Ecophysiological and genomic approaches to cyanobacterial hardening for restoration.

Journal of Phycology. 2024;60:465–482. | 465 wileyonlinelibrary.com/journal/jpy RESEARCH ARTICLE Ecophysiological and genomic approaches to cyanobacterial hardening for restoration BeatrizRoncero- Ramos1,2 | ValentinaSavaglia1,3 | BenoitDurieu1 | IsabelleVandeVreken4 | AuroreRichel4 | AnnickWilmotte1 Received: 7 September 2023 | Accepted: 15 December 2023 DOI: 10.1111/jpy.13436 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2024 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America. Abbreviations: CDS, coding sequence; COGs, clusters of orthologous groups; EPS, exopolysaccharide; GGPP, geranylgeranylpyrophosphate; KEGG, Kyoto encyclopedia of genes and genomes; LBEPS, loosely bound EPS; MAAs, mycosporinelike amino acids; OCP, orange carotenoidprotein; PAR, photosynthetic active radiation; ROS, reactive oxygen species; SOD, superoxide dismutase; TBEPS, tightly bound EPS; UN, United nations; UVR, ultraviolet radiation. 1InBios- Molecular Diversity and Ecology of Cyanobacteria, University of Liège, Liege, Belgium 2Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Sevilla, Spain 3Laboratory of Protistology & Aquatic Ecology, Ghent University, Ghent, Belgium 4TERRA- Biomass and Green Technologies, University of Liège, Gembloux, Belgium Correspondence Beatriz Roncero- Ramos, InBios- Molecular Diversity and Ecology of Cyanobacteria, University of Liège, 4000 Liège, Belgium. Email: [email protected] Funding information Université de Liège, Grant/Award Number: R.DIVE.0899- J- F- I; Junta de Andalucía, Grant/Award Number: PAIDIDOCTOR 21_00571 Editor: D. Casamatta Abstract Cyanobacteria inhabit extreme environments, including drylands, providing multiple benefits to the ecosystem. Soil degradation in warm drylands is increasing due to land use intensification. Restoration methods adapted to the extreme stress in drylands are being developed, such as cyanobacteria inoculation to recover biocrusts. For this type of restoration method to be a success, it is crucial to optimize the survival of inoculated cyanobacteria in the field. One strategy is to harden them to be acclimated to stressful conditions after laboratory culturing. Here, we analyzed the genome and ecophysiological response to osmotic desiccation and UVR stresses of an Antarctic cyanobacterium, Stenomitos frigidus ULC029, which is closely related to other cyanobacteria from warm and cold dryland soils. Chlorophyll a concentrations showed that preculturing ULC029 under moderate osmotic stress improved its survival during an assay of desiccation plus rehydration under UVR. Additionally, its sequential exposure to these stress factors increased the production of exopolysaccharides, carotenoids, and scytonemin. Desiccation, but not osmotic stress, increased the concentrations of the osmoprotectants trehalose and sucrose. However, osmotic stress might induce the production of other osmoprotectants, for which the complete pathways were observed in the ULC029 genome. In total, 140 genes known to be involved in stress resistance were annotated. Here, we confirm that the sequential application of moderate osmotic stress and dehydration could improve cyanobacterial hardening for soil restoration by inducing several resistance mechanisms. We provide a highquality genome of ULC029 and a description of the main resistance mechanisms (i.e., production of exopolysaccharides, osmoprotectants, chlorophyll, and carotenoids; DNA repair; and oxidative stress protection). KEYWORDS biocrusts, cyanobacteria, drylands, exopolysaccharide, genomics, resistance mechanisms, soil restoration, UV radiation 466 | RONCERORAMOS etal. INTRODUCTION Cyanobacteria are extremophiles that can survive in different ecosystems, including some of the most extreme on Earth, such as the McMurdo Dry Valleys in Antarctica (Zhang etal.,2015) or the Atacama Desert in Chile (Patzelt etal.,2014). In terrestrial ecosystems, they are often part of communities, known as biological soil crusts, or biocrusts, along with other organisms (lichens, mosses, fungi, heterotrophic bacteria, or microalgae), colonizing the first centimeter of soil surfaces (Weber etal.,2022). Biocrusts provide many ecosystem functions such as improving soil fertility (Barger et al., 2016), stability (Belnap et al., 2007; Chamizo et al., 2017), and biodiversity (Maestre et al., 2011), among others. The cyanobacteria located in these ecosystems have mechanisms to withstand environmental stresses, such as high and continuous light intensity and UV radiation (UVR), desiccation, freezing, or high salinity. The cyanobacteria produce an exopolysaccharide (EPS) matrix surrounding cells, which accumulates water, stabilizing the cell membranes during desiccation (Pereira etal.,2009). This EPS matrix also protects cell membranes from freezinginduced damage (Tamaru et al., 2005) and UVR (Chen et al., 2009), and it accumulates nutrients (Mager & Thomas,2011), which is important for survival in oligotrophic environments. In order to prevent the production of reactive oxygen species (ROS) by UVA radiation, some cyanobacteria accumulate the pigment scytonemin in the EPS matrix (Soule etal.,2013). In addition, cyanobacteria produce a wide range of mycosporinelike amino acids (MAAs) that accumulate inside cells and prevent DNA damage by absorbing UVB radiation (Gao & Garcia- Pichel,2011). Cyanobacteria in extreme environments are also exposed to high light intensity, which can lead to photooxidative damage by ROS formation (Muramatsu & Hihara,2012). To revert or avoid ROS formation, they produce oxygenscavenging proteins (Kvíderová et al., 2019) and the nonphotochemical quencher orange carotenoidprotein (OCP; Muramatsu & Hihara,2012). Another important stress factor in hot and cold deserts is salinity. The two major strategies to withstand the osmotic stress due to an increase of the external ion concentrations present in a highly saline environment or due to events of desiccation or freezing are the saltout strategy and the production/uptake of osmoprotectants (Kvíderová etal.,2019). The type of osmoprotectants, also called compatible solutes, that cyanobacteria may produce or uptake is roughly correlated to their natural habitat and their salt tolerance range (Kirsch etal.,2019), mainly sucrose and trehalose in low salt tolerance strains, glucosylglycerol in medium salt tolerance ones, and glycine betaine and glutamate betaine for high salt tolerance strains (Mackay etal.,1984). However, there are a few exceptions to these patterns (Kirsch et al., 2019). If these protective strategies do not succeed, the strategies to revert cellular damage are activated, such as DNA repair mechanisms (Pathak etal.,2019) or chaperone production (Chatterjee etal.,2020). Cyanobacterial ability to withstand extreme conditions makes these interesting organisms for different disciplines, such as astrobiology (Billi et al., 2022), or for soil restoration in drylands (Rossi etal.,2017). The publications focused on dryland soil restoration by cyanobacterial inoculation have increased during the last decades (Antoninka etal.,2020) because of the increase in degradation of dryland ecosystems due to climate change and anthropogenic impacts (Huang etal.,2016). In fact, the protection and restoration of terrestrial habitats have been included in the Sustainable Development Goals of the United Nations (UN) for 2030 (UN,2015). Because cyanobacteria are the first colonizers of soils and thus participate in the first steps of biocrusts succession, soil restoration aims to induce the formation of biocrusts on degraded soils by different cyanobacteria (Weber etal.,2016). In laboratory conditions, the inoculated cyanobacteria can survive and colonize different types of degraded soils and induce biocrust formation (Adessi et al., 2021) thanks to their capacity to withstand the environmental stresses. However, when the restoration is tested in natural conditions in degraded dryland soils, the success inducing biocrust formation has been limited, until now, to inoculated sand dunes in cold deserts in China (Park etal.,2017). Román, Chamizo, etal.(2021) did not observe an increase in soil chlorophyll a (chl a) content, as a proxy of biocrust formation, compared to controls after inoculating cyanobacteria in field, even after hardening cultures by dehydration combined with a progressive increase of light intensity. In Bowker etal.(2020), the authors showed that a hardening protocol based on desiccation events, exposure to high light over time, and fluctuating temperatures on inocula composed of cyanobacteria, mosses, and lichens did not perform better than nonhardened ones. Conversely, Giraldo- Silva etal.(2020) observed an increase in chl a content in inoculated soils when cyanobacteria were prehardened by wetdry cycles and a progressive increase in light intensity. However, they maintained a watering regime outdoors, ameliorating field conditions. This study investigates the resistance mechanisms of a cyanobacterial strain to stresses encountered in its natural environment, with the aim to develop a hardening method that could be applied in future field experiments with success. For this goal, we followed a physiological and genomic approach. The selected strain, ULC029, isolated in one of the most extreme regions on Earth, continental Antarctica, is a thin homocytous filamentous cyanobacterium that was identified as Leptolyngbya frigida ANT.LH52B.3 at the time of isolation (Taton etal.,2006) but was later renamed as Stenomitos frigidus, which 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 467 CYANOBACTERIAL RESISTANCE TO STRESS belongs to a genus commonly observed in dryland biocrusts (Muñoz- Martín etal.,2019; Patzelt etal.,2014; Pushkareva et al., 2015; Williams et al., 2016). This strain shows a high 16S rRNA gene similarity (99.4%; Shalygin et al., 2020) with several Leptolyngbya strains isolated in warm semiarid biocrusts in SE Spain (Roncero- Ramos etal.,2019). Moreover, it has been shown that the relative abundance of these strains increases in incipient biocrusts with ecosystem degradation (Roncero- Ramos et al., 2020). Also, other thin filamentous strains identified as Leptolyngbya have already shown promising results in inducing biocrusts after soil inoculation (Mugnai etal.,2018). Yet, authors studying the impact of stress have shown that the exposure to individual or combined stress factors may produce a different response (Joshi etal.,2018; Rai et al., 2013). Some stress factors might activate mechanisms usually associated with another one. For example, osmotic stress (Dillon et al., 2002) or desiccation (Fleming & Castenholz,2007) led to the production of the UV- screening pigment scytonemin. Therefore, we hypothesize that culturing cyanobacteria under multiple stress factors would improve the hardening method, leading to a better performance after inoculation in the field. To test our hypothesis, two ecophysiological assays were performed with cultures of ULC029 grown in the laboratory. First, we analyzed the influence of increasing salinity, and decreasing nutrient concentration on cyanobacterial growth, the efficiency of photosynthesis (Fv/Fm), and pigment production (chl a, carotenoids, and scytonemin). After characterizing the salinity tolerance of the strain, we analyzed the influence of a multiplestress environment that included osmotic, desiccation, and UVR stresses. In this second assay, we also quantified the osmoprotectants (sucrose and trehalose) and the exopolysaccharides. Finally, to identify all the potential resistance mechanisms of the strain ULC029, we sequenced and assembled the genome after a hybrid, which reduced the error rate and gaps and, thus, increased genome assembly quality (Koren etal.,2012). We identified the presence or absence of a selection of 140 genes known to be involved in cyanobacterial resistance mechanisms to environmental stress. MATERIALS AND METHODS Cyanobacterial strain and culture conditions The unicyanobacterial strain Stenomitos frigidus ULC029 (synonym Leptolyngbya frigida; FigureS1 in the Supporting Information), from the BCCM/ULC Cyanobacterial Collection (Liège, Belgium) was isolated from a benthic microbial mat in the lake 52 (76°23′ S, 69°24′ E) in the Larsemann Hills in the Prydz Bay region of East Antarctica in 1998 (Taton etal.,2006). It was identified as L. frigida by morphological analysis and sequencing of the 16S rRNA gene and the ITS rRNA region between the 16S and 23S rRNA genes (ITS rRNA region; Taton etal.,2006), but it was later reclassified to S. frigidus (Shalygin et al., 2020). A maximum likelihood tree (FigureS2 in the Supporting Information) built with all 16S rRNA gene sequences showing more than 96.3% similarity to ULC029 showed its close relatedness (circa 90%) with other strains from extreme biotopes (Antarctica and semiarid and dry environments). The climatic conditions in the Larsemann Hills where ULC029 was isolated are characterized by a maximum snow annual precipitation of 250 mm, strong katabatic winds, and air temperatures that range from maximum values of 4 or even 10°C in summer and mean monthly temperatures between −15 and −18°C in winter (Sabbe etal.,2004). When the lakes partially melt in summer, the water temperature can increase up to 8°C. The lake 52 where the mat sample was collected is oligotrophic and oligosaline (conductivity: 138 μS · cm−1). The pH was 6.7 and the alkalinity was 0.1 meq · L−1 (Sabbe etal.,2004). ULC029 was cultured at 12°C under a continuous irradiance of 6 μmol photons · m−2 · s−1 and shaking at 80 rpm (IKA KS 4000). The culture medium was BG11 (Rippka etal.,1979). Salinity thresholds of Stenomitos frigidus ULC029 To determine the salt tolerance of the strain, it was cultured in BG11 with increasing concentrations of NaCl: 0, 0.5, 0.2, 0.7, 1.8, and 2.5 M. A homogeneous starting inoculum was prepared by centrifuging the cyanobacterial biomass at 6360 g for 7 min. The supernatant was discarded, and the pellet was resuspended in BG11 to reach a proportion of 20:80 of ULC029 pellet (solid) in BG11 (liquid). Then, six different inocula were prepared by mixing 1 mL of the prepared suspension of 20% of ULC029 in BG11 and 1 mL of solutions with increasing concentrations of NaCl in sterilized milliQ H2O. The final concentration of all compounds in BG11 after mixing the suspension with the increasing concentrations of NaCl was half for all of them. Twelve multiwell plates were used to carry out the experiment. Each well was filled with 2 mL of inoculum, and four replicates were set up for each treatment and time point (0 h, 2 h, 21 d, and 32 d; FigureS3 in the Supporting Information). All multiwell plates were incubated at 12°C under an irradiance of 6 μmol photons · m−2 · s−1. At each time point, four replicates of each treatment were used to measure the efficiency of the photosynthesis (Fv/Fm) and the concentration of three pigments: chl a, carotenoids, and scytonemin. At the end of the experiment, 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 468 | RONCERORAMOS etal. the cultures were subjected to one of the two additional steps: either a UVR treatment or a recovery phase. The first treatment was carried out by placing the multiwell plates containing four replicates of each treatment at day 32 inside a dark box provided with UVA, UVB, and PAR lamps. The total irradiance received by the cultures was 10 and 2 W · m−2 of UVA and UVB respectively, and 11 μmol photons · m−2 · s−1 of PAR. The plates were left in the box for 3 days, and after that, the same measurements as before were performed. For the second treatment, the biomass of four replicates of each treatment was transferred at day 32 to new multiwell plates filled with 2 mL of BG11 and was allowed to recover for 33 days. Then, the same two measurements were performed. Note that the BG11 medium used in the recovery phase was not diluted in half. Would a mild salinity stress improve the resistance of Stenomitos frigidus ULC029 to desiccation and UVR? When ULC029 was cultivated in BG11/2 + 0.05 or 0.2 M NaCl, its growth and recovery appeared to improve, as did its resistance to UVR, following the data from Experiment 1. To better understand the potential effect of NaCl on the capacity of ULC029 to withstand stress, we carried out a second experiment with two media: BG11 (as a control) and BG11 + 0.2 M NaCl (the highest salt concentration that allowed ULC029 to grow in Experiment 1; FigureS3). During a preadaptation phase, ULC029 was cultured in flasks with either of the culture media (periodically refreshed) for more than 4 months. As in Experiment 1, the biomass was centrifuged and resuspended in fresh medium to obtain two homogeneous inocula with the same quantity of biomass. Two milliliters of each inocula were filtered on GF/F filters of Ø 25 mm (Whatman) for each replicate. Four replicates per treatment and measurement were set up. Three consecutive treatments were performed: the control, which consisted of analyzing samples just after filtering; the desiccation treatment; and the rehydration + UVR treatment. The desiccation treatment consisted of transferring the filters into a chamber filled with 100 g of silica gel at the bottom to keep an HR of 10%, which allowed us to desiccate the filters in less than 1 day. Temperature and % RH inside the desiccation box were monitored with an iButton Hygrochron logger (Maxim Integrated, USA) every 5 min (FigureS4 in the Supporting Information). The filters were kept desiccated for 1 week. The desiccation chamber was opened, and four replicates were kept for each measurement. The rest of the filters were transferred to multiwell plates filled with 2 mL of BG11 medium to rehydrate them. Plates were placed into the UV box with the same irradiances as in Experiment 1 and left inside for 4 days. After that, the last replicates were taken and all measurements were performed on them. For this experiment, additional variables were measured: the exopolysaccharides and the osmolytes (trehalose and sucrose) content. Culture monitoring and physiological measurements In both experiments at each time point (including the initial inoculum), the culture growth was monitored by quantifying the chl a content as a proxy of photosynthetic biomass. It was extracted by mixing the biomass of each replicate with acetone 90% in a proportion of 1:5, vortexing, and leaving it for 24 h in darkness at 4°C (following Castle et al., 2011; Giraldo- Silva etal.,2020). After that, the samples were centrifuged (6360 g, 7 min at 4°C), and the absorbance at 680 and 750 nm was measured with a UV–visible spectrophotometer (SPECORD 50, Analytik Jena). The quantity of two other pigments was also measured in the same extract at 490 (carotenoids) and 384 nm (scytonemin). The trichromatic equation was used to correct for the interferences of the other pigments (Garcia- Pichel & Castenholz,1991). The maximum photochemical quantum yield of photosystem II (Fv/Fm), considered as an indicator of photosynthesis efficiency and cellular stress, was measured with a PAM fluorometer (AquaPen AP- C, Photon Systems Instruments, CZ) after culture adaptation in the dark for 15 min. Three additional variables were measured in Experiment 2, using four replicates per treatment and time point, and the EPS was extracted to find out if production was affected by the addition of NaCl to the medium, desiccation, and/or the rehydration + UVR treatment. The extractions were divided into two categories, the loosely bound EPS (LBEPS) and the tightly bound EPS (TBEPS), following Rossi et al. (2018). Extracting the LBEPS was done by adding 3 mL of miliQ H2O, vortexing, and incubating the filters for 15 min at 20°C. After that, samples were centrifuged (4000 g, 20 min, 9°C), the supernatant was transferred to a new tube, and the same process was repeated twice, ending up with a final extract of 9 mL, classified as LBEPS fraction. After that, 3 mL of 0.1 M Na2EDTA were added to the filters, vortexed, and incubated overnight at 20°C. After centrifuging as described above, this step was repeated twice, decreasing the incubation time to 20 min. At the end, we had an extract of 9 mL, classified as the TBEPS fraction. All the extractions were quantified by the phenolsulfuric acid method (Dubois etal.,1956), measuring the absorbance (488 nm) with a spectrophotometer (SPECORD 50, Analytik Jena). The total quantity of EPS was obtained by summing up both fractions. The other two variables measured in Experiment 2 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 469 CYANOBACTERIAL RESISTANCE TO STRESS were the quantification of two osmolytes: trehalose and sucrose. Filters were freezedried, eluted, and measured with a Dionex System HPAEDPAD (Highperformance Anion Exchange Chromatography with Pulsed Amperometric Detection) equipped with a CarboPAC PA100 column of 4 × 250 mm. The eluents used were: (A) HPLC- grade water + 100 mM NaOH and (B) HPLC- grade water + 100 mM NaOH + 600 mM of sodium acetate. The system was operated at 30°C with a selected flow rate of 1 mL · min−1. DNA extraction and (meta) genome sequencing A fresh culture of ULC029 in BG11 was used to extract genomic DNA using the GenElute Bacterial Genomic DNA Kit (Sigma- Aldrich) following the recommendations of the manufacturer. Three tubes with 1 mL of the culture were centrifuged (4211 g for 5 min); the supernatant was discarded and washed again in BG11 several times to get rid of as many heterotrophic bacteria as possible, as it was not an axenic culture. In the end, we extracted the DNA from these three pellets. The integrity of the gDNA was checked by electrophoresis and the DNA quality and concentration were quantified with NanoVue (Biochrom). Finally, the three extractions were mixed to obtain enough DNA (>5 μg) and sent to the sequencing platform (GIGA Genomics, University of Liege). Both short and long reads of the gDNA were sequenced. Library preparation was performed using Nextera XT libraries (for Illumina sequencing) and Native barcoding genomic DNA kit (EXPNBD114/ LSK109) for Nanopore sequencing. Short reads (2 × 300 bp, 100× coverage) were sequenced by the Illumina Miseq v3 sequencing platform. Long reads sequences (coverage 100×) were obtained using a MinION Flow Cell R9.4.1 (Oxford Nanopore Technologies, UK). Bioinformatic analyses As the ULC029 culture was not axenic, its genomic dataset was treated as a metagenome and binned to obtain cyanobacterialspecific contigs. The Illumina reads were filtered and trimmed using iufilter- qualityminoche from Anvi'o (Eren et al., 2013), and their quality was checked with fastQC (Andrews, 2010). After checking the Nanopore sequences quality with NanoPlot (v1.29.0; De Coster et al., 2018), NanoFilt (v2.6; De Coster etal.,2018) was used to filter out reads with a quality score lower than 10 and a length shorter than 500 bp. Long reads were assembled using Flye (metagenome mode; v2.9; Kolmogorov et al., 2020). Afterward, filtered Illumina sequences were aligned to the assembled genome with bwa (v0.7.12; Li & Durbin,2009). The obtained sam files were converted to bam format and indexed using Samtools (v1.3.1; Danecek etal.,2021). Then, the assembled genome was polished with pilon (v1.23; Walker et al., 2014), using the indexed bam files as input. Binning was performed with CONCOCT (v1.1; Alneberg et al., 2014). Finally, we used GTDBtk (Parks etal.,2018) to determine the taxonomic placement of the bins and CheckM (v.1.1.6; Parks etal.,2014) to assess their completeness and contamination level. The assembly statistics were obtained with QUAST (default settings; v2.3; Gurevich etal.,2013). The genome was plotted using Proksee (Stothard & Wishart,2005) and manually edited with Inkscape v1.2. The genome assembly has been deposited in GenBank (Bioproject number: PRJNA1006388). Genome annotation A contig database was created using Anvi'o (Eren etal.,2015, 2021). Then, a first annotation was performed using a web version of Dfast (Tanizawa etal., 2016). Afterward, genes in the database were annotated with functions from the NCBI's Clusters of Orthologous Groups (COGs) using DIAMOND (fast mode; Buchfink et al., 2021) and with KEGG (Kyoto Encyclopedia of Genes and Genomes) identifiers using GhostKOALA (Kanehisa et al., 2016). Gene annotations and sequences are available in the Supporting Information: TablesS1 and S2 in the Supporting Information, respectively. Identifying genes related to stress resistance Genes known to be involved in resistance mechanisms against stress (Al- Hosani etal.,2015; Chatterjee etal.,2020; Chrismas etal.,2016, 2018; Ferreira & Garcia- Pichel,2016; Klicki etal.,2018; Kopf etal.,2015; Kvíderová etal.,2019; Mosca etal.,2021; Napoli etal.,2021; Pathak etal.,2019; Pereira etal.,2009, 2019; Rajeev etal.,2013; Shang et al., 2019; Shimura et al., 2015; Sinetova & Los, 2016; Soule et al., 2013; Ye et al., 2021) were searched in the generated contig database and classified in the following groups: (i) Production of the EPS matrix, (ii) Photoprotection, (iii) Oxidative stress protection, (iv) DNA repair, and (v) Osmotic stress protection. When genes were not located in the database, they were searched manually using tblastn against the ULC029 assembled genome with an evalue threshold of 1e- 5 and a score higher than 50 bits (following Pearson,2013). The queries used can be located in the supplementary material (TableS3 in the Supporting Information). Alignments were validated by checking if the Pfam functional protein domains determined with SMART (normal mode; Letunic & Bork,2020) were the same as those from the query. 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 470 | RONCERORAMOS etal. Statistical analyses In Experiment 1, results were analyzed to determine the effect of the increasing NaCl concentration on the pigments' concentrations at different time points individually. Values of each time point were tested for normality and homoscedasticity with the Shapiro–Wilk (shapiro. test function, stats package) and Levene (function leveneTest, car package) tests, respectively. A twoway ANOVA (anova function, car package) followed by the Tukey post hoc test (TukeyHSD function, stats package) was applied when data followed a normal distribution and was homoscedastic; otherwise, the Kruskal–Wallis test (package conover.test) was applied. Moreover, the pigments contents after incubating the cultures under UVR from days 32 to 35 were analyzed by applying a oneway ANOVA (anova function, car package) and the Tukey post hoc test (TukeyHSD function, stats package) for chl a and carotenoid values independently. Data obtained in Experiment 2 were also tested for normality and homocedasticity, as previously described. To determine the influence of the desiccation and rehydration+UVR treatments in cyanobacteria cultured in media with different NaCl concentrations, the following variables were analyzed: chl a, carotenoids, scytonemin, trehalose, sucrose, and EPS content. In all cases, we performed a twoway ANOVA (anova function, car package) followed by the Tukey post hoc test (package lsmeans). Figures were plotted using the ggplot2 package and manually edited using Inkscape v1.2. All analyses were performed with R 4.0.5 (R Core Team,2017). RESULTS Determining the salt tolerance of Stenomitos frigidus ULC029 Stenomitos frigidus ULC029 could grow and survive for 32 days in BG11/2 medium with a NaCl concentration up to 0.7 M (Figure1a). After 32 days, the highest chl a concentration was observed in the medium with 0.2 M NaCl, followed by 0.05 and 0 M. For NaCl concentrations of 1.8 and 2.5 M, the chl a concentration decreased with time and was null after 1 month. Moreover, there was no recovery, as no chl a was detected after transferring and incubating the cultures in BG11 without NaCl for another 33 days. Interestingly, the highest chl a values after recovery were in cultures growing in 0.05 and 0.2 M NaCl, followed by 0 M NaCl (Figure1a). Such high values of chl a concentration may be caused by the higher nutrient availability provided by the nondiluted BG11 used for recovery in comparison with the BG11/2 medium used in the previous phase. However, although S. frigidus kept producing chl a in BG11 + 0.7 M NaCl during the recovery, the amount of chl a was significantly lower than with lower salt concentrations (Figure1a). Therefore, 0.7 M NaCl is the maximum concentration that S. frigidus can withstand among the tested concentrations. In Figure1b, the carotenoid concentration pattern is very similar to the one followed by chl a. In conclusion, the highest concentrations of chl a were obtained in 0.2, 0.05, and 0 M of NaCl (Figure1b) after cultivation for 32 days, but also when a subsequent recovery was performed in 0 M NaCl for 33 days. Scytonemin was not detected in any treatments of Experiment 1. Fv/Fm values (∼0; FigureS5 in the Supporting Information) showed that Stenomitos frigidus was photosynthetically inactive in 0.7, 1.8, and 2.5 M NaCl during the growth phase. Only the strain cultured in 0.7 M NaCl could recover after 33 days in BG11 medium, and it became photosynthetically active again (Fv/Fm 0.3; Figure1). Cultures in BG11/2 with 0.2 M NaCl were also photosynthetically inactive at day 32 (∼0; FigureS5), but they could fully recover and reach the same values as at time 0 (∼0.2; FigureS5). Finally, cultures with a low salinity stress (0.05 M NaCl) and the controls (0 M NaCl) were always photosynthetically active, and both followed a similar pattern with the highest value at day 21 (0.3 and 0.4, respectively; FigureS5). Moreover, FigureS5 shows that culturing S. frigidus in diluted BG11 did not cause stress to the cyanobacterium, as the Fv/Fm values are similar to those shown 33 days after being transferred to nondiluted BG11. When cultures were exposed to UVR after 32 days of cultivation in media with different NaCl concentrations, both chl a and carotenoid concentrations were significantly lower compared to the nonexposed replicates (Figure1c,d). However, cultures previously grown in media with 0 and 0.05 NaCl did not show stress and were photosynthetically active even after UVR exposure (FigureS5). Cultures in 0.2 M NaCl had higher Fv/Fm values, and thus were more photosynthetically efficient, after 3 days of UVR compared to the control, which was measured before UVR exposure (FigureS5). Ecophysiological response of Stenomitos frigidus ULC029 to multiple stress factors (osmotic stress pretreatment, desiccation, and UVR) Contrary to the results obtained in Experiment 1, none of the variables measured showed significant differences between the cultures cultivated in medium with 0 or 0.2 M NaCl in the absence of other stresses (Control treatment; Figure2). However, significant differences were observed in most of variables after desiccating the cultures for 1 week and after the 4 days of rehydration and UVR exposure. Chlorophyll a concentration did not decrease compared to the control after maintaining the cultures 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 471 CYANOBACTERIAL RESISTANCE TO STRESS desiccated at 10% HR for 1 week after a pretreatment in BG11 with 0.2 M NaCl (Figure3). This pretreatment did not prevent the chl a from decreasing after the rehydration + UVR treatment; however, the concentration was significantly higher (0.4 μg · mL−1; Figure2) than in the absence of NaCl pretreatment (0.03 μg · mL−1; Figure2). The different pretreatments did not seem to influence the carotenoid concentrations after desiccation, but they were again significantly higher in the rehydrated- UVR exposed cultures pretreated with 0.2 M NaCl (Figure2). Interestingly, in this experiment, we detected the production of scytonemin for the first time (Figure2) after rehydrating and exposing the cultures to UVR (Figure2). A significantly higher concentration was observed in cultures preincubated with 0.2 M NaCl (0.31 μg · mL−1; Figure2) compared to 0 M NaCl. The production of EPS increased after desiccation in the absence of a NaCl pretreatment. However, it increased further after rehydration and UVR exposure regardless of the pretreatment (Figure2). The total content of EPS was always significantly lower when Stenomitos frigidus was pregrown in medium with 0.2 M NaCl, except for the control (Figure2). The loosely bound fraction of EPS was significantly different between salinity pretreatments (FigureS6 in the Supporting Information). This fraction was always significantly higher in cultures pretreated with 0 M NaCl, even in the control treatment (FigureS6). The only significant increase of EPS after desiccation compared to the control was overserved in the LBEPS fraction of the 0 M NaCl pretreatment (FigureS6). After the rehydration + UVR treatment, both EPS fractions significantly increased compared with the control and desiccation treatments (FigureS6). The production of the osmoprotectants trehalose and sucrose was significantly higher after the desiccation and rehydration + UVR treatments in the absence of a NaCl pretreatment, except for the control treatments in which there were no differences (Figure2). Their concentrations significantly increased after desiccation compared to the control for both preincubation types, followed by a significant decrease after rehydration + UVR, whatever the pretreatment type (Figure2). The highest concentrations were observed FIGURE 1 Pigment contents produced by Stenomitos frigidus ULC029 cultured at different concentrations of NaCl during 32 days of incubation and after subsequent recovery in BG11 medium for 33 days in Experiment 1: (a) chlorophyll a and (b) carotenoid concentrations during the 32 days of incubation; (c) chlorophyll a and (d) carotenoid concentrations after 32 days compared with the same cultures exposed to 3 additional days of UVR. All data are means (n = 4). The error bars represent ±SE and letters indicate significant differences (p < 0.05) among treatments. Test performed, df, and critical values are, respectively: (a) 32 days (TukeyHSD, 12, 4.20); (a) recovery and (b) 20 days and recovery (TukeyHSD, 18, 4.49) and 33 days (Conover- Iman, 18, 20.64); (c and d) (TukeyHSD, 12, 4.20). [Color figure can be viewed at wileyonlinelibrary.com] 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 472 | RONCERORAMOS etal. after desiccation of the strain pregrown without NaCl, namely for sucrose (1130.3 mg · mL−1), followed by trehalose (981 mg · mL−1; Figure2). The efficiency of the photosynthesis (Fv/Fm) did not show differences when Stenomitos frigidus had been precultured with or without 0.2 M NaCl in any treatment FIGURE 2 Influence of 1 week of desiccation and subsequent 4 days of rehydration combined with UVR exposure on Stenomitos frigidus ULC029 cultured in BG11 with 0 or 0.2 M NaCl compared with the control (initial values before desiccation) on different variables: chlorophyll a, carotenoids, scytonemin, and total exopolysaccharides contents; sucrose and trehalose concentrations; and the photosynthetic efficiency (Fv/Fm). All data are means (n = 4). The error bars represent ±SE and letters indicate significant differences (p < 0.05) among treatments. The test performed, df and critical values are in all cases: TukeyHSD, 18, and 4.49, respectively. [Color figure can be viewed at wileyonlinelibrary.com] 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 473 CYANOBACTERIAL RESISTANCE TO STRESS (Figure2). However, the strain was photosynthetically inactive after desiccation (∼0; Figure2), as expected, and started recovering its initial values of Fv/Fm after rehydration and UVR treatment (Figure2). Mechanisms to cope with stress potentially observed in the genome of ULC029 Genome assembly After sequencing and combining short and long reads of Stenomitos frigidus ULC029, we obtained eight bins (TableS4 in the Supporting Information). One of them was identified by CheckM as a cyanobacterium, with a completeness of 99.29% and a contamination of 0.35% (Table1). This genome was assigned to S. frigidus ULC029 and had a total length of 6,403,461 bp and a GC% of 50.68 (Table1). It was composed of 1 long contig (6,320,863 bp) and 2 short contigs (FigureS7 in the Supporting Information), with a coding ratio of 80.6% (Table1). Gene annotation by DFAST resulted in 5587 CDSs, six rRNAs, and 47 tRNAs (Table1). GTDBtk classification placed the genome in the Stenomitos cluster (see FigureS7) with S. frigidus ULC018 (GCF_003003795.1), which showed the closest ANI value (79.22%) and was isolated from a neighboring lake microbial mat in the same Antarctic region (Stenomitos sp. ANT.LH53B.2; Taton etal.,2006). Mechanisms to cope with osmotic, desiccation, and UVR stress Genome annotation was enhanced after using the COG and KEGG databases, which resulted in 4042 and 2026 annotated genes, respectively (TableS1). Among them, we identified 140 genes coding for proteins known to be involved in resistance mechanisms to desiccation, UVR, and salinity stress (TableS5 in the Supporting Information): production of the EPS matrix, photoprotection, oxidative stress protection, DNA repair, and osmotic stress protection. One hundred thirteen of these were annotated with the COGs and/or KEGGs databases, and 27 were identified using tblastn against the protein sequence of interest downloaded from NCBI (TableS3). We detected genes coding for proteins related to the production of the following monosaccharides FIGURE 3 Complete pathways related to stress resistance mechanisms observed in ULC029. [Color figure can be viewed at wileyonlinelibrary.com] TABLE 1 Genome characteristics obtained from results produced by Quast, DFAST, anvi'o, and CheckM software. Stenomitos frigidus ULC029 Contigs 3 Largest contig (bp) 6,320,863 Genome size (bp) 6,403,461 GC (%) 50.68 No. of CDSs 5587 No. of rRNA operons 3 No. of tRNA 47 No. of CRISPRS 15 Coding ratio (%) 80.6 COGs assignation 4042 KEGGs assignation 2026 CheckM completeness (%) 99.29 CheckM contamination (%) 0.35 Abbreviation: CRISPRS, Clustered Regularly Interspaced Short Palindromic Repeats. 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 480 | RONCERORAMOS etal. Kokabi, M., Yousefzadi, M., Soltani, M., & Arman, M. (2019). Effects of different UV radiation on photoprotective pigments and antioxidant activity of the hotspring cyanobacterium Leptolyngbya cf. fragilis. Phycological Research, 67, 215–220. Kolmogorov, M., Bickhart, D. M., Behsaz, B., Gurevich, A., Rayko, M., Shin, S. B., Kuhn, K., Yuan, J., Polevikov, E., Smith, T. P. L., & Pevzner, P. A. (2020). metaFlye: Scalable longread metagenome assembly using repeat graphs. Nature Methods, 17, 1103–1110. Kopf, M., Möke, F., Bauwe, H., Hess, W. R., & Hagemann, M. (2015). Expression profiling of the bloomforming cyanobacterium Nodularia CCY9414 under light and oxidative stress conditions. ISME Journal, 9, 2139–2152. Koren, S., Schatz, M. C., Walenz, B. P., Martin, J., Howard, J. T., Ganapathy, G., Wang, Z., Rasko, D. A., McCombie, W. R., Jarvis, E. D., & Phillippy, A. M. (2012). Hybrid error correction and de novo assembly of singlemolecule sequencing reads. Nature Biotechnology, 30(7), 693–700. Kvíderová, J., Elster, J., & Komárek, J. (2019). Ecophysiology of cyanobacteria in the polar regions. In A. K. Mishra, D. N. Tiwari, & A.N. Rai (Eds.), Cyanobacteria (pp. 277–302). Academic Press. Letunic, I., & Bork, P. (2020). SMART: Recent updates, new developments and status in 2020. Nucleic Acids Research, 49(1), 458–460. Li, C., Zhang, X., Ye, T., Li, X., & Wang, G. (2022). Protection and damage repair mechanisms contributed to the survival of Chroococcidiopsis sp. exposed to a marslike near space environment. Microbiology Spectrum, 10(6), e0344022. Li, H., & Durbin, R. (2009). Fast and accurate short read alignment with burrowswheeler transform. Bioinformatics, 25, 1754–1760. Lin, C. S., & Wu, J. T. (2014). Tolerance of soil algae and cyanobacteria to drought stress. Journal of Phycology, 50(1), 131–139. Llewellyn, C. A., Airs, R. L., Farnham, G., & Greig, C. (2020). Synthesis, regulation and degradation of carotenoids under low level UV- B radiation in the filamentous cyanobacterium Chlorogloeopsis fritschii PCC 6912. Frontiers in Microbiology, 11, 163. Mackay, M. A., Norton, R. S., & Borowitzka, L. J. (1984). Organic osmoregulatory solutes in cyanobacteria. Journal of General Microbiology, 130, 2177–2191. Maestre, F. T., Bowker, M. A., Cantón, Y., Castillo- Monroy, A. P., Cortina, J., Escolar, C., Escudero, A., Lázaro, R., & Martínez, I. (2011). Ecology and functional roles of biological soil crusts in semiarid ecosystems of Spain. Journal of Arid Environments, 75(12), 1282–1291. Mager, D. M., & Thomas, A. D. (2011). Extracellular polysaccharides from cyanobacterial soil crusts: A review of their role in dryland soil processes. Journal of Arid Environments, 75, 91–97. Mosca, C., Fagliarone, C., Napoli, A., Rabbow, E., Rettberg, P., & Billi, D. (2021). Revival of anhydrobiotic cyanobacterium biofilms exposed to space vacuum and prolonged dryness: Implications for future missions beyond low earth orbit. Astrobiology, 21(5), 541–550. Mugnai, G., Rossi, F., Felde, V. J. M. N. L., Colesie, C., Büdel, B., Peth, S., Kaplan, A., & Philippis, R. (2018). The potential of the cyanobacterium Leptolyngbya ohadii as inoculum for stabilizing bare sandy substrates. Soil Biology and Biochemistry, 127, 318–328. Mulders, K. J. M., Weesepoel, Y., Bodenes, P., Lamers, P. P., Vincken, J.- P., Martens, D. E., Gruppen, H., & Wijffels, R. H. (2015). Nitrogendepleted chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: A carotenoid metabolism study. Journal of Applied Phycology, 27, 125–140. Muñoz- Martín, M. A., Becerra- Absalón, I., Perona, E., Fernández- Valbuena, L., Garcia- Pichel, F., & Mateo, P. (2019). Cyanobacterial biocrust diversity in Mediterranean ecosystems along a latitudinal and climatic gradient. New Phytologist, 22, 123–141. Muramatsu, M., & Hihara, Y. (2012). Acclimation to highlight conditions in cyanobacteria: From gene expression to physiological responses. Journal of Plant Research, 125, 11–39. Napoli, A., Iacovelli, F., Fagliarone, C., Pascarella, G., Falconi, M., & Billi, D. (2021). Genomewide identification and bioinformatics characterization of superoxide dismutases in the desiccationtolerant cyanobacterium Chroococcidiopsis sp. CCMEE 029. Frontiers in Microbiology, 12, 660050. Pade, N., & Hagemann, M. (2014). Salt acclimation of cyanobacteria and their application in biotechnology. Life, 5(1), 25–49. Park, C. H., Li, X. R., Zhao, Y., Jia, R. L. & Hur, J. S. (2017). Rapid development of cyanobacterial crust in the field for combating desertification. PLoS One, 12(6), e0179903. https:// doi. org/ 10. 1371/ journ al. pone. 0179903 Parks, D. H., Chuvochina, M., Waite, D. W., Rinke, C., Skarshewski, A., Chaumeil, P.- A., & Hugenholtz, P. (2018). A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nature Biotechnology, 36, 996–1004. Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P., & Tyson, G. W. (2014). Assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Research, 25, 1043–1055. Pathak, J., Rajneesh, Singh, P. R., Häder, D. P., & Sinha, R. P. (2019). UV- induced DNA damage and repair: A cyanobacterial perspective. Plant Gene, 19, 100194. Patzelt, D. J., Hodač, L., Friedl, T., Pietrasiak, N., & Johansen, J. R. (2014). Biodiversity of soil cyanobacteria in the hyperarid Atacama Desert, Chile. Journal of Phycology, 50(4), 698–710. Pearson, W. R. (2013). An introduction to sequence similarity (“homology”) searching. Current Protocols in Bioinformatics, 3, 3.1.1–3.1.8. Pereira, S., Zille, A., Micheletti, E., Moradas- Ferreira, P., De Philippis, R., & Tamagnini, P. (2009). Complexity of cyanobacterial exopolysaccharides: Composition, structures, inducing factors and putative genes involved in their biosynthesis and assembly. FEMS Microbiology Reviews, 33, 917–941. Pereira, S. B., Sousa, A., Santos, M., Araújo, M., Serôdio, F., Granja, P., & Tamagnini, P. (2019). Strategies to obtain designer polymers based on cyanobacterial extracellular polymeric substances (EPS). International Journal of Molecular Sciences, 20(22), 5693. Pessi, I. S., Popin, R. V., Durieu, B., Lara, Y., Savaglia, V., Roncero- Ramos, B., Hultman, J., Verleyen, E., Vyverman, W., & Wilmotte, A. (2023). Novel diversity of polar cyanobacteria revealed by genomeresolved metagenomics. Microbial Genomics, 9(7), 001056. Potts, M. (1994). Desiccation tolerance of prokaryotes. Microbiology Reviews, 58, 755–805. Pushkareva, E., Pessi, I. S., Wilmotte, A., & Elster, J. (2015). Cyanobacterial community composition in Arctic soil crusts at different stages of development. FEMS Microbiology Ecology, 91, fiv143. R Core Team. (2017). R: A Language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Raanan, H., Oren, N., Treves, H., Berkowicz, S. M., Hagemann, M., Pade, N., Keren, N., & Kaplan, A. (2016). Simulated soil crust conditions in a chamber system provide new insights on cyanobacterial acclimation to desiccation. Environmental Microbiology, 18(2), 414–426. Rai, S., Singh, S., Shrivastava, A. K., & Rai, L. C. (2013). Salt and UV- B induced changes in anabaena PCC 7120: Physiological, proteomic and bioinformatic perspectives. Photosynthesis Research, 118, 105–114. Rajeev, L., da Rocha, U., Klitgord, N., Luning, E. G., Fortney, J., Axen, S. D., Shih, P. M., Bouskill, N. J., Bowen, B. P., Kerfeld, 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 481 CYANOBACTERIAL RESISTANCE TO STRESS C. A., Garcia- Pichel, F., Brodie, E. L., Northen, T. R., & Mukhopadhyay, A. (2013). Dynamic cyanobacterial response to hydration and dehydration in a desert biological soil crust. ISME Journal, 7, 2178–2191. Rego, A., Raio, F., Martins, T. P., Ribeiro, H., Sousa, A. G. G., Séneca, J., Baptista, M. S., Lee, C. K., Cary, S. C., Ramos, V., Carvalho, M. F., Leão, P. N., & Magalhães, C. (2019). Actinobacteria and cyanobacteria diversity in terrestrial Antarctic microenvironments evaluated by culturedependent and independent methods. Frontiers in Microbiology, 10, 1018. Rehakova, K., Capkova, K., Hrouzek, P., Koblizek, M., & Dolezal, J. (2019). Microbial photosynthetic and photoprotective pigments in Himalayan soils originating from different elevations and successional stages. Soil Biology and Biochemistry, 132, 153–164. Rippka, R., Deruelles, J., & Waterbury, J. B. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology, 111, 1–61. Román, J. R., Chamizo, S., Roncero- Ramos, B., Adessi, A., De Philippis, R., & Cantón, Y. (2021). Overcoming field barriers to restore dryland soils by cyanobacteria inoculation. Soil and Tillage Research, 207, 104799. Román, J. R., Roncero- Ramos, B., Rodríguez- Caballero, E., Chamizo, S., & Canton, Y. (2021). Effect of water availability on induced cyanobacterial biocrust development. Catena, 197, 104988. Roncero- Ramos, B., Muñoz- Martín, M. Á., Cantón, Y., Chamizo, S., Rodríguez- Caballero, E., & Mateo, P. (2020). Land degradation effects on composition of pioneering soil communities: An alternative successional sequence for dryland cyanobacterial biocrusts. Soil Biology and Biochemistry, 146, 107824. Roncero- Ramos, B., Muñoz- Martín, M. A., Chamizo, S., Fernández- Valbuena, L., Mendoza, D., Perona, E., Cantón, Y., & Mateo, P. (2019). Polyphasic evaluation of key cyanobacteria in biocrusts from the most arid region in Europe. PeerJ, 7, e6169. Rossi, F., Li, H., Liu, Y., & De Philippis, R. (2017). Cyanobacterial inoculation (cyanobacterisation): Perspectives for the development of a standardized multifunctional technology for soil fertilization and desertification reversal. Earth- Science Reviews, 171, 28–43. Rossi, F., Micheletti, E., Bruno, L., Adhikary, S. P., Albertano, P., & De Philippis, R. (2012). Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments. Biofouling, 28(2), 215–224. Rossi, F., Mugnai, G., & De Philippis, R. (2018). Complex role of the polymeric matrix in biological soil crusts. Plant and Soil, 429, 19–34. Sabbe, K., Hodgon, D. A., Verleyen, E., Taton, A., Wilmotte, A., Vanhoutte, K., & Viverman, W. (2004). Salinity, depth and the structure and composition of microbial mats in continental Antarctic lakes. Freshwater Biology, 49, 296–319. Shalygin, S., Shalygina, R. R., Redkina, V. V., Gargas, C. V., & Johansen, J. R. (2020). Description of Stenomitos kolaenensis and S. hiloensis sp. nov. (Leptolyngbyaceae, cyanobacteria) with an emendation of the genus. Phytotaxa, 440(2), 108–128. Shang, J. L., Chen, M., Hou, S., Li, T., Yang, Y. W., Li, Q., Jiang, H. B., Dai, G. Z., Zhang, Z. C., Hess, W. R., & Qiu, B. S. (2019). Genomic and transcriptomic insights into the survival of the subaerial cyanobacterium Nostoc flagelliforme in arid and exposed habitats. Environmental Microbiology, 21(2), 845–863. Shimura, Y., Hirose, Y., Misawa, N., Osana, Y., Katoh, H., Yamaguchi, H., & Kawachi, M. (2015). Comparison of the terrestrial cyanobacterium Leptolyngbya sp. NIES- 2104 and the freshwater Leptolyngbya boryana PCC 6306 genomes. DNA Research, 22(6), 403–412. Sinetova, M. A., & Los, D. A. (2016). Systemic analysis of stress transcriptomics of Synechocystis reveals common stress genes and their universal triggers. Molecular BioSystems, 12(11), 3254–3258. Soule, T., Gao, Q., Stout, V., & Garcia- Pichel, F. (2013). The global response of nostoc punctiforme ATCC 29133 to UVA stress, assessed in a temporal DNA microarray study. Photochemistry and Photobiology, 89(2), 415–423. Stothard, P., & Wishart, D. S. (2005). Circular genome visualization and exploration using CGView. Bioinformatics, 21, 537–539. Strunecký, O., Ivanova, A. P., & Mareš, J. (2022). An updated classification of cyanobacterial orders and families based on phylogenomic and polyphasic analysis. Journal of Phycology, 59(1), 12–51. Swapnil, P., & Rai, A. K. (2018). Physiological responses to salt stress of saltadapted and directly salt (NaCl and NaCl+Na2SO4 mixture)- stressed cyanobacterium anabaena fertilissima. Protoplasma, 255, 963–976. Tamaru, Y., Takani, Y., Yoshida, T., & Sakamoto, T. (2005). Crucial role of extracellular polysaccharides in desiccation and freezing tolerance in the terrestrial cyanobacterium Nostoc commune. Applied and Environmental Microbiology, 71, 7327–7333. Tanizawa, Y., Fujisawa, T., Kaminuma, E., Nakamura, Y., & Arita, M. (2016). DFAST and DAGA: Webbased integrated genome annotation tools and resources. Bioscience of Microbiota, Food and Health, 35(4), 173–184. Taton, A., Grubisic, S., Ertz, D., Hodgson, D. A., Piccardi, R., Biondi, N., Tredici, M. R., Mainini, M., Losi, D., Marinelli, F., & Wilmotte, A. (2006). Polyphasic study of Antarctic cyanobacterial strains. Journal of Phycology, 42(6), 1257–1270. Temraleeva, A. D. (2018). Cyanobacterial diversity in the soils of Russian dry steppes and semideserts. Microbiology, 87, 249–260. UN General Assembly. (2015). Transforming our world: the 2030 Agenda for Sustainable Development, A/RES/70/1. https:// www. refwo rld. org/ docid/ 57b6e 3e44 Walker, B. J., Abeel, T., Shea, T., Priest, M., Abouelliel, A., Sakthikumar, S., Cuomo, C. A., Zeng, Q., Wortman, J., Young, S. K., & Earl, A. M. (2014). Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE, 9(11), e112963. Weber, B., Belnap, J., Büdel, B., Antoninka, A. J., Barger, N. N., Chaudhary, V. B., Darrouzet- Nardi, A., Eldridge, D. J., Faist, A. M., Ferrenberg, S., Havrilla, C. A., Huber- Sannwald, E., Malam Issa, O., Maestre, F. T., Reed, S. C., Rodriguez- Caballero, E., Tucker, C., Young, K. E., Zhang, Y., … Bowker, M. A. (2022). What is a biocrust? A refined, contemporary definition for a broadening research community. Biological Reviews, 97, 1768–1785. Weber, B., Büdel, B., & Belnap, J. (2016). Biological soil crusts: An organizing principle in drylands. Springer. Williams, L., Loewen- Schneider, K., Maier, S., & Budel, B. (2016). Cyanobacterial diversity of western European biological soil crusts along a latitudinal gradient. FEMS Microbiology Ecology, 92(10), fiw157. Wu, S., Yu, K., Li, L., Wang, L., & Liang, W. (2021). Enhancement of exopolysaccharides production and reactive oxygen species level of Nostoc flagelliforme in response to dehydration. Environmental Science and Pollution Research, 28(26), 34300–34308. Ye, T., Wang, B., Li, C., Bian, P., Chen, L., & Wang, G. (2021). Exposure of cyanobacterium Nostoc sp. to the Marslike stratosphere environment. Journal of Photochemistry and Photobiology, 224, 112307. Zhang, L., Jungblut, A. D., Hawes, I., Andersen, D. T., Sumner, D. Y., & Mackey, T. J. (2015). Cyanobacterial diversity in benthic mats 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 482 | RONCERORAMOS etal. of the McMurdo Dry Valley lakes, Antarctica. Polar Biology, 38, 1097–1110. Zhou, K., Aertsen, A., & Michiels, C. W. (2014). The role of variable DNA tandem repeats in bacterial adaptation. FEMS Microbiology Reviews, 38(1), 119–141. SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. How to cite this article: Roncero- Ramos, B., Savaglia, V., Durieu, B., VandeVreken, I., Richel, A., & Wilmotte, A. (2024). Ecophysiological and genomic approaches to cyanobacterial hardening for restoration. Journal of Phycology, 60, 465–482. https://doi.org/10.1111/jpy.13436 15298817, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpy.13436 by Universidad De Sevilla, Wiley Online Library on [31/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License