Abstract
Cyanobacteria are photosynthetic oxygen-evolving prokaryotes that are distributed in diverse habitats. They synthesize the ultraviolet (UV)-screening pigments, scytonemin (SCY) and mycosporine-like amino acids (MAAs), located in the exopolysaccharide (EPS) matrix. Multiple roles for both pigments have gradually been recognized, such as sunscreen ability, antioxidant activity, and heat dissipation from absorbed UV radiation. In this study, a filamentous terrestrial cyanobacterium Nostoc flagelliforme was used to evaluate the potential stabilizing role of SCY on the EPS matrix. SCY (∼3.7 %) was partially removed from N. flagelliforme filaments by rinsing with 100 % acetone for 5 s. The physiological damage to cells resulting from this treatment, in terms of photosystem II activity parameter Fv/Fm, was repaired after culturing the sample for 40 h. The physiologically recovered sample was further desiccated by natural or rapid drying and then allowed to recovery for 24 h. Compared with the normal sample, a relatively slower Fv/Fm recovery was observed in the SCY-partially removed sample, suggesting that the decreased SCY concentration in the EPS matrix caused cells to suffer further damage upon desiccation. In addition, the SCY-partially removed sample could allow the release of MAAs (∼25 %) from the EPS matrix, while the normal sample did not. Therefore, damage caused by drying of the former resulted from at least the reduction of structural stability of the EPS matrix as well as the loss of partial antioxidant compounds. Considering that an approximately 4 % loss of SCY led to this significant effect, the structurally stabilizing potential of SCY on the EPS matrix is crucial for terrestrial cyanobacteria survival in complex environments.
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Cyanobacteria are primitive photosynthetic oxygen-evolving prokaryotes that appeared on Earth when the ozone shield was absent [1]. Today, they are ubiquitous in terrestrial, freshwater, and marine habitats, having adapted to various extreme environments. Many cyanobacteria are surrounded by a matrix of exopolysaccharides (EPSs) and synthesize ultraviolet (UV)-screening pigments, including mycosporine-like amino acids (MAAs) and scytonemin (SCY) [2–4]. Both pigments can accumulate within the EPS matrix, with the latter distributed in the peripheral region [4–6]. SCY is mainly induced by UV-A radiation and can prevent up to 90 % of incident UV-A from entering the cells [7]. In some tested cyanobacteria, MAA concentrations ranged from 0.61 up to 8.23 mg/g dry weight (DW) and SCY ranged from 0.76 to 79.84 mg/g DW [8]. In the terrestrial cyanobacterium Nostoc commune, DW percentages were estimated to be >50, 4, and 0.4 % for EPS, MAAs, and SCY, respectively [6]. With the accumulation of research data, the multi-functionality of both pigments has been recognized, such as sunscreen ability, antioxidant activity, and heat dissipation from absorbed UV radiation [9–11]. The last role may facilitate the optimization of photosynthesis by increasing the surface temperature of cyanobacteria grown in cold environments. In addition, the significant stability of SCY has been recognized [12, 13]. Because of its abundance and peripheral distribution in the EPS matrix of cyanobacteria, as well as its molecular stability, we speculate that SCY may play an extra eco-physiological role in stabilizing the EPS matrix.
The terrestrial cyanobacterium Nostoc flagelliforme, found in the arid or semi-arid steppes of some countries, has a hair-like colony form (filament) of 0.2–1 mm in diameter [14]. It undergoes frequent cycles of desiccation and rehydration in its native habitats. When fully rehydrated, the filaments expand 2–4-fold in diameter. During rehydration and desiccation, the cells embedded in the dense EPS matrix remain intact [15]. Light microscopy of N. flagelliforme sections clearly shows the peripheral distribution of SCY in the filaments [5]. Thus, this species serves as an ideal material for investigating the potential role of SCY in stabilizing the EPS matrix. The chemical reagent acetone was often used for SCY extraction [7, 13]. When fully rehydrated filaments were rinsed in 100 % acetone for 5 s, the loss of ∼3.7 % of total SCY was observed (Fig. 1a). SCY in the N. flagelliforme sample was calculated to be 0.21 mg/g fresh weight (FW), according to the extinction coefficient of 112.6 L g−1 cm−1 at 384 nm [16]. Meanwhile, this rapid rinsing process also destroyed some cells, as implied by the tiny peak of chlorophyll a (Chl a) observed (Fig. 1a). However, this peak only accounted for ∼0.3 % of total Chl a content (0.27 mg/g FW), as calculated by the extinction coefficient of 92.6 L g−1 cm−1 at 663 nm [16]. The peripheral distribution of SCY was also implied by the higher SCY/Chl a ratio (12.3) than the average ratio (0.78). As shown in Fig. 1b, SCY-partially removed sample showed physiological damage but fully recovered its physiological activity after a 40-h culture period, in terms of the widely used photosystem II (PSII) activity parameter Fv/Fm [17–19]. Cyanobacterial Fv/Fm is very indicative to quickly detect stressful conditions and monitor adaptation responses to surrounding environments [19]. The recovered samples were further desiccated by natural or rapid drying with hot air followed by 24 h of culture for recovery (Fig. 1c). The SCY concentration did not change during these short-term drying treatments or the recovery processes (data not shown). A relatively slower physiological recovery for SCY-partially removed sample, especially after rapid drying, was observed compared with the normal sample (Fig. 1c), implying that the removal of SCY could cause additional damage to cells subjected to desiccation stress.
Modulation of the structure and function of the dense EPS matrix has been taken to be crucial for desiccation resistance in terrestrial cyanobacteria [6]. The shrinking process of N. flagelliforme filaments upon desiccation can have two effects on the EPS matrix, a change in the structural rigidity/elasticity and an oxidative burst [6, 15, 20]. The secreted WSPA protein putatively plays an important role in maintaining dynamic structural stability [6]. Extracellular superoxide dismutase, MAAs, SCY, and even EPS itself are thought to scavenge reactive oxygen species [2, 21]. The effect of decreased SCY on the structural stability of the EPS matrix was evaluated by the release of MAAs (Fig. 2). Normal sample does not obviously release MAAs from the EPS matrix during the rehydration process, although the EPS matrix is expanded and also the dissociation of oligosaccharide-linked MAAs from the EPS cannot be excluded. n-acetylcysteine (NAC) can efficiently destabilize the EPS matrix of N. flagelliforme to allow the release of MAAs [5], an effect used for comparison purposes in this study. Acidic NAC solution may mediate the process of EPS hydrolysis to release MAAs, due to its acid hydrolysis effect. As shown in Fig. 2a, SCY-partially removed sample released partial MAAs following 1-h incubation in both solutions (BG110, BG110 supplemented with 10 mM NAC), and an additive effect was also observed for it in the NAC solution. Therefore, a structural relaxation of the EPS matrix was caused by the partial removal of SCY. Following incubation for 16 h (Fig. 2b), the maximum release of MAAs was calculated to be 2.03 mg/g FW for samples in the NAC solution, according to the extinction coefficient of 17 L g−1 cm−1 at 312 nm [22]. At this time, approximately, a quarter of total MAAs was released from the SCY-partially removed sample. These results implied that the aforementioned drying damage caused to the SCY-partially removed sample resulted at least from the altered structural stability of the EPS matrix as well as the partial loss of antioxidant compounds. Considering that approximately 4 % loss of SCY led to such a significant effect, the structurally stabilizing potential of SCY on the EPS matrix appears crucial for terrestrial cyanobacteria survival in arid regions.
Through rapid rinsing of N. flagelliforme samples with acetone, the potential stabilizing role of SCY on the EPS matrix was uncovered in this study. Due to its indispensability, this role cannot readily be uncoupled from its other key roles. Its hydrophobic feature, specific molecular structure, or even its oxidized state [23] might be associated with this novel function. However, a more appropriate experimental system is required to evaluate the functional mechanism of SCY with respect to the stabilizing of EPS matrix. This finding is consistent with our previous report showing that coating of aquatic-living N. flagelliforme colonies (which has lost a natural colony form) with the polymer polyvinylpyrrolidone, to potentially confer stability, endowed cells with desiccation resistance [20]. In contrast to N. flagelliforme, non-colonial cyanobacteria, such as Lyngbya sp. [24], contain a single trichome in their individual filaments. To avoid a potentially lethal crushing effect on the trichome upon acute desiccation, abundant SCY in the EPS matrix may play a more crucial role in maintaining the rigidity of the matrix. In general, multiple roles of secondary metabolites, including SCY, have provided crucial protection for cyanobacteria surviving in complex environments with simultaneous multiple stresses.
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Acknowledgments
This work has been supported in part by the Fundamental Research Funds for the Central Universities (No. CCNU16A02007) and the National Natural Science Foundation of China (No. 31670104). I also thank group members for some help in this work.
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Gao, X. Scytonemin Plays a Potential Role in Stabilizing the Exopolysaccharidic Matrix in Terrestrial Cyanobacteria. Microb Ecol 73, 255–258 (2017). https://doi.org/10.1007/s00248-016-0851-4
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DOI: https://doi.org/10.1007/s00248-016-0851-4