Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint

Monoterpene production in peppermint (Mentha x piperita L.) glandular trich omes is determined by the rate of biosynthesis, as determined by (CO2)-C-14 , incorporation, and is restricted to leaves 12 to 20 d of age. Using oil g lands isolated from peppermint leaves of different ages, in vitro assay of the eight sequential enzymes responsible for the biosynthesis of the princi pal monoterpene (-)-menthol indicated that all but one biosynthetic enzyme had a very similar developmental profile. Activities were highest in leaves 12 to 20 d of age, with a sharp peak centered at 15 d. The exception, (-)m enthone reductase, the last enzyme of the pathway, exhibited a later peak o f activity, which was centered at approximately 21 d. The correlation betwe en in vitro enzyme activity and the rate of biosynthesis measured in vivo s uggests that monoterpene formation is controlled mainly by the coordinately regulated activity of the relevant biosynthetic enzymes. Developmental imm unoblotting of limonene synthase, which catalyzes the committed step of the pathway, demonstrated a direct correlation between enzyme activity and enz yme protein, suggesting that the dynamic time course for the remaining path way enzyme activities also reflects the corresponding protein levels. RNA-b lot analyses indicated that the genes encoding enzymes of the early pathway steps are transcriptionally activated in a coordinated fashion, with a tim e course superimposible with activity measurements and immunoblot data. The se results demonstrating coincidental temporal changes in enzyme activities , enzyme protein level, and steady-state transcript abundances indicate tha t most of the monoterpene biosynthetic enzymes in peppermint are developmen tally regulated at the level of gene expression.