## 3 Results and Discussion
### Transcriptional Profiling of Genes Coding for Osmolyte Synthesis Enzymes
TPSA (Trehalose phosphate synthase), STPS (Heat shock trehalose phosphate synthase), CCG-9 (Trehalose phosphate synthase), TPP (Trehalose-6-P phosphatase), MTLD (Mannitol-1-phosphate 5-dehydrogenase), M2 DH (Mannitol 2-dehydrogenase), MPP (Manitol/Hexitol phosphatase), HK (Hexokinase), GPI (Phospho-glucose isomerase), PFK (Phospho-tructokinase), ALD (Aldolase), GPD (Glycerol-3-P dehydrogenase), GPP (Glycerol-3-P phosphatase), GUT1 (Glycerol-kinase), HAD1(Halo-acid dehalogenase), DAK1 (Dihydroxyacetone kinase) GLD1 (Glycerol dehydrogenase). TKT (Transketolase), TAD (Trans-aldolase), LAR (L-arabinose), ARDH (L-arabinitol dehydrogenase), D-XK (D-Xylose Kinase), ER (Erythrose reductase). The most common pathway of mannitol biosynthesis in filamentous fungi is the conversion from fructose-6-P to mannitol-1-P by the mannitol-1-P dehydrogenase and subsequently the dephosphorylation to obtain mannitol [92]. An alternative pathway converts fructose to mannitol by the mannitol dehydrogenase. In _A. nidulans_ the genes encoding these enzymes are named _mtld_ and _m2 dh_, respectively. The patterns of expression of _mtld_ gene and trehalose synthase genes were similar, showing higher expression in extreme conditions when compared to optimal salinity (Figure 3B). However, _mtld_ expression did not correlate with mannitol accumulation particularly without NaCl or 0.5 M NaCl. The _m2 dh_ gene, on the other hand, is highly expressed in the condition without NaCl, but it is downregulated in 0.5 M NaCl (Figure 3B). The expression pattern of these genes does not account for the accumulation of mannitol in 0.5 M NaCl. Although mannitol has been found in high abundance in several fungal species, accumulating evidence suggests that it is not required in all fungi for osmotic stress protection, oxidative stress prevention, or sporulation [92]. Therefore, while its role in fungal physiology is not completely clear, mannitol is not an essential polyol in fungi [93]. In _A. sydowii_, mannitol accumulation might result from active growth metabolism, as it does not seem to be regulated by salt stress response mechanisms. As mentioned before, glycerol is the canonical osmolyte produced under hyperosmotic stress by many microorganisms. Glycerol is produced by two routes from the dihydroxyacetone phosphate (DHAP) obtained during glycolysis. The first pathway involves the  
Figure 3: Expression analysis of transcripts related to osmolytes synthesis of _A. sydowii_ in different salinities after 7 d of culture. The values correspond to the average and standard deviations of three biological replicates (\(n=3\)) and two technical qPCR replicates. Analyzed genes were grouped according to the pathway: (**A**) Thehalose synthesis, (**B**) Mannitol synthesis, (**C**) Pentose phosphate pathway (for arabitol and erythroid synthesis) and (**D**) Glycerol synthesis. Statistical significance (\({}^{\star}\)) was assessed by a randomization test performed with the software Rest [46]. The dashed vertical lines correspond to a cutoff logFC = 2.  
conversion of DHAP to glycerol-3-P by the glycerol-3-P dehydrogenase (GPD) enzyme, which is the main regulatory point in the glycerol pathway. Glycerol-3-P is then converted to glycerol by the glycerol-3-P phosphatase (GPP), while the opposite reaction is catalyzed by the glycerol-kinase (GUT1). The alternative pathway involves the conversion of DHAP to dihydroxyacetone (DHA) by a halo-acid dehalogenase (HAD1) and the subsequent conversion to glycerol by the glycerol dehydrogenase (GLD1). In _A. sydowii_ the _gpd_ gene was highly expressed under hyperosmotic conditions, but not under the optimal salinity (Figure 3D). This coincides with the glycerol accumulation in 2.0 M NaCl. In fact, the _gpd_ gene was downregulated in the optimal condition when compared to the condition without salt. This reinforces our hypothesis that osmotic stress responses are not taking place when _A. sydowii_ is grown under optimal salinity but occur only under the hyperosmotic stress induced by 2.0 M NaCl.