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The highly phosphorylated nucleotide, guanosine tetraphosphate (ppGpp), functions as a secondary messenger in bacteria and chloroplasts. The accumulation of ppGpp alters plastidial gene expression and metabolism, which are required for proper photosynthetic regulation and robust plant growth. However, because four plastid-localized ppGpp synthases/hydrolases function redundantly, the impact of the loss of ppGpp-dependent stringent response on plant physiology remains unclear. We used CRISPR/Cas9 technology to generate an Arabidopsis thaliana mutant lacking all four ppGpp synthases/hydrolases and characterized its phenotype. The mutant showed over 20-fold less ppGpp levels than the wild type under normal growth conditions and exhibited leaf chlorosis and increased expression of defense-related genes as well as salicylic acid and jasmonate levels upon transition to nitrogen-starvation conditions. These results demonstrate that proper levels of ppGpp in plastids are required for controlling not only plastid metabolism but also phytohormone signaling, which is essential for plant defense. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The regulatory nucleotides, guanosine 5′-triphosphate 3′-diphosphate (pppGpp) and guanosine 5′-diphosphate 3′-diphosphate (ppGpp), were originally identified in Escherichia coli, and control a large set of gene expression and enzyme activities. The (p)ppGpp-dependent control of cell activities is referred to as the stringent response. A growing number of (p)ppGpp synthase/hydrolase homologs have been identified in plants, which are localized in plastids in Arabidopsis thaliana. We recently reported that the Arabidopsis mutant overproducing ppGpp in plastids showed dwarf chloroplasts, and transcript levels in the mutant plastids were significantly suppressed. Furthermore, the mutant showed more robust growth than the wild type (WT), especially under nutrient-deficient conditions, although the mechanisms are unclear. To better understand the impact of the ppGpp accumulation on plant responses to nutrient deficiency, photosynthetic activities and metabolic changes in the ppGpp-overproducing mutant were characterized here. Upon transition to the nitrogen-deficient conditions, the mutant showed reduction of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) contents, and effective and maximum quantum yield of photosystem II compared with WT. The mutant also showed more obvious changes in key metabolite levels including some amino acid contents than WT; similar metabolic change is known to be critical for plants to maintain carbon-nitrogen balance in their cells. These results suggest that artificially overproducing ppGpp modulates the organelle functions that play an important role in controlling photosynthetic performance and metabolite balance during nitrogen starvation. [ABSTRACT FROM AUTHOR]

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., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)