Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA–SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis

Abstract

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3′,5′-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA–SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

ppGpp functions as an alarmone in metazoa

“…3b), suggesting the existence of a ppGpp-specific hydrolase other than Mesh1. In Arabidopsis, ppGpp is rapidly accumulated upon light-to-dark transition and the accumulated ppGpp is degraded in several hours in the dark 32,43 . The efficient degradation of ppGpp in plants is achieved by not only RSHs, but also several nucleotide diphosphate linked some moiety X (Nudix) hydrolase enzymes, similar to the process that occurs in bacteria 44,45 .…”

In Silico Study of the RSH (RelA/SpoT Homologs) Gene Family and Expression Analysis in Response to PGPR Bacteria and Salinity in Brassica napus

“…Interestingly, this Ca 2+ -binding motif has not been identified in any bacterial or plant homolog [ 26 , 31 ]. It was confirmed in vitro that, for (p)ppGpp synthase activity, CRSH requires Ca 2+ [ 32 ]. The plant stringent response has been implicated in the stress response, flowering, seed development, photosynthesis, plant senescence, and nutrient remobilization [ 27 ].…”

A ppGpp-mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis

“…The accumulation of ppGpp is controlled by the ppGpp synthesis and hydrolysis activity of three families of chloroplast targeted RSH enzyme, RSH1, RSH2/3 and RSH4/CRSH (Atkinson et al, 2011;Ito et al, 2017;Avilan et al, 2019). In the angiosperm Arabidopsis, where ppGpp signalling is best characterized, RSH1 lacks ppGpp synthase activity and acts as the main ppGpp hydrolase (Sugliani et al, 2016), the closely related RSH2 and RSH3 act as the major ppGpp synthases during the day (Mizusawa et al, 2008;Maekawa et al, 2015;Sugliani et al, 2016), and the calcium activated RSH (CRSH) is responsible for ppGpp synthesis at night and in response to darkness (Ihara et al, 2015;Ono et al, 2020). RSH2 and RSH3 are bifunctional ppGpp synthase / hydrolase enzymes, and their hydrolase activity was recently shown to be necessary for constraining CRSH-mediated ppGpp production at night (Ono et al, 2020).…”

“…In the angiosperm Arabidopsis, where ppGpp signalling is best characterized, RSH1 lacks ppGpp synthase activity and acts as the main ppGpp hydrolase (Sugliani et al, 2016), the closely related RSH2 and RSH3 act as the major ppGpp synthases during the day (Mizusawa et al, 2008;Maekawa et al, 2015;Sugliani et al, 2016), and the calcium activated RSH (CRSH) is responsible for ppGpp synthesis at night and in response to darkness (Ihara et al, 2015;Ono et al, 2020). RSH2 and RSH3 are bifunctional ppGpp synthase / hydrolase enzymes, and their hydrolase activity was recently shown to be necessary for constraining CRSH-mediated ppGpp production at night (Ono et al, 2020). ppGpp itself has been shown to act as an inhibitor of the expression of certain chloroplast genes in vivo (Yamburenko et al, 2015;Maekawa et al, 2015;Sugliani et al, 2016;Ono et al, 2020).…”

“…Interestingly, the artificial accumulation of ppGpp also appears to protect plants against nitrogen deprivation (Maekawa et al, 2015;Honoki et al, 2018). In addition, a few studies have now demonstrated that ppGpp is required for different physiological processes; these include the regulation of plant growth and development (Sugliani et al, 2016;Ono et al, 2020), plant immunity (Abdelkefi et al, 2018), and photosynthesis under standard growth conditions (Sugliani et al, 2016).…”