Induction of a Longer Term Component of Isoprene Release in Darkened Aspen Leaves: Origin and Regulation under Different Environmental Conditions

Abstract

After darkening, isoprene emission continues for 20 to 30 min following biphasic kinetics. The initial dark release of isoprene (postillumination emission), for 200 to 300 s, occurs mainly at the expense of its immediate substrate, dimethylallyldiphosphate (DMADP), but the origin and controls of the secondary burst of isoprene release (dark-induced emission) between approximately 300 and 1,500 s, are not entirely understood. We used a fast-response gas-exchange system to characterize the controls of dark-induced isoprene emission by light, temperature, and CO 2 and oxygen concentrations preceding leaf darkening and the effects of short light pulses and changing gas concentrations during dark-induced isoprene release in hybrid aspen (Populus tremula 3 Populus tremuloides). The effect of the 2-C-methyl-D-erythritol-4-phosphate pathway inhibitor fosmidomycin was also investigated. The integral of postillumination isoprene release was considered to constitute the DMADP pool size, while the integral of dark-induced emission was defined as the "dark" pool. Overall, the steady-state emission rate in light and the maximum dark-induced emission rate responded similarly to variations in preceding environmental drivers and atmospheric composition, increasing with increasing light, having maxima at approximately 40°C and close to the CO 2 compensation point, and were suppressed by lack of oxygen. The DMADP and dark pool sizes were also similar through their environmental dependencies, except for high temperatures, where the dark pool significantly exceeded the DMADP pool. Isoprene release could be enhanced by short lightflecks early during dark-induced isoprene release, but not at later stages. Fosmidomycin strongly suppressed both the isoprene emission rates in light and in the dark, but the dark pool was only moderately affected. These results demonstrate a strong correspondence between the steady-state isoprene emission in light and the dark-induced emission and suggest that the dark pool reflects the total pool size of 2-C-methyl-D-erythritol-4-phosphate pathway metabolites upstream of DMADP. These metabolites are converted to isoprene as soon as ATP and NADPH become available, likely by dark activation of chloroplastic glycolysis and chlororespiration.

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

“…The initial rapid decay, 200 to 400 s after darkening, characterizes the pool of isoprene substrate (mainly DMADP and to some extent IDP that is rapidly converted to DMADP) that was present prior to darkening. In the darkness, there is a secondary rise of isoprene emission between approximately 400 and 1,200 s after darkening that results from conversion of phosphorylated intermediates to DMADP in the dark (dark pool; Li et al, 2011;Rasulov et al, 2011Rasulov et al, , 2014Li and Sharkey, 2013a). This secondary rise occurs earlier in bisphosphonate-inhibited leaves due to a greater contribution of IDP and changed affinity of reducing enzymes for alternative electron donors (Figs.…”

“…Illustration of the experimental protocol for measurements of steady-state isoprene emission rate in light and dark decay kinetics without inhibitors (0-3,000 s) and effects of application of bisphosphonate inhibitors (3,000-9,200 s) alendronate (A) and zoledronate (B) and subsequent determination of dark decay kinetics and light activation in leaves of hybrid aspen. The biphasic dark decay of isoprene emission was used to estimate the pool size of the immediate isoprene substrate DMADP and the pool size of upstream metabolites (dark pool; Li et al, 2011;Rasulov et al, 2011Rasulov et al, , 2014Li and Sharkey, 2013a). The integral of the initial rapid decrease of isoprene emission rate for 200 to 400 s after switching off the light provided the estimate for DMADP pool size, whereas the secondary rise of isoprene emission between approximately 400 and 1,200 s after darkening was defined as the dark pool.…”

“…3 for the sample plots). Both DMADP pool size and the dark pool size were determined from the biphasic dark decay of isoprene emission Rasulov et al, 2011Rasulov et al, , 2014Li and Sharkey, 2013a). The initial rapid decay, 200 to 400 s after darkening, characterizes the pool of isoprene substrate (mainly DMADP and to some extent IDP that is rapidly converted to DMADP) that was present prior to darkening.…”

“…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).…”

“…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.…”