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

Abstract

Guanosine tetraphosphate (G4P) and guanosine pentaphosphate (G5P) are signalling nucleotides found in bacteria and photosynthetic eukaryotes that are implicated in a wide-range of processes including stress acclimation, developmental transitions and growth control. Measurements of G4P/G5P levels are essential for studying the diverse roles of these nucleotides. However, G4P/G5P quantification is particularly challenging in plants and algae due to lower cellular concentrations, compartmentalization and high metabolic complexity. Despite recent advances the speed and accuracy of G4P quantification in plants and algae can still be improved. Here, we report a new approach for rapid and accurate G4P quantification which relies on the use of synthesized stable isotope-labelled as internal standards. We anticipate that this approach will accelerate research into the function of G4P signaling in plants, algae and other organisms.

A guanosine tetraphosphate (ppGpp) mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis

“…Therefore, ppGpp accumulation drives the depletion of the cellular GTP pools by other means, which could for example include the inhibition of enzymes involved in chloroplastic GTP biosynthesis such as guanylate kinase ( Nomura et al, 2014 ). Even more remarkable is that the ppGpp-driven GTP depletion occurs specifically under nitrogen deprivation because accumulation of ppGpp to high levels in OX:RSH3 plants does not affect the GTP pool under normal growth conditions ( Bartoli et al, 2020 ). The reduction in GTP levels may contribute to ppGpp signaling in two ways.…”

“…Nucleotides were extracted from about 150 mg of seedlings, and quantified by HPLC–MS/MS using stable isotope labeled ppGpp and GTP standards as described previously ( Bartoli et al, 2020 ).…”

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum

“…Interestingly, the concentrations of GTP were very similar in all lines, indicating that ppGpp synthesis does not deplete the cellular GTP pool. This result resembles the situation in Arabidopsis ppGpp overaccumulating lines (Bartoli et al ., 2020) and contrasts with Gram‐positive bacteria, where (p)ppGpp regulates transcription by decreasing the GTP pool in the cell (Krasny & Gourse, 2004).…”

“…Guanosine triphosphate (GTP) and ppGpp concentrations were determined using stable isotope labelled internal standards as described (Bartoli et al ., 2020). Briefly, 8 mg (DW equivalent) of P. tricornutum cells were harvested from cultures at 2 d after induction and nucleotides were directly extracted with 3 ml 2 M formic acid containing 12.5 pmol 13 C‐labelled ppGpp and 125 pmol 13 C‐labelled GTP.…”

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

“…Therefore, ppGpp accumulation drives the depletion of the cellular GTP pools by other means, which could for example include the inhibition of enzymes involved in chloroplastic GTP biosynthesis such as guanylate kinase (Nomura et al, 2014). Even more remarkable is that the ppGpp-driven GTP depletion occurs specifically under nitrogen deprivation because accumulation of ppGpp to high levels in OX:RSH3 plants does not affect the GTP pool under normal growth conditions (Bartoli et al, 2020). ppGpp can act as a competitive inhibitor of GTP dependent enzymes due to its structural resemblance to GTP (Pausch et al, 2018).…”