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bioRxiv · 10.1101/767004

Plastidial (p)ppGpp synthesis by the Ca2+-dependent RelA-SpoT homolog regulates the adaptation of chloroplast gene expression to darkness in Arabidopsis

Ono, S.·Suzuki, S.·Ito, D.·Tagawa, S.·Shiina, T.·MASUDA, S.
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Abstract

In bacteria, the hyper-phosphorylated nucleotides, guanosine 5-diphosphate 3-diphosphate (ppGpp) and guanosine 5-triphosphate 3-diphosphate (pppGpp), function as secondary messengers in the regulation of various metabolic processes of the cell, including transcription, translation, and enzymatic activities, especially under nutrient deficiency. The activity carried out by these nucleotide messengers is known as the stringent response. (p)ppGpp levels are controlled by two distinct enzymes, namely, RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants and algae 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 (p)ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent (p)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 of ppGpp was observed for 30 min in the wild type (WT) after light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyzes were performed with the rsh2rsh3 double and rsh1rsh2rsh3 triple mutants of Arabidopsis and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase of (p)ppGpp in the WT and rsh2rsh3 accompanied decrements in the mRNA levels of psbD transcribed by the plastid-encoded plastid RNA polymerase. These results indicated that the transient increase of intracellular ppGpp at night is due to CRSH-dependent ppGpp synthesis and the (p)ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2, and RSH3 to accustom plastidial gene expression to darkness.

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Ono, S., Suzuki, S., Ito, D., Tagawa, S., Shiina, T., MASUDA, S.. 2019-09-12. Plastidial (p)ppGpp synthesis by the Ca2+-dependent RelA-SpoT homolog regulates the adaptation of chloroplast gene expression to darkness in Arabidopsis. https://doi.org/10.1101/767004

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