## 3 Results
As for the calcineurin mutants, the _cnb1\(\Delta\)_ and _cna1\(\Delta\)_ strains presented 815 and 760 up-regulated DEGs, respectively, and 376 and 315 down-regulated DEGs, respectively; the _gpp2\(\Delta\)_ strain showed the largest number of DEGs (1396 up-regulated and 1226 down-regulated), as presented in Table 1. Figure 4A,B show the heat maps of induced (in blue) and repressed (in red) DEGs by GO biological function. Regarding induced categories (heat map in blue, Figure 4A), the carbohydrate metabolic process (GO: 0005975) is the only induced biological function common to all four strains under cationic stress. Transmembrane transport (GO:0055085) is the second category with the highest number of induced DEGs (Figure 4A in blue) in the wild type and calcineurin mutants. Only the wild-type strain presented induced genes categorized under lipid metabolic process, carbohydrate catabolic process, chitin biosynthetic process, microtubule-based movement, and mitotic spindle assembly checkpoint signaling (GO:0006629, GO:0016052, GO:0006031, GO:0007018, and GO:0007094, respectively). Biological functions with the highest number of DEGs induced specifically in all three mutants are autophagy, carboxylic acid metabolic process, glutathione metabolic process, glycerol-3-phosphate metabolic process, pentose-phosphate shunt, and response to oxidative stress (GO:0006914, GO:0019752, GO:0006749, GO:0006072, GO:0006098, and GO:0006979, respectively, Figure 4A). Biological functions specifically induced in the _gpp2\(\Delta\)_ mutant are related to protein catabolism (highlighted in italics): ubiquitin-dependent protein catabolic process, proteosome-mediated ubiquitin-dependent protein catabolic process, protein catabolic process, proteolysis, and proteolysis involved in protein catabolic process (GO:0006511, GO:0043161, GO:0030163, GO:0006508, and GO:0051603, respectively Figure 4A). In summary, three important elements arise from this analysis: (i) Carbon metabolism remodeling is important for counteracting cationic stress in all strains, probably by releasing energy and by changing cell wall composition; however, the carbon metabolism change does not seem to be controlled by calcineurin signaling, but indeed represents an important response to control damage caused by cationic stress. (ii) Transmembrane transport seems to be an important response to cationic stress for the wild type and calcineurin mutants, which may use metabolite transport to maintain homeostasis; however, _gpp2\(\Delta\)_ does not respond by enhancing membrane transport and instead seems to activate protein catabolism, proteolysis, and ubiquitin-dependent proteolysis as a way to handle misfolded proteins \begin{table}
\begin{tabular}{c c c} \hline \hline
**Comparison** & **Up-Regulated DEGs** & **Down-Regulated DEGs** \\ \hline Wild type OS vs. Wild type & 433 & 544 \\ \hline _cnb1\(\Delta\)_Os vs. Wild type OS & 815 & 376 \\ \hline _cna1\(\Delta\)_OS vs. Wild type OS & 760 & 315 \\ \hline _gpp2\(\Delta\)_ OS vs. Wild type OS & 1396 & 1226 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Number of DEGs (up- and down-regulated genes) in wild type and mutant transcriptomes (comparison). due to ionic imbalance. This difference in transcriptional response suggests that Gpp2 may have additional roles in cationic stress that are not related to glycerol biosynthesis since all three mutants have very similar glycerol content. (iii) The response to oxidative stress seems to play an important role in cationic stress for all three mutants, but not for the wild type. Figure 4: A heat map of GO according to biological function in the wild type and mutants (\(cna1\Delta\), \(cnb1\Delta\), \(gpp2\Delta\)). In (**A**) are the induced categories (in blue) during cationic stress, and in (**B**) are the repressed categories (in pink); \(p\) value < 0.05. More intense colors represent a higher number of genes in each category.