Desiccation Tolerance in Alpine Aeroterrestrial Green Algae

In the harsh alpine environment, factors such as low temperatures, high irradiation and periodical desiccation can severely limit plant life. However, aeroterrestrial green algae are successful in this environment and function as important components of alpine soil crusts, microbial communities that stabilize soils surfaces. One key to the success of green algae in alpine habitats is the ability to withstand desiccation in the vegetative state. This complex response to desiccation is achieved by protection of cellular integrity during the desiccation process as well as efficient repair of damage that is incurred during dehydration and rehydration. Very little is known about the molecular biological, cellular and physiological basis of desiccation protection in green algae. The general hypothesis that streptophyte algae are ancestors of terrestrial pants will be investigated under the light of their desiccation tolerance. For the proposed project streptophyte algae with different abilities to tolerate desiccation Klebsormidium crenulatum (Klebsormidiales, phylogenetic more basal and fully desiccation tolerant) and Zygogonium ericetorum (Zygnematales, phylogenetic more advanced and partially desiccation tolerant) will be in the centre of the investigations. The evolutionary question is - did basal, desiccation tolerant streptophyta give rise to poikilohydric land plants and advanced, less desiccation tolerant groups lead to homoiohydric plants? Three aspects of vegetative desiccation tolerance will be investigated in order to provide a more complete picture of this complex response. 1. Molecular biological investigations: In cooperation with leading research groups the following two questions will be addressed: A) The phylogenetic relationships between different desiccation tolerant strains of Klebsormidium sp., resp. Zyogonium ericetorum and various strains of Zygnema sp. will be investigated by rbcL and ITS sequence analysis to determine the phylogenetic placement of the alpine algae among aquatic relatives. B) A deep sequencing approach (Illumina RNA-seq) will be undertaken for transcriptome analysis of Klebsormidium crenulatum and Zygogonium ericetorum to allow an unbiased view on changes upon desiccation in these two species with different desiccation tolerance. Additionally expressed sequence tag (EST) analysis will be used to investigate particular proteins of interest. 2. Cell biological aspects of desiccation: Desiccation tolerant green algae will be exposed to different air humidities, which allow different degrees of desiccation. Survival rates will be determined with vital staining. Reorganization of cytoskeleton elements have been shown, and will be investigated during the desiccation and rehydration process. Transmission electron microscopic studies after high pressure freeze fixation will allow to investigate alterations of the cell wall and possible damage to cellular organelles. The different degrees of alteration will be investigated in relation to the phylogentic position of the algae and their different physiological abilities to tolerate desiccation. 3. Physiological aspects of desiccation: Photosynthesis and respiration measurements will provide information on the kinetics of the desiccation process in green algae. The determination of compatible solutes such as low-molecular weight carbohydrates will give further insights into protection mechanisms. Understanding the mechanisms of desiccation tolerance of aeroterrestrial green algae is of great importance. These organisms participate in soil surface stabilization, which is increasingly important in expanding arid regions globally.

Valuable results on desiccation tolerance in aeroterrestrial green algae were obtained in this project. (1) Composition of organisms in soil crusts were analyzed with molecular biological methods, (2) transcriptional changes were investigated after severe desiccation stress, (3) cell biological aspects, like cell wall modifications during desiccation and (4) physiological changes as a consequence of desiccation. A high biodiversity of soil crust organisms was observed, many of these organisms could be distinguished only by molecular phylogeny. The biodiversity is related to the different habitats with variable pH-value, mineral composition or the occurrence of iron and aluminum. The here investigated green algae (Streptophyta) are ancestors to land plants. Among the investigated objects are: Klebsormidium crenulatum (phylogenetically basal, fully desiccation tolerant), Zygnema sp., Zygogonium ericetorum (phylogenetically advanced, partially desiccation tolerant). For comparison the intertidal alga Ulva compressa (Chlorophyta), which is not related to land plants was investigated. Transcriptome analysis of Klebsormidium crenulatum showed a land plant like reaction to severe desiccation stress. Antioxidative protection mechanisms and contigs involved in production of osmotically active substances were upregulated. Other aspects were severely downregulated. This demonstrates that these algae are already capable of reactions to desiccation stress and show tolerance in the vegetative stage. In the genus Zygnema sp. the physiological condition (like the occurrence of akinetes) is crucial for desiccation tolerance. While young cells are sensitive to desiccation, mature, lipid rich akinetes are capable to tolerate desiccation. In the genus Zygogonium purple vacuolar pigments are found, which have UV-protective functions and are involved in iron detoxification mechanisms. Reorganizations of the cell walls, differentially regulated, were observed in all desiccation tolerant green algae. Cytoskeleton elements show a dynamic reorganization, which was investigated within this project for the first time in combination with desiccation in green algae.