In the present study, RNA-seq analysis was carried out in the common moss B. pseudotriquetrum and genes related to lipid metabolism and oil body formation were found to be highly expressed in FSs (Table 1; Fig. 3). In plant cells, lipid accumulation and change in fatty acid composition are important mechanisms for acquiring environmental stress tolerance. Thus, these genes may be involved in multiple stress tolerance in B. pseudotriquetrum grown in Antarctica (Fig. 2).
Oil body membrane proteins such as oleosin and REF/SRP are involved in regulation of the number and size of oil bodies and accumulation of lipids in cells9,10. In Arabidopsis thaliana and Oryza sativa, lipid contents in leaves or seeds were increased by overexpression of the oleosin gene11,12. Likewise, lipid accumulation was promoted by co-overexpression of the oleosin gene and genes involved in TAG-biosynthesis13,14. Oil body membrane proteins are also involved in acquisition of environmental stress tolerance through oil body formation. In seeds of A. thaliana, oleosin has been shown to contribute to freezing tolerance by preventing abnormal fusion of oil bodies15. Overexpression of REF/SRPP gene increased drought stress tolerance, whereas mutation or RNAi suppression of REF/SRPP gene reduced drought tolerance in A. thaliana16,17. In the present study, oleosin and REF/SRP genes were highly expressed and large amounts of fatty acids were accumulated in FSs (Tables 1 and 2). Therefore, oil body membrane proteins may be involved in environmental stress tolerance through promotion of oil body formation and lipid accumulation in cells of B. pseudotriquetrum grown under Antarctic field conditions.
Increase in membrane fluidity is essential for reducing damage of environmental stresses such as low temperatures to cell membrane function. Generally, higher proportions of unsaturated fatty acids to the total fatty acids increase the fluidity of cell membrane18. Unsaturated fatty acids are produced through elongation reactions and unsaturation by various elongases and FADs. Cytochrome b5 acts as an electron donor to FADs in this process. In Solanum lycopersicum, LeFAD3 (Δ15FAD gene of S. lycopersicum) expression was up-regulated by a cold stress treatment, and overexpression of LeFAD3 increased α-linolenic acids (C18:3n-3) contents and induced cold stress tolerance19. FAD2 is essential for proper function of the membrane-attached Na+/H+ antiporter and maintenance of low cytosolic Na+ levels, and A. thaliana fad2 mutant (mutant for endoplasmic reticulum-localized Δ12FAD) showed high sensitivity to salt stress20. In addition, the melting point of fatty acids decreases as the degree of their unsaturation increases, e.g. 13.4°C for oleic acid (18:1n-9, mono-unsaturated fatty acid), -5°C for linoleic acid (18:2n-6, di-unsaturated fatty acid), -11°C for α-linolenic acid (18:3n-3, tri-unsaturated fatty acid), and − 49.5°C for arachidonic acid (20:4n-6, tetra-unsaturated fatty acid). In the present study, several FAD genes were highly expressed and unsaturated fatty acids were mainly accumulated in FSs (Tables 1 and 2; Fig. 3). Therefore, unsaturation of lipids and change of fatty acid composition may be important adaptation mechanisms to harsh environments in Antarctic mosses.
Very long-chain fatty acids (VLCFAs; C26–C36) are substrates of a cuticle wax mixture covering the plant surface, and therefore, fatty acids are also involved in physical protection against environmental stresses. Lipid transfer proteins (LTPs) are involved in transport of a cuticular wax mixture to the cuticular layer. In many plant species, it has been reported that expression of LPT genes is up-regulated in response to abiotic/biotic stresses and contribute to multiple stress tolerance21,22,23,24. Among LTP family proteins, non-specific LTPs (nsLTPs) are also involved in cuticle formation, wax biosynthesis, plant growth and development25,26. In Nicotiana glauca, expression level of a nsLTP gene and cuticular wax accumulation increased in response to drought stress, resulting in increased drought resistance27. In the present study, some nsLPT genes were highly expressed in FSs (Table 1). Thus, physical protection may also be enhanced by reinforcement of extracellular matrix in mosses growing under Antarctic environments.
Expression of many of genes involved in lipid metabolism and accumulation, such as FADs and oleosin, increased after relatively long periods of various artificial stress treatments (> 1 week) (Fig. 2). This result indicates that acquisition of environmental stress tolerance in Antarctic mosses by changing fatty acid metabolism requires relatively prolonged exposure to environmental stresses. Although most previous studies using Antarctic mosses have examined changes in gene expression profiles after single artificial stress treatments for short periods (a few hours to several days)4,5,6,7, longer periods of stress treatments may be required to clarify actual mechanisms of environmental stress tolerance in Antarctic mosses.
In the present study, expression levels of photosynthesis-related genes, especially light-harvesting chlorophyll (LHC) protein genes, decreased in FSs grown under a unique daylength condition in Antarctic summer (midnight sun). It has been reported that LHC proteins are involved both in collecting light energy for driving the primary photochemical reactions of photosynthesis and in photoprotection when absorbed light energy exceeds the capacity of the photosynthetic apparatus28. In A. thaliana, expression levels of several LHC protein genes were decreased by excessive light treatments29. In the present study, ultraviolet-B receptor UVR8-like gene, which has been reported to be involved in UV-protective responses30, was also highly expressed in FSs (logFC: 6.294977, padj: 3.18E-06; data not shown). Likewise, artificial UV-B irradiation increased expression of UV-B signaling genes including UVR8-like genes in the Antarctic moss Leptobryum pyriforme5. These gene expression responses might be one of the common mechanisms of tolerance to light stresses and excessive UV exposure under midnight sun in Antarctica.
A number of PCESR genes have been reported to be related to early responses to environmental stress8. In the present study, however, expression of only a few PCESR genes were up-regulated in FSs (Table S2). In B. pseudotriquetrum grown in Antarctica, stress tolerances appear to have already been established by altering their secondary metabolism such as lipid accumulation and alteration of fatty acid composition.
The present study is the first one showing actual genes expression profiles in mosses grown under Antarctic field conditions. The study also showed a possibility that lipid accumulation and change in fatty acid composition are major mechanisms for acquiring environmental stress tolerance in Antarctic mosses. In natural fields, plants are exposed to complex environmental factors for a long time. Thus, on-site analysis is essential elucidating actual plant environmental responses and stress tolerance mechanisms.