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

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.

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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.

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.

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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).

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Authors

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.

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Correspondence to Conrad W. Mullineaux.

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The authors declare no competing interests.

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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.

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Supplementary Tables 1–6.

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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.

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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

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