Molecular Regulation of the Capacity for Isoprene Synthesis in Plants

异戊二烯是从许多植物，特别是树木中释放出来的。异戊二烯的排放对大气化学有很大的影响，因此它的排放已被广泛研究，但排放速率的调节机制尚未确定。植物的异戊二烯排放量随温度和光照变化很大。实验提出了几个假设，检验异戊二烯排放率从植物是如何调节。通过在光或温度变化之前、期间和之后测量代谢物来研究短期调节。通过分析（1）瞬态期间代谢物水平的变化和（2）对异戊二烯合成途径进料中间体的响应，评估控制异戊二烯瞬时排放速率的因素。为了跟踪来自内源光合作用代谢产物和来自外源中间体的异戊二烯，波恩大学的同事将使用一种基于激光的光声系统来检测来自氘代中间体的氘标记异戊二烯。将在（1）从未产生异戊二烯的叶片和（2）通过将植物移入低温和低光环境而降低异戊二烯排放能力的叶片中研究更长期的诱导现象。分析中将包括异戊二烯合成途径所选酶的前体代谢物水平、酶活性和转录本丰度的变化。最后，将研究异戊二烯排放能力演变所需的步骤。这将涉及两种办法。首先，将来自豆科植物的异戊二烯合酶在组成型启动子、其自身启动子和热激启动子上转化到拟南芥中。将评估异戊二烯合成途径中异戊二烯合酶和其他选定基因的表达和活性的调节，以确定非排放植物是否通常具有光和热依赖性异戊二烯排放所需的调节机制。其次，异戊二烯合酶和类似的倍半萜合酶之间的差异将通过分析异戊二烯合酶的晶体结构来确定。这些研究将提供调节异戊二烯合成的基本信息，以允许改变异戊二烯合成的植物。可能需要工程化植物或细菌以产生经济上有用的量的异戊二烯，或者可能有用的是工程化用于树木种植的树木，使得它们不产生异戊二烯。这些研究还将用于帮助预测植物的异戊二烯排放，这将提高我们预测大气臭氧产生的能力

Isoprene is emitted from many plants, especially trees. Isoprene emission has large effects on atmospheric chemistry and so its emission has been extensively studied but mechanisms of regulation of the rate of emission have not yet been determined. Isoprene emission from plants varies substantially with temperature and light. Experiments are proposed to test several hypotheses about how the rate of isoprene emission from plants is regulated. Short term regulation will be studied by measuring metabolites before, during, and after a change in light or temperature. The factors that control the instantaneous rate of isoprene emission will be assessed by analyzing (1) changes in metabolite levels during transients and (2) responses to feeding intermediates of the isoprene synthesis pathway. To keep track of isoprene coming from endogenous photosynthesis metabolites and that coming from exogenous intermediates, a laser-based photoacoustic system for detecting deuterium labeled isoprene from deuterated intermediates will be used by colleagues at the University of Bonn. Longer term induction phenomena will be studied in (1) leaves that have never made isoprene and in (2) leaves where the isoprene emission capacity has been reduced by moving the plants into low temperature and low light. Changes in precursor metabolite levels, enzyme activity, and transcript abundance for selected enzymes of the isoprene synthesis pathway will be included in the analysis. Finally, the steps required for the evolution of the capacity for isoprene emission will be studied. This will involve two approaches. First, isoprene synthase from the legume kudzu will be transformed into Arabidopsis on a constitutive promoter, on its own promoter, and on a heat-shock promoter. The regulation of the expression and activity of isoprene synthase and other selected genes in the isoprene synthesis pathway will be assessed to determine if non-emitting plants normally have the regulatory mechanisms needed for light and heat-dependent isoprene emission. Second, the differences between isoprene synthase and a similar sesquiterpene synthase will be determined by analyzing the crystal structure of isoprene synthase. These studies will provide basic information on regulation of isoprene synthesis to allow for altering isoprene synthesis by plants. It may be desirable to engineer plants or bacteria to make economically useful amounts of isoprene or it might be useful to engineer trees used in tree farming so that they do not produce isoprene. These studies will also be used to help predict isoprene emission from plants, which will improve our ability to predict atmospheric ozone production