Isoprene emission from plants is the largest non-methane hydrocarbon flux into the atmosphere. This large hydrocarbon flux from the biosphere has a profound effect on ozone chemistry, aerosol formation and greenhouse gas lifetime in the atmosphere. Understanding the biochemical and molecular regulation of isoprene emission in response to environmental drivers will allow us to build models that better predict future atmospheric conditions. In addition, isoprene is produced through the plastidic methylerythritol phosphate (MEP) pathway, an essential pathway in plants, and accounts for over 90% of the MEP pathway flux. Studies of regulation of isoprene emission, therefore, also provide a unique opportunity for understanding regulation of the MEP pathway. My graduate work presented in this dissertation focused on two aspects of short-term control of isoprene emission: responses of isoprene emission to changes in 1) temperatures and 2) photon flux density. DMADP measurements using post-illumination isoprene emission suggest that the temperature dependence of isoprene emission reflects combined regulation from both isoprene synthase (IspS) and upstream MEP pathway enzymes. Analysis of transient response to elevated temperature shows that emission levels increase initially due to an increase in IspS activity, and then decrease over time as limited by substrate levels. An unexpected discovery from this work was the observation of a highly temperature-dependent "post-illumination isoprene burst". It was hypothesized that this burst represents early metabolites in the MEP pathway that was trapped initially upon darkening, and then converted to isoprene in a later phase. To understand the nature of this phenomenon I employed high pressure liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) to measure metabolites in the MEP pathway under physiological conditions. Levels of intermediate metabolites, in particular methylerythritol cyclodiphosphate, were largely unaffected during phase I in darkness when isoprene emission declined exponentially. Quantitative comparison suggests this pool can account for the post-illumination isoprene burst. Steps in the MEP pathway that requires reducing power are likely the most susceptible steps in response to changes in light levels. This work represents one of the first studies that profiled MEP pathway metabolites in plants and it is suggested that the same technique can be used to understand short-term responses of isoprene emission to other environmental variables and to study metabolic control of the MEP pathway.
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