## 2 Results and Discussion
#### 2.1.2 Characteristic of Selected _Brassicae_ RSH Proteins  
In silico studies have shown that all analysed RSH proteins contain the (p)ppGpp hydrolase (HD) and (p)ppGpp synthetase (SYNTH) domains (Figures 3 and S2). RSH1 proteins also possess a TGS domain that is also present in bacterial stringent-response proteins. CRSH proteins contain an EF domain which is specific only for plant CRSH. On the other hand, bacterial ReLA and SpoT proteins contain an ACT domain that is not present in any group of plant RSH proteins (Figure 3). Figure 2: Intron-exon structure of _RSH_ genes in _A. thaliana_ and _B. napus_. White rectangles indicate UTRs, and black rectangles indicate coding sequence. Intron positions are marked by lines. The analysis was performed using the CIWOG tool. The analysed plant RSH1 proteins contain a functional HD domain, i.e., proteins belonging to this group possess alarmone hydrolytic activity, whereas they do not have a functional (p)ppGpp synthesis domain due to the substitution of functional glycine with serine (Figures 4 and S3). The proteins belonging to RSH2/3 have both (p)ppGpp hydrolase and synthetase activity. CRSH has a functional SYNTH domain, but the hydrolytic domain has lost its activity because of the substitution, conserved in bacterial and plant proteins, of histidine (H) and aspartic acid (D) with serine and glutamic acid, respectively. The _E. coli_ RelA protein is also characterized by the lack of a functional HD domain due to the substitution of His and Asp with phenylalanine and proline, respectively (Supplementary Figure S3). The catalytic activity of plant RSH proteins predicted by the in silico analysis of amino acid sequences could be confirmed by a complementation test in _E. coli_ relA\({}^{-}\) and _relA\({}^{-}\)/spoT\({}^{-}\)_ mutants. It was shown that RSH1 proteins from _A. thaliana_ and _I. nil_ do not possess (p)ppGpp synthase activity, whereas AtRSH2, AtRSH3, and InRSH2, are able to synthesise and hydrolyse alarmones [25, 26, 41]. The (p)ppGpp synthesis activity was confirmed also for RSH2/3 from _Suada japonica_[45], and for _Nicotiana tabacum_ RSH2 alarmone synthesis and hydrolysis activity was shown [44]. Interestingly, AtCSR has only (p)ppGpp synthase activity, as expected based on amino acid sequence analysis [51], whereas InCRSH complements both mutations suggesting that this protein is able also to hydrolyse alarmones, despite the crucial His and Asp in HD domain in InCRSH being substituted with Arg and Gln [25]. Figure 3: Predicted primary structures of RSH1, RSH2/RSH3, and CRSH proteins from _A. thaliana_ and _B. napus_. HD (hd contains HD-SE substitution) (p)ppGpp hydrolase domain; SYNTH (synth contains G-S substitution) (p)ppGpp synthase domain; ACT aspartate kinase chorismate mutase TyrA domain; EF Ca\({}^{2+}\)-binding domain; TGS: threnyl-tRNA synthetase, GTPase, SpoT domain. Plant RSH proteins such as bacterial Rel, RelA, and SpoT proteins belong to the so-called \"long RSH\" group. However, there are also \"short RSH\" proteins containing either a SYNTH domain (SAS) or an HD domain (SAH), without any regulatory domains. SAS and SAH are present in some bacteria together with long RSH. It was hypothesised that \"short RSH\" proteins allow different lineages of bacteria to expeditiously adapt to fluctuating environments, increasing their chance to survive harsh environmental conditions [36]. In metazozo, SpoT homolog 1 (Mesh) is a class of SAH and contains only (p)ppGpp hydrolytic domains [34]. However, in plants no representatives of \"short\" RSH proteins have been identified. In some plant species the degradation of the HD domain has been shown, however mostly in algae species [36]. Interestingly, one of the RSH3 proteins in _B. napus_ (Figure 3) contains only an HD domain that is an unprecedented feature of plant RSH. However, the functionality of this truncated protein remains to be confirmed.