## 3 Results and Discussion
### Transcriptional Profiling of Genes Coding for Osmolyte Synthesis Enzymes  
The metabolic pathways of osmolyte synthesis have been described in several fungi including model Aspergillus such as _A. niger_, _A. nidulans_, and _A. fumigatus_[76; 77; 78; 79; 80]. However, the genetic regulation of these pathways is diverse, and hence, a characterization of the transcriptional regulation of genes coding for osmolyte synthesis enzymes should be conducted in distinct species and under different conditions. The metabolic pathways leading to the synthesis of the identified osmolytes are depicted in Figure 2, highlighting the enzymes for which transcriptional regulation was evaluated (Figure 3). Trehalose, a glucose disaccharide, is synthesized from glucose-6-phosphate by two consecutive reactions catalyzed by the trehalose-6-phosphate synthase (TPS) and trehalose-6-P phosphatase (TPP) enzyme complex. In _S. cerevisiae_ the genes _tps1_ and _tps2_ encode the TPS and TPP subunits, respectively, while the genes _tps3_ and _ts11_ encode regulatory subunits. In _A. nidulans_ the orthologues of the _tps1-3_ genes are named _tpsA_, _orIA_, and _tps3_. All three genes contain a glycosyl transferase domain from family 20 (GT 20 domain, pfam: PF00982), while _orIA_ and _tps3_ additionally contain a TPP domain (pfam: PF02358). _A. nidulans_ has also a heat shock trehalose synthase gene (_stps_) that has been found in Aspergillus, a homolog of the _Neurospora crassa ccg-9_ gene which encodes a trehalose synthase with a glycosyl transferase family 1 domain, and a trehalose-6-P phosphatase gene containing only the TPP domain, which is putatively involved in the synthesis of trehalose from trehalose-6-P. The _A. sydowii_ homologs of these genes are listed in Supplementary Table S4. Among the tested metabolites in _A. sydowii_, we found that trehalose was accumulated to a lesser extent (Figure 1B). The highest intracellular concentration of trehalose occurred without NaCl at day 7 (38 umol/g) ant it was also produced in the condition with 2.0 M NaCl (at days 5 and 11). As shown in Figure 3, this is consistent with the expression of the _stps_ trehalose synthase gene, which was upregulated in both extreme conditions, but downregulated in optimal salinity. The _ccg-9_ gene has the same expression pattern but with lesser differential expression among the conditions (Figure 3A). Trehalose is suggested to function as a reserve carbohydrate, as an osmo-protectant, or for protection against protein denaturation by dehydration [81; 82]. For example, spores that have a high content of trehalose are more resistant to temperature stress, dehydration, freezing, oxidizing agents, or starvation [81; 83; 84; 85; 86]. _A. nidulans_ and _S. cerevisiae_ mutants lacking the _tps1_ gene are also less thermo- and halotolerant [87; 86; 69]. Nevertheless, _tps_ genes are also involved in the regulation of cell wall structure by modulating chitin synthase activity [88; 89; 90; 91], hence the effect of these genes might intertwine both physiological responses. In _A. sydowii_, the low amount of accumulated trehalose and the high differential expression of the _stps_ and _ccg-9_ genes suggest that they are involved in stress responses other than trehalose synthesis. Figure 2: Pathways for the synthesis of metabolites with possible osmolyte functions in response to salinity. The pathway has been modified from the _Aspergillus nidulans_ KEGG pathway. Enzymes involved in the regulation of osmolyte concentration and depicted with the color grey: trehalose pathway; green: mannitol; blue: glycerol; purple: erythroiditol, and red: arabitol were evaluated by qPCR. The enzymes depicted in light orange were not evaluated.