This study has revealed that naturally red, exposed growth forms of Antarctic C. purpureus have higher levels of cell wall UVAC and lower intracellular UVAC as well as anthocyanin concentrations than its green, more shaded growth form. However, both colour morphs contained similar total UVAC concentrations. Also, anthocyanin trends described here confirmed those reported in Robinson et al. [35] which were the reverse of a previous study [17], although similar extractions were undertaken. Upon further analysis the intense red colouration of this species was shown to be associated with the cell walls rather than being localised in vacuoles or other intracellular compartments. FT-IR spectra indicated these red cell walls lacked phenolic ester and pectin signatures that were otherwise present in spectra obtained from colourless cell walls of C. purpureus green leaves, although both contained strong signals that represented cellulose and phenol or aromatic compounds. In addition, there were no peaks that could distinguish differences in the compounds between extracts from the red and green shoots via HPLC analysis. Therefore, the pigment responsible for the red colouration in the cell wall of C. purpureus is yet to be identified.
From early (December 2011) to midseason (January 2012) red varieties of C. purpureus exhibited a dynamic increase in all UVAC concentrations, which was found to be significant for intracellular and cell wall UVAC. Consequently, total UVAC more than doubled in this species over the duration of this experiment. Similar significant results were found for red samples of S. antarctici for all extracts. By contrast, the species B. pseudotriquetrum seemed to significantly increase only its cell wall UVAC. UVAC concentrations declined for all species when the midseason red-brown morphs were grown in a low light, warmer and hydrated environment but species varied in the cellular location of this change. A significant decrease was observed in the cell wall UVAC concentrations for both C. purpureus and S. antarctici over the 2 weeks of growth. In contrast, B. pseudotriquetrum reduced its intracellular UVAC. After 2 weeks, all moss species showed healthy new, green growth suggesting the three Antarctic species were thriving under these conditions.
Cell wall UVAC are an important investment in exposed moss
Although significant differences in intracellular and cell wall UVAC were shown between the red (exposed) and green (shaded) Antarctic C. purpureus, these seemed to offset each other essentially resulting in similar combined UVAC levels (Fig. 1). This suggests C. purpureus growing in exposed sites produces the same total amount of UV-B-absorbing compounds as in shaded areas but these resources are integrated into a possibly more effective protective barrier in the cell wall rather than in the cytosol or intracellular compartments. The ability of C. purpureus to avoid UV-induced DNA damage in its desiccated state has been attributed to UVAC bound to its cell walls [1, 12], which is considered a better direct first defence against damaging UV rays than an intracellular location.
In Antarctic moss beds, desiccation events are more likely to occur for moss situated on ridges and in wind-exposed turfs where water is scarce and where exposure to high photosynthetically active radiation (PAR) and UVR is more likely than in shaded locations. Consequently, this microclimate subjects the moss to photosynthetically-stressful conditions [17], which appear to influence the localisation of the similar pool of UVAC. As the leakage of cytosolic solutes from cells can be quite substantial whilst moss is desiccating [36, 37], the cell wall is likely to be a better location in order to prevent loss of UVAC as long as the wall integrity is not compromised during desiccation. Antarctic mosses could localise these particularly important molecules within the cell walls as a preservation strategy where the compounds are less likely mobilised or leached during desiccation processes, thus preparing the tissue for other stresses like high UV light. This distribution of UVAC between cellular locations may also be affected by low temperatures and tissue age [38]. Hence, Antarctic C. purpureus moss might constitutively accumulate important UVAC in its cell walls ensuring protection against high radiation and desiccation.
The photoprotective strategy of red cell wall pigments
Red pigments may also be produced and incorporated in the cell walls in order to physically protect against excess visible light. This physical barrier would effectively mediate faster recovery of photosynthesis when dried moss has been rewetted by reducing the formation of reactive oxygen species and protecting the chloroplasts from photobleaching [39]. For example, red gametophytes of a liverwort Jamesoniella colorata recovered faster than the green morphs upon rehydration showing a higher degree of tolerance to desiccation [40]. The red liverworts were also better protected from oxidative damage during the rehydration process. Red growth forms of C. purpureus may also show similar characteristics to this liverwort and could be better prepared to recover from desiccation than the green, shaded moss.
The red colouration in the walls of C. purpureus may be reducing light stress resulting in similarly healthy chloroplasts to the green form, as was detected via light and confocal microscopy (Fig. 2). The red pigments may act as photoprotective barriers by directly absorbing more PAR than the green leaves in a comparable way to J. colorata and another liverwort Isotachis lyallii [41]. The red morphs of these liverworts absorbed more green and blue wavelengths than their green counterparts but the green leaves absorbed and reflected more red and far-red light. In addition, the authors found that the red leaves of these liverworts had higher carotenoids than the green leaves, which was similarly the case for exposed moss on microtopographic ridges in Antarctica [21]. This suggests that Antarctic mosses respond strongly to red light, a characteristic of many bryophytes [42]. It is possible that red light is an important signal for these mosses to indicate environments where PAR may be in excess so that they can enhance the production of cell wall red pigments as well as UVAC to protect existing tissue.
Cell wall UVAC decline under low radiation
A significant reduction of cell wall UVAC occurred when C. purpureus and S. antarctici from exposed sites were grown in low light (Fig. 4). This contrasts with B. pseudotriquetrum, which showed a significant decrease in intracellular UVAC. During the 2 weeks of growth, the original brown or red coloured gametophytes produced new green tissue showing that the laboratory conditions used were favourable for growth. New tissue development, in parallel with a reduction in cell wall UVAC, suggests that these wall compounds are present at significantly lower concentrations in young tissue and are probably laid down towards the end of cell maturation [43]. In addition, cell wall UVAC could be induced as new cells/tissues are exposed to changes in radiation, water and/or cold temperature stresses during their formation. This could be tested in the field in a similar way to a previous investigation in the Antarctic liverwort, Cephaloziella varians, where the authors studied changes in the dark pigmentation upon prolonged placement and subsequent removal of UV-BR filters [30]. It would be interesting to compare the studied mosses, especially C. purpureus, in this same way to see how their colouration responds to changes in UV light, desiccation and/or cold temperatures.
Bryum pseudotriquetrum showed a more dramatic response in the production of intracellular UVAC than the other two moss species over the 2-week laboratory experiment (Fig. 4). The extent of change is reflected in the time taken for new growth to emerge. For example, B. pseudotriquetrum produced bright green tissue earlier than the other two mosses, consistent with this species faster growth rate observed in the field [5, 44, 45]. Consequently, B. pseudotriquetrum would be expected to show more distinct changes in its intracellular UVAC mobilisation and/or production than C. purpureus and S. antarctici. Comparably, more subtle changes would be expected to occur for the other two species due to their slower growth rates [5, 44, 45]. If conditions are favourable, then it is likely that Antarctic B. pseudotriquetrum will be more responsive to environmental changes in the field [as seen in 14] and reflect these in its intracellular UVAC, whereas C. purpureus and S. antarctici probably show steady, efficient accumulation of UVAC in the cell walls during their slower active growth periods.
Stress increases red colouration in Antarctic mosses
Considering that red moss was found in exposed locations, which are affected by multiple stressors, and that the red-brown colouration was absent in new green growth thriving under less stressful conditions, it is reasonable to suggest that the red pigmentation is stimulated under stress. It is unclear at this stage whether one or more stressors are responsible and the response may be species-specific. Whilst Antarctic C. purpureus has often been found to exhibit red tissue [17, 46], as has temperate B. pseudotriquetrum [47], previously there were fewer reports of red S. antarctici in the Windmill Islands. Although, there have been increasing accounts of red-tipped S. antarctici in recent years [7, 48]. This apparent change in the endemic moss may be an indication that it is responding to increasing stress occurring as a result of changes to its microclimate.
The seasonal increase in UVAC for all species may be due to environmental stresses intensifying across the 2011/12 season (Fig. 3). These include high PAR, UVR, cold and drought stresses [17, 49, 50], which are generally common in Antarctic environments [5]. However, the mosses would need to have been sufficiently metabolically active to synthesise and store secondary metabolites including UVAC. This would require at least a short boost of fresh snow melt or possibly a longer period of rehydration to provide the carbon necessary for production of new compounds [30].
In search of the red compound in C. purpureus
Red or reddish-brown colour in C. purpureus was distinctly associated with pigments in the cell walls and our findings did not indicate chloroplast movement or chlorophyll a/b content changes. A cell wall pigment location is rarely found in higher plants [26] but has been increasingly reported in bryophyte studies [17, 28,29,30, 32, 41, 51, 52]. Previous investigations of C. purpureus have reported the colouration, but have not localised the red pigment or extracted the UVAC [17]. Several detailed attempts have been made to extract red pigments from bryophyte cell walls but without much success [41, 51].
FT-IR microspectroscopic techniques revealed that cell walls in red and green leaves were mainly composed of cellulose and pectin, which is expected for mosses [53]. In addition, the discovery of phenolic esters in the green C. purpureus species was not unusual as similar hydrolysed compounds were isolated from the cell wall, namely p-coumaric acid, trans-ferulic acid and p-hydroxybenzoic acids [13]. These were in their carboxylic acid form after extraction and isolation, but FT-IR analysis showed that they naturally exist as esters. These isolates are probably covalently linked to the cellulose strands during cell wall manufacture. Although phenolic esters were not detected in the red cell walls, a strong presence of phenolic ring signals was observed for both red and green leaves tested. These could be flavonoid or anthocyanidin derivatives [27 as cited in 28].
The identity of the red compound/s within C. purpureus is unresolved and it could be because they are very tightly bound to the cellulose architecture of the cell wall—so tightly bound that they could be very difficult to remove [28, 54]. Our findings suggest that the coloured compounds are strongly bound and incorporated within the cellulose as structural building blocks rather than loosely associated to the cell wall via hydrophobic interactions that would otherwise allow easy extraction using acidified methanol solvents [29]. Additionally, phenolics in plant cell walls could also form complexes with larger aromatic compounds, such as anthocyanins, reinforcing their binding to the cellulose [55, 56]. Similar to this study, Hooijmaijers and Gould [41] found it difficult to identify red cell wall pigments in the liverwort J. colorata. In contrast, an anthocyanidin called riccionidin A was identified as the dark purple/black pigment in the cell walls of the Antarctic liverwort, C. varians [30], but this pigment, which was removed using acidified methanol, could have been highly abundant in vacuoles as well and/or been weakly bound to the cell wall. Thus, anthocyanins or coloured phenolic compounds could be responsible for the colouration of C. purpureus; however, this is not yet confirmed and requires further investigation.
Future studies into the extraction of this tightly bound red pigment in C. purpureus are likely to require harsher extraction solvents as the current technique either did not extract a sufficient amount of the compounds of interest for identification; or they existed in polymeric or complex forms in the extract that were unable to be separated. The current method involved saponification (alkali hydrolysis) of cell wall residue at room temperature. As proposed in the FT-IR analysis, this hydrolysis probably facilitated the conversion of cell wall bound esters to carboxylic acids for their removal. Other approaches could include: digesting cell wall carbohydrates further using enzymes such as cellulase, targeting cellulose extraction using diglyme-HCl first and alkali hydrolysis second, or heating during the extraction process. For example, alkali hydrolysis at 200 °C was necessary to extract three phenolics from red cell walls of Sphagnum nemoreum moss [52]. The use of harsher solvents and reaction conditions however, risks severely altering the natural structure of the chemical/s responsible for the red/brown pigmentation within any plant species. Although investigations into the red pigments in B. pseudotriquetrum and S. antarctici were beyond the scope of the present study, identifying the red compounds for all these species remains an important avenue to pursue.