Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene  

Abstract

The plastidic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13 C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus 3 canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO 2 ]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-Dxylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-D-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. b-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.

Knocking Down of Isoprene Emission Modifies the Lipid Matrix of Thylakoid Membranes and Influences the Chloroplast Ultrastructure in Poplar

“…Similar to the dgd1 mutant (Dörmann et al, 1995), the NE poplar plants showed a lower DGDG content, modified chloroplastic ultrastructure, increased NPQ, restricted electron transport rate (Behnke et al, 2007), and decreased total chlorophyll content ( Fig. 5C; Behnke et al, 2013;Way et al, 2013;Ghirardo et al, 2014).…”

“…It has been proposed that leaf isoprene emission is an important adaptation for plants, conferring tolerance to different environmental constraints (Vickers et al, 2009;Loreto and Schnitzler, 2010;Loreto and Fineschi, 2014). However, biogenic isoprene emission represents a nontrivial carbon loss in plants, particularly under stress conditions (Fang et al, 1996;Brilli et al, 2007;Teuber et al, 2008;Ghirardo et al, 2014), and the reason(s) why plants emit isoprene are still ambiguous, and the true role of isoprene emission remains elusive. Different approaches and techniques have been used to determine whether and how the cost of this expensive carbon emission is matched by the accomplishment of the physiological function in planta.…”

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

“…Although DXS activity was increased more than 2-fold, we noted only sporadic increases in chlorophylls and carotenoids formed by the MEP pathway. One explanation for the lower-than-expected increases in end product accumulation may lie with negative feedback inhibition of DXS by IDP and DMADP Ghirardo et al, 2014), which allosterically bind to the thiamine diphosphate cofactor binding site as noncompetitive inhibitors. An increase in IDP and DMADP in vivo would then inhibit DXS activity, ultimately limiting the potential gains in flux expected by up-regulation of DXS.…”

“…Flux through metabolic pathways with complicated stoichiometry or whose end products are themselves substrates for other metabolic pathways is most conveniently measured using a stable isotopic label such as 13 C. The incorporated isotopic label can then be analyzed by mass spectrometry or NMR. Examples of 13 C-based measurements of metabolic flux include heterotrophic (Williams et al, 2008;Alonso et al, 2010;Allen et al, 2012) and mixotrophic (Kempa et al, 2009) plant cell culture systems, photoautotrophic microbial systems (Shastri and Morgan, 2007;Young et al, 2011), individual leaves of whole plants (Hasunuma et al, 2010;Ghirardo et al, 2014), and whole Arabidopsis rosettes (Szecowka et al, 2013).…”

“…In turn, we investigated the effect of DMADP concentration on DXS activity because DMADP (and IDP) have been shown to regulate DXS activity in poplar through negative feedback inhibition Ghirardo et al, 2014). Various concentrations of DMADP were added to wild-type Arabidopsis protein extracts and the effect on DXS activity was measured (Supplemental Fig.…”