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. 2015 Nov;56(11):2259-70.
doi: 10.1093/pcp/pcv139. Epub 2015 Sep 26.

Localization and Quantification of Callose in the Streptophyte Green Algae Zygnema and Klebsormidium: Correlation With Desiccation Tolerance

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Free PMC article

Localization and Quantification of Callose in the Streptophyte Green Algae Zygnema and Klebsormidium: Correlation With Desiccation Tolerance

Klaus Herburger et al. Plant Cell Physiol. .
Free PMC article

Abstract

Freshwater green algae started to colonize terrestrial habitats about 460 million years ago, giving rise to the evolution of land plants. Today, several streptophyte green algae occur in aero-terrestrial habitats with unpredictable fluctuations in water availability, serving as ideal models for investigating desiccation tolerance. We tested the hypothesis that callose, a β-d-1,3-glucan, is incorporated specifically in strained areas of the cell wall due to cellular water loss, implicating a contribution to desiccation tolerance. In the early diverging genus Klebsormidium, callose was drastically increased already after 30 min of desiccation stress. Localization studies demonstrated an increase in callose in the undulating cross cell walls during cellular water loss, allowing a regulated shrinkage and expansion after rehydration. This correlates with a high desiccation tolerance demonstrated by a full recovery of the photosynthetic yield visualized at the subcellular level by Imaging-PAM. Furthermore, abundant callose in terminal cell walls might facilitate cell detachment to release dispersal units. In contrast, in the late diverging Zygnema, the callose content did not change upon desiccation for up to 3.5 h and was primarily localized in the corners between individual cells and at terminal cells. While these callose deposits still imply reduction of mechanical damage, the photosynthetic yield did not recover fully in the investigated young cultures of Zygnema upon rehydration. The abundance and specific localization of callose correlates with the higher desiccation tolerance in Klebsormidium when compared with Zygnema.

Keywords: Aero-terrestrial green algae; Cell wall; Evolutionary biology; Imaging-PAM; Phylogeny; Terrestrialization.

Figures

Fig. 1
Fig. 1
Comparison of the callose content in two Zygnema and Klebsormidium strains (control and desiccated for 30 and 210 min) determined by colorimetric quantification (n = 4 ± SD). Callose content is expressed in µg of pachyman equivalents per mg of algal dry mass. Significant differences between groups are indicated by lower case letters. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test (P < 0.001).
Fig. 2
Fig. 2
Confocal laser scanning micrographs of Zygnema S (A, B), Zygnema E-A (C, D), Klebsormidium crenulatum (E–G) and Klebsormidium nitens (H–J). Controls (A, C, E, H) and desiccated (30 min at ambient humidity; B, D, F, G, I, J) algal cell filaments. Chl autofluorescence is shown in red. (A) Protoplasts closely attached to the cell wall, two stellate chloroplasts and a centrally located nucleus per cell (arrow). (B) Longitudinal cell walls convexly expanded with conspicuous undulations, cross cell walls not deformed; protoplasts retracted from the longitudinal cell walls. (C) Hydrated cells with two stellate chloroplasts and central nuclei. (D) Retracted protoplasts and deformed longitudinal cell walls. (E) Hydrated cells with parietal single chloroplasts, protuberances in cross walls (arrows). (F) Desiccated filament with reduced diameter, undulated cross cell walls (arrowhead), protoplasts closely attached to the cell wall (arrows). (G) The longitudinal cell walls appear frayed. (H) Protuberances of wall material in the cross cell walls are indicated by arrows. (I) Desiccated filament with reduced diameter. (J) Frayed longitudinal cell walls. Scale bar = 10 µm (A–D); 5 µm (E–J).
Fig. 3
Fig. 3
Light and corresponding Aniline blue-stained fluorescence (callose) micrographs of Zygnema S (A, B), Zygnema E-A (C, D), Klebsormidium crenulatum (E) and Klebsormidium nitens (F). (A) Callose in the cell corners between individual cells (arrowheads). (B) Abundant callose accumulation in the terminal cell wall. (C) Callose in the corners between individual cells (arrowheads); lateral wall with less signal (arrow). (D) Fragmented filament with callose accumulation in convex terminal walls; cross wall with less signal (arrow). (E) Abundant callose in cross walls, with a maximum in the center (arrows) and in the terminal wall. (F) Callose in the terminal cell wall and in protuberances of cross cell walls (arrows). Scale bar = 10 µm (A–D); 5 µm (E, F).
Fig. 4
Fig. 4
Micrographs of Zygnema S (A, B, E, G) and Zygnema E-A (C, D, F, H); hydrated cells (A–F), 210 min desiccated cells (G, H). (A–D) Live cell labeling (red, Chl autofluorescence); (E–H) the corresponding bright field image and labeling of semi-thin sections with the monoclonal antibody 400-2 (green). (A) Convex terminal cross wall stained abundantly. (B) Recently fragmented cells with massive staining in the new terminal wall. (C) Detaching filament; a strong signal in the terminal cells (asterisks) and weak labeling in longitudinal walls (arrow). (D) Filament with a deformed longitudinal cell wall abundantly stained (arrow). (E) Filament with clearly visible starch grains in pyrenoids (arrowheads); cell corners show strong labeling; only cross cell walls of young cells are stained (arrow). (F) Filament with a deformed cell (asterisk) and strong callose labeling in the area of deformation and in cell corners (arrowheads). (G) Desiccated filament with labeling restricted to the terminal cell wall and cell corners. (H) Desiccated filament with labeling in cell corners. Scale bar = 10 µm.
Fig. 5
Fig. 5
Micrographs of Klebsormidium crenulatum (A, E, G) and Klebsormidium nitens (B, C, D, F, H). (A–D) Live cell labeling (red, Chl autofluorescence), and labeling of sem-ithin sections (E–H) of hydrated (E, F) and desiccated filaments (G, H) with the monoclonal AB 400-2 (green). Bright field image and corresponding labeling is shown. (A) Callose labeling in the longitudinal cell walls between individual cells (arrowheads). (B) Filament fragment with callose labeling in the terminal cross cell walls. (C) Detached cells (asterisks) show labeling in the terminal walls (arrowhead). (D) 3D projection of a deformed cell filament with strong callose labeling. (E) Callose labeling in cross cell walls, with maximal labeling in the center of the cross cell walls and protuberances (arrows). (F) Callose in the center of cross cell walls (arrows). (G) Undulated cross cell walls with abundant callose staining. (H) Callose labeling in cross cell walls and longitudinal walls (arrows). Scale bar = 5 µm.
Fig. 6
Fig. 6
Immunogold localization of callose epitopes (AB 400-2) in transmission electron micrographs of Klebsormidium crenulatum (A) and Klebsormidium nitens (B). (A) Cross cell wall and protuberance (arrowhead) stained by 10 nm gold particles (arrows). (B) Terminal cross cell with abundant binding of 10 nm gold particles (arrows) throughout the terminal cell wall. Scale bar = 500 nm (A); 200 nm (B).
Fig. 7
Fig. 7
Imaging-PAM micrographs of Zygnema S (A) and Zygnema E-A (B), near infrared (NIR) remission image and corresponding Y(II) images (false colored) (control, after 30 min of desiccation at ambient humidity and after 10, 60 and 180 min recovery). The color bar at the top indicates the relative Y(II) as a percentage. (A) After recovery for 180 min, cells with thicker cross cell walls (arrowheads) show the highest Y(II) and in some cells the characteristic stellate chloroplasts appear again (asterisks). (B) The stellate chloroplasts appear again after 180 min (asterisks). Scale bar = 10 µm.
Fig. 8
Fig. 8
Imaging-PAM micrographs of Klebsormidium cernulatum (A) and Klebsormidium nitens (B), near infrared (NIR) remission image and corresponding Y(II) images (false color) of algal filaments (control, after 30 min of desiccation at AH and after 10, 60 and 180 min of recovery). The color bar at the top indicates the relative Y(II) as a percentage. Scale bar = 10 µm.

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