Plant Molecular Biology 34: 935–948, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Molecular cloning and characterization of
desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase
involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don
Felipe Vazquez-Flota, Emidio De Carolis
, Anne-Marie Alarco
and Vincenzo De Luca
Institut de Recherche en Biologie V
epartement de Sciences Biologiques, Universit
e de Montr
rue Sherbrooke est, Montr
ebec, Canada H1X 2B2 (
author for correspondence);
Present address: Pﬁzer
Canada Inc., Pharmaceutical Division, 17300 Trans-Canada Hwy., Kirkland, Qu
ebec, Canada H9J 2M5;
Present address: Institut de Recherches Cliniques de Montr
eal, 110 Ave. Des Pins Ouest, Montr
Canada H2L 4M1
Received 7 February 1997; accepted in revised form 7 May 1997
Key words: Catharanthus roseus, dioxygenases, indole alkaloids, molecular regulation, secondary metabolism
A 2-oxoglutarate-dependent dioxygenase (EC 18.104.22.168) which catalyzes the 4-hydroxylation of desacetoxyvin-
doline was puriﬁed to homogeneity. Three oligopeptides isolated from a tryptic digest of the puriﬁed protein
were microsequenced and one oligopeptide showed signiﬁcant homology to hyoscyamine 6
Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a
Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three
clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence
of all three peptides. Primer extension analysis suggestedthat cD4H-3, the longest cDNA clone, wasmissing 156 bp
at the 5
end of the clone and sequencing of the genomic clone, gD4H-8, conﬁrmed these results. Southern blot
analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the signiﬁcant
differences in the sequence of the 3
-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of
the same hydroxylase. The identity of the clone was further conﬁrmed when extracts of transformed Escherichia
coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated
molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing
family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to
the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and
1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those
for e8 protein, hyoscyamine-6
-hydroxylase and ethylene-forming enzyme. These results provide further support
that a common ancestral gene is responsible for the appearance of this family of dioxygenases.
Hydroxylase assays and RNA blot hybridization studies showed that enzyme activity followed closely the
levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits.
In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost undetectable
hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without
a signiﬁcant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest
that the activating effect of light may occur at a point downstream of transcription which remains to be elucidated.
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDB7 Nucleotide Sequence Databases under the accession
numbers U71605 (cD4H-1), U71604 (cD4H-3) and AF008597 (gD4H-8).
Generic names: desacetoxyvindoline, 16-methoxy-2,3-dihydro-3-hydroxy-
(1)-methyltaberosonine; desacetoxyvindorosine, 2,3-
(1)-methyltabersonine; deacetylvindoline, 16-methoxy-2,3-dihydro-3,4-dihidroxy-
16-methoxy-2,3-dihydro-3-hydroxy-4-acetoxy-tabersonine. The numbering system used is as for aspidospermine alkaloids in Chemical
Abstracts (Collective Substance Index vol. 106–115, 12CS3, p. 5731CS, 1987–1991).
GR: 201001988, Pips nr. 141193 BIO2KAP
pla435us.tex; 16/07/1997; 16:10; v.7; p.1