Germanium dioxide treated and non-treated field samples.

<p>Mean of BMAA concentration (±STD) in different samples of diatoms.</p><p><i>n</i>: Biological replicates.</p

Quantification of BMAA in diatoms.

<p>Mean of BMAA concentration (±STD) in different samples of diatoms.</p><p><i>n</i>: Biological replicates.</p

Multiple Modes of Cell Death Discovered in a Prokaryotic (Cyanobacterial) Endosymbiont

<div><p>Programmed cell death (PCD) is a genetically-based cell death mechanism with vital roles in eukaryotes. Although there is limited consensus on similar death mode programs in prokaryotes, emerging evidence suggest that PCD events are operative. Here we present cell death events in a cyanobacterium living endophytically in the fern <i>Azolla microphylla,</i> suggestive of PCD. This symbiosis is characterized by some unique traits such as a synchronized development, a vertical transfer of the cyanobacterium between plant generations, and a highly eroding cyanobacterial genome. A combination of methods was used to identify cell death modes in the cyanobacterium. Light- and electron microscopy analyses showed that the proportion of cells undergoing cell death peaked at 53.6% (average 20%) of the total cell population, depending on the cell type and host developmental stage. Biochemical markers used for early and late programmed cell death events related to apoptosis (Annexin V-EGFP and TUNEL staining assays), together with visualization of cytoskeleton alterations (FITC-phalloidin staining), showed that all cyanobacterial cell categories were affected by cell death. Transmission electron microscopy revealed four modes of cell death: apoptotic-like, autophagic-like, necrotic-like and autolytic-like. Abiotic stresses further enhanced cell death in a dose and time dependent manner. The data also suggest that dynamic changes in the peptidoglycan cell wall layer and in the cytoskeleton distribution patterns may act as markers for the various cell death modes. The presence of a metacaspase homolog (domain p20) further suggests that the death modes are genetically programmed. It is therefore concluded that multiple, likely genetically programmed, cell death modes exist in cyanobacteria, a finding that may be connected with the evolution of cell death in the plant kingdom.</p></div

BMAA in axenic diatom cultures.

<p>¹ Culture Collection of Algae and Protozoa.</p><p>D: Detected.</p><p><i>n</i>: Biological replicates.</p

LC-MS/MS chromatograms of (a) BMAA and its isomer standards (5 µg L^–1 for BAMA, BMAA, and AEG and 20 µg L^–1 for DAB), and chromatograms showing BMAA produced by axenic cultures of diatoms; (b) <i>Achnanthes</i> sp. CCAP 1095/1; (c) <i>Navicula pelliculosa</i> CCAP 1050/9; (d) <i>Skeletonema marinoi</i> SAAAE 08603; (e) <i>Skeletonema marinoi</i> ST28; (f) <i>Thalassiosira</i> sp. CCAP 1085/15; (g) <i>Proboscia inermis</i> CCAP 1064.

<p>LC-MS/MS chromatograms of (a) BMAA and its isomer standards (5 µg L^–1 for BAMA, BMAA, and AEG and 20 µg L^–1 for DAB), and chromatograms showing BMAA produced by axenic cultures of diatoms; (b) <i>Achnanthes</i> sp. CCAP 1095/1; (c) <i>Navicula pelliculosa</i> CCAP 1050/9; (d) <i>Skeletonema marinoi</i> SAAAE 08603; (e) <i>Skeletonema marinoi</i> ST28; (f) <i>Thalassiosira</i> sp. CCAP 1085/15; (g) <i>Proboscia inermis</i> CCAP 1064.</p

Actin distribution pattern in healthy cyanobacteria.

<p>(<b>A</b>) Bright field light microscopy of vegetative cells not affected by cell death. (<b>B</b>) DAPI (DNA) stained cells (UV filter set). (<b>C</b>) Autofluorescence by phycobiliproteins (green light filter set). (<b>D</b>) 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.</p

Amplification of cyanobacterial genomic DNA and sequence alignments.

<p>(<b>A</b>) PCR products FJ541186, FJ541185 and FJ541183 amplified from cyanobacterial DNA with primer pairs cas203, cas202 and HCW3/4, respectively (Methods). M, molecular weight. (<b>B</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).</p

Cellular actin patterns in heterocysts passing through four modes of cell death.

<p>(<b>A–D</b>) 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. (<b>E–H</b>) An apoptotic-like heterocyst with a shrunken cytoplasm (long arrows) and a polygonal partly disorganized actin pattern (short arrow) and weaker DAPI- and autofluorescence. (<b>I–L</b>) 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. (<b>M–P</b>) 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). (<b>Q–T</b>) 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.</p

Comparison between cell death modes in animals, plants and cyanobacteria.

<p>Definitions of PCD modes according to reference 24, and 1, 4, 6–8, 23 and 37.</p>a)<p>Au-vacuoles, autophagy vacuoles;</p>b)<p>ND, Not determined;</p>c)<p>-, unknown.</p

Multiple cell death modes in the cyanobacterium visualized by TEM.

<p>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. (<b>A–C</b>) <i>Apoptotic-like cell death mode</i>: (<b>A</b>) Cell shrinkage commences and the cellular content is slightly reduced. (<b>B</b>) Cytoplasmic shrinkage continues and the cell shape is irregular and invaginated, while the cell wall and cytoplasmic inclusions remain intact. (<b>C</b>) 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). (<b>D–F</b>) <i>Autophagic-like cell death mode:</i> (<b>D</b>) The cell is swollen, while the cytoplasm starts to condense and undergo vacuolization. The outer membrane and peptidoglycan layer are intact. (<b>E</b>) The plasma membrane ruptures and continued cytoplasm degradation. The outer membrane and the peptidoglycan layer are still intact (arrow). (<b>F</b>) The outer cell shape is retained, while the cellular content and the peptidoglycan layer disappear. (<b>G–I</b>) <i>Necrotic-like cell death mode</i>, <i>type I</i> (affects vegetative cells): (<b>G</b>) The cell shape is distorted, the cell wall disrupted and the cellular content released. The peptidoglycan layer is preserved (arrow). (<b>H</b>) Continued disintegration of cellular contents and disappearance of the peptidoglycan layer. (<b>I</b>) Only partial membranes (thylakoids) remain at this stage. (<b>J–L</b>): <i>Necrotic-like cell death mode, type II</i> (affects akinetes and heterocysts): death progression as for type I above, but the outer cell envelope of the cells remains until the late stage. (<b>M–O</b>) <i>Autolytic-like cell death mode:</i> (<b>M</b>) The degrading cytoplasm is surrounded by a unit membrane only, and no peptidoglycan layer. (<b>N</b>) The membrane continues to dissolve, as does the intracellular content. (<b>O</b>) 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.</p