Effect of plant growth regulators on the biosynthesis of vinblastine ...

Plant Growth Regul (2010) 60:133–141 DOI 10.1007/s10725-009-9429-1

ORIGINAL PAPER

Effect of plant growth regulators on the biosynthesis of vinblastine, vindoline and catharanthine in Catharanthus roseus Qifang Pan • Yu Chen • Quan Wang • Fang Yuan Shihai Xing • Yuesheng Tian • Jingya Zhao • Xiaofen Sun • Kexuan Tang

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Received: 25 May 2009 / Accepted: 1 December 2009 / Published online: 25 December 2009 Ó Springer Science+Business Media B.V. 2009

Abstract Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine. Keywords Catharanthus roseus Plant growth regulators Vinblastine Vindoline Catharanthine Blooming period Abbreviations C. roseus Catharanthus roseus PGRs Plant growth regulators TIA Terpenoid indole alkaloids

Q. Pan Y. Chen Q. Wang F. Yuan S. Xing Y. Tian J. Zhao X. Sun K. Tang (&) Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Dongchuan Road 800, Minhang District, Shanghai 200240, China e-mail: [email protected]

MeJA SA GA3 ABA CCC AVLBS DMRT ANOVA

Methyljasmonate Salicylic acid Gibberellic acid Abscissic acid Chlormequat chloride 30 , 40 -anhydrovinblastine synthase Duncan’s multiple range test Analysis of variance

Introduction Catharanthus roseus (L.) G. Don. (Madagascar periwinkle) is widely studied as a model of medical plants for its highly economic and pharmaceutical value which comes from its diversity of useful terpenoid indole alkaloids (TIAs). Among them the two most valuable bisindole alkaloids are vinblastine and vincristine for their antineoplastic activity in the treatment of many cancers (El-Sayed and Verpoorte 2007a, b; Zhao and Verpoorte 2007). The two important bisindole alkaloids are synthesized by the monomeric alkaloids catharanthine and vindoline in the vacuole of C. roseus leaves and stem cells (Roytrakul and Verpoorte 2007). Vindoline, after biosynthesized in the cytosol, is channeled to the vacuole where a basic peroxidase-like enzyme 30 , 40 -anhydrovinblastine synthase (AVLBS) couples vindoline to catharanthine (Sottomayor et al. 1998). The coupling process results into a-30 , 40 -anhydrovinblastine which is converted into vinblastine which further derives into vincristine (Verpoorte et al. 1997). The pathway of vindoline biosynthesis has been established with a sequence of six steps in the transformation from tabersonine while the information about catharanthine biosynthesis is very little (El-Sayed and Verpoorte 2007a, b).

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Wide use and production of these useful bisindole alkaloids is limited by their trace amounts in C. roseus plants. Great efforts have been made to produce them in a large scale by cultures of plant cell suspensions and diverse tissues (such as hairy roots). It was found C. roseus cell cultures lacked the part of biosynthesis pathway of vindoline and failed to form these valuable and complex compounds for commercial use (Zhao and Verpoorte 2007). So research hotspots shifted from biochemical engineering of the process to the regulation of the biosynthetic pathway. The role of plant growth regulators (PGRs) in regulation of C. roseus indole alkaloids biosynthesis has been extensively researched (El-Sayed and Verpoorte 2007a, b; Zhao and Verpoorte 2007; Roytrakul and Verpoorte 2007; Decendit et al. 1992; Pasquali et al. 1992). PGRs, such as methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), ethylene (ethephon) and gibberellic acid (GA3), exert significant influence on indole alkaloids production and enzymes of the biosynthesis pathways in C. roseus cell suspensions cultures, hairy roots and seedlings (Ruiz-May et al. 2008; Bulgakov et al. 2002; El-Sayed and Verpoorte 2004; Aerts et al. 1994; El-Sayed and Verpoorte 2005; LeeParsons et al. 2004). For commercial uses, C. roseus plants are harvested 3 months after germination when they reach the height of 45–55 cm and begin to flower. However, there are few reports about effect of PGRs on alkaloids production in C. roseus plants during the blooming period when it is the time for harvest. Pre-harvest PGRs application to C. roseus plants during the blooming time is carried out to collect plant materials with high yields of alkaloids for commercial use and production. Chlormequat chloride (CCC, 2-chloroethyl-trimethylammonium chloride), a growth retardant which inhibits the growth of stems, leaves and stolons but promotes tuberisation, is effective in stimulating the accumulation of secondary metabolites (Farooqi et al. 2005). CCC at 1 and 2 g L-1 increased pyrethrins level in Chrysanthemum cinerariaefolium (Haque et al. 2007). El-Sayed and Verpoorte (2007a, b) reported that CCC could increase serpentine accumulation in C. roseus cell cultures, but its effect on the biosynthesis of other indole alkaloids in C. roseus plants was rarely studied. Most previous researches were focused on regulating the production of ajmalicine, serpentine, tabersonine, ajmaline, vindoline and catharanthine by developmental or exogenous signals. But there are little reports about the regulation on vinblastine biosynthesis. In this study, a short-term application of CCC, MeJA, ABA, SA, ethephon and GA3 to mature plants of C. roseus during the blooming time is conducted to research their roles in regulating the biosynthesis of valuable bisindole alkaloid vinblastine, as well as vindoline and catharanthine. The correlation among

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Plant Growth Regul (2010) 60:133–141

vinblastine, vindoline and catharanthine accumulation are discussed and evaluated.

Materials and methods Plant materials and cultivation methods Seeds (named Pacifica cherry red) were bought from Pan American Seed Co, IL, USA; its website is http:// www.panamseed.com). Seeds were germinated in 50-cell plug tray containing soil and organic manure mixture in equal volumes in glass greenhouse. After germination, the plug trays were kept in the glass greenhouse for 2 more weeks at 25 ± 3°C and then were shifted into outside field. When C. roseus plants began to flower and the average height was around 50 cm, the treatment of PGRs was given. During the treatment, average temperature was between 20 and 26°C and relative humidity varied between 68.8 and 72.9%. Plant growth regulators and treatments MeJA, Ethephon, ABA, GA3, SA and CCC were purchased from Sigma–Aldrich. All PGRs stocks were prepared by dissolving them in ethanol to make a concentration of 1 M. The working solution of PGRs was 0.1 mM (diluted by water) and used for the treatments to study their effect on TIAs accumulation in C. roseus, respectively. After spraying the PGRs working solutions, leaves were collected at the following 3 days and the control leaves were collected before spraying. The experiments were conducted in randomized block design (RBD) with three replications. Sample preparation Collected leaf samples were dried at 60°C for 48 h and pulverized in a mortar. Dried sample (200 mg) were immersed and shaked in 2 mL Eppendorf tubes with 1 ml methanol for 10 min. Then the tube was put into Ultrasonic bath (DL-60D) with the power of 80 W for 60 min. The mixture was centrifuged at 12,000 rpm/min for 10 min at room temperature and the supernatant was filtered into 1.5 mL Eppendorf tubes with 4 lm filter membrane and processed for HPLC. The extracts were stored at 4°C. Quantification of alkaloids by HPLC The chromatography was carried out using YMC-Pack Pro C18 RS columns. The chromatographic system is Hitachi L-2000 series, consisting of a L-2000 Organizer, a L-2130 Pump, a L-2200 AutoSampler, a L-2300 Column Oven and a L-2455 Diode Array Detector. The injected samples


(20 lL) were detected from 190 to 400 nm by L-2455 Diode Array Detector. The mobile phase (acetonitrile and diethylamine buffer solution; 1:1) was used at a constant flow rate of 1 mL per min. A mixture of standards and alkaloids were detected (Fig. 1). After analysis of the UV absorb chromatograms (Hisiger and Jolicoeur 2007), Alkaloids—vindoline, catharanthine and, vinblastine were identified after UV analysis of absorbance chromatograms (Hisiger and Jolicoeur 2007) (Fig. 2). Samples were applied in triplicate for quantification of catharanthine, vindoline and vinblastine. The alkaloids were quantified by using regression equation of calibration curve. Statistical analysis All experiments were conducted with three replicates. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range (DMRT) test. The values are mean ± SD for three samples in each group. P values B 0.05 were considered as significant.

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Results and discussion Effect of MeJA on alkaloids accumulation in Catharanthus roseus The treatment of MeJA was shortly applied to C. roseus plants during the blooming time and showed no significant effect on the accumulation of vindoline and catharanthine (Fig. 3). Previous studies reported that the ability of MeJA to increase alkaloid biosynthesis decreased with age of the seedlings. The function of MeJA to stimulate the accumulation of alkaloids was confined to a narrow time interval where the developmentally regulated onset of alkaloid synthesis occurred (El-Sayed and Verpoorte 2004; Aerts et al. 1994). MeJA hardly affected alkaloid contents when it was applied at later developmental stages of the seedlings (Aerts et al. 1996). Our results indicated that MeJA may only affect alkaloids biosynthesis during the seedling stage of C. roseus and lose the ability to increase the monomeric alkaloids content in mature C. roseus plants during the blooming time.

Fig. 1 Chromatograms of mixed standards (a) and C. roseus extract (b); Peak 1: vindoline; Peak 2: catharanthine; Peak 3: vinblastine

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Fig. 2 UV absorb chromatograms of vinblastine (a), catharathine (b) and vindoline (c) standards

Vinblastine, the derivative of dimerization of vindoline and catharanthine, had little response to MeJA induction and significant change of its content was not observed in C. roseus plants during the blooming time after MeJA spraying (Fig. 3). In the previous works, MeJA treatment could highly increase the contents of vindoline and catharanthine but failed to enhance vinblastine accumulation in C. roseus seedlings (Aerts et al. 1996). Taken together, it is speculated that MeJA may not regulate the coupling process leading to vinblastine.

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Effect of ethephon on alkaloids accumulation of Catharanthus roseus Ethephon (0.1 mM) treatment on C. roseus plants during the blooming time significantly enhanced the production of vindoline and catharanthine (Fig. 4). These two monomeric alkaloids accumulation responded to ethephon inducement from the first 24 to 72 h after treatment. The maximum increase of their contents was observed at 72 h, 137% of vindoline and 138% of catharanthine over the control. In this


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Fig. 3 Effect of 0.1 mM MeJA on vindoline, catharanthine and vinblastine contents of C. roseus at different time after treatment

study, temporal application of ethephon to C. roseus plants had a fast and lasting effect on the production of monomeric alkaloids. Previously, exogenously applied ethylene (ethephon) greatly increased the ajmalicine accumulation in C. roseus cell lines (Yahia et al. 1998) and the production of serpentine, tabersonine, catharanthine and vindoline in C. roseus seedlings (El-Sayed and Verpoorte 2004). Vinblastine accumulation followed a similar development with its precursors under ethephon treatment (Fig. 4). The content of vinblastine reached the highest increase at 72 h, 91% over the control. There has been evident that the production of dimeric alkaloids may be linked to ageing of tissues in Catharanthus (Naaranlahti et al. 1991). Accumulation of anhydrovinblastine, the direct precursor of vinblastine, was much higher in old leaves than in young ones (El-Sayed and Verpoorte 2005). Ethephon which is converted into ethylene upon metabolism by plants is a powerful regulator of plant growth and leaf senescence (Abeles et al. 2004; Wingler and Roitsch 2008). It is supposed that the increase of vinblastine accumulation may result from the accelerated senescence of C. roseus leaves which is induced by ethephon. Effect of ABA on alkaloids accumulation of Catharanthus roseus Treatment of ABA at concentration of 0.1 mM during the blooming time resulted in a significant reduction of the three alkaloids accumulation in C. roseus (Fig. 5). The greatest reduction of vinblastine and catharanthine accumulation was observed at the first 24 h after spraying, decreasing by 54% and 62% each. Vindoline decreased to the lowest content at 48 h, 61% lower than the control. A significantly negative effect of ABA on vindoline and catharanthine production continued till the 72 h after treatment. Vinblastine content came back to the normal level at 72 h after treatment. Treatment of ABA, which modulates plant growth and development in various aspects, promoted the pathways towards ajmalicine,

serpentine, tabersonine, vindoline and catharanthine in C. roseus seedlings (El-Sayed and Verpoorte 2004). It has been reported that ABA added to cell suspensions of C. roseus significantly stimulated intracellular accumulation of catharanthine and ajmalicine (Smith et al. 1987). The application of ABA to C. roseus cell suspensions cultures fed with precursors delayed the catabolism of strictosidine but did not induce the ajmalicine accumulation (Smith et al. 1987; El-Sayed and Verpoorte 2002). In disagreement with the past work, our results suggested that ABA may have a different induction pattern on mature plants, seedlings and cell lines of C. roseus. Effect of SA on alkaloids accumulation of Catharanthus roseus Significant increase of the accumulation of the three alkaloids was observed after 0.1 mM SA spraying (Fig. 6). The content of vinblastine had a high increase of 22% at 24 h, an increase of 13% at 48 h and reduced to the level of untreated sample at 72 h. Catharanthine content was noted its maximum increase of 42% at 24 h, 31% at 48 h and 28% at 72 h higher than control. Vindoline was greatly enhanced by SA at 24 h, 22% over the control and returned to the normal level from 48 h after spraying. SA, as an important compound in the defense system of plants, has no strong induction on the yield of alkaloids in cell cultures but results in high increase of the alkaloid production in seedling of C. roseus (El-Sayed and Verpoorte 2004; Dutta et al. 2007; Godoy-Hernandez and Loyola-Vargas 1997). It was found that low enhancement of vindoline biosynthesis under SA inducement compared to other alkaloids (El-Sayed and Verpoorte 2004; Aerts et al. 1996). Effect of GA3 on alkaloids accumulation of Catharanthus roseus There was a significant reduction of vindoline, catharanthine and vinblastine content under 0.1 mM GA3 treatment

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Fig. 4 Effect of 0.1 mM ethephon on vindoline, catharanthine and vinblastine contents of C. roseus at different time after treatment

Fig. 5 Effect of 0.1 mM ABA on vindoline, catharanthine and vinblastine contents of C. roseus at different time after treatment

Fig. 6 Effect of 0.1 mM SA on vindoline, catharanthine and vinblastine contents of C. roseus at different time after treatment

(Fig. 7). Vindoline decreased 16% at 24 h and 72 h compared to the control. Catharanthine decreased 28% and vinblastine decreased 15% only at the first 24 h after treatment. Previous studies showed that GA3 decreased vindoline accumulation in C. roseus cells, roots and seedlings (Abdul Jaleel et al. 2009; Srivastava and Srivastava 2007) and had no effect on catharanthine and tabersonine accumulation in C. roseus seedlings (El-Sayed and Verpoorte 2004). Taken all the results together, GA3 was suggested to regulate the biosynthesis branch leading to vinblastine as a suppressor (Figs. 6, 7, 8).

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Effect of chlormequat chloride on alkaloids accumulation of Catharanthus roseus The inducement of CCC showed a different pattern of influence on alkaloids biosynthesis from other PGRs in C. roseus (Fig. 8). CCC treatment resulted in a significant decrease on vindoline and catharanthine accumulation with lower concentration (0.01 and 0.1 mM). The content of these two monomeric alkaloids showed a slow response to CCC induction and began to decline from the 48 h after treatment. The maximum decrease of vindoline and


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Fig. 7 Effect of 0.1 mM GA3 on vindoline, catharanthine and vinblastine contents of C. roseus at different time after treatment

Fig. 8 Effect of CCC with different concentrations on vindoline, catharanthine and vinblastine contents of C. roseus at different time after treatment. The different concentrations are 0.01, 0.1 and 1 mM

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catharanthine was 51% at 72 h and 37% each at 48 h after 0.01 mM CCC treatment. 1 mM CCC showed no significant effect on vindoline and catharanthine production. However, vinblastine content was observed a significant increase after CCC treatment. Its biosynthesis was strongly enhanced at 48 h with a significant increase of 21% after 0.1 mM CCC treatment and 15% after 1 mM CCC treatment over the control. The low concentration (0.01 mM) of CCC did not affect vinblastine accumulation. Application of CCC could result in an increase of catharnthine in C. roseus cell cultures (Smith et al. 1987). Interestingly, under 0.1 mM CCC treatment, although production of vindoline and catharanthine, the two parent molecules of vinblastine, was greatly decreased, more vinblastine was still formed with a significant increase of its content. Under 0.01 mM CCC, vinblastine production was not affected with the sharp decrease of its two precursors. 1 mM CCC strongly stimulated the biosynthesis of vinblastine but showed no significant effect on the accumulation of the two precursors. Such case also happened in the former research with MeJA treatment. In that case, the increase of the parent compounds accumulation did not improve the biosynthesis of vinblastine (Aerts et al. 1996). It may be considered that CCC did not regulate the pathway of vindoline and catharanthine biosynthesis but only stimulated the pathway of vinblastine accumulation. Alternatively, vinblastine accumulation may be not directly affected by the accumulation of the two precursors (vindoline and catharanthine). The amount of vindoline and catharanthine in C. roseus is supposed to be adequate for forming vinblastine. There may be some other stronger factors regulating and limiting the biosynthesis of vinblastine, which thus resulting in its low yield in the plant. Previous researches proved that vinblatine and its precursors are synthesized in different compartments of C. roseus cells (Roytrakul and Verpoorte 2007). Vindoline and catharanthine are transported into vacuoles to form vinblastine. Considered all these, rate-limiting step probably exists in the pathway from vindoline and catharanthine to vinblastine. Transporters involved in transporting vindoline and catharanthine into vacuoles may be a bottleneck of the biosynthesis of vinblastine.

Conclusions From the present study, it can be concluded that short-term application of ethephon and SA highly enhances the accumulation of vinblastine, vindoline and catharanthine while the application of ABA and GA3 have the opposite effects during the blooming time of C. roseus. MeJA, as a strong inducer to up-regulate many important enzymes and

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alkaloid biosynthesis in cell suspensions, cell cultures and seedlings of C. roseus, has no significant effect on the production of vinblastine, vindoline and catharanthine of C. roseus plants during the blooming period. C. roseus plants in different growth stages have different response patterns to MeJA induction. CCC significantly increases the vinblastine accumulation but greatly decreases the accumulation of its two precursors, vindoline and catharanthine. Acknowledgments This research was supported by China National High-Tech ‘‘863’’ Program (grant number 2007AA10Z189), China ‘‘973’’ Program (grant number 2007CB108805), Shanghai Science and Technology Committee (grant number 08391911800) and Shanghai Leading Academic Discipline Project (Project Number B209).

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