Results Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol149.Issue2.2000:

3. Results

3 . 1 . Isolation of a gene preferentially expressed in alkaloid-producing cells The periwinkle line used in the present study accumulates no alkaloid when grown in its mainte- nance medium MM. Culturing the cells in a 2,4-D-free medium IM triggers the production of ajmalicine and other TIAs [10]; adding zeatin at day 3 to the 2,4-D-free medium PM further increases the ajmalicine production [19], and Table 1. To isolate cDNAs that could encode enzymes involved in the regulation of the TIA pathway, we screened a non-oriented cDNA li- brary with + D, − D and + Z probes prepared with mRNAs from cells grown in MM, IM and PM, respectively. A partial cDNA giving higher hybridization with the + Z and − D probes than with the + D probe, was isolated. To generate the 5-missing region, we took advantage of an ori- ented-cDNA library. A set of three gene-specific primers Fig. 1 was used together with the M13 reverse universal primer in asymmetric PCRs fol- lowed by a series of PCR reamplifications see Section 2. The full-length cDNA designated CDS 1 encoded a putative 501 amino acid-long protein CDS1, Fig. 1 with a predicted molecular mass of 57.4 kDa. 3 . 2 . The CDS 1 cDNA corresponds to the CYP 96 C 1 gene Search for sequence homologies in databases showed the presence of the signature sequences of cytochrome P450 enzymes [20] in CDS1. In partic- ular the heme-binding domain FxxGxxxCxG, Fig. 1 characteristic of such enzymes was found at about 50 aminoacids upstream of the C-termi- nus [21]. Combining data from the hydropathy profile not shown, [22] and PSORT analysis sug- gested that the first 24 amino acids formed an endoplasmic reticulum membrane-anchoring se- quence Fig. 1. The highest homologies between CDS1 and other P450 were found with two P450s from Arabidopsis thaliana namely CYP96A1 Gen- bank accession no AC002391 and CYP96A2 Genbank accession no AL021811: 38 and 39 identity, respectively. There are also high homolo- gies with the Arabidopsis thaliana CYP86A1 Gen- bank accession no X90458, 31 identity, 49 homology, the Vicia sati6a CYP94A1 Genbank accession no AF030260, 32 identity, 50 homol- ogy and the Candida maltosa CYP52A2 Gen- bank accession no M63258, 27 identity, 44 homology Fig. 2. According to D.R. Nelson personal communication, CDS1 must be desig- nated as CYP96C1, the first member of a new subfamily of P450 enzymes. In a second step, we searched for 3-D structure homologies between P450s whose 3-D structures have been published and CYP96C1. We found the highest homologies between CYP96C1 and the bacterial CYP102, in spite of very poor homologies in primary struc- ture. Alignment of CYP96C1 with the 3-D struc- ture of CYP102 showed that the conservation was highest in both the a helices of the heme-binding domain and the a helices preceding this domain [23], data not shown. 3 . 3 . Molecular organization of the CYP 96 C 1 gene The molecular organization of the CYP 96 C 1 gene was determined by Southern analysis of ge- nomic DNA digested with two restriction endonu- cleases. Digestion with PstI, which does not cut the probe region, resulted in only one hybridiza- tion signal, whereas digestion with BclI, which cleaved within the probe region, resulted in two signals. This simple hybridization pattern is con- sistent with a single-copy of the CYP 96 C 1 gene Fig. 3. 3 . 4 . Accumulation of CYP 96 C 1 mRNA in periwinkle cells C20 cells were grown in MM, IM, PM and also in a fourth medium containing both 2,4-D and Table 1 Effects of 2,4-D and zeatin Z on alkaloid accumulation in C. roseus cells a Culture conditions PM IM MM+Z MM 2256 9 155 Ajmalicine accu- nd 743 9 70 nd mulation mgg DM a The C20D cells were cultured in 2,4-D-containing medium supplemented at day 3 with zeatin MM+Z, and in 2,4-D- free medium supplemented with zeatin PM or not IM. 2,4-D and zeatin are used at a concentration of 4.5 mM and 5 m M, respectively. Cells were harvested at day 7. Ajmalicine contents are given in mg per gram g of dry mass DM 9 SD 4 replicates, nd, not detectable. Fig. 2. Multiple sequence alignment of cytochrome P450s most similar to CYP96C1 from Blast searches. Dashes indicate gaps introduced to maximize alignment. Stars indicate identical or conserved residues in all sequences. ‘:’ indicates conserved substitution and ‘.’ semi-conserved substitution. Genbank accession nos are: AL021811 CYP96A2, Arabidopsis thaliana, X90458 CYP86A1, Arabidopsis thaliana, AF030260 CYP94A1, Vicia sati6a and M63258 CYP52A2, Candida maltosa. Fig. 3. Southern blot analysis of C. roseus genomic DNA. DNA 15 mg from C20 cells was totally digested with either BclI, PstI and the combination of BclI and PstI. The blot was hybridized with the CDS 1 probe, a 1113 bp-fragment of CDS 1 cDNA. Size markers bp, HindIII-EcoRI digested lambda-DNA are indicated on the left. zeatin MM + Z. Ajmalicine did not accumulate in the cells grown in MM and MM + Z, but was produced in cells grown in IM and higher amounts PM Table 1. Similarly, CYP 96 C 1 was not expressed in cells grown in MM or MM + Z. CYP 96 C 1 transcripts accumulated in cells grown in IM and slightly more in PM Fig. 4. The accumulation of CYP 96 C 1 messages was the highest at day 4 in the alkaloid-accumulating cells Fig. 4 and occurred before the alkaloid accumu- lation that previous studies have shown to begin at day 5 [24]. These data suggested that CYP 96 C 1 expression was associated directly or indirectly with the induction of TIA synthesis in C. roseus cells.

4. Discussion