Figure 1.
Life and death of an endosymbiotic cyanobacterium.
(A–G) Cyanobacteria isolated from leaf cavities of various developmental stages of the water fern Azolla microphylla. The leaves of the Azolla frond (∼1.5 cm long) are numbered along the main plant axis starting at the apex (left panel). Isolated cyanobacteria are visualized using bright field (middle panel) and fluorescence (right panel) microscopy (healthy cells fluoresce red). (A) Filaments of small-celled motile hormogonia, functioning as plant colonizing units, at the plant apex. Note the partly lysed cells lacking fluorescence, indicative of dead/dying cells (arrows). (B) Heterocystous filament in leaf no. 5. Dead vegetative cells (arrows) characterized by a reduction in cell volume and weak fluorescence. (C) Heterocystous filament in leaf no. 10 with dying vegetative cells (arrows), characterized by retraction of the cellular content from the cell wall and vacuolization (faint blue fluorescence). (D) Heterocystous filament in leaf no. 15, with a dead heterocyst fluorescing green (arrow). (E) Heterocystous filament in leaf no. 20, with a dying heterocyst (arrow) and differentiating pro-akinetes. (F) Heterocystous filament in leaf no. 25. The dying heterocysts have ruptured cell walls and are leaking cellular contents (blue). (G) Heterocystous filament in leaf no. 30 with a swollen and chlorotic dying akinete fluorescing green. Abbreviations: v, vegetative cell; h, heterocyst; pa, pro-akinete; a, akinete. (H–I) Diagram showing the percentage (H) and proportion of cell types (I) of living and dead cells in the leaf cavities along the main stem of Azolla fronds. Abbreviations: v, vegetative cell; h, heterocyst; a, akinete; pa, pro-akinete. Scale bars: 10 µm.
Figure 2.
Early stages of cyanobacterial cell death visualized by the Annexin V-EGFP assay.
The Annexin V-EGFP assay emits green fluorescence when phosphatidylserine (PS) is exposed at the outer membrane. (A) Cyanobacterial filaments (leaf no. 20) showing a distinct green Annexin V-EGFP signal at the vegetative cell plasma membrane (arrow) and on the external envelope of the heterocysts and akinete. (B) Magnification of the granulated akinete (boxed in A) under bright field LM (left) and fluorescence microscopy (right), showing the Annexin V-EGFP label (arrows). (C) A thick-walled heterocyst under bright field LM (left) and fluorescence microscopy (right), showing Annexin V-EGFP label and a ruptured cell wall (arrows). (D) A non-affected cyanobacterial filament (leaf no. 25) and a large granulated akinete displaying an Annexin V-EGFP signal (right) and leakage of cellular contents (arrows). (E) Cyanobacterial filaments (leaf no. 30) with green Annexin V-EGFP fluorescence (arrows) at heterocysts and the akinete. (F) Pro-akinetes in a sporocarp with an Annexin V-EGFP signal and a retracted cytoplasm (arrow). Abbreviations: a, akinete; h, heterocyst; v, vegetative cell. Scale bars: A-B 5 µm; C 4 µm; D-F 10 µm.
Figure 3.
Later stage cyanobacterial cell death visualized by the TUNEL assay.
The TUNEL assay emits green fluorescence in response to DNA fragmentation. (A) Hormogonium filaments at the plant apex, with groups of cells showing a positive TUNEL reaction (arrows). Non-affected cells exhibit red autofluorescence. (B) Chains of pro-akinetes in a megasporocarp which exhibits green fluorescence in apoptotic-like (arrow) and necrotic-like (double arrows) cells, the latter showing leaking cytoplasms. (C) A heterocytous filament (leaf no. 15) showing green TUNEL fluorescence in vegetative cells entering the later stages of the cellular death process: i) ∼10% of the DNA is fragmented (dots) and faintly stained in green-red, due to the red autofluorescence of the background pigmentation; ii) ∼50% of the DNA is stained and the fluorescing dots are larger; iii) ∼80% of the DNA is intensively stained; and ix) the DNA fragmentation is complete and the green fluorescence smeared (i.e., the cells are dead). (D) Positive control: homogonium filaments pre-treated with DNase I show a positive TUNEL reaction in all cells. (E) Negative control: chains of pro-akinetes with all cells displaying red fluorescence after omitting TdT, which detects the nicked DNA. Scale bars: A-E 10 µm; i-iv in C 1 µm.
Figure 4.
Actin distribution pattern in healthy cyanobacteria.
(A) Bright field light microscopy of vegetative cells not affected by cell death. (B) DAPI (DNA) stained cells (UV filter set). (C) Autofluorescence by phycobiliproteins (green light filter set). (D) The cellular meshed network of an actin-like cytoskeleton is visualized after staining with phalloidin and is apparent as a green fluorescence in healthy vegetative cells (blue light filter set). V, vegetative cells; H, heterocyst, Scale bars: 1 µm.
Figure 5.
Cellular actin patterns in heterocysts passing through four modes of cell death.
(A–D) Healthy heterocyst displaying phalloidin stained (PS) actin seen as a meshed network covering the cell (arrows), and a strong blue and red fluorescence of DNA (DAPI) and autofluorescing (AF) photosynthetic pigments, respectively. (E–H) An apoptotic-like heterocyst with a shrunken cytoplasm (long arrows) and a polygonal partly disorganized actin pattern (short arrow) and weaker DAPI- and autofluorescence. (I–L) An autophagic-like heterocyst with a retracted cytoplasm but with an intact plasma membrane (short arrow) and a condensed but mostly retained actin pattern (long arrow) and strong DAPI- and autofluorescence. (M–P) A necrotic-like heterocyst with a disruptered cell wall and plasma membrane (black arrow) resulting in leakage of cellular contents (DNA and pigments), and a more or less disrupted actin (long white arrow). (Q–T) An autolytic-like degraded heterocyst lacking most cellular inclusions including the actin network. A, E, I, M and P, bright field; B, F, J, N and R, phalloidin staining; C, G, K, O and S, DAPI-DNA staining; D, H, L, P and T, pigment autofluorescence. Scale bars: 1 µm.
Figure 6.
Multiple cell death modes in the cyanobacterium visualized by TEM.
Four death modes are illustrated at an early, intermediate and late stage. Figures S3, S4 and S6A depict the ultrastructural appearance of healthy cyanobacterial cell types. The boxed and magnified cell wall area in the TEM micrographs is given to the right of each micrograph. (A–C) Apoptotic-like cell death mode: (A) Cell shrinkage commences and the cellular content is slightly reduced. (B) Cytoplasmic shrinkage continues and the cell shape is irregular and invaginated, while the cell wall and cytoplasmic inclusions remain intact. (C) Dendritic-like cell morphology and loss of cellular contents are apparent at the later stage, but the cell wall, including the peptidoglycan layer, is still intact (arrow). Peptidoglycan layer intact (arrows). (D–F) Autophagic-like cell death mode: (D) The cell is swollen, while the cytoplasm starts to condense and undergo vacuolization. The outer membrane and peptidoglycan layer are intact. (E) The plasma membrane ruptures and continued cytoplasm degradation. The outer membrane and the peptidoglycan layer are still intact (arrow). (F) The outer cell shape is retained, while the cellular content and the peptidoglycan layer disappear. (G–I) Necrotic-like cell death mode, type I (affects vegetative cells): (G) The cell shape is distorted, the cell wall disrupted and the cellular content released. The peptidoglycan layer is preserved (arrow). (H) Continued disintegration of cellular contents and disappearance of the peptidoglycan layer. (I) Only partial membranes (thylakoids) remain at this stage. (J–L): Necrotic-like cell death mode, type II (affects akinetes and heterocysts): death progression as for type I above, but the outer cell envelope of the cells remains until the late stage. (M–O) Autolytic-like cell death mode: (M) The degrading cytoplasm is surrounded by a unit membrane only, and no peptidoglycan layer. (N) The membrane continues to dissolve, as does the intracellular content. (O) The membrane vesiculates and the cytoplasm disintegrates and leaks out. Abbreviations: b, bacterium; cg, cyanophycin granule; cs, carboxysome; en, envelope; t, thylakoid membrane. Scale bars: A-O: 1 µm; in boxed area 50 nm.
Figure 7.
Cyanobacterial cells death modes along the plant developmental gradient.
The apoptosis- and autophagy-like death modes dominate the initial plant developmental stages, while the autolysis- and necrotic-like dominate at later developmental stages. aup: autophagy-like; ap:, apoptosis-like; aut: autolysis-like; nec: necrotic-like.
Figure 8.
Stresses enhance cell death in the cyanobacterium.
(A) Percentage of cyanobacterial cells affected by the various death modes identified in relation to two plant developmental stages. (B) Cyanobacterial filaments (leaf no. 13) showing a strong Annexin V-EGFP signal in most cells and in all cell types, indicating a strong enhanced cell death in the majority of cells. C, control cells; DK, dark treated; ND, nutrient deprived; RT, radiation treated. v, vegetative cells; h, heterocysts; A, akinetes. Scale bar: 10 µm.
Figure 9.
Amplification of cyanobacterial genomic DNA and sequence alignments.
(A) PCR products FJ541186, FJ541185 and FJ541183 amplified from cyanobacterial DNA with primer pairs cas203, cas202 and HCW3/4, respectively (Methods). M, molecular weight. (B) Multiple sequence alignment of the partial protein sequence ACM79336.1, the predicted translation product of FJ541186, and significant homologs in cyanobacteria, plants and fungi. The numbers above the alignment and in the margins denote alignment position and residue position ranges for each sequence, respectively. The modal consensus is shown below the alignment. Residue color indicates hydrophobicity (from red [hydrophobic] to blue [hydrophilic]) and the shading intensity of a column is proportional to conservation (at 30 percent cutoff). The conserved caspase domain (pfam00656) spans this region of the alignment. The conserved catalytic His-Cys dyad is replaced with Y-S in the cyanobacterial sequences (indicated by arrows).
Table 1.
Comparison between cell death modes in animals, plants and cyanobacteria.