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J Plant Res (2015) 128:511518 DOI 10.1007/s10265-015-0711-1
TECHNICAL NOTE
A highly sensitive quantication method for the accumulation of alarmone ppGpp in Arabidopsis thaliana using UPLCESIqMS/MS
Yuta Ihara Hiroyuki Ohta Shinji Masuda
Abstract Recently, a bacterial second messenger, guano-sine 5-diphosphate 3-diphosphate (ppGpp), has been detected in chloroplasts. However, because ppGpp concentration in plants is much lower than that in bacteria, detailed analysis of ppGpp in plants has not been performed. A highly sensitive quantication method is required for further characterization of ppGpp function in chloroplasts. Here, we report a new method that allows for the highly sensitive and selective high-throughput quantication of ppGpp by ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI). This method requires only ~100 mg of plant tissue for ppGpp quantication. We used this method to measure ppGpp levels in Arabidopsis thaliana under different light conditions. A. thaliana accumulated ppGpp during dark periods. This method will be helpful to further characterize the stringent response in higher plants.
Keywords Arabidopsis Chloroplast Plastid ppGpp Stringent response UPLC-ESI-qMS/MS
Received: 6 November 2014 / Accepted: 4 January 2015 / Published online: 10 March 2015 The Botanical Society of Japan and Springer Japan 2015
Introduction
Guanosine 5-diphosphate 3-diphosphate (ppGpp) is a bacterial second messenger that helps bacteria survive in variable environments (Cashel et al. 1996). ppGpp-dependent metabolic control is termed the stringent response. In Escherichia coli, ppGpp accumulates under several stress conditions, including amino-acid starvation (Cashel et al. 1996). The ppGpp molecule affects several metabolic processes, such as transcription, translation, and DNA replication (Cashel et al. 1996; Hernandez and Bremer 1993;Rojas et al. 1984; Schreiber et al. 1991; Svitil et al. 1993;Wang et al. 2007; Traxler et al. 2008).
In E. coli, cellular ppGpp levels are regulated by RelA and SpoT (Cashel et al. 1996). Specically, RelA associates with ribosomes, and when the ribosomes halt protein synthesis because of amino-acid starvation, the RelAribo-some interaction is altered, causing RelA to increase ppGpp synthase activity (Wendrich et al. 2002). When amino acids again become available, SpoT dehydrates the accumulated ppGpp (Cashel et al. 1996). Additionally, SpoT has ppGpp synthase activity under certain conditions (Gentry and Cashel 1995). RelA/SpoT homologs (RSHs) are present in almost all bacterial species, indicating that the...