Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots

Abstract

The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

Fig 1. Terpenoid indole alkaloid (TIA) biosynthesis in C. roseus.

Solid arrows indicate single step, whereas dashed arrows represent multi-step enzymatic conversions. Enzymes activated by ORCA3 and/or repressed by ZCT1 (based on either promoter binding, transactivation, or overexpression studies) are indicated by a green arrow or red stop, respectively [–16]. *Binding/transactivation studies show ORCA2 and ORCA3 are activators of TDC, but only activate TDC in cell suspensions, not hairy roots. DXS = 1-deoxy-D-xylulose-synthase; CPR = cytochrome P450 reductase; G10H (G8O) = geraniol-10-hydroxylase (or geraniol 8-oxidase); SLS = secologanin synthase; AS = anthranilate synthase, α/β subunits; TDC = tryptophan decarboxylase; STR = strictosidine synthase; SGD = strictosidine β-D-glucosidase; T16H = tabersonine 16-hydroxylase; 16OMT = 16-O-methyltransferase; T3O = tabersonine 3-oxygenase; T3R = tabersonine 3-reductase; NMT = N-methyltransferase; D4H = desacetoxyvindoline 4-hydroxylase; DAT = deacetylvindoline 4-O-acetyltransferase.

Fig 2. Zct1 expression in Zct1hp-36, Zct1hp-38, Zct1hp-40, and GFPhp-29 transgenic lines with 5μM 17β-estradiol treatment for 24 h.

Fold change is calculated with respect to each line’s untreated control. Error bars represent standard deviations of biological triplicates. Statistical significance calculated used Student’s t-test; * p < 0.05, ** p < 0.005.

Fig 3. Zct1 expression in Zct1hp-38 over 48 h.

17β-estradiol (5μM) was added for 24 h, then 250μM or 1000μM MJ was added for the time specified (8, 24, and 48 h). Error bars represent standard deviations of qPCR triplicates.

Fig 4. Average TIA metabolite levels across six Zct1hp lines (Zct1hp-36, -38, -40, -42, -43, -44).

17β-estradiol (5μM) was added for 24 h, then 250μM MJ was added for 5 days. The TIAs were separated by HPLC and quantified by UV absorbance. Each data point represents the TIA level of one biological replicate; variability between biological replicates for a specific line is shown in Fig 5. Bars represent the mean and standard deviations of the six lines. Statistical significance calculated used Student’s t-test; * p < 0.05, ** p < 0.01, *** p < 0.0005.

Fig 5. TIA metabolite levels in Zct1hp-38.

17β-estradiol (5μM) was added for 24 h, then 250μM or 1000μM MJ was added for the time specified (3 and 5 d). The TIAs were separated by HPLC and quantified by UV absorbance. Error bars represent standard deviations between two biological replicates.

Fig 6. G10h, Tdc, and Str expression in Zct1hp-38.

17β-estradiol (5μM) was added for 24 h, then 250μM or 1000μM MJ was added for the time specified (8, 24, and 48 h). Error bars represent standard deviations of qPCR triplicates.

Fig 7. Orca2, Orca3, Zct2, and Zct3 expression in Zct1hp-38.

17β-estradiol (5μM) was added for 24 h, then 250μM or 1000μM MJ was added for the time specified (8, 24, and 48 h). Error bars represent standard deviations of qPCR triplicates.