Comparative Transcriptomic and Proteomic Analyses Provide New Insights into the Tolerance to Cyclic Dehydration in a Lichen Phycobiont.

Desiccation tolerance (DT) is relatively frequent in non-vascular plants and green algae. However, it is poorly understood how successive dehydration/rehydration (D/R) cycles shape their transcriptomes and proteomes. Here, we report a comprehensive analysis of adjustments on both transcript and protein profiles in response to successive D/R cycles in Coccomyxa simplex (Csol), isolated from the lichen Solorina saccata. A total of 1833 transcripts and 2332 proteins were differentially abundant as a consequence of D/R; however, only 315 of these transcripts/proteins showed similar trends. Variations in both transcriptomes and proteomes along D/R cycles together with functional analyses revealed an extensive decrease in transcript and protein levels during dehydration, most of them involved in gene expression, metabolism, substance transport, signalling and folding catalysis, among other cellular functions. At the same time, a series of protective transcripts/proteins, such as those related to antioxidant defence, polyol metabolism and autophagy, was upregulated during dehydration. Overall, our results show a transient decrease in most cellular functions as a result of drying and a gradual reactivation of specific cell processes to accommodate the hydration status along successive D/R cycles. This study provides new insights into key mechanisms involved in the DT of Csol and probably other dehydration-tolerant microalgae. In addition, functionally characterising the high number of genes/proteins of unknown functions found in this study may lead to the discovery of new DT mechanisms.

The Under-explored Extracellular Proteome of Aero-Terrestrial Microalgae Provides Clues on Different Mechanisms of Desiccation Tolerance in Non-Model Organisms.

Trebouxia sp. (TR9) and Coccomyxa simplex (Csol) are desiccation-tolerant lichen microalgae with different adaptive strategies in accordance with the prevailing conditions of their habitats. The remodelling of cell wall and extracellular polysaccharides depending on water availability are key elements in the tolerance to desiccation of both microalgae. Currently, there is no information about the extracellular proteins of these algae and other aero-terrestrial microalgae in response to limited water availability. To our knowledge, this is the first report on the proteins associated with the extracellular polymeric substances (EPS) of aero-terrestrial microalgae subjected to cyclic desiccation/rehydration. LC-MS/MS and bioinformatic analyses of the EPS-associated proteins in the two lichen microalgae submitted to four desiccation/rehydration cycles allowed the compilation of 111 and 121 identified proteins for TR9 and Csol, respectively. Both sets of EPS-associated proteins shared a variety of predicted biological functions but showed a constitutive expression in Csol and partially inducible in TR9. In both algae, the EPS-associated proteins included a number of proteins of unknown functions, some of which could be considered as small intrinsically disordered proteins related with desiccation-tolerant organisms. Differences in the composition and the expression pattern between the studied EPS-associated proteins would be oriented to preserve the biochemical and biophysical properties of the extracellular structures under the different conditions of water availability in which each alga thrives.

Disentangling the role of extracellular polysaccharides in desiccation tolerance in lichen-forming microalgae. First evidence of sulfated polysaccharides and ancient sulfotransferase genes.

Trebouxia sp. TR9 and Coccomyxa simplex are desiccation-tolerant microalgae with flexible cell walls, which undergo species-specific remodelling during dehydration-rehydration (D/R) due to their distinct ultrastructure and biochemical composition. Here, we tested the hypothesis that extracellular polysaccharides excreted by each microalga could be quantitatively and/or qualitatively modified by D/R. Extracellular polysaccharides were analysed by size exclusion and anion exchange chromatography, specific stains after gel electrophoresis and gas chromatography/mass spectrometry of trimethylsilyl derivatives (to determine their monosaccharide composition). The structure of a TR9-sulfated polymer was deduced from nuclear magnetic resonance (NMR) analyses. In addition, sugar-sulfotransferase encoding genes were identified in both microalgae, and their expression was measured by RT-qPCR. D/R did not alter the polydispersed profile of extracellular polysaccharides in either microalga but did induce quantitative changes in several peaks. Furthermore, medium-low-sized uronic acid-containing polysaccharides were almost completely substituted by higher molecular mass carbohydrates after D/R. Sulfated polysaccharide(s) were detected, for the first time, in the extracellular polymeric substances of both microalgae, but only increased significantly in TR9 after cyclic D/R, which induced a sugar-sulfotransferase gene and accumulated sulfated ß-D-galactofuranan(s). Biochemical remodelling of extracellular polysaccharides in aeroterrestrial desiccation-tolerant microalgae is species-specific and seems to play a role in the response to changes in environmental water availability.

The chloroplast genome of the lichen-symbiont microalga Trebouxia sp. Tr9 (Trebouxiophyceae, Chlorophyta) shows short inverted repeats with a single gene and loss of the rps4 gene, which is encoded by the nucleus.

The Trebouxiophyceae is the class of Chlorophyta algae from which the highest number of chloroplast genome (cpDNA) sequences has been obtained. Several species in this class participate in symbioses with fungi to form lichens. However, no cpDNA has been obtained from any Trebouxia lichen-symbiont microalgae, which are present in approximately half of all lichens. Here, we report the sequence of the completely assembled cpDNA from Trebouxia sp. TR9 and a comparative study with other Trebouxio-phyceae. The organization of the chloroplast genome of Trebouxia sp. TR9 has certain features that are unusual in the Trebouxiophyceae and other green algae. The most remarkable characteristics are the presence of long intergenic spacers, a quadripartite structure with short inverted repeated sequences (IRs), and the loss of the rps4 gene. The presence of long intergenic spacers accounts for a larger cpDNA size in comparison to other closely related Trebouxiophyceae. The IRs, which were thought to be lost in the Trebouxiales, are distinct from most of cpDNAs since they lack the rRNA operon and uniquely includes the rbcL gene. The functional transfer of the rps4 gene to the nuclear genome has been confirmed by sequencing and examination of the gene architecture, which includes three spliceosomal introns as well as the verification of the presence of the corresponding transcript. This is the first documented transfer of the rps4 gene from the chloroplast to the nucleus among Viridiplantae. Additionally, a fairly well-resolved phylogenetic reconstruction, including Trebouxia sp. TR9 along with other Trebouxiophyceae, was obtained based on a set of conserved chloroplast genes.

Ultrastructural and biochemical analyses reveal cell wall remodelling in lichen-forming microalgae submitted to cyclic desiccation-rehydration.

BACKGROUND AND AIMS: One of the most distinctive features of desiccation-tolerant plants is their high cell wall (CW) flexibility. Most lichen microalgae can tolerate drastic dehydration-rehydration (D/R) conditions; however, their mechanisms of D/R tolerance are scarcely understood. We tested the hypothesis that D/R-tolerant microalgae would have flexible CWs due to species-specific CW ultrastructure and biochemical composition, which could be remodelled by exposure to cyclic D/R. METHODS: Two lichen microalgae, Trebouxia sp. TR9 (TR9, adapted to rapid D/R cycles) and Coccomyxa simplex (Csol, adapted to seasonal dry periods) were exposed to no or four cycles of desiccation [25-30 % RH (TR9) or 55-60 % RH (Csol)] and 16 h of rehydration (100 % RH). Low-temperature SEM, environmental SEM and freeze-substitution TEM were employed to visualize structural alterations induced by D/R. In addition, CWs were extracted and sequentially fractionated with hot water and KOH, and the gel permeation profile of polysaccharides was analysed in each fraction. The glycosyl composition and linkage of the main polysaccharides of each CW fraction were analysed by GC-MS. KEY RESULTS: All ultrastructural analyses consistently showed that desiccation caused progressive cell shrinkage and deformation in both microalgae, which could be rapidly reversed when water availability increased. Notably, the plasma membrane of TR9 and Csol remained in close contact with the deformed CW. Exposure to D/R strongly altered the size distribution of TR9 hot-water-soluble polysaccharides, composed mainly of a ß-3-linked rhamnogalactofuranan and Csol KOH-soluble ß-glucans. CONCLUSIONS: Cyclic D/R induces biochemical remodelling of the CW that could increase CW flexibility, allowing regulated shrinkage and expansion of D/R-tolerant microalgae.