobacterium Synechocystis sp. PCC 6803.MethodsThe approach was to quantitate 94 pre-selected target proteins associated with various metabolic functions using SRM in Synechocystis. The evaluation was conducted in response to sufR deletion under different iron conditions, and complemented with EPR analysis on the functionality of the photosystems I and II as well as with RT-qPCR to verify the effects of SufR also on transcript level.ResultsThe results on both protein and transcript levels show that SufR acts not only as a repressor of the suf operon when iron is available but also has other direct and indirect functions in the cell, including maintenance of the expression of pyruvate:ferredoxin oxidoreductase NifJ and other Fe-S cluster proteins under iron sufficient conditions. Furthermore, the results imply that in the absence of iron the suf operon is repressed by some additional regulatory mechanism independent of SufR.ConclusionsThe study demonstrates that Fe-S cluster metabolism in Synechocystis is stringently regulated, and has complex interactions with multiple primary functions in the cell, including photosynthesis and central carbon metabolism.General significanceThe study provides new insight into the regulation of Fe-S cluster biogenesis via suf operon, and the associated wide-ranging protein-level changes in photosynthetic cyanobacteria.</p

Emerging from the darkness:interplay between light and plastid signaling during chloroplast biogenesis

Chloroplast biogenesis is a highly complex process that requires carefully coordinated communication between the nucleus and the chloroplast to integrate light signaling and information about the state of the plastid through retrograde signals. Most studies on plastid development have been performed using dark-grown seedlings and have focused on the transition from etioplast to chloroplast in response to light. Some advances are now also being made to understand the transition directly from proplastids to chloroplasts as it occurs in the shoot apical meristems. Recent reports have highlighted the importance of repressive mechanisms to block premature chloroplast development in dark, both at the transcriptional and post-transcriptional level. A group of new proteins with dual plastid and nuclear localization were shown to take part in the light triggered degradation of PHYTOCHROME INTERACTING FACTORs (PIFs) in the nucleus and thereby release the suppression of the nuclear photosynthesis associated genes. These dually localized proteins are also required to activate transcription of photosynthesis genes in the plastid in response to light, emphasizing the close link between the nucleus and the plastids during early light response. Furthermore, development of a fully functional chloroplast requires a plastid signal but the nature of this signal(s) is still unknown. GENOMES UNCOUPLED1 (GUN1) is a plastid protein pivotal for retrograde signal(s) during early seedling development, and recent reports have revealed multiple interactors of GUN1 from different plastid processes. These new GUN1 interactors could reveal the true molecular function of the enigmatic character, GUN1, under naturally occurring adverse growth conditions

SRM dataset of the proteome of inactivated iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803

This article contains SRM proteomics data related to the research article entitled”Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses” (L. Vuorijoki, A. Tiwari, P. Kallio, E.M. Aro, 2017) [1]. The data described here provide comprehensive information on the applied SRM assays, together with the results of quantifying 94 Synechocystis sp. PCC 6803 proteins. The data has been deposited in Panorama public (https://panoramaweb.org/labkey/SufR) and in PASSEL under the PASS00765 identifier (http://www.peptideatlas.org/PASS/PASS00765)

GENOMES UNCOUPLED1 plays a key role during the de-etiolation process in Arabidopsis

One of the most dramatic challenges in the life of a plant occurs when the seedling emerges from the soil and exposure to light triggers expression of genes required for establishment of photosynthesis. This process needs to be tightly regulated as premature accumulation of light harvesting proteins and photoreactive chlorophyll precursors cause oxidative damage when the seedling is first exposed to light. Photosynthesis genes are encoded by both nuclear and plastid genomes and to establish the required level of control, plastid-to-nucleus (retrograde) signalling is necessary to ensure correct gene expression. We herein show that a negative GUN1-mediated retrograde signal restricts chloroplast development in darkness and during early light response by regulating the transcription of several critical TFs linked to light response, photomorphogenesis, and chloroplast development, and consequently their downstream target genes in Arabidopsis. Thus, the plastids play an essential role during skotomorphogenesis and the early light response and GUN1 acts as a safeguard during the critical step of seedling emergence from darkness