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

Abstract

Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula 3 Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3-to 4-fold, upstream of DMADP in bisphosphonateinhibited leaves, but the DMADP pool was enhanced much less, 1.3-to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

Temperature controls on the basal emission rate of isoprene in a tropical tree Ficus septica: exploring molecular regulatory mechanisms

“…We previously examined dynamics of DXS and IDI mRNA levels together with DMADP pool size for possible explanation of changes in basal emission rate of isoprene and found very weak relationships (Mutanda et al 2016). However, studies with poplar suggest that other MEP pathway intermediates upstream of DMADP might accumulate under physiological conditions (Li & Sharkey 2013a), raising a possibility that huge changes in total metabolites might be masked by accumulating upstream intermediates, with only slight changes reflecting in DMADP pool as observed by Rasulov et al (2015). In light of this, we conducted a more comprehensive analysis of MEP pathway genes and key metabolites in this study (Fig.…”

Spectacular Oscillations in Plant Isoprene Emission under Transient Conditions Explain the Enigmatic CO2 Response

“…Two-year-old hybrid aspen (Populus tremula 3 Populus tremuloides clone H200) plants were used as in our previous experiments (Rasulov et al, 2009a(Rasulov et al, , 2011(Rasulov et al, , 2015. The plants were grown in 4-liter pots filled with an 1:1 mixture of commercial planting soil and sand with slow-release balanced fertilizer in Fitoclima S600 PLLH climatic chambers (Aralab).…”

“…However, isoprene synthase responsible for isoprene synthesis from DMADP has an exceptionally large K m value with respect to its substrate DMADP, two to three orders of magnitude greater than the K m values for mono-and sesquiterpene synthases to their substrates (Köksal et al, 2010). Thus, isoprene-emitting species typically support very high chloroplastic pools of DMADP and upstream metabolites that can sometimes reach impressive magnitudes (Li et al, 2011;Rasulov et al, 2011Rasulov et al, , 2015Li and Sharkey, 2013a). In fact, these pools can support isoprene emission in the darkness for extended periods of time (Li et al, 2011;Rasulov et al, 2011Rasulov et al, , 2015Li and Sharkey, 2013a).…”

“…Thus, isoprene-emitting species typically support very high chloroplastic pools of DMADP and upstream metabolites that can sometimes reach impressive magnitudes (Li et al, 2011;Rasulov et al, 2011Rasulov et al, , 2015Li and Sharkey, 2013a). In fact, these pools can support isoprene emission in the darkness for extended periods of time (Li et al, 2011;Rasulov et al, 2011Rasulov et al, , 2015Li and Sharkey, 2013a). These large pools can potentially buffer changes in photosynthetic energy supply such that oscillations in the MEP/DOXP pathway remain masked in transitory conditions that lead to oscillations in photosynthesis, although certain transitory dynamics in isoprene emission have been sometimes observed (Monson and Fall, 1989;Fall and Monson, 1992).…”

“…These large pools can potentially buffer changes in photosynthetic energy supply such that oscillations in the MEP/DOXP pathway remain masked in transitory conditions that lead to oscillations in photosynthesis, although certain transitory dynamics in isoprene emission have been sometimes observed (Monson and Fall, 1989;Fall and Monson, 1992). Here, we have developed protocols to study transitory processes in conditions of significantly reduced chloroplastic DMADP pool size in a hybrid aspen (Populus tremula 3 Populus tremuloides) plant model system (Rasulov et al, 2009a(Rasulov et al, , 2011(Rasulov et al, , 2015. Reduced DMADP pool size was achieved by two different approaches:…”