High temperature acclimation of isoprene emission in date palm is associated with enhanced substrate availability and reduction in synthase activity.

Heatwaves enhance plant isoprene emissions, but the relative contributions of instantaneous temperature effects on rate-limiting enzymes and longer-term acclimation remain unclear. We explored the controls on isoprene emission by isoprene synthase (IspS) activity and MEP pathway intermediates, dimethylallyl diphosphate (DMADP) and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP) pool size in Phoenix dactylifera cv. Medjool subjected to a temperature transient: stabilization at 25°C (Phase 1), an increase to 37°C (Phase II), and return to 25°C (Phase III). The rapid temperature rise increased isoprene emission due to immediate effects on IspS activity, followed by sustained increases driven by expanded DMADP and MEcDP pools. Upon cooling (Phase III), isoprene emissions dropped below initial levels due to reduced IspS activity, but recovered as substrate pool sizes increased. Acclimation to elevated temperature was driven by increased DMADP availability, which persisted after cooling, while slower MEcDP acclimation maintained carbon flux toward DMADP. The data indicate that the sustained moderate heat stress inhibits IspS, but increases substrate availability for isoprene synthesis. Thus, beyond the immediate IspS response, longer-term rises in isoprene emissions result from reprogrammed DMADP-consuming reactions, enhancing substrate availability. These findings provide new insight into the regulation of isoprene under thermal stress and underscore the need to integrate both intermediate pool dynamics and IspS activity into predictive models of isoprene emission and deepen our understanding of the MEP pathway's role under fluctuating environmental conditions.