Induction of early mevalonate pathway enzymes and biosynthesis of end products in potato ( Solanum tuberosum) tubers by wounding and elicitation

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In plants, several important classes of terpenoid compounds are synthesized via the mevalonate pathway. In addition to essential constitutive metabolites, potato ( Solanum tuberosum L.) tubers synthesize antifungal sesquiterpenoid phytoalexins in response to fungal infection or arachidonic acid elicitation, and toxic steroid glycoalkaloids in response to wounding. The activity of the early pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has previously been shown to increase rapidly and then decrease in response to these stimuli. During an investigation of the possible post-translational control of this enzyme, it was found that the inclusion of the cysteine protease inhibitors leupeptin and E-64 { N-[N-( l -3-trans- carboxyran-2-carbonyl)- l-leucyl]agmatine } in the enzyme extraction buffer increased nine-fold the total HMGR activity recovered in the microsomal fraction and greatly increased the ratio of microsomal to soluble activity. Incubation of microsomal HMGR preparations with soluble protein extracts, Mg 2+ and ATP caused an apparent inhibition of HMGR, consistent with published reports of post-translational inactivation of HMGR by phosphorylation. The apparent inhibition was completely reversed, however, by 5 mM mevalonate and was found to be an artefact caused by the presence of mevalonate kinase, the next enzyme in the pathway, in the soluble fraction. HPLC assays for mevalonate kinase and mevalonate phosphate kinase were developed and used to measure the activities of these enzymes following wounding and elicitation. While HMGR levels increased 30-fold following arachidonic acid treatment and 15-fold following wounding, mevalonate kinase and mevalonate phosphate kinase only increased two- to four-fold following these treatments, and the levels in arachidonic acid treated tubers were only 20–40% higher than in wounded tubers. While HMGR levels are extremely low in untreated tissues, the activities of the two kinases are ralatively high, suggesting that they do not serve as control points for the synthesis of terpenoids.