Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post‐illumination isoprene burst from leaves

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 physiological conditions. In this study, we combined gas exchange techniques and high-performance liquid chromatographytandem 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 accumulation 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 eventually under prolonged darkness. In contrast, a strong inhibition 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.

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

“…This explains a new regulatory mechanism that modulates the synthesis of one of the key intermediates of the MEP pathway. It has also been shown that the steps using reducing power (steps involving DXR, hydroxymethylbutenyl-diphosphate synthase, and hydroxymethylbutenyldiphosphate reductase enzymes) act as strong regulatory points of the MEP pathway under different environmental conditions (37). This suggests the presence of a regulatory mechanism at the middle of the pathway.…”

“…Measurement of the chloroplastic DMADP pool by postillumination isoprene emission estimated a concentration of ϳ43 M in oak leaves (53,54). Metabolic profiling studies by LC-MS/MS estimated the chloroplastic IDP/DMADP pool to be ϳ30 M in poplar leaves (37). Considering the variability in the measurement of the metabolites by these methods, the physiological concentration for DMADP/IDP can certainly be assumed in the range of K i of these metabolites for PtDXS.…”

“…Li and Sharkey (37) found that the physiological concentration of 4-hydroxy-3-methylbut-2-enyl diphosphate under normal conditions is very low (ϳ4.2 M). Therefore, we believe that 4-hydroxy-3-methylbut-2-enyl diphosphate plays little or no role in feedback within the MEP pathway.…”

“…Liquid chromatography was performed using a Merck SeQuant TM ZIC -pHILIC (50 ϫ 2.1 mm; 5 m; 200 Å; polymeric beads; poly(etherether ketone)) column (The Nest Group, Inc., Southborough, MA) fitted to two LC-20AD HPLC pumps and an SIL-HTc autosampler (Shimadzu, Kyoto, Japan) as described (37). The rest of the instru-mental setup for the mass spectrometer coupled to this chromatography system was done as described (37). The assay mixture was filtered through a Whatman syringeless filter device (Mini-Uniprep TM polytetrafluoroethylene filter medium), and 5 l of the sample was injected into the column.…”

“…1, inset) at a flow rate of 0.15 ml/min. Mass spectrometry was performed as described (37). Multiple reaction monitoring mode was used to acquire the precursor/product ion pairs for DXP, [ 13 C 2 ]DXP, and DHAP.…”