## 3 Results
### Glycerol-3-Phosphate Metabolic Flow Is Altered in Calcineurin and gpp2 Mutants  
Since glycerol accumulation is affected by calcineurin and Gpp2 mutations (Figure 1B) and glycerol-3-phosphate metabolism (GO:0006072) is a biological process altered in the transcriptome during cationic stress (Figure 4A), the expression levels of differentially expressed genes (DEGs) in this pathway were evaluated (Figure 5A). As shown in Figure 5A,B, in the wild-type strain under cationic stress (WT OS), the metabolic flow is directed toward glycerol biosynthesis by a slight increase in gene expression of the GPD1/2 and GPP1/2 genes, whereas the genes (_GUT_1 and _GUT_2) involved in generating dyhydroxyacetone phosphate (DHAP) and glycerol-3-phosphate (G3P) are repressed (Figure 5B,C); likewise, the transformation of glycerol to DHA and DHAP by _GCY_1 and _DAK_1/2, respectively, is also repressed, since these genes are down-regulated (Figure 5B,C). Finally, _GPT_2/_SCT_1, which is the first committed step of G3P conversion to glycerolipids, is down-regulated. These observations suggest that glyceraldehyde phosphate (GAP) and glycolysis are not favored in this condition; along the same lines, the metabolic flow is not directed toward glycerolipid biosynthesis. In summary, during cationic stress, the transcriptional response of the wild-type strain is directed toward glycerol biosynthesis (pink arrows in Figure 5A). The _cna1\(\Delta\)_, _cnb1\(\Delta\)_, and _gpp2\(\Delta\)_ strains have similar expression profiles, as shown in the heat map (Figure 5B). Glycerol biosynthesis is no longer favored in the calcineurin mutants since the GPP1/GPP2 genes are down-regulated in these strains (_cna1\(\Delta\)_ and _cnb1\(\Delta\)_); however, GPD1, which is necessary to generate glycerol-3-phosphate (G3P), is up-regulated in all three mutants (Figure 5A, pink arrow), increasing G3P biosynthesis; the _GUT_1 and _GUT_2 genes are also highly induced in all three mutants, especially _GUT_2, suggesting that G3P is being used to generate DHAP. The conversion of glycerol to DHA and DHAP is also repressed since the GCY1 and DAK1/DAK2 genes are repressed in the calcineurin mutants, but they are induced in the gpp2\(\Delta\) strain (Figure 5A, green arrows). The branch of the pathway that leads to glycerolipids from G3P is activated since the GPT2 gene is up-regulated in all three mutants. Lack of Glycerol Modulates Carbon Metabolism, Translation, Transmembrane Transport, and Protein Degradation at the Transcriptional Level  
Since carbohydrate metabolism (GO: 0005975) is a GO category that seems important for the cationic stress response, we analyzed the DEGs in this category in mutant and wild-type strains in NaCl. The wild-type strain showed more repressed genes (19 genes in green, first column, Figure 6A) than the mutants; the _cna_1\(\Delta\) and _cnb_1\(\Delta\) strains had just 1 repressed DEG, while the _gpp_2\(\Delta\) strain presented 4 repressed DEGs. Figure 6A shows that the DEGs in this category (GO:0005975) are involved in cell wall remodeling and chitin metabolism (red and orange letters), gluconeogenesis (medium blue), glycogen mobilization (light blue), glycosyl hydrolases (yellow), pentose phosphate pathway (gray), sphingolipid and sterol biosynthesis (lilac), and amino sugar and nucleotide sugar metabolism (dark blue). In general, wild-type and calcineurin-deficient strains were able to remodel their cell walls by enhancing the expression of glucan 1,3-beta-glucosidase and endo-1,3(4)-beta-glucanase (CNAG_06336 and CNAG_05458); the first is a major protein required for cell wall biosynthesis (_BCL2_, YGR282C in _S. cerevisiae_), involved in the incorporation of newly synthesized mannoprotein molecules into the cell wall in _S. cerevisiae_[31; 32], and the second codes for a glycosyl hydrolase important for the spore wall, required for normal spore wall assembly (_CRR_1, YLR213C in _S. cerevisiae_), possibly for cross-linking the glucan and chitosan layers [33].