Showing papers by "Philippe Hugueney published in 1992"

Identification of a cDNA for the plastid‐located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening

Abstract: Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.

Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and zeta-carotene in Capsicum chromoplasts.

Abstract: In plants, zeta-carotene is the first visible carotenoid formed in the biosynthetic pathway through the following two-step desaturation reaction: phytoene-->phytofluene--> zeta-carotene. Using Capsicum annuum chromoplast membranes and the reconstitution system previously described [Camara, B., Bardat, F. & Moneger, R. (1982) Eur. J. Biochem. 127, 255-258], we have attempted to purify the desaturase(s) catalyzing these reactions. The two activities were coincidental during all the purification procedures. Only a single polypeptide with 56 +/- 2 kDa was detected by SDS/PAGE of all active fractions. The enzyme contained protein-bound FAD. Antibodies raised against the purified polypeptide selectively precipitated the phytoene and the phytofluene desaturase activities, thus demonstrating that the enzyme is a bifunctional flavoprotein. The antibodies were used to isolate a full-length cDNA clone from which was deduced the primary structure of the desaturase which contains a characteristic dinucleotide-binding site. Overexpression of the cDNA in Escherichia coli allowed the production of a recombinant desaturase which had all the properties of the chromoplast desaturase. The phytoene/phytofluene desaturase mRNA levels were extremely low in green fruits and increased slightly before detectable carotenoid synthesis and remained constant throughout ripening. However, the desaturase activity and protein levels were found to increase significantly during the chloroplast to chromoplast transition in C. annuum fruits.

Carotenoid Biosynthesis and Regulation in Plants

Abstract: Great impetus to the investigation of carotenoid biosynthesis was given by the introduction of Porter and Lincoln (1950) of the hypothesis that coloured carotenoids were formed by the sequential dehydrogenation of the unsaturated polyenes detected in several tomato fruit lines. Since then the implications of these studies have led to a massive exploration which now allows to describe the different steps of the biosynthetic pathway. Several reviews (Porter and Spurgeon, 1979; Camara and Moneger, 1982; Porter and Spurgeon, 1983; Jones and Porter, 1986; Bramley and Mackenzie, 1988; Britton, 1988) present different pictures of our advances in this field, but they also inevitably point to persistent gaps in our understanding. A search of the literature leaves the impression that as far as the enzymology and the underlying basis of its control are concerned, little progress has been made there. The lag can be accounted for by the exceptionally great difficulties that such studies must face. In this chapter, we wish to consider some of the recent development in this area, focusing mainly on higher plants.