Quantification of guanosine tetraphosphate and other nucleotides 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, compartmentation 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, applicable to plants and algae, which relies on the use of synthesised 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.

ppGpp influences protein protection, growth and photosynthesis inPhaeodactylum tricornutum

“…Interestingly, the levels of GTP were very similar in all lines, indicating that ppGpp accumulation does not affect the total GTP cellular pool. This result resembles the situation in Arabidopsis ppGpp over-accumulating lines (Bartoli et al , 2019) and contrasts with Gram-positive bacteria, where (p)ppGpp regulates transcription by decreasing the GTP pool in the cell (Krasny & Gourse, 2004).…”

“…Low ppGpp levels could be detected in WT cells under normal growth conditions. These ppGpp levels correspond to 0.25% of total GTP, which is similar to Arabidopsis where ppGpp accumulates to 0.36% of total GTP under normal growth conditions (Bartoli et al , 2019). While nitrogen deprivation did not affect ppGpp levels, extended dark treatment caused a 4-fold increase in WT and SYN control lines (Fig.…”

“…GTP and ppGpp levels were determined using stable isotope labelled internal standards as described (Bartoli et al , 2019). Briefly, 8 mg (dry weight equivalent) of P. tricornutum cells were harvested from cultures 2 days after induction and nucleotides directly extracted with 3 ml 2M formic acid containing 12.5 pmoles 13 C-labelled ppGpp and 125 pmoles 13 C labelled GTP.…”

Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis

“…Nucleotides were extracted by repeated vortexing of the formic acid containing cells extracts over 30 min at 4 °C. Samples were then centrifuged at 7000 g for 10 min at 4 °C, and the nucleotides in the supernatant were extracted as previously described by Ihara et al in 2015 and employed for bacteria as well. , For solid-phase extraction (SPE) purification, 3 cm 3 OASIS WAX Vac cartridges with 60 mg of sorbent and 30 μm particle size (Waters Corp. USA) were used. The OASIS WAX column was equilibrated first with 1 mL of methanol, followed by 1 mL of 50 mM ammonium acetate, pH 4.5.…”

“…In light of the comparably poor sensitivity of UV detection, large amounts of biological samples are usually required for monitoring the abundance of MSN in vivo . Mass spectrometry facilitates the detection of much lower quantities, ,, but AEX- and IPRP-based separation methods have limited compatibility with MS detectors. Moreover, AEX methods exhibit low column efficiency and suffer from unstable retention and analysis time. ,, IPRP Chromatography is more efficient, but the resolution of ppGpp ( 1 ) is poor.…”