Abstract
The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring.
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Abbreviations
- C. roseus :
-
Catharanthus roseus
- DAPI:
-
Diamidino-2-phenylindole
- DIC:
-
Differential interference contrast
- DMAPP:
-
Dimethylallyl diphosphate
- DXR:
-
1-Deoxy-d-xylulose 5-phosphate reductoisomerase
- DXS:
-
1-Deoxy-d-xylulose 5-phosphate synthase
- ER:
-
Endoplasmic reticulum
- GFP:
-
Green fluorescent protein
- G10H:
-
Geraniol 10-hydroxylase (CYP76B6)
- HDS:
-
Hydroxymethylbutenyl 4-diphosphate synthase
- IPAP:
-
Internal phloem-associated parenchyma
- IPP:
-
Isopentenyl diphosphate
- MECS:
-
Methyl-d-erythritol 2,4-diphosphate synthase
- MEP:
-
Methyl-d-erythritol 4-phosphate
- MIA:
-
Monoterpene indole alkaloids
- NAA:
-
Naphthalene acetic acid
- OSER:
-
Organized smooth ER
- PRX1:
-
Class III peroxidase isoenzyme
- SGD:
-
Strictosidine β-d-glucosidase
- SLS:
-
Secologanin synthase (CYP72A1)
- spG10H:
-
ER targeting signal peptide and anchoring transmembrane helix of G10H
- STR:
-
Strictosidine synthase
- TDC:
-
Tryptophan decarboxylase
- TEM:
-
Transmission electron microscopy
- YFP:
-
Yellow fluorescent protein
- ΔspG10H:
-
Truncated variant of G10H lacking the ER anchoring transmembrane helix
References
Ahn CS, Pai HS (2008) Physiological function of IspE, a plastid MEP pathway gene for isoprenoid biosynthesis, in organelle biogenesis and cell morphogenesis in Nicotiana benthamiana. Plant Mol Biol 66:503–517. doi:10.1046/j.1365-313x.2000.00764.x
Araki N, Kusumi K, Masamoto K, Niwa Y, Iba K (2000) Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-d-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis. Physiol Plant 108:19–24
Barry C, Faugeron G, Rossignol JL (1993) Methylation induced premeiotically in Ascobolus: coextension with DNA repeat lengths and effect on transcript elongation. Proc Natl Acad Sci USA 90:4557–4561
Batoko H, Zheng HQ, Hawes C, Moore I (2000) A rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants. Plant Cell 12:2201–2218
Bourett TM, Czymmek KJ, Howard RJ (1999) Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing. Planta 208:472–479
Bouvier F, Suire C, d’Harlingue A, Backhaus RA, Camara B (2000) Molecular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells. Plant J 24:241–252. doi:10.1046/j.1365-313x.2000.00875.x
Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B (2004) Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites. Plant J 38:131–141. doi:10.1111/j.1365-313X.2004.02030.x
Carretero-Paulet L, Ahumada I, Cunillera N, Rodríguez-Concepción M, Ferrer A, Boronat A, Campos N (2002) Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-d-erythritol 4-phosphate pathway. Plant Physiol 129:1581–1591. doi:10.1104/pp.003798
Chahed K, Oudin A, Giglioli-Guivarc’h N, Hamdi S, Chénieux JC, Rideau M, Clastre M (2000) 1-Deoxy-d-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells. Plant Physiol Biochem 38:559–566
Collu G, Unver N, Peltenburg-Looman AM, van der Heijden R, Verpoort R, Memelink J (2001) Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett 508:215–220. doi:10.1016/S0014-5793(01)03045-9
Contin A, van der Heijden R, Lefebre AW, Verpoorte R (1998) The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a Catharanthus roseus cell culture. FEBS Lett 4:413–416. doi:10.1016/S0014-5793(98)01022-9
Costa MM, Hilliou F, Duarte P, Pereira LG, Almeida I, Leech M, Memelink J, Barceló AR, Sottomayor M (2008) Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant Physiol 146:403–417. doi:10.1104/pp.107.107060
Courdavault V, Burlat V, St-Pierre B, Giglioli-Guivarc’h N (2005a) Characterisation of CaaX-prenyltransferases in Catharanthus roseus: relationships with the expression of genes involved in the early stages of monoterpenoid biosynthetic pathway. Plant Sci 168:1097–1107. doi:10.1016/j.plantsci.2004.12.010
Courdavault V, Thiersault M, Courtois M, Gantet P, Oudin A, Doireau P, St-Pierre B, Giglioli-Guivarc’h N (2005b) CaaX-prenyltransferases are essential for expression of genes involved in the early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells. Plant Mol Biol 57:855–870. doi:10.1007/s11103-005-3095-0
Courdavault V, Burlat V, St-Pierre B, Giglioli-Guivarc’h N (2009) Proteins prenylated by type I protein geranylgeranyltransferase act positively on the jasmonate signalling pathway triggering the biosynthesis of monoterpene indole alkaloids in Catharanthus roseus. Plant Cell Rep 28:83–93. doi:10.1007/s00299-008-0610-1
Daniell H, Vivekananda J, Nielsen BL, Ye GN, Tewari KK, Sanford JC (1990) Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proc Natl Acad Sci USA 87:88–92
De Luca V, Cutler AJ (1987) Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 85:1099–1102
De Luca V, Marineau C, Brisson N (1989) Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases. Proc Natl Acad Sci USA 86:2582–2586
de Vetten N, ter Horst J, van Schaik HP, de Boer A, Mol J, Koes R (1999) A cytochrome b5 is required for full activity of flavonoid 3′,5′-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci USA 96:778–783
de Waal A, Meijer AH, Verpoorte R (1995) Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms. Biochem J 306:571–580
Facchini PJ, St-Pierre B (2005) Synthesis and trafficking of alkaloid biosynthetic enzymes. Curr Opin Plant Biol 8:657–666. doi:10.1016/j.pbi.2005.09.008
Geerlings A, Ibañez MM, Memelink J, van Der Heijden R, Verpoorte R (2000) Molecular cloning and analysis of strictosidine beta-d-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J Biol Chem 275:3051–3056
Gray JC, Sullivan JA, Hibberd JM, Hansen MR (2001) Stromules: mobile protrusions and interconnections between plastids. Plant Biol 3:223–233. doi:10.1055/s-2001-15204
Guirimand G, Courdavault V, St-Pierre B, Burlat V (2009) Biosynthesis and regulation of alkaloids. In: Pua EC, Davey MR (Eds) Plant developmental biology—biotechnological perspectives, vol 2. Springer-Verlag GmbH, Berlin (in press)
Hans J, Hause B, Strack D, Walter MH (2004) Cloning, characterization, and immunolocalization of a mycorrhiza-inducible 1-deoxy-d-xylulose 5-phosphate reductoisomerase in arbuscule-containing cells of maize. Plant Physiol 134:612–622. doi:10.1104/pp.103.032342
Hanson MR, Kwok EY (2004) Plastids and stromules interact with the nucleus and cell membrane in vascular plants. Plant Cell Rep 23:188–195. doi:10.1007/s00299-004-0824-9
Hilliou F, Christou P, Leech MJ (1999) Development of an efficient transformation system for Catharanthus roseus cell cultures using particle bombardment. Plant Sci 140:179–188
Holzinger A, Buchner O, Lütz C, Hanson MR (2007) Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana. Protoplasma 230:23–30. doi:10.1007/s00709-006-0222-y
Holzinger A, Kwok EY, Hanson MR (2008) Effects of arc3, arc5 and arc6 mutations on plastid morphology and stromule formation in green and nongreen tissues of Arabidopsis thaliana. Photochem Photobiol 84:1324–1335. doi:10.1111/j.1751-1097.2008.00437.x
Hsieh MH, Chang CY, Hsu SJ, Chen JJ (2008) Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis. Plant Mol Biol 66:663–673. doi:10.1007/s11103-008-9297-5
Irmler S, Schroder G, St-Pierre B, Crouch NP, Hotze M, Schmidt J, Strack D, Matern U, Schroder J (2000) Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J 24:797–804. doi:10.1111/j.1365-313X.2000.00922.x
Kim SM, Kuzuyama T, Chang YJ, Kwon HJ, Kim SU (2006a) Cloning and functional characterization of 2-C-methyl-d-erythritol 4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba. Phytochemistry 67:1435–1441. doi:10.1016/j.phytochem.2006.05.034
Kim SM, Kuzuyama T, Chang YJ, Kim SU (2006b) Cloning and characterization of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MECS) gene from Ginkgo biloba. Plant Cell Rep 25:829–835. doi:10.1007/s00299-006-0136-3
Kim SM, Kuzuyama T, Kobayashi A, Sando T, Chang YJ, Kim SU (2008) 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes in gymnosperms Ginkgo biloba and Pinus taeda. Planta 227:287–298. doi:10.1007/s00425-007-0616-x
Klein TM, Fromm M, Weissinger A, Tomes D, Schaaf S, Sletten M, Sanford JC (1988) Transfer of foreign genes into intact maize cells with high-velocity microprojectiles. Proc Natl Acad Sci USA 85:4305–4309
Kodja H, Liu D, Mérillon J-M, Andreu F, Rideau M, Chénieux JC (1989) Stimulation of indole alkaloid accumulation in suspension cell cultures of Catharanthus roseus (G. Don) by cytokinins. C R Acad Sci Life Sci 309:453–458
Köhler RH, Hanson MR (2000) Plastid tubules of higher plants are tissue-specific and developmentally regulated. J Cell Sci 113:81–89
Köhler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997) Exchange of protein molecules through connections between higher plant plastids. Science 276:2039–2042
Koroleva OA, Tomlinson ML, Leader D, Shaw P, Doonan JH (2005) High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J 41:162–174. doi:10.1111/j.1365-313X.2004.02281.x
Krysiak C, Mazus B, Buchowicz J (1999) Relaxation, linearization and fragmentation of supercoiled circular DNA by tungsten microprojectiles. Transgenic Res 8:303–306
Kutchan TM (2005) A role for intra- and intercellular translocation in natural product biosynthesis. Curr Opin Plant Biol 8:292–300
Kwok EY, Hanson MR (2004) Stromules and the dynamic nature of plastid morphology. J Microsc 214:124–137
Leménager D, Ouelhazi L, Mahroug S, Veau B, St-Pierre B, Rideau M, Aguirreolea J, Burlat V, Clastre M (2005) Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus. J Exp Bot 56:1221–1228. doi:10.1093/jxb/eri116
Levac D, Murata J, Kim WS, De Luca V (2008) Application of carborundum abrasion for investigating the leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase. Plant J 53:225–236
Lois LM, Rodríguez-Concepción M, Gallego F, Campos N, Boronat A (2000) Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-d-xylulose 5-phosphate synthase. Plant J 22:503–513. doi:10.1046/j.1365-313x.2000.00764.x
Madyastha KM, Meehan TD, Coscia CJ (1976) Characterization of a cytochrome P-450 dependent monoterpene hydroxylase from the higher plant Vinca rosea. Biochemistry 15:1097–1102
Maggio C, Barbante A, Ferro F, Frigerio L, Pedrazzini E (2007) Intracellular sorting of the tail-anchored protein cytochrome b5 in plants: a comparative study using different isoforms from rabbit and Arabidopsis. J Exp Bot 58:1365–1379. doi:10.1093/jxb/erl303
Mahroug S, Courdavault V, Thiersault M, St-Pierre B, Burlat V (2006) Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs. Planta 223:1191–1200. doi:10.1007/s00425-005-0167-y
Mahroug S, Burlat V, St-Pierre B (2007) Cellular and sub-cellular organization of the monoterpenoid indole alkaloid pathway in Catharanthus roseus. Phytochem Rev 6:363–381
Mazuś B, Krysiak C, Buchowicz J (2000) Tungsten particle-induced nicking of supercoiled plasmid DNA. Plasmid 44:89–93
McKnight TD, Bergey DR, Burnett RJ, Nassler CL (1991) Expression of an enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants. Planta 185:148–152
Mérillon JM, Ouelhazi L, Doireau P, Chénieux JC, Rideau M (1989) Metabolic changes and alkaloid production in habituated and non-habituated cells of Catharanthus roseus grown in hormone-free medium. Comparing hormone-deprived non-habituated cells with habituated cells. J Plant Physiol 134:54–60
Mitsui M, Murohashi Y, Asano Y, Masada M, Kodama H (2003) Transient assay for in vivo splicing in tobacco leaf cells by particle bombardment. Plant Mol Biol Rep 21:21–30
Montiel G, Breton C, Thiersault M, Burlat V, Jay-Allemand C, Gantet P (2007) Transcription factor Agamous-like 12 from Arabidopsis promotes tissue-like organization and alkaloid biosynthesis in Catharanthus roseus suspension cells. Metab Eng 9:125–132. doi:10.1016/j.ymben.2006.10.001
Murata J, Roepke J, Gordon H, De Luca V (2008) The leaf epidermome of Catharanthus roseus reveals its biochemical specialization. Plant Cell 20:524–542
Nandadeva YL, Lupi CG, Meyer CS, Devi PS, Potrykus I, Bilang R (1999) Microprojectile-mediated transient and integrative transformation of rice embryogenic suspension cells; effect of osmotic cell conditioning and of physical configuration of plasmid DNA. Plant Cell Rep 18:500–504
Natesan SKA, Sullivan JA, Gray JC (2005) Stromules: a characteristic cell-specific feature of plastid morphology. J Exp Bot 56:787–797. doi:10.1093/jxb/eri088
Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136. doi:10.1111/j.1365-313X.2007.03212.x
Okada K, Kasahara H, Yamaguchi S, Kawaide H, Kamiya Y, Nojiri H, Yamane H (2008) Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant development in Arabidopsis. Plant Cell Physiol 49:604–616. doi:10.1093/pcp/pcn032
Oudin A, Mahroug S, Courdavault V, Hervouet N, Zelwer C, Rodriguez-Concepcion M, St-Pierre B, Burlat V (2007) Spatial distribution and hormonal regulation of gene products from methyl erythritol phosphate and monoterpene-secoiridoid pathways in Catharanthus roseus. Plant Mol Biol 65:13–30. doi:10.1007/s11103-007-9190-7
Paszty C, Lurquin PF (1987) Improved plant protoplast plating/selection technique for quantification of transformation frequencies. BioTechniques 5:716–718
Phillips MA, D’Auria JC, Gershenzon J, Pichersky E (2008) The Arabidopsis thaliana type I isopentenyl diphosphate isomerases are targeted to multiple subcellular compartments and have overlapping functions in isoprenoid biosynthesis. Plant Cell 20:677–696. doi:10.1105/tpc.107.053926
Querol J, Campos N, Imperial S, Boronat A, Rodríguez-Concepción M (2002) Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid isoprenoid biosynthesis. FEBS Lett 514:343–346. doi:10.1016/S0014-5793(02)02402-X
Rasco-Gaunt S, Riley A, Barcelo P, Lazzeri PA (1999) Analysis of particle bombardment parameters to optimize DNA delivery into wheat tissues. Plant Cell Rep 19:118–127
Ro DK, Ehlting J, Douglas CJ (2002) Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar. Plant Physiol 130:1837–1851. doi:10.1104/pp.008011
Russell JA, Roy MK, Sanford JC (1992) Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation. Plant Physiol 98:1050–1056
Ruzin SE (1999) Plant microtechnique and microscopy. Oxford University Press, New York. doi:10.1016/S0968-4328(00)00007-X
Sanford JC, Smith FD, Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods Enzymol 217:483–509
Sapir-Mir M, Mett A, Belausov E, Tal-Meshulam S, Frydman A, Gidoni D, Eyal Y (2008) Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes. Plant Physiol 148:1219–1228. doi:10.1104/pp.108.127951
Schuler MA, Werck-Reichhart D (2003) Functional genomics of P450s. Annu Rev Plant Biol 54:629–667. doi:10.1146/annurev.arplant.54.031902.134840
Siang Tee C, Maziah M (2005) Optimization of biolistic bombardment parameters for Dendrobium Sonia 17 calluses using GFP and GUS as the reporter system. Plant Cell Tiss Org Cult 80:77–89
Snapp EL, Hegde RS, Francolini M, Lombardo F, Colombo S, Pedrazzini E, Borgese N, Lippincott-Schwartz J (2003) Formation of stacked ER cisternae by low affinity protein interactions. J Cell Biol 163:257–269. doi:10.1083/jcb.200306020
Stevens LH, Blom TJM, Verpoorte R (1993) Subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata. Plant Cell Rep 12:573–576
St-Pierre B, Vazquez-Flota FA, De Luca V (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell 11:887–900
Ueki S, Lacroix B, Krichevsky A, Lazarowitz SG, Citovsky V (2009) Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat Protoc 4:71–77. doi:10.1038/nprot.2008.217
van der Fits L, Memelink J (1997) Comparison of the activities of CaMV 35S and FMV 34S promoter derivatives in Catharanthus roseus cells transiently and stably transformed by particle bombardment. Plant Mol Biol 33:943–946
van der Heijden R, Jacobs D, Snoeijer W, Hallard D, Verpoorte R (2004) The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr Med Chem 11:607–628
Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M (2000) Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus. Biochim Biophys Acta 1517:159–163
Waters MT, Fray RG, Pyke KA (2004) Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell. Plant J 39:655–667. doi:10.1111/j.1365-313X.2004.02164.x
Wellmann H, Kaltschmidt B, Kaltschmidt C (1999) Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J Neurosci Methods 92:55–64
Xu Y, Ishida H, Reisen D, Hanson MR (2006) Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflata. BMC Plant Biol 6:8. doi:10.1186/1471-2229-6-8
Yamasaki Y, Akimitsu K (2007) In situ localization of gene transcriptions for monoterpene synthesis in irregular parenchymic cells surrounding the secretory cavities in rough lemon (Citrus jambhiri). J Plant Physiol 164:1436–1448. doi:10.1016/j.jplph.2006.10.008
Yoonram K, Takahashi S, Rattanapittayaporn A, Koyama T, Wititsuwannakul D, Wititsuwannakul R (2008) cDNA, from Hevea brasiliensis latex, encoding 1-deoxy-d-xylulose-5-phosphate reductoisomerase. Plant Sci 175:694–700. doi:10.1016/j.plantsci.2008.07.005
Zhang M, Li K, Zhang C, Gai J, Yu D (2008) Identification and characterization of class 1 DXS gene encoding 1-deoxy-d-xylulose-5-phosphate synthase, the first committed enzyme of the MEP pathway from soybean. Mol Biol Rep (in press). doi:10.1007/s11033-008-9258-8
Ziegler J, Facchini PJ (2008) Alkaloid biosynthesis: metabolism and trafficking. Ann Rev Plant Biol 59:735–769
Acknowledgments
This research was financially supported by the Ministère de l’Enseignement Supérieur et de la Recherche (MENRT) and by the Ligue contre le Cancer (Comité du Loir et Cher). G. Guirimand was financed by a MENRT fellowship. We thank Dr. Chris Hawes (Oxford Brookes University) who kindly provided the ER-YFP marker. We thank Marie-Antoinette Marquet, Evelyne Danos and Céline Melin for help in maintaining cell cultures. We also thank Dr. B. Arbeille and the staff of the Electron Microscopy Platform (CHU Bretonneau, Tours, France) for providing excellent working conditions for ultramicrotomy and TEM studies.
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Guirimand, G., Burlat, V., Oudin, A. et al. Optimization of the transient transformation of Catharanthus roseus cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase. Plant Cell Rep 28, 1215–1234 (2009). https://doi.org/10.1007/s00299-009-0722-2
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DOI: https://doi.org/10.1007/s00299-009-0722-2