In planta transient expression analysis of monoterpene synthases

In vitro-based analyses of monoterpene synthase (mono-TPS) enzymes have led to a wealth of knowledge regarding their catalytic behavior, the mechanistic principles governing their product specificity, and the molecular basis for their evolution. However, the efficient production of active enzymes in Escherichia coli or yeast can be challenging. Agrobacterium-mediated transient expression in tobacco leaves is increasingly being used as a viable alternative to in vitro-based approaches for the production and functional analysis of a wide range of plant proteins. Transient expression is well suited for qualitative and semiquantitative analyses of mono-TPS enzyme product specificity and, in conjunction with standard volatile analysis techniques, provides an efficient tool for screening mono-TPS function in planta. The primary advantages of this system for mono-TPS analysis are that both mono-TPS genomic clones and cDNAs can be cloned directly into plant expression vectors without modification and expressed enzymes can be analyzed without the need for purification or endogenous precursor addition. Here, we describe a simple and cost-effective method for the in planta functional analysis of plant mono-TPS enzymes. This method can accommodate both the analysis of single genes and the scaling for more high-throughput functional screening of mono-TPS gene families or mutant libraries.