Skip to main content

Main menu

  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
  • About
    • Editorial Board and Staff
    • About the Journal
    • Terms & Privacy
  • More
    • Alerts
    • Contact Us
  • Submit a Manuscript
    • Instructions for Authors
    • Submit a Manuscript
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Teaching Tools in Plant Biology
    • ASPB
    • Plantae

User menu

  • My alerts
  • Log in

Search

  • Advanced search
Plant Cell
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Teaching Tools in Plant Biology
    • ASPB
    • Plantae
  • My alerts
  • Log in
Plant Cell

Advanced Search

  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
  • About
    • Editorial Board and Staff
    • About the Journal
    • Terms & Privacy
  • More
    • Alerts
    • Contact Us
  • Submit a Manuscript
    • Instructions for Authors
    • Submit a Manuscript
  • Follow PlantCell on Twitter
  • Visit PlantCell on Facebook
  • Visit Plantae
Research ArticleResearch Article
You have accessRestricted Access

The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria

Jens Georg, Dennis Dienst, Nils Schürgers, Thomas Wallner, Dominik Kopp, Damir Stazic, Ekaterina Kuchmina, Stephan Klähn, Heiko Lokstein, Wolfgang R. Hess, Annegret Wilde
Jens Georg
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dennis Dienst
bHumboldt-University Berlin, Institute of Biology, 10115 Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nils Schürgers
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Thomas Wallner
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dominik Kopp
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Damir Stazic
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ekaterina Kuchmina
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stephan Klähn
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Heiko Lokstein
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wolfgang R. Hess
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Annegret Wilde
aUniversity of Freiburg, Faculty of Biology, D-79104 Freiburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: annegret.wilde@biologie.uni-freiburg.de

Published September 2014. DOI: https://doi.org/10.1105/tpc.114.129767

  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Additional Files
  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1.

    Selected PsrR1 Homologs in the Cyanobacterial Phylum.

    A sequence alignment of putative PsrR1 homologs from unicellular and multicellular cyanobacteria is shown. Only the conserved part is shown, including a highly conserved region involved in interaction with target mRNAs and an imperfect palindromic region able to fold into a hairpin secondary structure, followed by a T-rich sequence, hallmarks of a Rho-independent terminator of transcription. Experimentally verified transcripts are indicated by asterisks and are in boldface. In M. aeruginosa NIES-843, two copies of PsrR1 were found. The alignment includes the nucleotides 50 to 131 from the Synechocystis 6803 PsrR1 homolog.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2.

    Accumulation of PsrR1 sRNA.

    The expression kinetic of PsrR1 is shown after a shift from 50 μmol photons m−2 s−1 (moderate light; ML) to 300 μmol photons m−2 s−1 (high light; HL) and back to moderate light. The bottom part shows PsrR1 RNA gel blot analysis, and the top part shows densitometric analysis of the RNA gel blot. nt, nucleotides.

    [See online article for color version of this figure.]

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3.

    Phenotypes of the psrR1 Mutant Strains.

    (A) RNA gel blot analysis of PsrR1 accumulation in the psrR1 overexpressor strain (psrR1+) upon removal of copper after 24 and 48 h in comparison with the wild type and the knockout strain (ΔpsrR1) grown under the same conditions. 5S rRNA was used as a loading control.

    (B) Overexpression of psrR1 leads to cell bleaching. Whole cell absorption spectra of a psrR1+ culture are shown before as well as 16, 24, and 48 h after induction of psrR1 expression from the PpetJ promoter by copper removal. At 0 h, cells were harvested, washed twice with copper-free medium, and then incubated for a further 48 h in copper-free medium. The peak at 625 nm resembles mainly absorption from phycocyanin, whereas allophycocyanin absorbs at 655 nm. The peak at 680 nm originates from chlorophyll. Absorption spectra were normalized to OD750. PBS, phycobilisomes.

    (C) Pigment analysis of the psrR1+ mutant strain under inducing (5 d under copper-limiting conditions) and noninducing conditions in comparison with the isogenic wild-type control. Data are means ± sd from three replicate analyses.

    (D) Whole cell absorption spectra of wild-type and ΔpsrR1 strains grown under moderate light conditions (ML) and then shifted to high-light conditions (HL) for 48 h. Spectra were measured using a spectrophotometer equipped with an integrating sphere to reduce light scattering and then normalized to cell density.

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4.

    PsrR1 Is Predicted to Control Genes for Proteins Involved in Photosynthesis and Tetrapyrrole Metabolism.

    (A) Visualization of the functional enrichment analysis for targets of PsrR1 (orange squares) predicted by the CopraRNA tool (Wright et al., 2013). All top 15 CopraRNA target predictions are shown plus selected predictions from the top 85 candidates that were functionally enriched and three additional candidates (ccmk1, ilvB, and livf). Targets subsequently verified experimentally (Figure 5) are labeled by black squares, and targets detected by microarray analysis (Supplemental Table 1) are labeled with blue squares.

    (B) Visualization of the predicted interaction domains within the predicted mRNA targets of PsrR1.

    (C) Visualization of the predicted interaction domains in PsrR1 for the different targets.

    In (B) and (C), the density plots at the top give the relative frequency of a specific nucleotide position in the predicted PsrR1–target interactions. The plots combine all predictions with a CopraRNA P value ≤ 0.01 in all included homologs. Local maxima indicate distinct interaction domains and are marked with vertical lines. The schematic alignments of PsrR1 homologs and of targets at the bottom show the predicted interaction domains. The aligned regions are displayed in gray, gaps in white, and predicted interaction regions in color (color differences are for contrast only). The positions of start codons are annotated, and locus tags and gene names of a representative cluster member are given on the right, if available from Synechocystis 6803. The region covered by the alignment in Figure 1 is indicated by the gray box. The numbering at the x axes refers to the alignment position.

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5.

    Verification of PsrR1–mRNA Interactions in a Heterologous Reporter Assay.

    (A) Translational repression of 5′ UTR–sgfp fusions when overexpressing PsrR1 in E. coli. The wild-type form of PsrR1 was compared against two versions with point mutations in the targeting region (Mut4 and Mut1 [B]) in its interaction with the 5′ UTR of genes psaL, psaL*, cpcA, cpcA*, chlN, chlN*, psaJ, psaJ*, and psbB. The 5′ UTR–sgfp fusions for genes psaK1 and hemA were tested with the wild-type PsrR1 only. Point mutations in the UTRs are indicated by asterisks. The fold reduction is the ratio of the GFP fluorescence of the respective translational 5′ UTR–sgfp fusion in the presence of the control plasmid pJV300 and a plasmid for the overexpression of the respective PsrR1 variant, after subtraction of the background fluorescence.

    (B) Predicted interactions of the tested targets with PsrR1. Point mutations are indicated by arrows and red and blue letters. The putative ribosome binding sites and the start codons are boxed.

  • Figure 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 6.

    Interaction of PsrR1 with the 5′ UTR of psaL mRNA in Vitro.

    (A) EMSA of PsrR1 with the 5′ UTR of psaL mRNA. At a constant final concentration of 1 nM, labeled PsrR1 was incubated for 30 min at 30°C with increasing concentrations (0 to 100 nM) of unlabeled psaL RNA segments.

    (B) Comparative EMSA of PsrR1 and a PsrR1 mutant version (PsrR1 Mut4) with psaL-5′UTR-(+9). For the in vitro synthesis of PsrR1 Mut4, a nucleotide exchange was introduced to the anti-RBS sequence as depicted in Figure 5B.

    (C) EMSA of PsrR1 with the 5′ UTR of psaC mRNA as a negative control. PsaC was not predicted as a target of PsrR1.

    (D) Schematic representation of the fragments tested in (A) to (C). The 5′ UTR and the proposed −10 region of the promoter are indicated by boxes. The black bar indicates the predicted interaction site. Numbers denote nucleotide positions with respect to the first nucleotide of the start codon.

    [See online article for color version of this figure.]

  • Figure 7.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 7.

    Analysis of PSI Complexes from PsrR1 Mutant Strains.

    (A) Immunoblot analysis of PsaL and HemH in cell extracts of psrR1 mutant strains. The Synechocystis 6803 isogenic wild type as well as psrR1 overexpressor (psrR1+) and knockout (ΔpsrR1) strains were grown in BG-11 medium to exponential growth phase and incubated in medium lacking CuSO4 (inducing psrR1 expression) or containing 5 µM CuSO4 (repressing psrR1 expression) for 48 h. Proteins of whole cell extracts were subjected to immunoblot analysis of PsaL accumulation. Probing for ferrochelatase (HemH) served as a loading control. For independent quantification, see Supplemental Figure 7.

    (B) The 77K fluorescence emission spectra of psrR1+ cultures after induction of PsrR1 overaccumulation for 48 h in comparison with the wild-type and ΔpsrR1 strains. Spectra were normalized to the peak at 695 nm, reflecting fluorescence originating mainly from PSII. The peak at 725 nm originates from PSI complexes.

    (C) BN-PAGE of solubilized thylakoids isolated from the wild type as well as psrR1 overexpressor (psrR1+) and ΔpsrR1 mutant strains grown in copper-free medium 48 h after copper step down. Green protein complexes that were seen on the unstained gel are labeled.

    (D) After separation of the protein complexes on the blue-native gel (shown above each respective blot), the lanes were excised and subjected to Tris-Tricine PAGE (second dimension). Gels from the second dimension were electroblotted and probed sequentially with PsbA and PsaC antisera. For identification of the protein complexes, a Tris-Tricine gel that originated from the same first dimension of a wild-type sample was subjected to silver staining (bottom panel).

    (E) Wild-type and ΔpsrR1 cells were grown under moderate light conditions (time 0) and then shifted for at least 2 h to high light. The isolated RNA was subjected to RNA gel blot analysis and probed with psaL and 23S rRNA as a control.

    (F) Signals from the RNA gel blot were quantified with Quantity One software. Levels of psaL at 0, 0.5, 1, 1.5, and 2 h after a shift from 40 to 400 μmol photons m−2 s−1 are shown.

  • Figure 8.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 8.

    Half-Life Estimation of psaL and psaL 5′ UTR RNAs.

    (A) RNA gel blot analysis for psaL and the psaL 5′ UTR (same probe) and the 23S rRNA control hybridization. Wild-type and psrR1+ cells were treated with rifampicin (final concentration of 300 μg/mL) after 24 h of copper depletion under moderate light conditions. RNA was extracted prior to and 2, 4, 8, 16, 32, and 64 min after rifampicin addition.

    (B) The intensity of the psaL signal was quantified with Quantity One software and normalized with the respective 23S rRNA signal for the wild type and the psrR1+ mutant. The half-life time (t1/2) was estimated by fitting the formula N(t) = N0 × 0.5^(t/t1/2), where t = time after rifampicin addition, N(t) = normalized amount of transcript at time point t, and N0 = normalized amount of transcript at time point t = 0, to the experimental data with the nonlinear least squares function of R. The figure gives the experimental data points for the wild type (black) and the mutant (red) with the sd from two biological replicates. The theoretical curve for the calculated t1/2 is plotted as a broken line. The reference lines indicate the respective half-lives for RNAs in the wild type and mutant.

    (C) Quantified data for the psaL 5′ UTR. The t1/2 was calculated only for the psrR1+ mutant, because the UTR does not accumulate in the wild type under standard conditions.

  • Figure 9.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 9.

    Conditional Processing of the psaL Transcript.

    (A) PsrR1 mediates the RNase E–dependent destabilization of psaL mRNA in vitro. Shown is an 8 M urea, 10% PAA gel of in vitro–transcribed single-stranded psaL RNA (left panel), psaL RNA + NsiR4 (middle panel), and duplex psaL RNA:PsrR1 (right panel) incubated without (−) and with (+) recombinant Synechocystis 6803 RNase E. psaL RNA cleavage fragments were identified by RNA gel blot hybridization using a 5′ [γ-32P]ATP–labeled oligonucleotide probe complementary to the 5′ end of the psaL mRNA. The psaL fragment used is shown in (B). An ethidium bromide–stained version of the gel is shown in Supplemental Figure 8.

    (B) The processed psaL 5′ fragment is present in vivo under high-light conditions (HL). Normalized Solexa transcriptome sequencing reads are for the psaL 5′ region for the exponential growth phase and high-light conditions (470 µmol photons m−2 s−1 for 30 min; raw data from Kopf et al., 2014). The 5′ UTR of psaL and the interaction site with PsrR1 are indicated. Sequence coverage in the data from high light shows a sharp drop, which indicates the processing site, marked by the arrow and the black vertical line.

    (C) Accumulation of the psaL 5′ fragment correlates with the abundance of PsrR1 in vivo under different stress conditions. As a measure for the amount of processed 5′ fragment, we compared the read coverage from a nucleotide position 5′ of the processing site with the read coverage from a nucleotide position 3′ of the processing site (Pos1 and Pos2 in [B], respectively). To get roughly the relative amount of 5′ fragments from all psaL transcripts (i.e., the amount of processed psaL), we divided the difference from the read count at Pos1 and Pos2 by the read count at Pos1. This value is calculated for all 10 conditions tested in the Kopf et al. (2014) transcriptome study and plotted against the respective read count for PsrR1. The Pearson correlation of this ratio with the PsrR1 abundance is ∼0.86 (Spearman correlation of ∼0.7).

    [See online article for color version of this figure.]

  • Figure 10.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 10.

    Simplified Scheme of the High-Light Response in Synechocystis 6803.

    The model for the activity of transcription factors and their targets explains most of the physiological observations during acclimation to high light. The two-component system DspA-RpaB activates PSI and likely also phycobilisome (PBS) gene expression under low-light conditions, whereas it represses psbA. PedR activates some chlorophyll (Chl) biogenesis genes under low-light conditions. Under high-light (HL) conditions, PsrR1 is induced by a so far unknown mechanism and posttranscriptionally represses chlorophyll biogenesis and phycobilisome and PSI genes. The effect of PsrR1 on PSII is so far unclear. High-light acclimation is achieved by the interplay of transcriptional regulation by transcription factors (gray lines), σ-factors (not included in the model), and posttranscriptional regulation by PsrR1 (blue lines).

Additional Files

  • Figures
  • Supplemental Data

    Files in this Data Supplement:

    • Supplemental Figures and Tables
    • Supplemental Dataset 2
    • Supplemental Dataset 1
    • Supplemental Dataset 3
    • Supplemental Dataset 4
PreviousNext
Back to top

Table of Contents

Print
Download PDF
Email Article

Thank you for your interest in spreading the word on Plant Cell.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria
(Your Name) has sent you a message from Plant Cell
(Your Name) thought you would like to see the Plant Cell web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria
Jens Georg, Dennis Dienst, Nils Schürgers, Thomas Wallner, Dominik Kopp, Damir Stazic, Ekaterina Kuchmina, Stephan Klähn, Heiko Lokstein, Wolfgang R. Hess, Annegret Wilde
The Plant Cell Sep 2014, 26 (9) 3661-3679; DOI: 10.1105/tpc.114.129767

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria
Jens Georg, Dennis Dienst, Nils Schürgers, Thomas Wallner, Dominik Kopp, Damir Stazic, Ekaterina Kuchmina, Stephan Klähn, Heiko Lokstein, Wolfgang R. Hess, Annegret Wilde
The Plant Cell Sep 2014, 26 (9) 3661-3679; DOI: 10.1105/tpc.114.129767
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • METHODS
    • Acknowledgments
    • AUTHOR CONTRIBUTIONS
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

In this issue

The Plant Cell Online: 26 (9)
The Plant Cell
Vol. 26, Issue 9
Sep 2014
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Index by author
  • Advertising (PDF)
  • Front Matter (PDF)
View this article with LENS

More in this TOC Section

  • Temporal Regulation of the Metabolome and Proteome in Photosynthetic and Photorespiratory Pathways Contributes to Maize Heterosis
  • Chloroplast Chaperonin-Mediated Targeting of a Thylakoid Membrane Protein
  • Ectopic Expression of the Transcriptional Regulator silky3 Causes Pleiotropic Meristem and Sex Determination Defects in Maize Inflorescences
Show more RESEARCH ARTICLES

Similar Articles

Our Content

  • Home
  • Current Issue
  • Plant Cell Preview
  • Archive
  • Teaching Tools in Plant Biology
  • Plant Physiology
  • Plant Direct
  • Plantae
  • ASPB

For Authors

  • Instructions
  • Submit a Manuscript
  • Editorial Board and Staff
  • Policies
  • Recognizing our Authors

For Reviewers

  • Instructions
  • Peer Review Reports
  • Journal Miles
  • Transfer of reviews to Plant Direct
  • Policies

Other Services

  • Permissions
  • Librarian resources
  • Advertise in our journals
  • Alerts
  • RSS Feeds
  • Contact Us

Copyright © 2021 by The American Society of Plant Biologists

Powered by HighWire