## 2 Results and Discussion
### Effect of Salinity and Rhizobacteria on the Expression of BnRSH Genes
Although, in promoters of _BnRSH_ genes, several putative regulatory _cis_-elements involved in response to varied abiotic stresses, possibly including salinity stress, have been identified (Supplementary Table S1), the stable expression of _BnRSHs_ in response to salt has been observed. This may be due to the concentrations of NaCl used in this study. Using an NaCl solution, up to 200 mM mimics non saline, slightly saline, and medium saline soils, whereas a concentration above 250 mM is typical for highly saline soils [67]. Moreover, the observed, almost changeless expression of _BnRSH_ genes in response to NaCl, and the differences in expression of _RSH  
Figure 7: Expression analysis of _BnRSH_ in the _B. napus_ cotyledons and roots of 6-day-old seedlings. Chart shows the relative transcript level of analysed genes (_BnRSH1\(b\), _BnRSH2\(b\), _BnRSH3\(a\), and _BnCRSH_) with respect to the expression of the reference gene (_BnAc_). Different letters indicate statistically significant changes according to one-way ANOVA test at \(p\) < 0.05. Bars represents means \(\pm\) SD. genes response to salinity among plants, might be caused by the different developmental stages of the analysed plants, and/or varied sampling time points. Figure 8: Expression analysis of _BnRSHs_ in salt stress and in the presence of PGPR bacteria using sqRT-PCR. Charts show the relative transcript level of _BnRSH1\(b\), _BnRSH2\(b\), _BnRSH3\(a\), and _BnCRSH_ genes with respect to the expression of a reference gene (_BnAc_). Bars represent means \(\pm\) SD. Control (black bars) are plants grown in in different NaCl concentrations (0 mM, 50 mM, 100 mM, 150 mM, and 200 mM NaCl) but without inoculation with bacteria. Mt (green bars)—_M. timonae_, SI (red bars)—_S. liquefaciens_, Sp (yellow bars)—_S. plymuttica_. Statistical analysis was performed using two-way ANOVA followed by Scheffe post-hoc test. Asterisks indicate statistically significant differences in comparison to the control (i.e., expression of a particular \(RSH\) gene in plants grown in the same concentration of salt but without bacteria—black bars) at \(p\)-value < 0.001 (**), \(p\)-value < 0.01 (**), and \(p\)-value < 0.05 (*). Full statistical analysis data are available as Supplementary Materials (Supplementary Tables S4–S11). The effects of the rhizobacteria, _S. plymuthica_, _S. liputhica_, _S. liquefaciens_, and _M. timonae_, on the expression of _BnRSHs_ in leaves and roots was investigated (Figure 8). Using plant growth-promoting bacteria to improve plant tolerance to environmental stresses, including salt stress, in order to obtained a high yield even in adverse environmental conditions, is considered an economically and environmentally friendly approach [9]. Earlier reports have shown that PGPR bacteria mitigate salt stress via varied mechanisms including the production of indole acetic acid (IAA) [6], induction of potassium and calcium accumulation in plants, increased content of osmolytes including proline [68], and activation of plant antioxidant enzymes [69]. Using two-way ANOVA, significant interactions between salt concentration and species of bacteria for all analysed genes, besides _BnRSH2_ in roots, has been found (Supplementary Tables S4-S11). Therefore, we examined the bacteria simple main effect, i.e., the differences between the expression of _BnRSHs_ in plants inoculated with different bacteria, for each salt concentration. Among all analysed bacteria _S. plymuthica_ had the greatest impact on the expression of all _BnRSH_ genes in all tested salt concentrations, both in cotyledons and roots. The expression of _BnRSH1_ was upregulated by _S. plymuthica_ and _S. liquefaciens_ in both cotyledons and roots, whereas _M. timonae_ increased the expression of _BnRSH1_ in roots only (Figure 8). _S. plymuthica_ increased the expression of and _BnRSH2_ and _BnRSH3_ in cotyledons and roots, while _S. liquefaciens_ increased the expression of _BnRSH2_ in roots only. The expression of _BnRSH_ in roots is mostly unaffected by PGPR bacteria, whereas _S. plymuthica_ and _S.