Postillumination Isoprene Emission: In Vivo Measurements of Dimethylallyldiphosphate Pool Size and Isoprene Synthase Kinetics in Aspen Leaves

Abstract

The control of foliar isoprene emission is shared between the activity of isoprene synthase, the terminal enzyme catalyzing isoprene formation from dimethylallyldiphosphate (DMADP), and the pool size of DMADP. Due to limited in vivo information of isoprene synthase kinetic characteristics and DMADP pool sizes, the relative importance of these controls is under debate. In this study, the phenomenon of postillumination isoprene release was employed to develop an in vivo method for estimation of the DMADP pool size and to determine isoprene synthase kinetic characteristics in hybrid aspen (Populus tremula 3 Populus tremuloides) leaves. The method is based on observations that after switching off the light, isoprene emission continues for 250 to 300 s and that the integral of the postillumination isoprene emission is strongly correlated with the isoprene emission rate before leaf darkening, thus quantitatively estimating the DMADP pool size associated with leaf isoprene emission. In vitro estimates demonstrated that overall leaf DMADP pool was very large, almost an order of magnitude larger than the in vivo pool. Yet, the difference between total DMADP pools in light and in darkness (light-dependent DMADP pool) was tightly correlated with the in vivo estimates of the DMADP pool size that is responsible for isoprene emission. Variation in in vivo DMADP pool size was obtained by varying light intensity and atmospheric CO 2 and O 2 concentrations. From these experiments, the in vivo kinetic constants of isoprene synthase were determined. In vivo isoprene synthase kinetic characteristics suggested that isoprene synthase mainly operates under substrate limitation and that short-term light, CO 2 , and O 2 dependencies of isoprene emission result from variation in DMADP pool size rather than from modifications in isoprene synthase activity.

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

“…Application of fosmidomycin, a specific inhibitor of the DXP reductoisomerase (Kuzuyama et al, 1998;Zeidler et al, 1998), resulted in strongly reduced isoprene emissions after 30-min application, in agreement with previous observations (Loreto et al, 2004;Rasulov et al, 2009aRasulov et al, , 2011Li and Sharkey, 2013a). The reduction of isoprene emission was associated with almost complete depletion of the DMADP pool ( Fig.…”

“…The first pool, defined as the DMADP pool, was obtained by integrating the initial rapid decay 200 to 400 s after darkening. It provides the immediate pool of isoprene substrate (mainly DMADP and to some extent IDP that is rapidly converted to DMADP) that was present prior to darkening (Rasulov et al, 2009a;Weise et al, 2013). In the darkness, a secondary process is activated providing DMADP that results in the rise of isoprene emission between approximately 400 and 1,200 s after darkening Rasulov et al, 2011Rasulov et al, , 2014Li and Sharkey, 2013a).…”

“…In addition, more hydrophobic zoledronate likely penetrates the membranes more feasibly than alendronate (Russell et al, 2008). The first reaction of the DXP/MEP pathway, condensation of pyruvate and glyceraldehyde 3-phosphate resulting in formation of DXP, is catalyzed by DXP synthase, a thiamin diphosphate (TDP)-dependent transketolase (Rohmer et al, 1996;Li and Sharkey, 2013b , without any signs of feedback inhibition (Rasulov et al, 2009a(Rasulov et al, , 2011(Rasulov et al, , 2014Li et al, 2011;Ghirardo et al, 2014). This suggests that relatively high DMADP concentrations are needed for an operational feedback at least in vivo (i.e.…”

“…1) were used to estimate the sizes of intermediate pools of chloroplastic isoprenoid synthesis (Rasulov et al, 2009a(Rasulov et al, , 2011(Rasulov et al, , 2014Li et al, 2011;Li and Sharkey, 2013a). The first pool, defined as the DMADP pool, was obtained by integrating the initial rapid decay 200 to 400 s after darkening.…”

“…Zoledronate operates similarly to alendronate, but is a much stronger inhibitor, being operationally active in concentrations several orders of magnitude less than alendronate (Lange et al, 2001;Henneman et al, 2011;Wasko, 2011). A unique in vivo method was used to study dynamic changes in DMADP and phosphorylated intermediate pool sizes (Rasulov et al, 2009a(Rasulov et al, , 2011Li et al, 2011), and different inhibitors were applied alone or in sequence to study the regulation of the pathway flux in conditions when the flux out of the pathway or into the pathway is curbed and when both the input and the output are curbed. Dynamic model calculations were used to quantitatively evaluate the significance of the cytosolic intermediate input into chloroplastic isoprenoid synthesis under different conditions of the DXP/MEP pathway entrance and exit, and to evaluate the possible nonspecific inhibition of other steps controlling DXP/MEP pathway flux.…”