Ferredoxin C2 is required for chlorophyll biosynthesis and accumulation of photosynthetic antennae in Arabidopsis.

Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import.

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 Chl precursors causes 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 GENOMES UNCOUPLED1 (GUN1)-mediated retrograde signal restricts chloroplast development in darkness and during early light response by regulating the transcription of several critical transcription factors 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.

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.

Acclimation of Oxygenic Photosynthesis to Iron Starvation Is Controlled by the sRNA IsaR1.

Oxygenic photosynthesis crucially depends on proteins that possess Fe2+ or Fe/S complexes as co-factors or prosthetic groups. Here, we show that the small regulatory RNA (sRNA) IsaR1 (Iron-Stress-Activated RNA 1) plays a pivotal role in acclimation to low-iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe2+-containing proteins involved in photosynthetic electron transfer, detoxification of anion radicals, citrate cycle, and tetrapyrrole biogenesis. IsaR1 is essential for maintaining physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (1) directly, via posttranscriptional repression of gene expression; (2) indirectly, via suppression of pigment; and (3) Fe/S cluster biosynthesis. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a critically important riboregulator. These findings provide a new perspective for understanding the regulation of iron homeostasis in photosynthetic organisms.

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).

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

BACKGROUND: Iron-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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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 SIGNIFICANCE: The 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.

Development of a Quantitative SRM-Based Proteomics Method to Study Iron Metabolism of Synechocystis sp. PCC 6803.

The cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) is a well-established model species in oxygenic photosynthesis research and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study, we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with a targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short- and long-term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short- and long-term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low-abundance proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.