## 2 Results and Discussion
#### 2.1.2 Characteristic of Selected _Brassicae_ RSH Proteins
The degradation of the HD or SYNTH domains in plant RSH proteins suggests subfunctionalization similar to that found in bacteria specialised RSHs which may be needed to strengthen the stringent response [24]. Although plant RSHs are nuclear-encoded proteins they contain chloroplast transit peptides at their N-terminus [31]. In silico analysis of putative amino acid sequences of RSH proteins from the _Brassicaceae_ family also showed that the chloroplast is the most probable subcellular localisation (Table 1). Interestingly, in the case of CRSH, the presence of a chloroplast signal peptide is less probable than for other types of RSH protein. In fact, the chloroplast localization has been shown for many of these proteins belonging to all types of  
Figure 4: Amino acid alignments for the (p)ppGpp hydrolase HD (upper part) and synthetase SYNTH (lower part) domains of the RSH1, RSH2, RSH3, and CRSH proteins in _A. thaliana_ and _B. napus_. In the HD domain the His (H) and Asp (D), important for its hydrolysis activity, are highlighted in blue. In the SYNTH domain the Gly (G), important for its synthetase activity, is highlighted in pink. plant RSH groups [26; 31; 44; 51; 52; 53]. There is a paucity of reports of the direct measurement of (p)ppGpp in whole plants, and in particular, in isolated chloroplasts. [PERSON] et al. [54] showed that the level of ppGpp in pea chloroplasts is 13 times higher than in shoots, which confirmed, that the majority of alarmones in plants are localized in chloroplasts. Later reports have determined the level of (p)ppGpp only in whole plants [56; 41; 57]. The phylogenetic analysis of RSH proteins from selected _Brassicaceae_ species (Figure 5) showed the presence of three separate RSH groups. RSH2 and RSH3 could be distinguished but, due to sequence similarity, they are grouped on one branch of the phylogenetic tree. In _A. thaliana_, true _RSH3_ homologs are missing since AtRSH2 and AtRSH3 are the result of recent duplication of the ancestral _RSH2_ gene with a 75% amino acid sequence similarity [36]. True RSH3 homologs are, however, present in other plants. Interestingly, the amino acid sequence similarities between RSH2 and RSH3 in other plant species analysed in this study are very high (ranging from 74% to even 80%), which may suggest that, similar to _A. thaliana_, a true RSH3 homolog is also missing from other plants belonging to the _Brassicaceae_ family. Figure 5: The phylogenetic analysis of RSH proteins based on predicted amino acid sequences given in Table 1. The evolutionary history was inferred using the Neighbor-Joining method by MEGA7.0 software. The optimal tree, with the sum of branch length = 3.01054035, is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson-correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. _B. napus_ RSH sequences are indicated in bold.