Bacterial Alarmone, Guanosine 5′‐Diphosphate 3′‐Diphosphate (ppGpp), Predominantly Binds the β′ Subunit of Plastid‐Encoded Plastid RNA Polymerase in Chloroplasts

Abstract

It's alarming: Bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp), which is a key regulatory molecule that controls the stringent response, also exists in chloroplasts of plant cells. Cross-linking experiments with 6-thioguanosine 5'-diphosphate 3'-diphosphate (6-thioppGpp) and chloroplast RNA polymerase indicate that ppGpp binds the beta' subunit of plastid-encoded plastid RNA polymerase that corresponds to the Escherichia coli beta' subunit. Chloroplasts, which are thought to have originated from cyanobacteria, have their own genetic system that is similar to that of the bacteria from which they were derived. Recently, bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp, 1), a key regulatory molecule that controls the stringent response, was identified in the chloroplasts of plant cells. Similar to its function in bacteria, ppGpp inhibits chloroplast RNA polymerase; this suggests that ppGpp mediates gene expression through the stringent response in chloroplasts. However, a detailed mechanism of ppGpp action in chloroplasts remains elusive. We synthesized 6-thioguanosine 5'-diphosphate 3'-diphosphate (6-thioppGpp) as a photoaffinity probe of ppGpp; this probe thus enabled the investigation of ppGpp binding to chloroplast RNA polymerase. We found that 6-thioppGpp, as well as ppGpp, inhibits chloroplast RNA synthesis in vitro in a dose-dependent manner. Cross-linking experiments with 6-thioppGpp and chloroplast RNA polymerase indicated that ppGpp binds the beta' subunit (corresponding to the Escherichia coli beta' subunit) of plastid-encoded plastid RNA polymerase composed of alpha, beta, beta', beta'', and sigma subunits. Furthermore, ppGpp did not inhibit transcription in plastid nucleoids prepared from tobacco BY-2 cells; this suggests that ppGpp does not inhibit nuclear-encoded plastid RNA polymerase.

An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis

“…ppGpp appears to act principally through the inhibition of chloroplast transcription to reduce the quantities of individual transcript available for translation and the total translational capacity of the chloroplast by reducing rRNA and tRNA transcript levels ( Figures 4A and 4C). These results are broadly in agreement with previous in vitro data and a recent study with the phytohormone ABA that suggested a link between RSH gene function and chloroplast gene transcription (Sato et al, 2009;Nomura et al, 2012;Yamburenko et al, 2015). A less extensive study that leads to similar conclusions was also published during the final preparation of this manuscript (Maekawa et al, 2015).…”

“…Despite the absence of a homolog of DksA, in vitro studies on chloroplast extracts have shown that ppGpp specifically binds to and inhibits the bacterial-like polymerase encoded by the chloroplast genome (Plastid-Encoded Polymerase [PEP]) (Takahashi et al, 2004;Sato et al, 2009). However, the 50% inhibitory concentrations (IC50s) are rather high (1 mM [Sato et al, 2009]; 2 mM [Takahashi et al, 2004]). Chloroplasts also contain an alternative Nucleus-Encoded Polymerase (NEP), which plays a minor role in green tissues and is not inhibited by ppGpp.…”

“…Studies using purified chloroplast enzymes and chloroplast extracts suggest that ppGpp may function in planta by inhibiting translation and/or transcription in a manner analogous to the bacterial stringent response (Takahashi et al, 2004;Sato et al, 2009;Masuda, 2012;Nomura et al, 2012;Nomura et al, 2014). However, there remains substantial uncertainty about both the principal targets and effects of ppGpp in the plant under physiological conditions.…”

Diversity in Guanosine 3′,5′-Bisdiphosphate (ppGpp) Sensitivity among Guanylate Kinases of Bacteria and Plants

“…We have previously shown that the chloroplast translation system is negatively regulated by ppGpp in vitro (12), and others have suggested that chloroplast RNA polymerase is a target of ppGpp by demonstrating a physical interaction between a ppGpp analog and the polymerase (13). It remains unclear, however, whether translation and transcription are primary targets of ppGpp in chloroplasts, given that the estimated inhibitory concentrations for ppGpp of ϳ250 M (12) and Ͼ1 mM (6), respectively, are relatively high.…”

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp

“…Although the mechanism of ppGpp synthesis is rather poorly understood Tozawa & Nomura, 2011;Tozawa et al, 2007), there is accumulating evidence regarding the function of ppGpp in reorienting cellular metabolism and its physiological consequences, including research into sporulation (Balzer & McLean, 2002;Lemos et al, 2004;Ochi et al, 1981), competence (Inaoka & Ochi, 2002), fruiting body formation (Harris et al, 1998), antibiotic production (Bibb, 2005;Hesketh et al, 2001;Hoyt & Jones, 1999;Inaoka et al, 2003;Ochi, 1987aOchi, , 2007Sun et al, 2001), development of persistence (Dahl et al, 2003;Korch et al, 2003), quorum sensing (Baysse et al, 2005;Harris et al, 1998;van Delden et al, 2001), biofilm formation (Balzer & McLean, 2002), pathogenesis (Erickson et al, 2004;Gaynor et al, 2005;Godfrey et al, 2002;Haralalka et al, 2003;Pizarro-Cerdá & Tedin, 2004;Song et al, 2004) and symbiosis (Moris et al, 2005;Wells & Long, 2002;Zhang et al, 2004). The RNA polymerase is the primary target for ppGpp, as confirmed recently by X-ray crystallographic analysis of an RNA polymerase-ppGpp complex (Artsimovitch et al, 2004;Chatterji et al, 1998;Sato et al, 2009). In Bacillus subtilis, the stringent response is directly elicited by an abrupt decrease in GTP level.…”