## 2 Results and Discussion
### Regulatory Elements Present in Promoter Regions of B. napus RSH Genes  
The expression of plant _RSH_ genes is tissue/organ-dependent; it depends on the stage of development as well as on the type of the _RSH_ gene. It is generally thought, that (p)ppGpp affects gene expression in chloroplasts at transcriptional, translational, and post-translational level, and thus alarmones regulate plant growth and development, and response to stress stimuli [55]. In fact, the expression of _RSH_ genes, and thus the level of alarmones, is up-regulated by different factors, including abscisic acid [53; 56], salt stress [5; 25; 26; 59], oxidative stress [57], drought [25], and the presence of plant growth promoting bacteria [5]. Interestingly, it was also shown that the overaccumulation of (p)ppGpp in plants has some negative effects. For instance, _Arabidopsis_ plants overexpressing _RSH_2 and _RSH_3 were smaller, contained less chlorophyll, and their seeds had lower vigour [41]. The increased level of (p)ppGpp in _Arabidopsis_ led to dwarf chloroplasts, and reduction of metabolites, however, the mutant plants were more tolerant to nutrient-deficient conditions than wild-type plants [52]. Moreover, the increased level of alarmones increased the susceptibility of plants to turnip mosaic virus, whereas for plants with a decreased level of (p)ppGpp, reduced susceptibility was observed [58]. These results clearly show that the level of (p)ppGpp is tightly controlled, since alarmones are critical not only for plastid development and metabolism, but also for the fine-tuning of plant growth and development. Promoters are responsible for controlling the efficiency, timing, and location of gene expression via clusters of short sequences, including _cis_-regulatory elements (CREs). CREs provide binding sites for transcription factors [37; 62; 63] and their presence may reflect multiple pathways of gene expression regulation. In order to gain some insight into the putative roles of _BnRSH_, in silico analysis of promoter regions, using the PlantCare database, was performed. This kind of bioinformatical analysis provides a background for further research [59; 60; 61; 62]. A promoter analysis of the _BnRSH_ genes revealed the presence of several putative _cis_-acting elements involved in light signalling, in plant development, in response to plant hormones, as well as in plant response to abiotic and biotic stress (Supplementary Table S1). The most abundant elements in all _BnRSH_ genes were those related to the abiotic stress response, followed by light- and hormone-responsive elements. Only 1% of all identified CREs in _BnRSH_ genes were related to the biotic stress response, and this kind of element was not identified in _BnRSH2/3_ genes (Figure 6 and Supplementary Table S2). The highest number of elements was identified in the _BnRSH3\(b\) gene (69), and the lowest in the _BnRSH1\(b\) gene (25) (Supplementary Table S1 and Supplementary Figure S4). The most abundant of the abiotic stress response elements were the drought and ABA response element MYB (41), followed by MYC (27), which is a drought, ABA, and cold response element, and the general stress-response element, STRE (22). Among hormone-responsive elements the ethylene response element was the most frequently occurring (36) (Supplementary Table S1). The frequencies of the types of CRE in _BnCRSH_ genes were different from the frequencies observed in _BnRSH1_-3_ genes. In the promoter region of _BnCRSH_, the most abundant elements were those related to response to light, followed by hormone responsive elements. Only 9% of CREs were abiotic stress response elements (Figure 6). This observation may imply that CRSH plays a significantly different physiological role than RSH1-3. In fact, the expression of _CRSH_ was not changed by salt stress, osmotic stress, or drought in _I. nil_[25].