(425c) The Transient Effect of Jasmonic Acid Feeding along with Orca3 Overexpression in Catharanthus roseus Hairy Roots

Jasmonic acid has been shown to activate the transcription factor ORCA3 which regulates numerous terpenoid indole alkaloid (TIA) genes and causes the increase of TIA pools within Catharanthus roseus. Two important TIAs produced in small quantities within C. roseus are vinblastine and vincristine which are used clinically as anticancer drugs. This work explores the transient effects of overexpressing ORCA3 under the control of the glucocorticoid inducible promoter in C. roseus hairy roots along with the simultaneous feeding of jasmonic acid (JA). Time profiles of both mRNA and alkaloid levels were analyzed by sampling 0, 6, 12, 24, 48, and 72 hours after feeding of JA and/or induction of the overexpression of ORCA3.

Analysis of the alkaloid levels showed that JA treatment alone caused the greatest increase in TIA pools with significant increases occurring around 24 hours. ORCA3 overexpression with or without JA feeding caused a significant increase in the pools of hörhammericine, lochnericine, and tabersonine. Q RT PCR was used to analyze mRNA levels of anthranilate synthase, tryptophan decarboxylase, 1-deoxy-D-xylulose synthase, geraniol 10-hydroxylase, NADPH:cytochrome P-450 reductase, secologanin synthase, strictosidine synthase, and strictosidine beta-glucosidase at each time point. In general, JA treatment alone caused the largest increase in mRNA levels with the maximal levels occurring around 12 hours (for genes early in the indole pathway) or 24 hours and decreasing to values close to the un-treated samples by 72 hours. ORCA3 overexpression with or without JA feeding had little effect on mRNA levels as compared to the untreated samples, with the JA feeding contributing to slightly higher mRNA levels than the unfed samples.

These results show that a coordinate overexpression of many enzymes may be necessary to increase the production of the terpenoid indole alkaloids and point us towards a multigene strategy to engineer C. roseus hairy roots for increased production of TIAs. These results also suggest that ORCA3 overexpression with or without JA feeding is not an effective means of coordinately overexpressing many enzymes within C. roseus hairy roots.