JXB Advance Access originally published online on February 28, 2005
Journal of Experimental Botany 2005 56(414):1221-1228; doi:10.1093/jxb/eri116
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus*
1EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, 31 Avenue Monge, F-37200 Tours, France
2Departamento de Fisiologia Vegetal, Universidad de Navarra, c/Irunlarrea s/n, 31008 Pamplona, Spain
3Laboratoire de Biochimie Végétale et Symbiotes, Institut National de la Recherche Scientifique et Technique, BP 95, 2050 Hammam-Lif, Tunisie
To whom correspondence should be addressed at Laboratoire de Biologie Moléculaire Végétale, Faculté des Sciences Pharmaceutiques, 31 Avenue Monge, F-37200 Tours, France. Fax: +33 247 27 66 60. E-mail: marc.clastre{at}univ-tours.fr
Received 14 October 2004; Accepted 14 January 2005
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Abstract |
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Identification of molecular markers of monoterpenoid indole alkaloid (MIA) accumulation in cell-suspension cultures of Madagascar periwinkle (Catharanthus roseus (L.) G. Don) was performed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the protein patterns from alkaloid-producing and non-producing cells showed the specific occurrence of a 28 kDa polypeptide restricted to cells accumulating MIAs. The polypeptide was purified by preparative two-dimensional gel electrophoresis, digested with trypsin, and microsequenced by the Edman degradation method. Cloning of the corresponding cDNA revealed that the protein which has been named CrPS (Catharanthus roseus Protein S) is a member of the
/ß hydrolase superfamily. Time-course expression studies by northern blot analysis confirmed that CrPS gene expression was associated with MIA accumulation in cell suspension cultures. In the whole plant, multicellular compartmentation is required for alkaloid biosynthesis. In situ mRNA hybridization on developing leaves revealed that CrPS mRNA and transcripts encoding the first enzymes of the MIA pathway were co-localized in internal phloem parenchyma cells. The possible implication of the alkaloid-accumulation associated protein CrPS in the signal transduction pathway leading to MIA production is discussed.
Key words:
/ß hydrolase, Catharanthus roseus, in situ RNA hybridization, monoterpenoid indole alkaloid, phytohormone, two-dimensional gel electrophoresis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The Madagascar periwinkle Catharanthus roseus (L.) G. Don (Apocynaceae) produces a wide range of monoterpenoid indole alkaloids (MIAs). Some of these secondary metabolites possess therapeutical value (van der Heijden et al., 2004
). Monomeric MIAs, ajmalicine and serpentine are used in the treatment of hypertension, while the dimeric MIAs, vincristine and vinblastine, are powerful antitumour drugs in widespread use in cancer chemotherapy. These compounds are obtained by extracting the leaves of the plant.
Cell cultures of C. roseus have been extensively investigated as an alternative source of valuable therapeutic alkaloids. This laboratory has developed a C. roseus cell-suspension culture system as a biotechnological tool to study MIA biosynthesis. This periwinkle line does not accumulate alkaloids under normal growth conditions, whereas culturing cells in 2,4-D-free medium supplemented with zeatin triggers the production of ajmalicine and other MIAs (Décendit et al., 1992).
In order to elucidate the molecular basis for alkaloid biosynthesis, this model system was used to discover genes whose expression was highly correlated with MIA accumulation. Several strategies were applied to identify candidate genes. A targeted approach, based on the isolation of genes by PCR, has been used to characterize cDNAs encoding enzymes of the methyl-erythritol phosphate (MEP) pathway (Chahed et al., 2000; Veau et al., 2000) that leads to the terpenoid branch of the MIA biosynthetic pathway (Contin et al., 1999). Various non-targeted approaches were also developed. They allowed cDNAs encoding a cyclophilin (Clastre et al., 1995) and a novel cytochrome P450 enzyme (Oudin et al., 1999) to be isolated through differential screening of cDNA libraries, as well as complete or partial cDNAs of unknown function through mRNA differential display (Oudin, 2000).
In addition, systematic analysis of total proteins by two-dimensional gel electrophoresis has been carried out on alkaloid-producing and non-producing cells (Ouelhazi et al., 1993). Changes in protein patterns showed that some polypeptides were differentially expressed, including one protein called polypeptide S with a Mr of 28 kDa that is expressed only in cells accumulating MIAs.
This paper reports on the purification of the polypeptide S and the characterization of the corresponding full-length cDNA clone (designated CrPS). The deduced protein sequence exhibited similarities to several plant /ß hydrolase superfamily members. It is shown that the expression of the CrPS gene is associated with MIA accumulation in cell-suspension cultures. Furthermore, it is demonstrated that, in the whole plant, the CrPS gene displays identical cell-specific expression pattern as that of the genes involved in the early steps of MIA biosynthesis.
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Materials and methods |
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Plant material and growth conditions
The cell line (C20D) of Catharanthus roseus (L). G. Don was maintained on a 7 d growth cycle in 100 ml Erlenmeyer flasks each containing 25 ml B5 medium of Gamborg et al. (1968)
supplemented with 58 mM sucrose and 4.5 µM 2,4-D (maintenance medium, MM). The cultures were grown on shakers (100 rpm) at 25 °C in the dark.
For experimental purposes, 7-d-old cells were subcultured in MM or in a 2,4-D-free B5 medium supplemented with 5 µM of the cytokinin zeatin added at day 3 of culture (production medium, PM) (Décendit et al., 1992). Treatment with methyl viologen (2 µM) was performed on the third day of culture in PM. The cells were harvested by vacuum filtration, frozen in liquid nitrogen, and either stored at –80 °C (for northern blot analysis) or freeze-dried before dry mass determination and ajmalicine quantitation.
Mature C. roseus plants, grown in a greenhouse, were used for in situ hybridization studies.
Determination of alkaloid content
The cells (25 mg of dry mass) were extracted for 2 h with 1 ml methanol, then 1 µl of crude extract was spotted onto a silica gel 60 TLC plate (Ref 5553, Merck) using the TLC III Autosampler from Camag (Muttenz, Switzerland). The plates were developed for 2 min in a horizontal developing chamber using ethyl acetate/diethylamine (9:1, v/v) as eluant, then UV-irradiated for 4 min. Ajmalicine was chosen as a marker of alkaloid accumulation and was quantified directly on the TLC plate by densitometry (TLC Scanner III in the fluorescence emitting mode, 
exc, 254 nm).
Extraction and quantitation of total proteins
For protein extraction, 1 g of freeze-dried cells was ground in 2 ml of 2D-MH buffer (according to Mayer et al., 1987; as modified by Ouelhazi et al., 1993) using a motor-driven homogenizer. Homogenates were transferred into microtubes, treated with protamine (1.5 mg ml–1), shaken for 10 min at room temperature, then centrifugated at 15 000 g for 15 min. The supernatants were precipitated with acetone (final concentration 80%) at –20 °C. The resulting pellets were dried in a desiccator then resuspended in the 2D-MH buffer adjusted to 9 M urea. Aliquots were stored at –80 °C until further use. Protein contents were determined according to Bradford's (1976) procedure modified by Ramagli and Rodriguez (1985). Bovine serum albumin dissolved in extraction buffer adjusted to 9 M urea was used as a standard.
Two-dimensional gel electrophoresis
Two-dimensional polyacylamide gel electrophoresis (2D-PAGE) was carried out with a mini-electrophoresis system (Miniprotean II, Bio-Rad) for protein pattern analysis or with a standard system (Protean II, Bio-Rad) for preparative electrophoresis. IEF (first dimension) and SDS-PAGE (second dimension) were performed as previously described (Ouelhazi et al., 1993).
After electrophoresis, the minigels were fixed in methanol/acetic acid/water (40:10:50, by vol.), then silver-stained according to Oakley et al. (1980). Patterns were compared through visual estimation of the relative intensities of the spots corresponding to each polypeptide. To improve the reliability of the results, equal amounts of proteins (10 µg) extracted from cells grown in MM and PM harvested on day 5, were simultaneously subjected to electrophoresis.
Purification of protein and microsequencing
Total protein was extracted from 5-d-old cells grown in PM, then separated by preparative 2D-PAGE. 400 µg of total proteins were loaded per gel. The separated proteins were electroblotted onto polyvinylidene difluoride (PVDF) membranes using a semi-dry blotter (MilliBlot-SDE system; Millipore). Protein spots were visualized using Amido black staining. Proteolytic digestion, peptide separation, and amino-acid sequencing were performed as described by Bauw et al. (1989, 1990). For the determination of internal sequences, the membrane pieces carrying the protein were excised, and subjected to trypsin digestion. The formed peptides were separated by reverse-phase HPLC on a 4.6x250 mm C18 column (DuPont, Wilmington, USA) and sequenced using an amino-acid sequencer (model 470A, Applied Biosystem).
cDNA isolation and analysis
Isolation of the full-length cDNA encoding the polypeptide S was performed by RT-PCR strategy followed by asymmetric PCR and 3'-RACE PCR.
RT-PCR was performed on total RNA extracted from 7-d-old cells of C. roseus subcultured in PM. The degenerate, inosine-containing primers PS9 5'-TT(C,T)ACIGC(A,T,C,G)GTIAA(C,T)(C,T)TIGC (sense orientation), PS6 5'-CC(A,G)AAICCGTCGTCIGT(A,G)AA (antisense orientation) and PS8 5'-ACIGG(A,G)TT(A,G)TT(C,T)TC(G,A,T)ATCAT (antisense orientation) were derived from the peptides FTAVNLA, FTEEGFG, and MIENNPV, respectively. First-strand cDNA was generated from 1 µg total RNA by the use of AMV reverse transcriptase (Promega) and the PS8 primer. The cDNA was amplified by a first PCR using the primers PS9 and PS8, then by a second nested PCR with primers PS9 and PS6. The thermal profiles consisted of denaturation at 94 °C for 1 min, annealing at 44 °C for 1 min, and extension at 72 °C for 1 min. The 35 cycles were preceded by a denaturation step of 94 °C for 2 min and followed by a final extension of 72 °C for 5 min. An internal cDNA fragment of 500 bp was amplified, which was cloned into pGEM-T Easy vector (Promega) and sequenced in order to design primers for the amplification of the 3' and 5' ends of the corresponding cDNA.
All PCR amplifications were performed according to the above-mentioned programme, except that the annealing temperature used was 57 °C.
The 3'-end cDNA was characterized through 3'-RACE-PCR (Gibco BRL). Following the reverse transcription, a first PCR was primed by the sense oligonucleotide S5 5'-CTCCACCAGAAGCATGGAAGGAT and the antisense abridged anchor primer (AUAP). The amplicon was reamplified by a second nested PCR with the sense oligonucleotide S6 5'-ACTCTGTATCACCTCTCCCCTAT and AUAP. The amplified cDNA of 550 bp was purified and sequenced directly. To isolate the 5'-upstream terminal region of the cDNA, an asymmetric PCR was performed on a -ZAPII-oriented cDNA library with the antisense primer S4 5'-TCGGTATCAGGCATTATAGCATT and the universal M13 reverse primer (sense orientation). The resulted amplicon of 500 bp was cloned into pGEM-T Easy vector (Promega) and sequenced. Finally, the full-length cDNA was amplified using specific primers from the 5' and 3' untranslated regions and sequenced directly.
DNA sequencing was carried out by Genome express SA (France). Nucleotide and amino acid sequences were analysed using the BLAST and FASTA network service. Protein sequence alignment was performed using Clustal W from the Mac Vector program (Oxford Molecular Ltd).
Northern blot analysis
Frozen cells (3 g fresh mass) were ground to a fine powder in liquid nitrogen. Total RNA were extracted using an RNA isolation kit (RNAeasy Plant Mini Kit, Qiagen). For northern blot analysis, 15 µg of total RNA were fractionated on a 1.5% agarose gel containing 2.2 M formaldehyde, capillary transferred onto a nylon membrane (Hybond-N+, Amersham-Pharmacia-Biotech) and subsequently baked for 2 h at 80 °C. The membranes were prehybridized at 42 °C for 1 h in UltraHYB solution (Ambion). cDNAs were labelled with the ‘Prime-a-gene’ labelling kit (Amersham-Pharmacia-Biotech). Hybridization was carried out for 12 h at 42 °C in UltraHYB solution. The membranes were washed for 30 min at 42 °C in 0.1x saline sodium phosphate EDTA and 0.5% (w/v) SDS and autoradiographed. Equal loading of RNA was checked by ethidium bromide-staining of the gel.
In situ RNA hybridization
Tissue fixation and embedding, in situ hybridization and microscopy were performed according to Burlat et al. (2004). The cDNA cloned into pGEM-T Easy vector (Promega) were used for the synthesis of sense and antisense RNA probes. DXR cDNA clone contained a 1740 bp fragment (Veau et al., 2000; Genbank Accession number AF250235). CrPS cDNA clone contained a 488 bp internal fragment.
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Results |
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Purification and protein sequencing
It was previously found that adding zeatin to C20D cells grown without 2,4-D led to the accumulation of alkaloids and polypeptide S (Ouelhazi et al., 1993
). Indeed, analysis through 2D-PAGE showed that polypeptide S was undetectable in cells grown in MM and was highly expressed in cells grown in PM (Fig. 1A). The polypeptide migrated in 2D gels with an apparent pI of approximately 5.7 and an apparent molecular mass of 28 kDa. Total proteins were extracted from 5-d-old cells cultivated in PM, then separated by preparative 2D-PAGE, and blotted onto PVDF membranes. By combining six experiments, sufficient amounts of Amido black-stained S spots were obtained for microsequencing. After tryptic digestion, the fragments were resolved by HPLC and selected peptide peaks were sequenced. As illustrated in Table 1, four sequences of peptide fragments were obtained.
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Molecular cloning of the full-length cDNA
Based on the amino acid sequence of the polypeptide S digest, degenerate oligonucleotides were designed. With two primer pairs (PS9/PS6 and PS9/PS8) used in nested PCR, a 500 bp internal cDNA fragment was amplified. The 5' and 3' flanking sequences of this partial cDNA were isolated by asymmetric PCR and 3' RACE-PCR, respectively. A final PCR was used to verify the entire sequence of the full-length cDNA (designated CrPS for Catharanthus roseus Protein S).
The CrPS cDNA had a size of 891 bp with an open reading frame of 777 bp. The deduced amino acid sequence contained the four sequenced peptide fragments of the polypeptide S and encoded a protein with 258 amino acids. The calculated molecular mass (28.5 kDa) and the theoretical pI (5.4) of the CrPS product matched the data derived from 2D gel analysis.
A similarity search in sequence databases revealed that the CrPS protein is a member of the /ß hydrolase superfamily, whose members contain a catalytic triad consisting of the conserved amino acids Ser, Asp, and His (Fig. 2). CrPS exhibited significant similarity to several members of this superfamily, including plant lyases, esterases, and lipases. CrPS showed 41% and 43% identity with hydroxynitrile lyases (HNL) from Hevea brasiliensis (Hasslacher et al., 1996) and Manihot esculenta (Hughes et al., 1994), respectively. The same range of identity (around 42%) was found with the methyl jasmonate esterase (MJE) from Lycopersicon esculentum (Stuhlfelder et al., 2004) and the product of the Pir7b gene from Oryza sativa (Wäspi et al., 1998). CrPS showed highest identities with the polyneuridine aldehyde esterase (PNAE) (50% identity) from Rauwolfia serpentina (Dogru et al., 2000), an ethylene-induced esterase (EIE) (55% identity) from Citrus sinensis (Zhong et al., 2001), and the salicylic acid-binding protein 2 (SABP2) (55% identity) from Nicotiana tabacum (Kumar and Klessig, 2003). In addition, 19 genes similar to CrPS, with identities ranging from 32–53%, have been found in the genome of Arabidopsis thaliana.
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Transcript accumulation and alkaloid biosynthesis in cell-suspension cultures
The expression of the CrPS gene was investigated by northern blot analysis in periwinkle cell-suspension cultures. Cells grown in MM and PM were harvested from days 3 to 7 for RNA extraction and alkaloid quantitation. CrPS gene expression was studied in both culture conditions. As shown in Fig. 1B no CrPS transcripts were detected in cells grown in MM. In cells cultured in PM, CrPS messages accumulated from day 4 onwards. This induction preceded MIA production that started at day 5 (Fig. 1B).
The correlation between MIA accumulation and CrPS gene activation was further confirmed using methyl viologen (paraquat). This herbicide causes oxidative stress by generating reactive oxygen species (Bus and Gibson, 1984). Treatment with methyl viologen strongly decreased the content of both MIA and CrPS transcripts (Fig. 1C), without affecting cell growth (data not shown).
Cell-specific gene expression
The localization of CrPS mRNA was investigated in developing leaves using in situ hybridization.
It has recently been shown that early steps of the MIA pathway specifically occurred in adaxial (internal) phloem parenchyma cells of C. roseus aerial organs (Burlat et al., 2004). An example of the in situ hybridization signal to localize the expression of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene is illustrated in Fig. 3A and D. DXR encodes the second enzyme of the MEP pathway. Its expression is representative of that of the MEP pathway genes as well as the geraniol 10-hydroxylase gene, which are involved in the early steps of the MIA pathway (Burlat et al., 2004).
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Expression of the DXR gene in adaxial phloem parenchyma cells was more pronounced in the young revoluted base of the leaves (Fig. 3A, D) with a basipetal gradient decreasing toward the tips of older leaves (Fig. 3A). Interestingly, CrPS transcripts (Fig. 3B, E) accurately co-localized with the DXR mRNA and displayed a similar basipetal gradient.
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Discussion |
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Thorough analysis of the molecular and biochemical mechanisms underlying the biosynthesis of secondary metabolites is of major importance for future plant biotechnological applications. In this way, C. roseus cell-suspension cultures provide a good model system to investigate changes of gene and protein expression associated with MIA biosynthesis. Comparing 2D-PAGE patterns from alkaloid-producing and non-producing cells allowed the identification of a 28 kDa polypeptide that is highly and specifically expressed in cells accumulating MIAs. Digestion of this polypeptide, followed by microsequencing of the generated peptides, led to the characterization of a full-length cDNA, referred to as CrPS.
Northern blotting experiments confirmed the lack of expression of CrPS in non-producing cells and, by contrast, the strong expression of the gene in alkaloid-producing cells. High accumulation of CrPS transcripts was observed during the late phase of culture (from day 4 to day 7 of culture) and occurred 1 d before MIA accumulation. This result fitted well with the time-course of polypeptide S accumulation previously observed through 2D-PAGE analysis of total protein extracts (Ouelhazi et al., 1993) and with the de novo synthesis of polypeptide S, as studied through in vivo protein labelling (Ouelhazi et al. 1994). A good correlation between the amount of polypeptide S and the production of MIAs was also previously noticed after induction by salt stress, or after inhibition by gibberellic acid or hypoxia (Carpin et al., 1997). In agreement with these studies, a concomitant decrease in both MIA production and CrPS transcript accumulation, in cells grown under oxidative stress caused by methyl viologen, has been shown in the present study. The effect of the herbicide on isoprenoid biosynthesis has been reported by Lange et al. (2001). The authors showed that the pools of prenyl diphosphate related to monoterpene biosynthesis strongly decreased in isolated peppermint oil gland secretory cells treated with methyl viologen. Together, the above data on the expression of polypeptide S or its corresponding mRNA suggested that CrPS could be directly or indirectly involved in MIA biosynthesis.
Comparing the CrPS sequence with those in the data bank led to the conclusion that CrPS belongs to the extremely divergent superfamily of /ß hydrolases. The members of this class of proteins display a large range of enzymatic activities (Holmquist, 2000). CrPS showed high sequence similarity with /ß hydrolases exhibiting esterase activities and one of them (PNAE) was reported to be involved in MIA accumulation. PNAE catalyses the conversion of polyneuridine aldehyde into epi-vellosimine during the formation of ajmaline and sarpagine in the medicinal plant Rauvolfia serpentina (Dogru et al., 2000). However, the view that CrPS is a PNAE-like enzyme is not favoured for two reasons. The first one is that ajmaline/sarpagine-type alkaloids are not synthesized in C. roseus; the second one can be inferred from results on tissue-specific gene expression. PNAE activity takes place in intermediate steps of the MIA pathway whereas CrPS expression was localized to a tissue associated with the early steps of alkaloid biosynthesis. Indeed, other works from this group have reported multicellular compartmentation of MIA biosynthesis in leaves of C. roseus. The terminal steps of the MIA pathway are associated with specialized alkaloid-accumulating cells, i.e. the laticifers and idioblasts (St-Pierre et al., 1999). The intermediate steps occur in the epidermis (St-Pierre et al., 1999; Irmler et al., 2000) while the first steps, including the MEP pathway reactions and the synthesis of 10-hydroxygeraniol are present in the internal phloem parenchyma (Burlat et al., 2004). In this report, RNA in situ hybridization showed that the expression of the DXR gene from the MEP pathway and the CrPS gene are co-localized to internal phloem parenchyma cells (Fig. 3). Therefore, this co-expression pattern supports the idea that CrPS could be associated with the first steps of MIA biosynthesis. However, CrPS is unlikely to be an enzyme of the alkaloid pathway since there was no esterase activity in the MEP branch and probably not in the terpenoid branch either (i.e. from isopentenyl diphosphate to loganin), even if the latter remains less characterized. Indeed, some steps are not well known, but the biosynthesis of their metabolic intermediates by as yet uncharacterized mechanisms does not require esterase enzymes. Thus, CrPS may have another role than a direct involvement in the MIA pathway.
Besides PNAE, the /ß hydrolase superfamily contains proteins involved in plant defence and phytohormone-mediated responses. CrPS showed similarity with HNL involved in the process of cyanogenesis response against herbivores and phytopathogens (Jones, 1998). CrPS is unlikely to be a HNL-like protein since periwinkle is not cyanogenic. Two other defence-related proteins similar to CrPS have been found in rice and tobacco. The rice protein exhibits esterase activity and is specified by the Pir7b gene (Wäspi et al., 1998). The gene is activated by inoculation of rice plants with the non-host pathogen Pseudomonas syringae pv. syringae which confers acquired resistance of rice against the rice blast fungus Pyricularia oryzae. The tobacco SABP2 protein is required for full local resistance and also acquired resistance to pathogen infection (Kumar and Klessig, 2003). SABP2 has salicylic acid-stimulated lipase activity and the authors suggested that the protein acts as a salicylic acid receptor involved in the transmission of the defence signal. Other phytohormone signals bring into play /ß hydrolase protein family members. Thus, ethylene up-regulates the expression of the EIE gene characterized in Citrus sinensis (Zhong et al., 2001) whereas methyl jasmonate is the substrate of the MJE protein isolated from tomato cell cultures (Stuhlfelder et al., 2002, 2004). Some of the above data could be related to the results obtained on C. roseus cell suspensions. Various treatments with phytohormones have been applied to this plant cell system. Salicylic acid had no effect on MIA accumulation in C. roseus cell suspensions cultured in an auxin-starved medium (data not shown), but ethylene and methyl jasmonate greatly increased the level of alkaloids in the same range as cytokinins (Yahia et al., 1998; Gantet et al., 1998).
Thus (i) the involvement of /ß hydrolases in response to phytohormone signals, of which some could induce alkaloid accumulation, (ii) the identical cell-specific expression profiles of CrPS and genes involved of the early steps of MIA pathway, and (iii) the strong correlation between CrPS gene expression and MIA accumulation, led to the hypothesis that CrPS may participate in the signal transduction pathway triggering MIA biosynthesis.
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Acknowledgements |
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We kindly thank Professor Dirk Inzé (Gent University, Belgium) for protein microsequencing. This research was supported by the ‘Ministère de l'éducation nationale, de l'enseignement supérieur et de la recherche’ (France).
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Footnotes |
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* The sequence of CrPS was deposited in the Genbank database under the accession number AY751530.
This paper is in memory of Dr Lazhar Ouelhazi.
Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; CrPS, Catharanthus roseus Protein S; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; EIE, ethylene-induced esterase; HNL, hydroxynitrile lyase; MEP, methyl-erythritol phosphate; MIA, monoterpenoid indole alkaloid; MJE, methyl jasmonate esterase; MM, maintenance medium; PM, production medium; PNAE, polyneuridine aldehyde esterase; SABP2, salicylic acid-binding protein 2.
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