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The filamentous green alga Zygogonium ericetorum (Zygnematophyceae, Streptophyta) was collected in a high-alpine rivulet in Tyrol, Austria. Two different morphotypes of this alga were found: a purple morph with a visible purple vacuolar content and a green morph lacking this coloration. These morphotypes were compared with respect to their secondary metabolites, ultrastructure, and ecophysiological properties. Colorimetric tests with aqueous extracts of the purple morph indicated the presence of soluble compounds such as phenolics and hydrolyzable tannins. High-performance liquid chromatography-screening showed that Z. ericetorum contained several large phenolic peaks with absorption maxima at ~280 nm and sometimes with minor maxima at ~380 nm. Such compounds are uncommon for freshwater green microalgae, and could contribute to protect the organism against increased UV and visible (VIS) irradiation. The purple Z. ericetorum contained larger amounts (per dry weight) of the putative phenolic substances than the green morph; exposure to irradiation may be a key factor for accumulation of these phenolic compounds. Transmission electron microscopy of the purple morph showed massive vacuolization with homogenous medium electron-dense content in the cell periphery, which possibly contains the secondary compounds. In contrast, the green morph had smaller, electron-translucent vacuoles. The ecophysiological data on photosynthesis and desiccation tolerance indicated that increasing photon fluence densities led to much higher relative electron transport rates (rETR) in the purple than in the green morph. These data suggest that the secondary metabolites in the purple morph are important for light acclimation in high-alpine habitats. However, the green morph recovered better after 4 d of rehydration following desiccation stress.
Zygogonium ericetorum Kützing is a filamentous zygnematophycean green alga, which occurs in extreme habitats such as open soils or acidic ponds (Lynn and Brock 1969, Hoppert et al. 2004, Kleeberg et al. 2006), waterbodies with high contents of heavy metals (Boyd et al. 2009), and high-alpine ephemeral streamlets (Holzinger et al. 2010). At high altitudes, the alga is exposed to harsh environmental conditions, including intense photosynthetically active radiation (PAR) and ultraviolet radiation (UVR), as investigated for Z. ericetorum by Cockell and Rothschild (1999); regular periods of desiccation (Holzinger et al. 2010); a short growing season; and sharp temperature fluctuations with occasional frost events, even during summer (for meteorological details see Karsten et al. 2010). Being subject to these different abiotic stresses, Z. ericetorum must possess a range of protective mechanisms to guarantee long-term survival. For example, photoprotection is needed to allow for the harmless dissipation of excess absorbed radiation energy, which otherwise would saturate photosynthetic electron transport and drive the transfer of electrons to oxygen (Mehler reaction), thereby generating reactive oxygen species (ROS; Saibo et al. 2009). Excess ROS inactivate photosystem II (PSII) reaction centers by causing chronic photoinhibition, can induce oxidation and depolymerization of nucleic acids and the breakage of peptide bonds (Kranner et al. 2010), and have other destructive intracellular effects (Hadacek et al. 2011). Rothschild (1994) showed in Z. ericetorum that the addition of CO2 enhanced carbon fixation rates during daylight hours, which led to the conclusion that the naturally occurring midday depression in photosynthesis was unlikely to be due to photoinhibition.
Currently, the phylogenetic position of the genus Zygogonium remains unclear, with no features that clearly separate Zygogonium from Zygnema (Hall et al. 2008, Stancheva et al. 2012). However, for this study we use the species name Z. ericetorum, which has marked vacuolar-confined purple pigmentation (e.g., West and Starkey 1915, Fritsch 1916, Transeau 1933, Lynn and Brock 1969, Hoppert et al. 2004, Holzinger et al. 2010, Newsome and van Breemen 2012); this name is traditionally used in the literature. The main goal of this study was to provide insights into the abundance of phenolic compounds in Z. ericetorum, as well as to provide a general ecophysiological description of the performance of this alga.
Zygogonium ericetorum is frequently exposed to desiccation stress (Holzinger et al. 2010) and forms conspicuous macroscopic mat-like sheets that can recover physiologically upon rewetting (e.g., Hoppert et al. 2004). Cells from the top layer of these sheets may provide an additional protective function for cells in lower layers. These cells possess thick, rigid cell walls that protect against high PAR and UV-irradiation by self-shading, as described for other filamentous conjugating green algae, such as members of Zygnema (Harrison and Smith 2009, Holzinger et al. 2009, Pichrtová et al. 2013). Macroalgal canopies of the chlorophyte Ulva sp. form multiple-layered sheet-like structures in the upper littoral zone when the blades are exposed. The top layer of this sheet usually bleaches due to strong insolation, desiccation, and other abiotic stresses, thereby providing photoprotection and moisture for the sub-canopy thalli (Bischof et al. 2002). A similar self-protecting strategy seems to be widespread among the filamentous algae.
Probably the most striking visual aspect of Z. ericetorum is the abundance of purple pigmentation stored in the vacuoles. The earliest report, by Lagerheim (1895), described the nature of the purple cell sap as “phycoporphyrin.” Alston (1958) analyzed the pigment in more detail and proposed that the compound is an “iron-tannin”; this compound was also formed when gallic acid was mixed with the bog water collected from the algae's habitat. Most recently, the purple pigment was suggested to be a highly branched polymer of glucose, containing traces of ester-linked polyphenolic moieties such as gallic acid, which exhibits a purple color when complexed by ferric iron (Newsome and van Breemen 2012).
Galloyl glucose derivatives have been identified in the filamentous green alga Spirogyra varians (Zygnematales) (Nishizawa et al. 1985, Cannell et al. 1988) and their synthesis increased after cold stress (Han et al. 2009). A galloylglucopyranose lends a brownish color to the vacuoles of the freshwater ice alga Mesotaenium berggrenii (Remias et al. 2012b). Mesotaenium berggreni is also a member of the Zygnematales, which raises the possibility that this derived group of streptophycean algae has evolved the potential to synthesize certain phenols. These compounds are well known in higher plants (e.g., flavonoids) and have been proposed to serve as photoprotectants against PAR and UVR, antioxidants, repellents against grazers, and organic osmolytes that preserve intracellular activities during freezing (Remias et al. 2012a,b). Among algae, only marine Phaeophyceae are known to synthesize large amounts of tannins; these compounds are suggested to serve multiple functions including cell-wall strengthening, feeding deterrents, antimicrobial agents, and UV sunscreens (Targett and Arnold 1998, Swanson and Druehl 2002, Schönwälder 2008, Holzinger et al. 2011a). Mycosporine-like amino acids (MAAs) are common photoprotective sunscreens in many, but not all algae taxa. These biomolecules have a high absorptivity for UV-A and UV-B radiation, but are not found in most green algae except in the aeroterrestrial representatives of the Trebouxiophyceae (Karsten et al. 2005, 2007).
The ultrastructure of Z. ericetorum during desiccation was evaluated by Holzinger et al. (2010). The present study compared green and purple morphs of Z. ericetorum with respect to pigmentation and light-dependent photosynthesis, as well as to the features of their water-soluble secondary compounds, using spectrophotometric assays, high-performance liquid chromatography (HPLC), and mass-spectrometry (LC-MS). Our main goal was to evaluate the levels of the secondary photoprotective metabolites in the purple and green morphs, and to test whether elevated levels of these compounds were correlated with a higher tolerance to enhanced solar irradiation.
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This study compared the cell structure and ultrastructure, differences in the abundance of phenolic compounds, and ecophysiological performance of purple and green morphs of Z. ericetorum. To our knowledge, this is the first description of the ultrastructure of Z. ericetorum after high-pressure freeze fixation and freeze substitution. Several unusual phenolic compounds were detected, molecular masses of several compounds were determined, and spectrophotometric tests revealed higher amounts of phenolics as well as hydrolyzable tannins in the purple morph. Measurements of the rETR demonstrated that the purple morph was substantially better protected against higher irradiation levels; however, this morph was sensitive to desiccation.
Structure and ultrastructure
Earlier studies by Hoppert et al. (2004) and Holzinger et al. (2010) investigated the structure of field-grown Z. ericetorum by classical chemical fixation procedures. While the gross appearance of the cellular structure is similar after chemical fixation, some details were preserved better by the high-pressure freeze-fixation technique.
The most prominent compartments, likely containing high amounts of phenolics and tannins, were the large vacuoles surrounding the central parts of the cells. The likelihood that these compounds are present in the vacuoles is further supported by the observation that these vacuoles showed higher electron-density in the purple than in the green morph. However, the degree of electron density varied, depending on the degree of osmium tetroxide reduction. It appears particularly interesting that the freshwater ice algae Ancylonema nordenskiöldii (Remias et al. 2012a) and M. berggrenii (Remias et al. 2009) also have structurally similar vacuoles with varying electron densities. Other compartments, with particularly high-electron contrast were round and only about 1–2 μm in diameter; similar structures have been described in Zygnema sp. (Holzinger et al. 2009, Pichrtová et al. 2013), M. berggrenii (Remias et al. 2009), and A. nordenskiöldii (Remias et al. 2012a). These structures seem to be similar to the brown-algal phlorotannins containing physodes (Schönwälder 2008).
Remarkably, only a single peroxisome was found in the center of each cell, located between the nucleus and the chloroplast. This is particularly interesting, as a similar phenomenon was observed in other streptophycean algae of the Klebsormidiales (Honda and Hashimoto 2007, Holzinger et al. 2011b). Whether this observation has phylogenetic significance remains to be demonstrated. The electron-dense compartments covering the chloroplasts are of high ecophysiological significance. Similar structures were observed in other members of the Zygnematales, such as Zygnema sp., and were suggested to contain the phenolic compounds (Holzinger et al. 2010, Pichrtová et al. 2013).
Ecological importance of phenolic compounds
As described by Holzinger et al. (2010), the top layers of the Z. ericetorum mats appeared especially intensely purple in the field; this coloration was lost under culture condition, in the absence of UV irradiation (unpublished observation). Since the purple morph significantly absorbs in the VIS region, the pigmentation may prevent damage from intense light to the chloroplast caused, for example, by photoinhibition or the generation of ROS. Other abiotic stresses such as desiccation and temperature fluctuations could also be responsible for increasing the content of phenolics, due to their high-antioxidative potential. While phenolics are less common in non-streptophycean green algae, in Zygnematophyceae they have been repeatedly reported (Han et al. 2007, Remias et al. 2012a,b, Pichrtová et al. 2013).
A previous study in Z. ericetorum showed that primary production increased when UV-A and UV-B were screened out (Cockell and Rothschild 1999). The present finding of higher amounts of UV-A and UV-B absorbing compounds (RT 13.9 and 25.2 min) in the purple morph compared to the green morph, indicates that protection against excessive irradiation is important for these algae. Interestingly, the largest increase was found in compound RT 7.2 min (molecular mass: 332 u), which was hypothesized to be galloylglucopyranose, a glucogallin (see below). Most likely, this substance plays a key role in the formation of the purple pigmentation. A recent conference contribution of Newsome and van Breemen (2012) demonstrated that the purple color of a specimen of Z. ericetorum, collected in Yellowstone National Park (WY, USA), was due to complexation of polyphenolic moieties, such as gallic acid, with ferric iron.
Using a different HPLC protocol (230 nm DAD-chromatogram, resin column instead of C18), Holzinger et al. (2010) found in Z. ericetorum two major peaks with a spectral absorption maximum at 270 nm and a shoulder at 380 nm, and another peak with absorption only at less than 330 nm and a maximum at 280 nm. Analyzing Arctic and Antarctic species of Zygnema, Pichrtová et al. (2013) showed four major phenolic compounds, at different RT (RT 6.5, 8.3, 16.1 and 18.8 min), as described in this study, but detected with an almost identical RP C18 method. For M. berggrenii, Remias et al. (2012b) reported three UV-absorbing phenolic compounds at RTs of 6.6, 17.2 and 18.1 min. Obviously, screening mechanisms exist in zygnematophycean freshwater ice algae, which probably down-regulate photosynthesis upon exposure to high irradiances, as opposed to other reported protective strategies, such as photochemical quenching and cell movement (Yallop et al. 2012); similar strategies can therefore also be expected in Z. ericetorum.
The absence of UV-absorbing compounds such as MAAs or secondary carotenoids suggests that mainly phenolic compounds are responsible for the photo-protection in Z. ericetorum. The production of phenolic compounds incurs lower metabolic costs than the production of MAAs, because phenolics do not contain nitrogen (Carreto and Carignan 2011). This is particularly important for the Zygnematales, which are mostly found in oligotrophic environments such as spring waters in the high Alps, the source of the species investigated here.
The implications of our finding of the unusual phenolic compounds in Z. ericetorum might be broader than simply an interaction between plants and abiotic conditions. Phenolic compounds can also discourage natural enemies of plants, including fungi, insects and mammalian herbivores (Dudt and Shurer 1994, Lattanzio et al. 2006). Although hydrolyzable tannins can inhibit the activity of alpha-glucosidase (Cannell et al. 1988), no negative effects on herbivores were revealed (Alonso et al. 2002), indicating that the higher production of phenolics and tannins may function predominantly as photo-protectants.
Functions and sources of hydrolyzable tannins
The positive result of the FC-assay and the characteristic absorbance shifts in different alkalinities (Harborne 1998) clearly demonstrated the occurrence of phenolic compounds in the hydrophilic extracts. The positive rhodanine assay and the negative vanillin- and DMACA assays provided good evidence that the compounds detected are indeed hydrolyzable tannins.
The most likely source of hydrolyzable tannins is the shikimate pathway (SAP), due to the close similarities in chemical structure between SAP products and phenolic precursors (Ossipov et al. 2003). All genes responsible for this pathway have been identified in brown, green, and red algae and diatoms such as Thalassiosira pseudonana (Richards et al. 2006). Moreover, a bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQ/SDH) was isolated in S. varians, which is phylogenetically closely related to Z. ericetorum (Han et al. 2009). However, besides brown algae, which contain phenolic compounds such as phlorotannins that are stored in physodes or incorporated into the cell wall (e.g., Schönwälder and Clayton 1998, Schönwälder 2008, Holzinger et al. 2011a), only a few groups of red and green algae are known to contain phenolics (e.g., Pérez-Rodríguez et al. 2001, Schmidt et al. 2012, Pichrtová et al. 2013). Similar to the function of phloroglucinol as a precursor of phlorotannins, gallic acid, glycosylated with D-glucose, acts as a precursor for hydrolyzable tannins, so-called gallotannins (Haslam 2007).
The complex mixture of phenolics and hydrolyzable tannin compounds detected in Z. ericetorum in the present study, and the purple polymer recently characterized by Newsome and van Breemen (2012), chemotaxonomically support the phylogenetic position of the Zygnematophyceae as a sister group to land plants (Wodniok et al. 2011). In addition, these poorly investigated phenolics could be ancestors of or substitutes for flavonoids, which have not yet been found in algae.
Photosynthesis and desiccation tolerance
The PI curves of Z. ericetorum clearly indicated significant differences between the green and purple morphs in the Ik and ETRmax values, as well as in the degree of photoinhibition under moderate photon fluence rates. The ETRmax values for the purple morph correlate well with findings in several Antarctic and one Arctic species of Zygnema (Kaplan et al. 2013). In contrast, the green morph of Z. ericetorum showed a much lower photosynthetic performance (3-fold lower ETRmax) and a lower Ik-value, which point to pronounced low-light requirements, and strong photoinhibition above ~150 μmol photons · m−2 · s−1 compared to the purple morph. The latter morph was not photoinhibited under the photon fluence rates applied, indicating much higher light requirements for photosynthesis and obviously better photoprotection. This conspicuously high light sensitivity of the green morph of Z. ericetorum is interesting because this alga occurs in high-alpine ephemeral streamlets (Holzinger et al. 2010). Very close (about 200 m) to the collecting site of Z. ericetorum in this study, biological soil crusts with the filamentous streptophycean algae Klebsormidium crenulatum and K. dissectum were collected and ecophysiologically characterized (Karsten et al. 2010, Holzinger et al. 2011b, Karsten and Holzinger 2012). Both species of Klebsormidium are typical aeroterrestrial taxa, and both exhibited, in contrast to the green morph of Z. ericetorum, high photophysiological plasticity, as reflected in the low-light requirements for photosynthesis combined with a lack of photoinhibition under enhanced irradiances. Karsten et al. (2010) argued that this high photophysiological plasticity seems to be essential for living under high-alpine conditions, because of the extreme fluctuations in abiotic factors. The steep environmental gradients include strong diurnal temperature fluctuations between day and night, occasional frost in summer, high photon fluence rates even at low temperatures, a large increase in UV-B with altitude, high impact by wind or storms resulting in drought and physical abrasion, and reduced partial pressure of carbon dioxide as an inorganic carbon source for photosynthesis. Organisms such as the aeroterrestrial Klebsormidium species living in alpine regions seem to be well adapted to these extreme conditions. In the case of the green morph of Z. ericetorum, the photophysiological properties do not support an exposed lifestyle in high-alpine streamlets, and hence the adaptive strategy seems to be to occur in shaded conditions such as underneath the purple morph. The purple morph of Z. ericetorum exhibited a high degree of photoprotection, which can be explained by the presence of secondary phenolic compounds.
The green and purple morphs of Z. ericetorum are sensitive to desiccation, and both types exhibited a sharp decrease in the optimum quantum yield after only 1–2 h air-drying. After only 2.5 h exposure to the atmosphere, the plants were rewetted and then recovered over the succeeding 8 d. Although the purple morph died, the green morph showed moderate, but very slow recovery. These observations are in strong contrast to the performance of K. crenulatum, which was air-dried and investigated under a similar experimental design. In this species, air-drying for 3 h also resulted in strong inhibition of the maximum PSII quantum efficiency (Fv/Fm), but full recovery after rehydration of the dried samples occurred after only 2 h (Karsten et al. 2010), indicating a capacity for rapid recovery and thus a rather high-desiccation tolerance. In addition to the physiological properties, the cellular ultrastructure also contributed to desiccation tolerance, as it remained more or less intact under drying conditions, and was additionally supported by flexible cross walls, resulting in a decrease in cell volume under water loss and vice versa (Holzinger et al. 2011b). Similarly, Proctor et al. (2007) reported a very rapid recovery of photosynthesis and respiration following rehydration of desiccated samples of the moss Polytrichum formosum. This recovery did not depend on protein synthesis and repair mechanisms, but, rather, was related to the structural integrity of the cytoskeleton. These data suggest that the reactivation of cell biological systems may have a physical component, and point to a significant role of the ultrastructure in desiccation tolerance (Holzinger et al. 2010, 2011b). In the case of Z. ericetorum, study of the ultrastructure of desiccated samples revealed that the vacuoles and cytoplasmatic portions appeared destroyed, whereas the nucleus and chloroplasts generally remained intact (Holzinger et al. 2010). These observations may explain why photosynthesis only partially recovered or failed to recover in the morphs of Z. ericetorum, after desiccation.
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Members of the Zygnematophyceae have been previously shown to accumulate complex phenolic substances that are uncommon in other freshwater microalgae (e.g., Han et al. 2009, Remias et al. 2012b, Pichrtová et al. 2013). The relatively high contents of phenolics and hydrolyzable tannins in the purple morph of Z. ericetorum are likely involved in photo-protection. Moreover, electron-dense vacuoles and electron-dense compartments were observed on the outside of the small chloroplasts in the purple morph of Z. ericetorum by TEM. On the one hand, screening of the phenolic compounds and hydrolyzable tannins against excessive or harmful irradiation (PAR and UV-A/B) could be the key factor in photoprotection; on the other hand, the phenolics could serve as a pool of antioxidants. Probably both processes are interacting, in such a way that increased irradiation stress may trigger increased production of phenolic substances through the shikimate pathway. Highly antioxidant active metabolites such as glucogallin are produced as first steps in the biosynthesis of complex phenolic compounds. The latter can additionally screen UV-A irradiation; however, the chemical structure of these compounds that produces the purple pigmentation could not elucidated by the methods used in this study. It is likely that harsh conditions occurring in the alpine regions stimulate microevolutionary processes, which result in the development of special physiological and morphological adaptations. The formation of algal mats could be seen as a morphological adaptation to intense light and desiccation stress, where the upper layers shade and protect algal filaments in the lower layers.