Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria

Nature Plants volume 6, pages 1179–1191 (2020)Cite this article

Subjects

Abstract

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 fluorescence in situ hybridization to probe the subcellular location of messenger RNA 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 probably play roles in targeting photosynthetic proteins to the thylakoid membrane.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Location of mRNAs encoding PsbA proteins in Synechocystis, with effects of ribosome inhibitors.
Fig. 2: Distribution of psbA mRNA in Synechococcus under different conditions.
Fig. 3: Location of mRNAs encoding PsaA proteins in Synechocystis and Synechococcus, with effects of ribosome inhibitors.
Fig. 4: Location of mRNAs that do not encode TM-integral proteins: cpcAB in Synechocystis and rbcL in Synechococcus.
Fig. 5: RBPs in Synechocystis GT-I and their effects on mRNA localization.
Fig. 6: Model for TM-targeting based on recognition of thylakoid-mRNA molecules by specific RBPs with affinity for the thylakoid membrane surface.

Similar content being viewed by others

Data availability

The datasets analysed during the current study are available from the corresponding author on reasonable request. Source data are provided with this paper.

References

  1. Pisareva, T. et al. Model for membrane organization and protein sorting in the cyanobacterium Synechocystis sp. PCC 6803 inferred from proteomics and multivariate sequence analyses. J. Proteome Res. 10, 3617–3631 (2011).

    CAS  PubMed  Google Scholar 

  2. Mullineaux, C. W. Co-existence of photosynthetic and respiratory activities in cyanobacterial thylakoid membranes. Biochim. Biophys. Acta – Bioenerg. 1837, 503–511 (2014).

    CAS  Google Scholar 

  3. Baers, L. L. et al. Proteome mapping of a cyanobacterium reveals distinct compartment organization and cell-dispersed metabolism. Plant Physiol. 181, 1721–1738 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Nevo, R. et al. Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria. EMBO J. 26, 1467–1473 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Liberton, M., Austin, J. R., Berg, R. H. & Pakrasi, H. B. Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography. Plant Physiol. 155, 1656–1666 (2011).

    CAS  PubMed  Google Scholar 

  6. van de Meene, A. M. L., Hohmann-Marriott, M. F., Vermaas, W. F. J. & Roberson, R. W. The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803. Arch. Microbiol. 184, 259–270 (2006).

    CAS  PubMed  Google Scholar 

  7. Rast, A. et al. Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane. Nat. Plants 5, 436–446 (2019).

    CAS  PubMed  Google Scholar 

  8. Mullineaux, C. W. & Liu, L.-N. Membrane dynamics in phototrophic bacteria. Annu. Rev. Microbiol. 74, 633–654 (2020).

    Google Scholar 

  9. Zhao, L.-S. et al. Structural variability, coordination, and adaptation of a native photosynthetic machinery. Nat. Plants 6, 869–882 (2020).

    CAS  PubMed  Google Scholar 

  10. Zak, E. et al. The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes. Proc. Natl Acad. Sci. USA 98, 13443–13448 (2001).

    CAS  PubMed  Google Scholar 

  11. Selão, T. T., Zhang, L., Knoppová, J., Komenda, J. & Norling, B. Photosystem II assembly steps take place in the thylakoid membrane of the cyanobacterium Synechocystis sp. PCC6803. Plant Cell Physiol. 57, 95–104 (2015).

    PubMed  Google Scholar 

  12. Heinz, S., Liauw, P., Nickelsen, J. & Nowaczyk, M. Analysis of photosystem II biogenesis in cyanobacteria. Biochim. Biophys. Acta – Bioenerg. 1857, 274–287 (2016).

    CAS  Google Scholar 

  13. Nixon, P. J., Michoux, F., Yu, J., Boehm, M. & Komenda, J. Recent advances in understanding the assembly and repair of photosystem II. Ann. Bot. 106, 1–16 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Chidgey, J. W. et al. A cyanobacterial chlorophyll synthase-HliD complex associates with the Ycf39 protein and the YidC/Alb3 insertase. Plant Cell 26, 1267–1279 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Rengstl, B., Oster, U., Stengel, A. & Nickelsen, J. An intermediate membrane subfraction in cyanobacteria is involved in an assembly network for Photosystem II biogenesis. J. Biol. Chem. 286, 21944–21951 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Stengel, A. et al. Initial steps of Photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis. Plant Cell 24, 660–675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Frain, K. M., Gangl, D., Jones, A., Zedler, J. A. Z. & Robinson, C. Protein translocation and thylakoid biogenesis in cyanobacteria. Biochim. Biophys. Acta – Bioenerg. 1857, 266–273 (2016).

    CAS  Google Scholar 

  18. Luirink, J. & Sinning, I. SRP-mediated protein targeting: structure and function revisited. Biochim. Biophys. Acta – Mol. Cell Res. 1694, 17–35 (2004).

    CAS  Google Scholar 

  19. Mulo, P., Sakurai, I. & Aro, E.-M. Strategies for psbA gene expression in cyanobacteria, green algae and higher plants: from transcription to PSII repair. Biochim. Biophys. Acta – Bioenerg. 1817, 247–257 (2012).

    CAS  Google Scholar 

  20. Tyystjärvi, T., Herranen, M. & Aro, E.-M. Regulation of translation elongation in cyanobacteria: membrane targeting of the ribosome nascent-chain complexes controls the synthesis of D1 protein. Mol. Microbiol. 40, 476–484 (2001).

    PubMed  Google Scholar 

  21. Skinner, S. O., Sepúlveda, L. A., Xu, H. & Golding, I. Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization. Nat. Protoc. 8, 1100–1113 (2013).

    PubMed  PubMed Central  Google Scholar 

  22. Trautmann, D., Voß, B., Wilde, A., Al-Babili, S. & Hess, W. R. Microevolution in cyanobacteria: re-sequencing a motile substrain of Synechocystis sp. PCC 6803. DNA Res. 19, 435–448 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Mullineaux, C. W. & Sarcina, M. Probing the dynamics of photosynthetic membranes with fluorescence recovery after photobleaching. Trends Plant Sci. 7, 237–240 (2002).

    CAS  PubMed  Google Scholar 

  24. Liu, L.-N. Distribution and dynamics of electron transport complexes in cyanobacterial thylakoid membranes. Biochim. Biophys. Acta – Bioenerg. 1857, 256–265 (2016).

    CAS  Google Scholar 

  25. Liu, L.-N. et al. Control of electron transport routes through redox-regulated redistribution of respiratory complexes. Proc. Natl Acad. Sci. USA 109, 11431–11436 (2012).

    CAS  PubMed  Google Scholar 

  26. Casella, S. et al. Dissecting the native architecture and dynamics of cyanobacterial photosynthetic machinery. Mol. Plant 10, 1434–1448 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Mohamed, A., Eriksson, J., Osiewacz, H. D. & Jansson, C. Differential expression of the psbA genes in the cyanobacterium Synechocystis 6803. Mol. Gen. Genet. MGG 238, 161–168 (1993).

    CAS  PubMed  Google Scholar 

  28. Kulkarni, R. D., Schaefer, M. R. & Golden, S. S. Differential expression of members of a cyanobacterial psbA gene family in response to light. J. Bacteriol. 171, 3973–3981 (1989).

    Google Scholar 

  29. Debus, R. J., Nguyen, A. P. & Conway, A. B. in Current Research in Photosynthesis (ed. Baltscheffsky, M.) 829–832 (Springer Netherlands, 1990); https://doi.org/10.1007/978-94-009-0511-5_191

  30. Schuergers, N. et al. Cyanobacteria use micro-optics to sense light direction. eLife 5, e12620 (2016).

    PubMed  PubMed Central  Google Scholar 

  31. Gustafsson, N. et al. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations. Nat. Commun. 7, 12471 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Salem, K. & van Waasbergen, L. G. Photosynthetic electron transport controls expression of the High Light Inducible gene in the cyanobacterium Synechococcus elongatus strain PCC 7942. Plant Cell Physiol. 45, 651–658 (2004).

    CAS  PubMed  Google Scholar 

  33. Herranen, M., Aro, E. & Tyystja, T. Two distinct mechanisms regulate the transcription of photosystem II genes in Synechocystis sp PCC 6803. Physiol. Plant 112, 531–539 (2001).

    CAS  PubMed  Google Scholar 

  34. Constant, S., Perewoska, I., Alfonso, M. & Kirilovsky, D. Expression of the psbA gene during photoinhibition and recovery in Synechocystis PCC 6714: inhibition and damage of transcriptional and translational machinery prevent the restoration of photosystem II activity. Plant Mol. Biol. 34, 1–13 (1997).

    CAS  PubMed  Google Scholar 

  35. Olive, J., Ajlani, G., Astier, C., Recouvreur, M. & Vernotte, C. Ultrastructure and light adaptation of phycobilisome mutants of Synechocystis PCC 6803. Biochim. Biophys. Acta – Bioenerg. 1319, 275–282 (1997).

    CAS  Google Scholar 

  36. Savage, D. F., Afonso, B., Chen, A. H. & Silver, P. A. Spatially ordered dynamics of the bacterial carbon fixation machinery. Science 327, 1258–1261 (2010).

    CAS  PubMed  Google Scholar 

  37. Faulkner, M. et al. Direct characterization of the native structure and mechanics of cyanobacterial carboxysomes. Nanoscale 9, 10662–10673 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Sun, Y., Wollman, A. J. M., Huang, F., Leake, M. C. & Liu, L.-N. Single-organelle quantification reveals stoichiometric and structural variability of carboxysomes dependent on the environment. Plant Cell 31, 1648–1664 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Sun, Y. et al. Light modulates the biosynthesis and organization of cyanobacterial carbon fixation machinery through photosynthetic electron flow. Plant Physiol. 171, 530–541 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Huang, F. et al. Roles of RbcX in carboxysome biosynthesis in the cyanobacterium Synechococcus elongatus PCC7942. Plant Physiol. 179, 184–194 (2019).

    CAS  PubMed  Google Scholar 

  41. Kanesaki, Y. et al. Identification of substrain-specific mutations by massively parallel whole-genome resequencing of Synechocystis sp. PCC 6803. DNA Res. 19, 67–79 (2011).

    PubMed  PubMed Central  Google Scholar 

  42. Tyystjärvi, T., Sirpiö, S. & Aro, E.-M. Post-transcriptional regulation of the psbA gene family in the cyanobacterium Synechococcus sp. PCC 7942. FEBS Lett. 576, 211–215 (2004).

    PubMed  Google Scholar 

  43. Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).

    CAS  PubMed  Google Scholar 

  44. Nevo-Dinur, K., Nussbaum-Shochat, A., Ben-Yehuda, S. & Amster-Choder, O. Translation-independent localization of mRNA in E. coli. Science 331, 1081–1084 (2011).

    CAS  PubMed  Google Scholar 

  45. Weis, B. L., Schleiff, E. & Zerges, W. Protein targeting to subcellular organelles via mRNA localization. Biochim. Biophys. Acta – Mol. Cell Res. 1833, 260–273 (2013).

    CAS  Google Scholar 

  46. Riediger, M., Spät, P., Bilger, R., Macek, B. & Hess, W. R. Analysis of a photosynthetic cyanobacterium rich in internal membrane systems via gradient profiling by sequencing (Grad-seq). Preprint at https://doi.org/10.1101/2020.07.02.184192 (2020).

  47. Uniacke, J. & Zerges, W. Chloroplast protein targeting involves localized translation in Chlamydomonas. Proc. Natl Acad. Sci. USA 106, 1439–1444 (2009).

    CAS  PubMed  Google Scholar 

  48. Castenholz, R. W. Culturing methods for cyanobacteria. Methods Enzymol. 167, 68–93 (1988).

    CAS  Google Scholar 

  49. Zhang, L., McSpadden, B., Pakrasi, H. B. & Whitmarsh, J. Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803. J. Biol. Chem. 267, 19054–19059 (1992).

    CAS  PubMed  Google Scholar 

  50. Kopf, M. et al. Comparative analysis of the primary transcriptome of Synechocystis sp. PCC 6803. DNA Res. 21, 527–539 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  52. Clerico, E. M., Ditty, J. L. & Golden, S. S. in Circadian Rhythms: Methods and Protocols (ed. Rosato, E.) 155–171 (Humana Press, 2007); https://doi.org/10.1007/978-1-59745-257-1_11

  53. Beyer, H. M. et al. AQUA cloning: a versatile and simple enzyme-free cloning approach. PLoS ONE 10, e0137652–e0137652 (2015).

    PubMed  PubMed Central  Google Scholar 

  54. Ferreira, E. A. et al. Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters. Synth. Biol. 3, ysy014 (2018).

    CAS  Google Scholar 

  55. Pinto, F. L., Thapper, A., Sontheim, W. & Lindblad, P. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria. BMC Mol. Biol. 10, 79 (2009).

    PubMed  PubMed Central  Google Scholar 

  56. Meeks, J. C. & Castenholz, R. W. Growth and photosynthesis in an extreme thermophile, Synechococcus lividus (Cyanophyta). Arch. Mikrobiol. 78, 25–41 (1971).

    CAS  PubMed  Google Scholar 

  57. Schindelin, J. et al. Fiji—an Open platform for biological image analysis. Nat. Methods 9, 676–682 (2009).

    Google Scholar 

  58. Watanabe, S., Sato, M., Nimura-Matsune, K., Chibazakura, T. & Yoshikawa, H. Protection of psbAII transcript from ribonuclease degradation in vitro by DnaK2 and DnaJ2 chaperones of the cyanobacterium Synechococcus elongatus PCC 7942. Biosci. Biotechnol. Biochem. 71, 279–282 (2007).

    CAS  PubMed  Google Scholar 

  59. Nimura, K., Takahashi, H. & Yoshikawa, H. Characterization of the dnaK multigene family in the cyanobacterium Synechococcus sp. strain PCC7942. J. Bacteriol. 183, 1320–1328 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge support from the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska Curie Grant Agreement no. 675006 (SE2B), Biotechnology and Biological Sciences Research Council grant nos. BB/P001807/1 and BB/R003890/1, Biotechnology and Biological Sciences Research Council ALERT17 grant no. BB/R01390X/1, Royal Society University Research Fellowships UF120411 and URF\R\180030 to L.-N.L. and Deutsche Forschungsgemeinschaft Research Training Group MeInBio-322977937/GRK2344 to A.W. and W.R.H. We thank W. Bigott (University of Freiburg) for northern blot analysis, the Liverpool Centre for Cell Imaging for technical assistance and equipment provision, and J. Trinugoho and P. Nixon (Imperial College, London) for supplying the Synechocystis psbA triple mutant (originally from R. Debus, University of California Riverside).

Author information

Authors and Affiliations

  1. School of Biological and Chemical Sciences, Queen Mary University of London, London, UK

    Moontaha Mahbub, Yuxiao Yang, Ramanpreet Kaur, Helder Carmen, Christoph Engl & Conrad W. Mullineaux

  2. Department of Botany, Jagannath University, Dhaka, Bangladesh

    Moontaha Mahbub

  3. Institute of Biology III, University of Freiburg, Freiburg, Germany

    Luisa Hemm, Matthias Riediger, Annegret Wilde & Wolfgang R. Hess

  4. Institute of Integrative Biology, University of Liverpool, Liverpool, UK

    Tuomas Huokko & Lu-Ning Liu

  5. Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan

    Satoru Watanabe

  6. College of Marine Life Sciences, and Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao, China

    Lu-Ning Liu

Authors
  1. Moontaha Mahbub
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luisa Hemm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuxiao Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ramanpreet Kaur
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Helder Carmen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christoph Engl
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tuomas Huokko
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Matthias Riediger
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Satoru Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lu-Ning Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Annegret Wilde
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Wolfgang R. Hess
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Conrad W. Mullineaux
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.M. carried out the bulk of the experimental work and data analysis. L.H., M.R., S.W. and W.R.H. generated and characterized RBP mutants and analysed RBP-mRNA association. Y.Y. and R.K. carried out cpcAB and rbcL FISH measurements. H.C. assisted with data analysis. C.E. helped to establish the RNA–FISH technique in the laboratory and discussed data. T.H. and L.-N.L. performed super-resolution microscopy and data analysis and provided the rbcL-gfp mutant. A.W. designed and analysed northern blot hybridization experiments and discussed data. C.W.M. and M.M. devised the study and wrote the paper, with input from all authors.

Corresponding author

Correspondence to Conrad W. Mullineaux.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Benjamin D. Engel, Jörg Nickelsen, Wim Vermaas and Wojciech Wietrzynski for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Background fluorescence in fixed cells of Synechocystis and Synechococcus.

a, Confocal fluorescence images of photosynthetic pigments in Synechocystis cells showing thylakoid membrane organisation without and with the fixation and permeabilisation used for mRNA-FISH probing. b, statistics for cell diameter in fixed vs live cells (n = 50 cells: no adjustments for multiple comparisons). c, Confocal fluorescence images of photosynthetic pigments in Synechococcus cells showing thylakoid membrane organisation in live vs. fixed cells. d, e, statistics for cell width and length in fixed vs live cells (n = 50 cells: no adjustments for multiple comparisons). f, g, Confocal micrographs of Synechocystis (f) and Synechococcus (g) cells (fixed and permeabilised but not probed) showing the background signal (green) in the TAMRA detection channel in comparison to fluorescence from the photosynthetic pigments (TM, in red). Line profiles drawn from representative cells of both Synechocystis (f. i, ii) and Synechococcus (g, i) confirm that the background signal (green line) colocalises with the TM (black dashed line). TAMRA channel shown without background correction. Images are representative of at least 2 independent experiments. Error bars in the box plots indicate the range of values recorded, the centre line shows the median and the box spans the interquartile range. p values are from unpaired two-tailed Student’s t-tests. Scale bars: 2 µm.

Extended Data Fig. 2 Correcting for background fluorescence in RNA-FISH micrographs.

Step-by-step illustration of the procedure used to subtract background fluorescence from the FISH images, illustrated with psbA mRNA in Synechocystis as an example. a, demonstration that the signal in the TAMRA channel is a predictable fraction of the signal in the TM channel in unprobed cells (n = 50 cells); b, use of this principle to remove background signal from FISH images in probed cells. Images are representative of at least 2 independent experiments.

Extended Data Fig. 3 Controls for specificity of psbA mRNA-labelling in Synechocystis.

a, Variation in psbA mRNA signal intensity between WT grown under standard conditions (first row) and three control samples: unprobed, ΔpsbA2 mutant (probed with psbA2 mRNA probes) and high light (HL: 600 µmol photons m-2s-1, 1 hour) -treated cells similarly probed. Micrographs showing the FISH channel (green) vs. TM (magenta). b, Mean intensity of the mRNA signal in the different samples. Analysis was done after smoothing the images (below optical resolution) and correcting the background signal in the FISH detection channel (detail in Methods): n = 100 cells; p=4x10-27 for WT probed vs unprobed, 5x10-9 for ΔpsbA2 vs WT, 4x10-5 for HL vs normal growth. c, Confirmation of full segregation status of ΔpsbA2 mutant by PCR amplification using the primer pair illustrated at the right hand side of the gel image (detail in Methods); d, Micrographs showing the triple psbA knockout29 ± psbA probe; e, Quantification of FISH signal from the triple psbA knockout ± psbA probe (n = 100 cells; p= 0.9). Analysis was done after smoothing the images (below optical resolution) and correcting the background signal in the FISH channel (detail in Methods). Images are representative of at least 2 independent experiments. Error bars in the box plots indicate the range of values recorded, the centre line shows the median and the box spans the interquartile range, n: number of cells measured, *: significant difference from the untreated cells, at p< 0.001, measured by unpaired two-tailed Student’s t-test; ns= p-value non-significant; scale bars: 2µm.

Extended Data Fig. 4 Controls for specificity of psbA mRNA-labelling in Synechococcus.

a, Comparison of psbA mRNA signals in cells grown under standard conditions (first row) and unprobed and rifampicin-treated cells. Micrographs of the mRNA FISH signal (green) and TM (magenta). b, Mean fluorescence intensity per cell of the mRNA signal in cells from the different samples: p=6x10-41 for probed vs unprobed, 5x10-40 for rifampicin vs untreated. Analysis was done after smoothing the images (below optical resolution) and correcting the background signal in the FISH channel (detail in Methods). Error bars in the box plots indicate the range of values recorded, the centre line shows the median and the box spans the interquartile range. Images are representative of at least 2 independent experiments. n: number of cells measured, *: significant difference, at p< 0.001, measured by unpaired two-tailed Student’s t-test; scale bar: 2µm.

Extended Data Fig. 5 Localisation of psbA FISH signals relative to thylakoid membrane (TM) and nucleoids (DAPI-stained) in Synechocystis and Synechococcus.

a, psbA mRNA in Synechocystis relative to TM and DAPI. Line profiles (a, i-ii) across representative cells show the distribution of TM, FISH and DAPI signals in Synechocystis. b, psbA mRNA in Synechococcus relative to TM and DAPI. Line profiles (b, i-ii) across the short axis of representative cells show the distribution of TM, FISH and DAPI signals in Synechocystis. Images are representative of at least 2 independent experiments. Scale bars: 2µm.

Extended Data Fig. 6 Effects of puromycin and lincomycin treatments.

a, b, Controls showing that puromycin and lincomycin treatments do not increase background autofluorescence. Confocal fluorescence micrographs and mean fluorescence intensity per cell of the background signal in the TAMRA channel for unprobed cells of (a) Synechocystis and (b) Synechococcus, showing similar intensity of the background autofluorescence with and without inhibitor treatment. Error bars in the box plots indicate the range of values recorded, the centre line shows the median and the box spans the first to third quartiles; none of the differences are significant. n: number of cells measured; Scale bars: 2 µm. c, d, Effects of puromycin on growth of Synechococcus and Synechocystis cells. Ten-fold serial dilutions of three independent cultures were spotted on BG11 plates ± puromycin (50 μg/ml). Plates were photographed after seven days of growth. Images are representative of 2 independent experiments.

Extended Data Fig. 7 Effects of ribosome inhibitors on psbA2 and psaA transcript levels.

RNA isolated from Synechocystis cells treated with lincomycin or puromycin was separated by electrophoresis, blotted onto a nylon membrane and hybridized with radioactively labeled probes against the 5’ UTR of psbA2 mRNA and psaA. A probe against the RNA subunit of the ribonuclease P (RnpB) was used as a control. a, b, Representative gel images. See Source Data for full-length gel images. Results shown are representative of 2 biological replicates, each with 2 technical replicates. c, d, Plots showing psbA2 (c) and psaA (d) mRNA levels (normalised to the RnpB signal) in inhibitor-treated cells relative to untreated cells, combining data from all 4 replicates. Error bars in the box plots indicate the range of values recorded, the centre line shows the median and the box spans the first to third quartiles. The puromycin-treated sample hybridized with psaA is not significantly different from the untreated sample (p = 0.245). Other differences are all significant at p < 0.05 (p = 0.036 for psbA2/lincomycin; 0.021 for psbA2/puromycin; 0.009 for psaA/lincomycin), measured by unpaired two-tailed Student’s t-tests.

Extended Data Fig. 8 Controls for specificity of psaA mRNA labelling in Synechocystis.

a, Variation in psaA mRNA signal intensity between cells grown under standard conditions (first row) and 3 control samples: unprobed, high light (HL)-treated cells (600 µmol photons m-2s-1, 1 hour) and Rifampicin-treated (400 µg ml-1, 1 hour) cells. Confocal fluorescence micrographs showing FISH signal in green and TM in magenta. An overlay between the DAPI-stained nucleoid region (blue) and FISH signal (yellow) is shown for rifampicin-treated cells. Line profiles from representative cells are shown below the corresponding micrographs. b, comparison of mRNA FISH signals per cell in the different conditions (p=3x10-29 for probed vs unprobed, 8x10-25 for HL vs normal growth and 2x10-9 for rifampicin vs untreated). Images smoothed (below optical resolution) and corrected for the background signal in the FISH channel. Error bars in the box plots indicate the range of values recorded, the centre line shows the median and the box spans the first to third quartiles. n: number of cells measured, *: significant difference from the untreated cells, at p< 0.001, measured by unpaired two-tailed Student’s t-test; scale bars: 2µm.

Extended Data Fig. 9 Controls to demonstrate specificity of psaA mRNA labelling in Synechococcus.

a, Variation in psaA mRNA signal intensity between cells grown under standard conditions (first row) and the three control samples: unprobed, Rifampicin-treated (400 µg ml-1, 1 hour) and high light (HL)-treated cells (600 µmol photons m-2s-1, 1 hour). Confocal fluorescence micrographs showing FISH signal in green and thylakoid membrane region in magenta. b, Comparison of mRNA signal in the control samples compared with the experimental sample. Images smoothed (below optical resolution) and corrected for the background signal in the FISH channel (p=2x10-50 for probed vs unprobed; 8x10-42 for rifampicin vs untreated; 8x10-44 for HL vs normal growth). Error bars in the box plots indicate the range of values recorded, the centre line shows the median and the box spans the interquartile range. Images are representative of at least 2 independent experiments. n: number of cells measured, *: significant difference from the untreated cells, at p< 0.001, measured by unpaired two-tailed Student’s t-test; scale bars: 2µm.

Extended Data Fig. 10 Synechocystis Rbp3 location and photosynthetic phenotype of the ∆rbp2/3 mutant.

a, Western blotting showing that Rbp3 is associated with the membrane fraction. See Source Data for full-length gel images with molecular weight markers. Data shown are representative of 2 biological replicates with similar results. b, growth curves for ∆rbp2/3 vs. the wild-type (GT-I) background in different conditions: 50 µmol photons m-2s-1 (low light), 80 µmol photons m-2s-1 (high light), or low light in the presence of 10 mM glucose. Error bars indicate standard deviations from 3 replicate cultures. c, absorption spectra of cell suspensions, normalised to turbidity at 750 nm. ∆rbp2/3 has lower pigment per OD750. d, fluorescence emission spectra at 77K with chlorophyll excitation (435 nm) normalised to PSI emission at 725 nm. e, fluorescence emission spectra at 77K with phycocyanin excitation (600 nm) normalised to phycobilin emission at 660 nm. ∆rbp2/3 has lower PSI emission (725 nm) relative to PSII (peaks at 685 and 695 nm). All spectra are representative of similar results from 3 independent cultures.

Supplementary information

Source data

Source Data Extended Data Fig. 7

Full-length gel images.

Source Data Extended Data Fig. 10

Full-length gel images with molecular weight markers.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mahbub, M., Hemm, L., Yang, Y. et al. mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria. Nat. Plants 6, 1179–1191 (2020). https://doi.org/10.1038/s41477-020-00764-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-020-00764-2

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing