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Abstract Key MessageTheC. roseus ZCTsare jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. AbstractCatharanthus roseusis the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known asZinc fingerCatharanthusTranscription factors (ZCTs).We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression ofZCTsin seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). TheZCTsdiffered in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except forZCT5). We showed significant activation of thepZCT1andpZCT3promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of theZCTscan be mediated by CrMYC2a. In summary, theC. roseus ZCTsare jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed. 

Abstract Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect ofAgrobacterium‐infiltration parameters on the transient transformation efficiency ofCatharanthus roseusseedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to theAgrobacteriumgrowth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold.Agrobacteriumin exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in ourAgrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and rawRenillaluciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation ofAgrobacteriuminfiltration inC. roseusseedlings will facilitate the study of this important medicinal plant and will expand the application ofAgrobacterium‐mediated transformation methods in other plant species.