mRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120

Abstract

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

Keywords: RNA binding protein; cyanobacteria; heterocyst; mRNA; membrane proteins; thylakoid membrane.

Fig 1

Cartoon to illustrate the layout of thylakoid membranes in Anabaena. The cell on the left is a heterocyst, and the cell on the right is a vegetative cell. During heterocyst differentiation, thylakoid membranes (shown in green) are remodeled to form two distinct domains: P thylakoids (peripheral thylakoids) and H thylakoids (honeycomb thylakoids). The heterocyst-specific Cox2 and Cox3 terminal oxidases are located in the H thylakoids in mature heterocysts (6–8). CPG, cyanophycin granule; HGL, heterocyst specific glycolipid; HEP, heterocyst envelope polysaccharide; OM, outer membrane; PM, plasma membrane. Figure created in BioRender.com

Fig 2

Localization of photosynthetic mRNAs in Anabaena. Confocal images showing TAMRA-labelled FISH probes for psaA and psbA in green, photosynthetic pigments in the thylakoids (TM) in magenta. Cells were grown in BG11 medium. Scale bars: 3 µm. (A) Comparison of psaA and psbA signals; (B) effect of puromycin on the psbA signal, with averaged line profiles across 20 cells shown below.

Fig 3

Effect of nitrogen step-down on psbA and cox2 mRNAs. Confocal images showing TAMRA-labelled FISH probes for psbA in green, photosynthetic pigments in the thylakoids in magenta. Cells were fixed at 6 h or 8 h after transfer to BG11₀ medium. Arrows indicate likely pro-heterocysts. Scale bars: 3 µm.

Fig 4

Localization of cox mRNAs and oxidase activity in developing Anabaena heterocysts. Confocal images showing TAMRA-labelled FISH probes for cox2 and cox3 in green, photosynthetic pigments in the thylakoids in magenta. Arrows indicate pro-heterocysts or heterocysts. (A) cox2 and cox3 mRNA localization 12 h after transfer to BG11₀ medium. (B) Location of cox2 mRNA (top panel) and oxidase activity from diaminobenzidine (DAB) staining (bottom panel) at different times after transfer to BG11₀ medium. A line profile of the cox2 mRNA signal along the long axis of a heterocyst is shown below. (C) Oxidase activity was revealed by DAB staining in heterocysts of wild-type Anabaena and the Δcox2cox3 and ΔfraH mutants. Cells were collected after 24 h in BG11₀ medium. Scale bars: 3 µm.

Fig 5

Phenotype of the ΔrbpG mutant. (A) Confocal images of PC and Chl fluorescence. Scale bars: 3 µm. (B) Mean cellular PC and Chl fluorescence intensities in the wild type and the ΔrbpG mutant (n = 100 cells). (C) Absorption spectra normalized to turbidity at 750 nm (averaged from three biological replicates). (D) 77K fluorescence emission spectra at 77K with 435 nm (Chl) and 600 nm (PC) excitation, normalized to the Photosystem I peak (averaged from three biological replicates). (E) Cell dimensions: length (L), width (W), and L:W ratio (n = 100 cells). **** indicates statistical significance (P < 0.0001).

Fig 6

Thylakoid membrane conformation of the ΔrbpG mutant at higher resolution. (A) Super-resolution (SIM²) imaging of chlorophyll fluorescence in Anabaena wild type and ΔrbpG cells. The arrows highlight dense spots of chlorophyll fluorescence. Scale bars: 2 µm. (B) Representative thin-section electron micrographs of Anabaena wild type and ΔrbpG cells. (C) Quantitation of a number of thylakoid membrane layers from thin-section electron micrographs (30 cells of each strain). **** indicates statistical significance (P < 0.0001). (D) Representative examples of thin-section electron micrographs of ΔrbpG cells that show highly curved and condensed thylakoid membrane regions that may correspond to the bright patches seen in fluorescence micrographs (arrows highlight these regions). Scale bars: 2 µm.

Fig 7

Effects of the ΔrbpG mutation on mRNA localization and expression. TAMRA FISH signals are shown in green and fluorescence from the photosynthetic pigments in magenta. All scale bars: 3 µm. (A) Localization and expression of three photosynthetic mRNAs in ΔrbpG vs wild-type Anabaena (cells grown in standard BG11 medium). (B) Mean cellular fluorescence intensity of FISH signals for the three photosynthetic mRNAs in wild type vs ΔrbpG. n = Number of cells measured; **** = significant difference with P < 0.0001; ** = significant difference with P < 0.01. (C) cox2 mRNA FISH signals in wild-type Anabaena and ΔrbpG (cells fixed 16 h after transfer to BG11₀ medium).

Fig 8

PSII activity and efficiency of the PSII repair cycle in Anabaena wild type and ΔrbpG. O₂ evolution was measured at saturating light in the presence of excess PSII electron acceptors. Cells were exposed to high light (HL) for 20 min, then returned to growth light (GL). The bars indicate rates of O₂ evolution per chlorophyll, and the percentages above indicate rates relative to the initial rate before exposure to HL. All values are means from three technical replicates, with SD indicated by the error bars.