Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis

Abstract

The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13 CO 2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-Cmethylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3-to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen

“…This secondary rise of isoprene emission in the dark has been suggested to result from dark conversion of intermediates of the DXP/MEP pathway upstream of DMADP synthesized in light; these intermediates are converted to DMADP as soon as ATP and NADPH become available in the dark Rasulov et al, 2011Rasulov et al, , 2014Li and Sharkey, 2013a). The dark pool has been shown to mainly reflect the concentration of ME-cDP (Li and Sharkey, 2013a;Wright et al, 2014).…”

“…It has further been shown that 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-cDP) is the metabolite accumulating in the plastids, and this accumulation can buffer DMADP and IDP changes in the case of varying DXP/MEP pathway input and consumption (Li and Sharkey, 2013a;Wright et al, 2014). A significant part of ME-cDP might even escape to cytosol, implying the existence of another interesting link between cytosolic and chloroplastic processes in isoprenoid synthesis .…”

“…In isoprene-emitting species, there is a concomitant use of the primary substrate DMADP between the plastidic synthesis of isoprene and isoprenoids with a larger molecular size, such as phytol residue of chlorophyll (C20) and carotenoids (C40; Ghirardo et al, 2014;Rasulov et al, 2014), and different from nonemitting species, isoprene emitters seem to support a much larger pool of DMADP without the onset of feedback inhibition Wright et al, 2014). However, it is poorly understood how inhibition of one branch of the pathway (isoprene versus larger isoprenoids) affects the other, to what extent it can lead to accumulation of phosphorylated intermediates, how it affects the overall pathway flux through the feedback regulation, and what is the possible role of cytosolic import and export of intermediates.…”

“…However, the strength of such a feedback regulation can be importantly modified by accumulation of phosphorylated intermediates of the pathway, such that DMADP and IDP do not accumulate. Previous studies have demonstrated that there is a certain pool of phosphorylated intermediates in vivo, and that this pool can strongly increase under certain conditions, including experimental and genetic modification of DXP/MEP pathway input and output Rasulov et al, 2011;Li and Sharkey, 2013a;Ghirardo et al, 2014;Wright et al, 2014).…”

“…Recent studies using genetically modified plants accumulating end products of the DXP/MEP pathway or using natural variation in product accumulation have demonstrated the existence of a potentially important feedback regulation of the DXP/MEP pathway flux by primary end products of the pathway Ghirardo et al, 2014;Wright et al, 2014). In particular, binding of DMADP and perhaps IDP to DXP synthase, the first enzyme in the DXP/MEP pathway, leads to downregulation of the pathway flux when the end products cannot be used, such as under stress conditions.…”