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It has been suggested that isoprene synthesis by isoprene synthase (IspS) proceeds via a substrate-assisted mechanism. The authors observed a non-enzymatic isoprene formation by Mn2+, which represents the basis of IspS enzyme reaction. Because IspS and many other terpene synthases require Mn2+ metal ions as cofactor, this study characterized the formation reaction for the first time. Metal ions including Mn2+ non-enzymatically produced both isoprene and 2-methyl-3-buten-2-ol (2-MBO) from dimethylallyl pyrophosphate (DMADP). Isoprene formation was most enhanced by Fe2+ and, to a lesser extent, by Mn2+ or Cu2+. Ni2+, Co2+, Mg2+, and Ba2+ exhibited a low activity to generate both isoprene and 2-MBO. The proportion of isoprene and 2-MBO varied with the Mn2+ concentration: isoprene predominated over 2-MBO at a higher Mn2+ concentration. Similarly, isoprene formation by Mn2+ increased exponentially as temperature increased with predominance of isoprene over 2-MBO at higher temperature. Both isoprene and 2-MBO formation was enhanced by acidic and neutral pH compared to alkaline conditions. Molecular dynamic simulation of DMADP suggested that the formation reaction is initiated by deprotonation of hydrogen on allyl terminal carbon by phosphate oxygen and generates carbocation and allyl anion intermediates. This is followed by quenching to produce isoprene or by hydroxyl addition to form 2-MBO. Thus, this study provided an insight into reaction mechanism of isoprene and 2-MBO biosynthesis and highlighted some parts of isoprene emission from terrestrial plants, which could be formed by non-enzymatic mechanism.

Plant hormone signalling and the circadian clock have been implicated in the transcriptional control of isoprene biosynthesis. To gain more insight into the hormonal control of isoprene biosynthesis, the present study measured plant hormone concentrations in jasmonic acid (JA)‐treated leaves of our previous model study, examined their relationship with gene expression of isoprene synthase (IspS) and hormone signalling transcription factors. Of the plant hormones, IAA and JA‐Ile and their related transcription factors (MYC2 and SAUR21) were significantly correlated with IspS gene expression. Concentrations of cytokinins, isopentenyladenine (iP), trans‐zeatin riboside (tZR) and cis‐zeatin riboside (cZR), were similarly significantly correlated with IspS expression. However, there was no significant correlation between their related transcription factor (ARR‐B) and IspS expression. The circadian clock‐related gene PRR7, but not the transcription factor LHY, was highly correlated with IspS expression. These results suggest that the hormonal balance between JA‐Ile and IAA plays a central role in transcriptional regulation of IspS through the transcription factors MYC2 and SAUR21, the early auxin responsive genes. The putative cis‐acting elements for SAUR on the IspS promoter (TGTCNN and CATATG), in addition to the G‐box for MYC2, support the above proposal. These results provide insightful information on the core components of plant hormone‐related regulation of IspS under coordination with the circadian clock genes.

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