ANALYTICAL SCIENCES DECEMBER 2011, VOL. 27 1243 2011 © The Japan Society for Analytical Chemistry

Enhancement of Vindoline Production in Suspension Culture of the Catharanthus roseus Cell Line C20hi by Light and Methyl Jasmonate Elicitation

Lihong HE,*,** Li YANG,* Ronghui TAN,* Shujuan ZHAO,*† and Zhibi HU*†

*The SATCM Key Laboratory for New Resources & Quality Evaluation of Chinese Medicines, The Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China **Department of Cell Biology, Medical College of Soochow University, Suzhou 215123, China

The effects of light and methyl jasmonate (MJ) on the transcription of biosynthetic genes as well as the accumulation of vindoline and catharanthine in Catharanthus roseus C20hi cell suspensions were studied. t16h (the gene encoding tabersonine 16-hydroxylase) could be induced by light and MJ, whereas d4h (the gene encoding deacetoxyvindoline 4-hydroxylase) could only be induced by light. Quantification by UPLC-MS showed that light significantly increased vindoline production in C20hi cells by about 0.49 – 5.51-fold more than that in controls, with the highest yield being 75.3 ng/g of dry weight. The biosynthesis of vindoline was further enhanced by combining MJ with light. The accumulation of catharanthine was not improved by either light or MJ elicitation. These results suggested that light and MJ could promote vindoline, but not catharanthine accumulation in C20hi cells.

(Received July 25, 2011; Accepted October 18, 2011; Published December 10, 2011)

4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase Introduction (DAT) catalyze the last two steps. Among them, T16H has reportedly been expressed in cell and hairy root cultures, while Catharanthus roseus produces more than 130 terpenoid indole the other four known enzymes have only been reported in alkaloids (TIAs), most of which have pharmacological activities. seedlings, and the aerial organs of C. roseus.3 Genes of T16H, The most medically significant of these are the potent antitumor D4H and DAT have been cloned, but transcripts of D4H and drugs vinblastine and vincristine. A variety of in vitro DAT have not been detected in cell and hairy root cultures.10–12 micropropagation systems, such as cell suspensions and hairy Genetic engineering and expression of DAT in C. roseus hairy roots, have been established to produce TIAs.1 However, the root cultures has led to high levels of DAT enzymatic activity yields of TIAs are commonly very limited, and the production and hoerhammericine accumulation in DAT-expressing cell of vinblastine and vincristine have failed in cell and hairy root lines in comparison to control lines, but not vindoline cultures, though various biochemical and bioengineering protocols have been applied to manipulate indole alkaloid production.2–5 16-hydroxylase (T16H) Vinblastine and vincristine are dimeric alkaloids formed by tabersonine 16-hydroxytabersonine the peroxidase-catalyzed condensation of vindoline and catharanthine. Catharanthine can be isolated at high concentrations from most cell cultures, but stable production of O-methyltransferase (OMT) vindoline has not been observed. Thus, the lack of vindoline hydration biosynthesis becomes the major obstacle of using cell cultures 16-methoxytabersonine 16-methoxy-2,3-dihydro-3- to produce vinblastine and vincristine.2,6,7 However, vindoline hydroxytabersonine biosynthesis was restored in shoots that regenerated from a N-methyltransferase (NMT) non-producing callus and tabersonine precursor accumulated in cell cultures, indicating that vindoline may be acquired by 4-hydroxylase (D4H) 16-methoxy-2,3-dihydro-3-hydroxy deacetylvindoline fulfilling its regulatory requirements.8,9 As illustrated in Fig. 1, N-methyltabersonine the vindoline pathway contains six enzymes: tabersonine 16-hydroxylase (T16H) starts the reaction; O-methyltransferase Acetyltransferase (DAT) (OMT) and N-methyltransferase (NMT) catalyze O-methylation and N-methylation, respectively; and deacetoxyvindoline vindoline

† To whom correspondence should be addressed. Fig. 1 Schematic pathway for vindoline biosynthesis in Catharanthus E-mail: [email protected]; [email protected] roseus. 1244 ANALYTICAL SCIENCES DECEMBER 2011, VOL. 27 accumulation,2 suggesting that the expression of genes upstream CGGATGCTGAGGATATTC-3′), actin-reverse (5′-TCCAGA- from dat (such as d4h) is needed for vindoline production. GTCCAGAACAATACCA-3′). PCR was carried out in a It has been reported that d4h is transcriptionally blocked in Bio-RAD Peltier thermal cycler (Techne, Burkhardtsdorf, C. roseus cell cultures, which may result in a failure of vindoline Germany) with Taq DNA polymerase (Sangon, Shanghai, biosynthesis.7 Therefore, external regulatory strategies China). The PCR cycle numbers were optimized for each gene, activating d4h transcription may contribute to the accumulation so that product accumulation was in the exponential range. The of vindoline, where elicitation is a very promising strategy. PCR reaction cycles varied as following: 25 cycles for actin and In C. roseus, a variety of physical and chemical elicitors have 34 cycles for t16h and d4h. PCR conditions were as follows: been found to induce the expression of enzymes leading to denaturing at 94°C for 5 min, followed by various cycles with vindoline biosynthesis. Light can induce the expression of different genes of denaturing at 94°C for 30 s, annealing at 55°C T16H in cells and the expression of D4H and DAT in seedlings for 45 s, extension at 72°C for 60 s, and final extension for at the transcription, translation and enzymatic activity 5 min at 72°C. Amplified cDNA fragments were separated on levels.5,6,9–14 The protein level and enzymatic activity of D4H 1.0% agarose gels and visualized under an ImageMaster®VDS increased by up to 9- and 8-fold, respectively, in etiolated (Pharmacia Biotech) imagining system after staining the gel seedlings after light treatment. These studies have indicated that with ethidium bromide. light plays an important role in the conversion of tabersonine to vindoline. Methyl jasmonate (MJ) is most often used as a Alkaloid extraction and determination chemical elicitor that can enhance TIA accumulation, including The extraction of total alkaloids from the C20hi cells and the vindoline in C. roseus plants.15,16 In this study, we elucidated culture media, and the quantification of vindoline and whether light and MJ treatments could induce the expression of catharanthine with ultra-performance liquid chromatography biosynthetic enzymes and the production of vindoline in stable coupled with mass spectrometry (UPLC-MS) were performed cell cultures thereafter. as previously described.18 Before UPLC-MS quantification, the A fully habituated C. roseus cell line, C20hi, has been stably existence of vindoline and catharanthine was first confirmed cultured in our laboratory for about 10 years. It has produced using liquid chromatography/electrospray ionization tandem much more ajmalicine and serpentine than the original C20D mass spectrometry (LC/ESI-MSn). cells,17 but the production of vindoline and catharanthine has not The LC/ESI-MSn analysis was performed to identify alkaloids been studied. This paper deals with the ability of C20hi to in the m/z range of 250 – 1000 on a Surveyor LCQ DECA XPplus produce vindoline and catharanthine after light and MJ system (Thermo Finnigan, San Jose, CA) equipped with an ESI treatments. interface and an ion-trap mass analyzer. Xcalibur Ver. 1.3 software was used for data analysis. Chromatographic separation was performed on a 250 mm × 4.6 mm × 5 μm Experimental CAPCELL PAK C18 column (Shiseido, Japan) at a column temperature of 25°C. The injection volume was 5 μl. The Cell culture and elicitation mobile phase consisted of acetonitrile (A) and 0.1% formic acid C. roseus C20hi cells were cultured as described by Zheng in water (B). The flow rate was 1 ml/min. Gradient elution was and Wu (2004) in the B5 basic medium in the dark. Cells as follows: from 5 to 10% A in 5 min, 10 to 50% A in 15 min, cultured in liquid media on a shaker at 120 rpm under dark hold at 50% A for 10 min, then from 50 to 80% A in 5 min, conditions for eight days were used for light- and MJ-induction finally from 80 to 5% A in 10 min. For the ESI-MS condition, experiments. the split ratio of the LC eluent was 1:3. The tuning parameters Light induction was performed with four white light lamps in positive mode were optimized and set as follows: capillary (Philips 40W/18) located 15 cm above the shakers. The light temperature, 300.00°C; source voltage, 5.00 kV; capillary intensity was 6000 lux and continuous irradiation was applied voltage, 9.00 V; tube lens offset, 15.00 V; sheath gas flow (N2), throughout the induction period. C20hi cells in flasks wrapped 32 A.U.; aux/sweep gas flow (N2), 10 A.U. with packaging film for photosensitive material were used as negative controls. MJ stock solution (100 mM) was sterile-filtrated through a 0.22-μm membrane and stored at 4°C before use. The Results final concentrations of MJ were 10, 50 and 100 μM, respectively. Cells treated with equal volumes of sterile buffer solution (9% Effects of light and MJ elicitation on the transcription of ethanol, v:v) were used as negative controls. biosynthetic genes in C20hi cells After six days of induction, the cells and culture media were It has been assumed that the transcriptional block of genes collected by filtration, immediately frozen in liquid nitrogen and leading to vindoline biosynthesis resulted in a lack of vindoline stored at –80°C for further use. in C. roseus cell cultures.7 Variations at gene transcription levels reflected an early regulatory step in a biosynthetic RNA extraction and RT-PCR pathway; therefore, we first tested the transcription of t16h and The total RNA was isolated from different cell samples using d4h in C20hi cells after elicitation. As can be seen in Fig. 2, the Trizol Reagent® (Invitrogen, Carlsbad, CA). Reverse transcription level of t16h was very low in the dark, but increased transcription was carried out using M-MLV reverse transcriptase significantly after light treatment, and reached the maximum at (Takara, Otsu, Japan). Semi-quantitative RT-PCR was used to 12 h, indicating that light could activate the transcription of t16h measure the transcription levels of t16h and d4h. Actin was in C20hi suspension cultures. used to calibrate the cDNA samples and as an internal reference. When the cells were exposed to continuous light, the Gene-specific primers were as follows: t16h-forward transcription levels of d4h in C20hi cells gradually increased, (5′-ATGACTTTGAAGCCCCACTG-3′), t16h-reverse (5′-CC- indicating that light induced the transcription of d4h. ACCAAATGGTAGATACTCG-3′); d4h-forward (5′-TAGGAT- Interestingly, transcripts of d4h could also be observed in dark CCGAGCAAGAAAGAAAAATG-3′), d4h-reverse (5′-ATCT- control samples; and also, at some time-points they were even GCAGGACGCTTATTTAGTCCAAC-3′); actin-forward (5′-GG- higher than those under light conditions, but then decreased at ANALYTICAL SCIENCES DECEMBER 2011, VOL. 27 1245

36 h (Fig. 2A), and became undetectable after several days in transcription of the first and second to last enzymes in the the dark. These results implied that light could induce the vindoline pathway (Fig. 1), which may lead to the biosynthesis transcription of d4h in C20hi cells, but it may not be necessary. of vindoline in C20hi cells. A similar conclusion was obtained from etiolated seedlings of The MJ treatment increased the levels of t16h transcripts C. roseus, where the levels of d4h transcripts were very low, and compared to the controls, indicating an induction of t16h gradually declined until being hardly detectable after 12 days in transcription by MJ. However, the transcription of d4h could the dark. However, the light treatment could stop and reverse not be detected either in MJ-treated cells or in the controls, the decline of d4h transcripts at later stages of seedling indicating that d4h transcription was not induced by MJ development.10 Our study has shown that light could induce the (Fig. 2B). This result was similar to that in seedlings, where jasmonate could not induce d4h transcription.16

Alkaloids in C20hi detected by HPLC/ESI-MS2 after light and MJ treatment Total alkaloids in C20hi cells were analyzed using high-performance liquid chromatography (HPLC)/ESI-MS2. About 28 indole alkaloids were detected, including 17 monomers and 11 dimeric indole alkaloids (Table 1). According to the retention time and ESI-MS-MS collision characteristics compared with those of the standards, compounds 5, 7, and 16 were identified as being catharanthine, ajmalicine, and vindoline, respectively. Compounds 1 and 2 were identified as 19S-vindolinine and vindolinine according to their retention behavior and mass-spectrometric data.19 The fragment ion of 305 at m/z 337 may suggest the existence of tabersonine, and the fragment ion of 321 at m/z 353 may suggest the existence of an isomer of ajmalicine.20 These results confirm that C20hi cells have the ability to produce vindoline and catharanthine. Fig. 2 Transcript levels of t16h and d4h in suspension-cultured There were no alkaloids detected in the culture media. C20hi after light (A) and MJ (50 μM) induction (B). The panels of Therefore, only the alkaloids in C20hi cells were further actin were used as controls. D, Controls that cultured under the dark analyzed. condition; L, groups that cultured under continuous light.

Table 1 Indole alkaloids identified from cultured cells of C. roseus

No. RT/min Ions for MS2 [M+H]+ Major fragment ion Identifi cation Sample

1 14.5 337 320(B), 308, 291 19S-Vindolinine L, D 2 15.2 337 320(B), 308, 291 Vindolinine L, D 3 17.1 337 319, 307, 174(B), 173, 144 D, MJ, E 4 17.6 337 337(B), 320, 319, 305, 222, 180, 144 L, D 5 18.2 337 319, 305, 173, 165, 144(B) Catharanthine L, D, MJ 6 15.8 353 335(B), 321, 320, 318, 308, 303, 229, 159 L, D, MJ 7 17.9 353 335, 321, 293, 222, 210, 178, 144(B) Ajmalicine L 8 18.5 353 336, 322, 293, 210, 144(B) L 9 25.2 353 335, 307, 279, 261(B), 227, 187, 165 L, D 10 28.4 353 335, 308, 263, 261(B), 245, 243, 177 L, D 11 29.6 353 335, 308, 263, 261(B), 243, 187, 173 L, D 12 17.4 369 238, 187(B), 170, 144 MJ, E 13 21.5 369 352, 309, 226(B), 144 L, D, MJ, E 14 22.2 369 351(B), 309, 277, 144 L, MJ 15 25.2 369 351, 338, 310, 306, 277, 226(B), 144, 115 D 16 19.3 457 439, 398, 397(B), 379, 365, 188 Vindoline L 17 19.2 457 439, 398, 397, 365, 314(B), 188 D 18 20.0 751 733, 718, 716, 691(B) L 19 32.8 755 737(B), 720, 323, 309, 255 L, D, MJ, E 20 28.0 769 751, 709, 684(B) D 21 28.3 769 751(B), 742, 709, 684(B), 307 L, MJ, E 22 32.9 769 751(B), 723, 709, 707, 309, 281, 253 MJ, E 23 28.6 813 795(B), 777, 754, 307 L, D, MJ, E 24 29.5 825 807(B), 763, 719, 569, 309 L, D, E 25 30.5 825 807(B), 766, 765, 719, 569, 309 D 26 33.5 825 807(B), 765, 735, 309 D, MJ, E 27 32.3 827 810, 809(B), 791, 333, 307 D 28 33.5 827 810, 809(B), 796, 762, 691, 353, 311, 309 L

L, C20hi cultured under light; D, C20hi cultured under dark; MJ, C20hi cultured with 50 μM MJ; E, C20hi cultured with 50 μl buffer solution of 9% ethanol; B, base peak. 1246 ANALYTICAL SCIENCES DECEMBER 2011, VOL. 27

Table 2 Catharanthine contents (ng/g DW) in C20hi suspension cultured under continuous light and dark conditions

Culture day 0 3 6 9 12

Light 97.5 ND 293.7 ND ND Dark (control) 97.5 ND 40.0 71.5 ND

Values are the highest determinations from triplicate cell samples. The production in the other samples was very low or not detected. ND, not detected.

Table 3 Combined induction of C20hi with light and varied concentrations of MJ

Blank 100 mM MJ (M)/ 9% Ethanol (E)/ Treatment control (B)/ μl μl μl

Light (L) 10 50 100 10 50 100 0 Dark (D) 10 50 100 10 50 100 0

Combination of light and MJ further enhanced vindoline accumulation Though MJ showed no significant improvement in vindoline and catharanthine accumulation, its enhancement on the accumulation of some intermediate alkaloids and d4h enzymatic activity have been reported.15,16 The increase of t16h transcription Fig. 3 Vindoline contents (ng/g DW) in the suspension cultured levels in C20hi cells led to a hypothesis that the combined C20hi cells induced by continuous light (A) and 50 μM of MJ (B). (A) D, Controls that cultured under dark condition; L, groups that application of light and MJ may further promote vindoline cultured under continuous light; values represent mean ± SD from biosynthesis in C20hi cells. Thus, we conducted a combined triplicate samples; *, statistically significant (p < 0.05, t-test). (B) E, induction experiment to test this hypothesis. In the experiment, Controls that treated with the buffer solution of 9% ethanol; M, groups C20hi cells were induced by light combined with 10, 50 or that treated with 50 μM MJ; values represent means from triplicate 100 μM MJ. Samples cultured under dark, or with different samples; standard deviations were less than 10% of the means. volumes of buffer solution (9% ethanol, v:v), were used as controls (Table 3). The contents of vindoline in C20hi cells cultured under light gradually increased with time, and reached a maximum concentration on the 12th day (Fig. 4). Light alone significantly Light treatment promotes vindoline, but not catharanthine increased the vindoline yield at 6 d (p < 0.02, t-test), though the accumulation in C20hi cells average yield was very low. The addition of MJ to C20hi cells Since vindoline and catharanthine could not be detected in cultured under light conditions further increased the vindoline some cell samples with HPLC/ESI-MS, we considered that the yield (p < 0.001, t-test) with an average increase of about 366% concentrations of vindoline and catharanthine were too low to (55 – 519%, n = 12) compared to those under the dark conditions be analyzed by traditional HPLC. Therefore, a more sensitive with MJ controls (DM), and an average increase of about 349% and rapid UPLC method was developed to quantify vindoline (145 – 539%, n = 12) was achieved compared to those under and catharanthine after elicitation, in which the limit of detection light conditions with 9% ethanol (LE). The addition of MJ to was 0.70 ng/ml for vindoline and 0.46 ng/ml for catharanthine.18 C20hi cells cultured in the dark (DM) also significantly As shown in Fig. 3A, the light treatment significantly increased increased the yield of vindoline by an average of 77% (n = 12) vindoline production in C20hi cells by about 0.49 – 5.51-fold compared to controls (dark with 9% ethanol, DE) (p < 0.05, compared to the controls (n = 3, p < 0.01, t-test). The highest t-test). Among the three concentrations of MJ, 50 and 100 μM yield of vindoline was 75.3 ng/g of dry weight (DW). These were much more effective than 10 μM. In C20hi cells that were results indicated that the light treatment can promote vindoline treated with 50 μM MJ and light for 12 days (LM50, 12 d), the accumulation in C20hi cells. content of vindoline was 43.10 ng/g, about 365% higher than The highest content of catharanthine was 293.7 ng/g DW in the controls (DM50, 12 d). These results confirmed that the C20hi cells grown under light for six days, but it was very low, combined applications of MJ and light significantly promoted or even undetectable, in most other cell samples (Table 2). This vindoline biosynthesis. result suggested that light could not activate catharanthine biosynthesis in C20hi. As shown in Fig. 3B, the contents of vindoline in C20hi cells Discussion treated with MJ were very low, and showed no obvious differences from those of the controls, which suggested that MJ Light is crucial for plant growth. It can induce the formation of had little effect on vindoline accumulation in C20hi cells. thylakoids, in which light-dependent reactions of photosynthesis take place. Chlorophyll molecules are specifically arranged in thylakoid membranes. In the absence of light, proplastids in the ANALYTICAL SCIENCES DECEMBER 2011, VOL. 27 1247

observation, we deduced that NMT activity may exist in C20hi cells after light treatment, because NMT has been reported to locate to the thylakoids of chloroplasts.21 Besides, light treatment can also induce the transcription of d4h (Fig. 2) as well as dat (data not shown) in C20hi cells. These combined results provided molecular support for the induced production of vindoline (Table 1, Fig. 3A) in C20hi cells by light treatment. A single application of MJ cannot improve the production of vindoline in C20hi cells, but the combined use of MJ and light can significantly increase the production of vindoline. This result may be related to the improved production of alkaloid precursors involved in the vindoline pathway by MJ, including tabersonine and 16-hydroxytabersonine, since MJ has been reported to increase the tabersonine content in C. roseus seedlings15 and the transcription of enzyme T16H, leading to 16-hydroxytabersonine (Fig. 2B). Additionally, MJ was also found to induce the expression of dat genes,22 since several motifs in the dat promoter were involved in the response to MJ signals.23 The molecular analysis in this study provided evidence of vindoline production in C20hi cells induced by light and MJ elicitation. However, using HPLC/ESI-MS2 we could not detect vindoline in all samples. We assumed that the traditional HPLC/ESI-MS method was not sensitive enough for any further quantification of vindoline in C20hi cells. In addition, analysis by traditional HPLC/ESI-MS required about 45 min for one sample, making it more laborious and time consuming. Therefore, we developed a more sensitive and rapid method by UPLC-MS. As described, the limit of detection for vindoline by UPLC-MS was about 1000-times lower than that reported by traditional HPLC methods, and the total analysis time was reduced to 6.0 min.18,24 Using the UPLC-MS method, we detected concentrations of as low as 1.63 ng/ml vindoline in unelicitated C20hi cells (Fig. 4), and found significantly increased vindoline production after elicitation with light or light combined with MJ (Figs. 3A and 4). Our study suggested that light can induce the biosynthesis of vindoline in undifferentiated C20hi cells, and its combination with MJ further promoted this process. Although the actual yield of vindoline is too low to be of any valuable use at present, light elicitation combined with MJ provided a promising method to promote vindoline biosynthesis in undifferentiated cell cultures, and is worthy of further study.

Acknowledgements Fig. 4 Vindoline contents (ng/g DW) in C20hi cells induced by light combined with 0 (A), 10 (B), 50 (C) and 100 μM (D) of MJ. L, Light; This study was supported by grants from the National Natural D, dark; M, MJ; E, 9% ethanol (buffer solution); B, blank control. Science Foundation of China (No. 30200358) and the Doctorate Values represent means from triplicate samples; standard deviations Specialized Research Fund from China Ministry of Education were less than 10% of the means; *, statistically significant (p < 0.05, (20070268008). t-test).

References plant cells develop into etioplasts that contain semicrystalline 1. A. Pietrosiuk, M. Furmanowa, and B. Łata, Phytochem. membrane structures, called prolamellar bodies. When exposed Rev., 2007, 6, 459. to light, these prolamellar bodies develop into thylakoids, which 2. M. Magnotta, J. Murata, J. Chen, and V. De Luca, then form stacks of disks, referred to as grana, in the chloroplasts. Phytochemistry, 2007, 68, 1922. This does not happen in plants grown in the dark, which undergo 3. S. Mahroug, V. Burlat, and B. St-Pierre, Phytochem. Rev., etiolation. Undifferentiated cells cultured under dark conditions 2007, 6, 363. may also undergo the etiolation processes. In this study, when 4. J. Zhao and R. Verpoorte, Phytochem. Rev., 2007, 6, 435. C20hi cells cultured under dark conditions were transferred to 5. A. K. Shukla, A. K. Shasany, R. K. Verma, M. M. Gupta, A. the light, their color gradually turned greenish, suggesting the K. Mathur, and S. P. Khanuja, Protoplasma, 2010, 242, 35. biogenesis of chlorophylls and thylakoids. From this 6. B. St-Pierre and V. De Luca, Plant Physiol., 1995, 109, 131. 1248 ANALYTICAL SCIENCES DECEMBER 2011, VOL. 27

7. F. Vázquez-Flota, V. De Luca, M. Carrillo-Pech, A. 15. R. J. Aerts, D. Gisi, E. De Carolis, V. De Luca, and T. W. Canto-Flick, and M. D. Miranda-Ham, Mol. Biotechnol., Baumann, Plant J., 1994, 5, 635. 2002, 2, 1. 16. F. A. Vázquez-Flota and V. De Luca, Phytochemistry, 1998, 8. F. Constabel, P. Gaudet-LaPrairie, W. G. W. Kurz, and J. P. 38, 284. Kutney, Plant Cell Rep., 1982, 1, 139. 17. Z. G. Zheng and M. Wu, Plant Sci., 2004, 166, 507. 9. G. Schröder, E. Unterbuscha, M. Kaltenbacha, J. Schmidt, 18. L. He, L. Yang, A. Xiong, S. Zhao, Z. Wang, and Z. Hu, D. Strack, V. De Luca, and J. Schröder, FEBS Lett., 1999, Anal. Sci., 2011, 27, 433. 458, 97. 19. H. Zhou, Y. P. Tai, C. R. Sun, and Y. J. Pan, Phytochem. 10. F. A. Vázquez-Flota and V. De Luca, Plant Physiol., 1998, Anal., 2005, 16, 328. 117, 1351. 20. F. Ferreres, D. M. Pereira, P. Valentao, J. M. A. Oliveira, J. 11. B. St-Pierre, P. Laflamme, A. M. Alarco, and V. De Luca, Faria, L. Gaspar, M. Sottomayor, and P. B. Andrade, J. Plant J., 1998, 14, 703. Pharm. Biomed., 2010, d51, 65. 12. B. St-Pierre, F. Vázquez-Flota, and V. De Luca, Plant Cell, 21. V. De Luca and A. J. Cutler, Plant Physiol., 1987, 85, 1099. 1999, 11, 887. 22. L.Van der Fits and J. Memelink, Science, 2000, 289, 295. 13. F. A. Vázquez-Flota, B. St-Pierre, and V. De Luca, 23. Q. Wang, F. Yuan, Q. Pan, M. Li, G. Wang, J. Zhao, and K. Phytochemistry, 2000, 55, 531. Tang, Plant Cell Rep., 2010, 29, 185. 14. J. Zhao, W. H. Zhu, and Q. Hu, Plant Growth Regul., 2001, 24. C. Tikhomiroff and M. Jolicoeur, J. Chromatogr., A, 2002, 33, 43. 955, 87.