Desiccation tolerance in the chlorophyte green alga Ulva compressa: does cell wall architecture contribute to ecological success?

MAIN CONCLUSION: Desiccation leads to structural changes of the inner pectic cell wall layers in Ulva compressa. This contributes to protection against mechanical damage due to desiccation-rehydration cycles. Ulva compressa, characterized by rbcL phylogeny, is a common species in the Mediterranean Sea. Ulva as an intertidal species tolerates repeated desiccation-rehydration cycles in nature; the physiological and structural basis were investigated under experimental conditions here. Desiccation to 73% relative water content (RWC) led to a significant decrease of the maximum quantum yield of photosystem II (F v/F m) to about half of the initial value. A reduction to 48 or 27% RWC caused a more drastic effect and thalli were only able to recover fully from desiccation to 73% RWC. Relative electron transport rates were stimulated at 73% RWC, but decreased significantly at 48 and 27% RWC, respectively. Imaging-PAM analysis demonstrated a homogenous desiccation process within individual thallus discs. The different cell wall layers of U. compressa were characterized by standard staining procedures, i.e. calcofluor white and aniline blue for structural components (cellulose, callose), ruthenium red for pectins and toluidine blue for acidic polysaccharides. Already a reduction to 73% RWC caused severe changes of the cell walls. The inner pectin-rich layers followed the shrinkage process of the cytoplasm, while the outer denser fibrillar layers maintained their shape. In this way, the thalli were not plasmolyzed during water loss, and upon recovery not negatively influenced by any mechanical damage. Transmission electron microscopy corroborated the arrangement of the different layers clearly distinguishable by their texture and electron density. We suggest the flexibility of the pectin-rich cell wall layers as a major contribution to desiccation tolerance in Ulva.

Transcriptomics of desiccation tolerance in the streptophyte green alga Klebsormidium reveal a land plant-like defense reaction.

BACKGROUND: Water loss has significant effects on physiological performance and survival rates of algae. However, despite the prominent presence of aeroterrestrial algae in terrestrial habitats, hardly anything is known about the molecular events that allow aeroterrestrial algae to survive harsh environmental conditions. We analyzed the transcriptome and physiology of a strain of the alpine aeroterrestrial alga Klebsormidium crenulatum under control and strong desiccation-stress conditions. PRINCIPAL FINDINGS: For comparison we first established a reference transcriptome. The high-coverage reference transcriptome includes about 24,183 sequences (1.5 million reads, 636 million bases). The reference transcriptome encodes for all major pathways (energy, carbohydrates, lipids, amino acids, sugars), nearly all deduced pathways are complete or missing only a few transcripts. Upon strong desiccation, more than 7000 transcripts showed changes in their expression levels. Most of the highest up-regulated transcripts do not show similarity to known viridiplant proteins, suggesting the existence of some genus- or species-specific responses to desiccation. In addition, we observed the up-regulation of many transcripts involved in desiccation tolerance in plants (e.g. proteins similar to those that are abundant in late embryogenesis (LEA), or proteins involved in early response to desiccation ERD), and enzymes involved in the biosynthesis of the raffinose family of oligosaccharides (RFO) known to act as osmolytes). Major physiological shifts are the up-regulation of transcripts for photosynthesis, energy production, and reactive oxygen species (ROS) metabolism, which is supported by elevated cellular glutathione content as revealed by immunoelectron microscopy as well as an increase in total antiradical power. However, the effective quantum yield of Photosystem II and CO2 fixation decreased sharply under the applied desiccation stress. In contrast, transcripts for cell integrative functions such as cell division, DNA replication, cofactor biosynthesis, and amino acid biosynthesis were down-regulated. SIGNIFICANCE: This is the first study investigating the desiccation transcriptome of a streptophyte green alga. Our results indicate that the cellular response is similar to embryophytes, suggesting that embryophytes inherited a basic cellular desiccation tolerance from their streptophyte predecessors.

Osmotic stress in Arctic and Antarctic strains of the green alga Zygnema (Zygnematales, Streptophyta): effects on photosynthesis and ultrastructure.

The osmotic potential and effects of plasmolysis on photosynthetic oxygen evolution and chlorophyll fluorescence were studied in two Arctic Zygnema sp. (strain B, strain G) and two Antarctic Zygnema sp. (strain E, strain D). Antarctic strain D was newly characterized by rbcL sequence analysis in the present study. The two Antarctic strains, D and E, are most closely related and may represent different isolates of the same species, in contrast, strain B and G are separate lineages. Incipient plasmolysis in the cells was determined by light microscopy after incubating cells in sorbitol solutions ranging between 200 mM and 1000 mM sorbitol for 3, 6 and 24h. In Zygnema strain B and G incipient plasmolysis occurred at ~600 mM sorbitol solution (720 mOsmol kg(-1), ψ=-1.67 MPa) and in strains D and E at ~300 mM (318 mOsmol kg(-1), ψ=-0.8 MPa) sorbitol solution. Hechtian strands were visualized in all plasmolysed cells, which is particularly interesting, as these cells lack pores or plasmodesmata. Ultrastructural changes upon osmotic stress were a retraction of the condensed cytoplasm from the cell walls, damages to chloroplast and mitochondrial membranes, increasing numbers of plastoglobules in the chloroplasts and membrane enclosed particles in the extraplasmatic space. Maximum photosynthetic rates (P(max)) in light saturated range were between 145.5 µmol O(2) h(-1)mg(-1)Chl a in Zygnema G and 752.9 µmol O(2) h(-1)mg(-1)Chl a in Zygnema E. After incubation in 800 mM sorbitol for 3h P(max) decreased to the following percentage of the initial values: B: 16.3%, D: 16.8%, E: 26.1% and G: 35.0%. Osmotic stress (800 mM sorbitol) decreased maximum photochemical quantum yield of photosystem II (F(v)/F(m)) when compared to controls. Maximum values of relative electron transport rates of photosystem II (rETR(max)) decreased after incubation in 400 mM sorbitol in Zygnema D and E, while they decreased in Zygnema B and G only after incubation in 800 mM sorbitol. The kinetics of the rETR curves were similar for the Arctic strains Zygnema B and G, but distinct from the Antarctic strains Zygnema D and E, which were similar when compared with each other. This suggests that the investigated Arctic Zygnema sp. strains might be better adapted to tolerate osmotic water stress than the investigated strains from the Antarctic.

Plasmolysis effects and osmotic potential of two phylogenetically distinct alpine strains of Klebsormidium (Streptophyta).

The osmotic potential and effects of plasmolysis were investigated in two different Klebsormidium strains from alpine habitats by incubation in 300-2,000 (3,000) mM sorbitol. Several members of this genus were previously found to tolerate desiccation in the vegetative state yet information was lacking on the osmotic potentials of these algae. The strains were morphologically determined as Klebsormidium crenulatum and Klebsormidium nitens. These species belong to distinct clades, as verified by phylogenetic analysis of the rbcL gene. K. crenulatum is part of to the K. crenulatum/mucosum ('F' clade) and K. nitens of the 'E2' clade. Plasmolysis occurred in K. crenulatum at 800 mM sorbitol (961 mOsmol kg(-1), Ψ = -2.09 MPa) and in K. nitens at 600 mM sorbitol (720 mOsmol kg(-1), Ψ = -1.67 MPa). These are extraordinarily high osmotic values (very negative osmotic potentials) compared with values reported for other green algae. In K. crenulatum, the maximum photosynthetic rate (Pmax) in the light-saturated range was 116 µmol O(2) h(-1) mg(-1) chl a. Incubation in 1,000 mM sorbitol decreased Pmax to 74.1% of the initial value, whereas 2,000 mM sorbitol (Ψ = -5.87 MPa) lead to an almost complete loss of oxygen production. In K. nitens, Pmax was 91 µmol O(2) h(-1) mg(-1) chl a under control conditions and incubation in 800 mM sorbitol did not decrease Pmax, 2,000 mM sorbitol decreased Pmax only to about 62.6% of the initial value whereas 3,000 mM sorbitol stopped oxygen evolution. This indicated a broader amplitude for photosynthesis in the examined strain of K. nitens. Control samples and samples plasmolysed for 3 h in 800 mM sorbitol (K. nitens), 1,000 mM sorbitol (K. crenulatum), or 2,000 mM sorbitol were investigated by transmission electron microscopy after chemical or high-pressure freeze fixation. In cells undergoing plasmolysis the protoplasts were retracted from the cell wall, the cytoplasm appeared dense, vacuoles were small and fragmented, and the cytoplasm was filled with ribosomes. Thin cytoplasmic strands were connected to the cell wall; 2,000 mM sorbitol increased the effect. The content of soluble carbohydrates in these two strains was investigated by HPLC, as this is one known mechanism for cells to maintain high osmotic pressure of the cytosol. Both Klebsormidium species contained diverse soluble carbohydrates, including a dominant mixed peak of unidentified oligosaccharides, and more minor amounts of raffinose, sucrose, glucose, xylose, galactose, mannose, inositol, fructose, glycerol, mannitol, and sorbitol. The total content of soluble carbohydrates was approximately 1.2% of the dry weight, indicating that this is not a major factor contributing to the high osmotic potential in these strains of Klebsormidium.