Christie A.M. Peebles

Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time.

Jasmonic acid (JA) activates the transcriptional regulator ORCA3, which has a role in regulating the terpenoid indole alkaloid (TIA) pathway within Catharanthus roseus. The TIA pathway leads to the production of the anticancer drugs vinblastine and vincristine. This work explores the transient effects of overexpressing ORCA3 under the control of a glucocorticoid-inducible promoter system in C. roseus hairy roots along with the simultaneous feeding of JA. The changes in TIA metabolites and in mRNA transcripts of pathway genes and regulators were tracked for 72h. Upon induction of ORCA3 expression and elicitation with JA, ORCA3 transcripts increased 170-fold whereas ORCA3 expression caused an 89-fold increase and JA elicitation caused a 5-fold increase in ORCA3 transcripts. JA treatment caused the largest increase in TIA metabolites and transcripts of pathway genes. These transcripts displayed a transient response with the maximum expression reached between 12 and 24h. In the samples overexpressing ORCA3, the largest increase in the transcripts of ZCT1 and ZCT2 (ZCT-zinc finger-binding protein), TIA transcriptional repressors, coincided with the largest increase in ORCA3 transcripts. This counter response of transcriptional repressors may explain why the large increase in ORCA3 transcripts do not correspond with larger increases in transcripts of TIA pathway genes.

The expression of 1-deoxy-d-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

The effects of UV-B stress on the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots.

In nature, plants generate protective secondary metabolites in response to environmental stresses. Such metabolites include terpenoid indole alkaloids (TIAs), which absorb UV‐B light and serve putatively to protect the plant from harmful radiation. Catharanthus roseus plants, multiple shoot cultures, and cell suspension cultures exposed to UV‐B light show significant increases in the production of TIAs, including precursors to vinblastine and vincristine, which have proven effective in the treatment of leukemia and lymphoma. Here, the effect of UV‐B light on C. roseus hairy roots was examined. Analysis of alkaloid concentrations up to 168 h after UV‐B exposure shows significant increases in the concentrations of lochnericine and significant decreases in the concentration of hörhammericine over time (ANOVA, P < 0.05). Our results also indicate that increasing UV‐B exposure time up to 20 min caused significant increases in lochnericine, serpentine, and ajmalicine and a decrease in hörhammericine (t‐test, p < 0.05).

Transient effects of overexpressing anthranilate synthase α and β subunits in Catharanthus roseus hairy roots

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback‐insensitive anthranilate synthase α subunit under the control of the glucocorticoid‐inducible promoter system and the anthranilate synthase β subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, hörhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.

Long-term maintenance of a transgenic Catharanthus roseus hairy root line

Stably transformed transgenic hairy root cultures have the potential to be a valuable production platform for a variety of secondary metabolites. This study reports that a transgenic hairy root culture of Catharanthus roseus has been stably maintained for over 4.5 years. This culture carries a transgene that expresses the green fluorescent protein under the control of the glucocorticoid‐inducible promoter. Genomic PCR confirmed the presence of the GFP insert within the hairy roots, and induction with dexamethasone caused a significant (p < 0.02) increase in GFP levels.

The role of the octadecanoid pathway in the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots under normal and UV-B stress conditions.

The octadecanoid pathway is responsible for producing jasmonic acid an important signaling molecule in plants, which controls the production of a variety of secondary metabolites. Previously the exogenous addition of jasmonic acid to Catharanthus roseus hairy roots caused an increase in terpenoid indole alkaloid (TIA) accumulation. The role of the endogenous production of jasmonic acid by the octadecanoid pathway in the production of TIAs in C. roseus hairy roots is examined. Feeding of octadecanoid pathway inhibitors suggests that the octadecanoid pathway does not actively control TIA production under normal growth conditions or during the UV-B stress response in C. roseus hairy roots.

Effect of sodium nitroprusside on growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root cultures.

Nitric oxide (NO) is known as a signaling molecule involved in elicitor‐induced defense responses of plants. Sodium nitroprusside (SNP), a donor of NO, stimulates catharanthine formation in Catharanthus roseus cells. 1 Two important terpenoid indole alkaloids produced in small quantities within C. roseus are vinblastine and vincristine which are being used clinically as anticancer drugs. We are interested in engineering C. roseus hairy roots to increase the production of the TIAs. The present work investigates the effects of treating different concentrations of SNP to the hairy root cultures from line LBE‐6‐1. The alkaloid concentrations were analyzed 9, 14, 17, 20, 23, 26, and 30 days after treatment of SNP on day 0. We also studied the transient effects of SNP treatment during the exponential phase in C. roseus hairy roots. Analysis of the results showed that treatment of 0.1‐mM SNP did not affect the growth of hairy roots, whereas 1‐mM SNP suppressed the growth significantly, and 10‐mM SNP almost completely inhibited the growth of hairy roots. 0.1‐mM SNP treatment on day 0 caused a significant increase in the concentration of serpentine, catharanthine, ajmalicine, lochnericine and tabersonine production. SNP treatment on day 12 stimulated the formation of serpentine, catharanthine, ajmalicine, hörhammericine, lochnericine and tabersonine by day 21. After the initial stimulation, serpentine, horhammericine and lochnericine concentrations returned to the basal level by day 28. Treatment of 0.1‐mM SNP on day 0 caused significant decrease in the mRNA levels for TDC, ASA, STR, ORCA3, ZCT1, and Crgbf1 on day 23. Treating 0.1‐mM SNP on day 12 caused decreases in the expression levels of STR, ORCA3, ZCT1, and Crgbf1 on day 21 and day 28. Compared with day 28, the mRNA transcript of ZCT1 on day 21 is about twofold higher. Expression levels of G10H increased significantly.

Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots.

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid‐inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol‐inducible promoter within C. roseus hairy roots is reported. This ethanol‐inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6‐fold increase in CAT reporter activity after 24 h of induction. The ethanol‐inducible CAT activity increased 24‐fold over a 72‐h induction period with 0.5% ethanol.

Five year maintenance of the inducible expression of anthranilate synthase in Catharanthus roseus hairy roots

Transgenic hairy root cultures have the potential to be an industrial production platform for a variety of chemicals. This report demonstrates the long-term stability of a transgenic Catharanthus roseus hairy root line containing the inducible expression of a feedback-insensitive anthranilate synthase (AS). After 5 years in liquid culture, the presence of the inserted AS gene was confirmed by genomic PCR. The inducible expression of AS was confirmed by enzyme assay and by changes in terpenoid indole alkaloid concentrations. This report also demonstrates that it may take as long as 2 years for the metabolite profile to stabilize.

Effect of the Engineered Indole Pathway on Accumulation of Phenolic Compounds in Catharanthus roseus Hairy Roots

Catharanthus roseus has been well‐known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback‐resistant anthranilate synthase holoenzyme (ASαβ) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASαβ hairy roots. Uninduced and induced ASαβ hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72‐h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5‐fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.