Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves ZIRU LI & THOMAS D. SHARKEY Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA ABSTRACT The methylerythritol phosphate (MEP) pathway in plants produces the prenyl precursors for all plastidic isoprenoids, including carotenoids and quinones. The MEP pathway is also responsible for synthesis of approximately 600 Tg of isoprene per year, the largest non-methane hydrocarbon flux into the atmosphere. There have been few studies of the regulation of the MEP pathway in plants under physiologi- cal conditions. In this study, we combined gas exchange techniques and high-performance liquid chromatography– tandem mass spectrometry (HPLC-MS-MS) and measured the profile of MEP pathway metabolites under different conditions.We report that in the MEP pathway, metabolites immediately preceding steps requiring reducing power were in high concentration. Inhibition of the MEP pathway by fosmidomycin caused deoxyxylulose phosphate accumula- tion in leaves as expected. Evidence is presented that accumulation of MEP pathway intermediates, primarily methylerythritol cyclodiphosphate, is responsible for the post-illumination isoprene burst phenomenon. Pools of intermediate metabolites stayed at approximately the same level 10 min after light was turned off, but declined eventu- ally under prolonged darkness. In contrast, a strong inhibi- tion of the second-to-last step of the MEP pathway caused suppression of isoprene emission in pure N2. Our study suggests that reducing equivalents may be a key regulator of the MEP pathway and therefore isoprene emission from leaves. Key-words: dimethylallyl diphosphate (DMADP); hydroxymethylbutenyl diphosphate synthase (HDS); iso- prenoids; MEP pathway; methylerythritol cyclodiphosphate (MEcDP). INTRODUCTION The methylerythritol phosphate (MEP) pathway (Fig. 1) is one of two pathways in plants responsible for the biosyn- thesis of dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP), the building blocks of all isoprenoids including carotenoids, monoterpenes, and prenyl chains of chlorophylls and quinones. The MEP pathway in plants is essential as it has been shown that mutants lacking this pathway were unable to develop func- tional chloroplasts (Mandel et al. 1996; Lois et al. 1998). MEP pathway-derived DMADP also leads to the produc- tion of approximately 600 Tg of isoprene (C5H8) per year, or about 1/3 of global hydrocarbon emission from all sources (Guenther et al. 2006). This biogenic isoprene con- tributes to tropospheric ozone production and affects for- mation of aerosols (Went 1960; Chameides et al. 1988; Kiendler-Scharr et al. 2009). However, relatively little is known about the regulation of the MEP pathway in plants. A useful probe of the MEP pathway flux in vivo is iso- prene emission. In emitting species, isoprene emission accounts for well over 90% of the flux through the MEP pathway (Sharkey, Loreto & Delwiche 1991).When light is turned off, isoprene emission from a leaf quickly declines to almost zero within 10 min, presumably because the MEP pathway requires energetic cofactors from the light reac- tions of photosynthesis (three ATP- and three NADPH- equivalents per C5 molecule) (Sharkey, Wiberley & Donohue 2008). The integral of post-illumination isoprene emission has been proposed to reflect the pool size of plas- tidic DMADP (Rasulov et al. 2009). Interestingly, it had been observed in poplars and oaks that isoprene emission rises again in darkness before eventually falling off to zero on a longer time scale (‘post-illumination burst’) (Monson et al. 1991; Rasulov et al. 2010, 2011; Li, Ratliff & Sharkey 2011). In a revisit to this phenomenon, we hypothesized that a pool of intermediate metabolites in the MEP pathway may be trapped upon an almost instantaneous depletion of reducing power during the first few moments of darkness, and that these metabolites were later converted to isoprene as reducing power becomes available (Li et al. 2011). The size of the pool of metabolites giving rise to the post- illumination burst is comparable with the size of the DMADP pool and these two pools responded similarly to environmental variables (Li et al. 2011; Rasulov et al. 2011). To understand the nature of post-illumination isoprene burst and gain insights into regulation of the MEP pathway, it would be useful to measure levels of leaf MEP pathway metabolites under physiological conditions. Some of the best efforts to date include studies using radioisotopes and 31 P-NMR studies. Using 31 P-NMR, it has been shown that Correspondence:T. D. Sharkey. E-mail: tsharkey@msu.edu Plant, Cell and Environment (2012) doi: 10.1111/j.1365-3040.2012.02584.x © 2012 Blackwell Publishing Ltd 1