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

Abstract

Abbreviations: CRSH, Ca 2+ -activated RSH; flg22, flagellin 22; (p)ppGpp, 5'-di(tri)phosphate 3'-diphosphate; PAMPs; pathogen-associated molecular patterns; RSH, RelA-SpoT homolog; qRT-PCR, quantitative real-time PCR 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 Ca 2+ -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. expression Plants were grown on 0.8% agar-solidified 1/2 MS medium under continuous light (40 µmol photons m −2 s −1 ). Fourteen-day-old plants were transferred to the dark and ~0.1 g shoots were harvested at each of the time points indicated (Fig. 3). Extraction and quantification of ppGpp were as described previously . Western blot analysisProtein content was measured using the Bradford assay Kit (Bio Lab). Total proteins (6 µg each) were applied to an SDS-PAGE gel and electroblotted onto a polyvinylidene difluoride membrane (GE-Healthcare). The membrane was incubated with the CRSH-specific antibody . Immunoreactive proteins were detected using the Alkaline Phosphatase Substrate Kit II (Vector Laboratories).

Quantification of guanosine triphosphate and tetraphosphate in plants and algae using stable isotope-labelled internal standards

“…We also observed only a very small increase in G4P levels in Chlamydomonas following dark treatment. This dark-induced G4P increase is substantially smaller than the 4-6-fold dark-induced increase in G4P that is observed in Arabidopsis and which is dependent on the Ca2+ activated G4P synthase CRSH [8,28]. A CRSH homologue is also present in C. reinhardtii.…”

Quantification of guanosine tetraphosphate and other nucleotides in plants and algae using stable isotope-labelled internal standards

“…We also observed only a very small increase in G4P levels in Chlamydomonas following dark treatment. This dark-induced G4P increase is substantially smaller than the 4-6-fold dark-induced increase in G4P that is observed in Arabidopsis and which is dependent on the Ca2+ activated G4P synthase CRSH (Ihara et al, 2015;Ono et al, 2019). A CRSH homologue is also present in C. reinhardtii.…”

ppGpp influences protein protection, growth and photosynthesis inPhaeodactylum tricornutum

“…S1). Dark treatment also causes an increase in ppGpp levels in Arabidopsis in a CRSH dependent manner (Ihara et al , 2015; Ono et al , 2019). However, P. tricornutum lacks a direct orthologue of CRSH (Avilan et al , 2019), suggesting differences in the underlying mechanism controlling (p)ppGpp synthesis in the dark.…”