Chromoplasts, isolation

Bouvier, F. et ah, Xanthophyll biosynthesis in chromoplasts isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocar-otenoid. Plant J. 6, 45, 1994. 

Mettal, U., Boland, W., Beyer, P. and Kleinig, H. (1988) Biosynthesis of monoterpene hydrocarbons by isolated chromoplasts from daffodil flowers. Eur.. Biochem., 170, 613-6. 

A cell-free system from photosynthetically active species has to be developed that is able to produce substantial amounts of intermediates and desaturated carotenes. Noteworthy a recent report using isolated chromoplasts from daffodil flowers. Narcissus pseudonarcissus > and isopentenyl pyrophosphate as substrate noted increased levels of phytoene and geranylgeraniol vs. control in the presence of high concentration (50 j,M) of SAN 6706 (27). These cell-free assays should be investigated further with regard to possible different sensitivity against inhibitors because of the species used. 

Tomato (Lycopersicon esculenturn cv Platense) chloroplasts (9) and chromoplasts (3) were isolated by previously published procedures. Pt DNA and total RNA were prepared as described in Ref. 3 and 9, respectively. Conditions for agarose electrophoresis of DNA restriction fragments, electrophoresis of RNA in formaldehyde/formamide/agarose gels Southern and Northern hybridizations were esentially those of Ref. 10. 

In some instances, the ability to produce prephytoene pyrophosphate (10) is closely linked to the production of phytoene (8) and appears to involve one enzyme with two activities (Britton, 1993 Dogbo et al., 1988). With an enzyme isolated from Capsicum chromoplast preparations, biosynthesis precedes via condensation of two molecules of geranylgeranyl pyrophosphate (9) directly to phytoene (8) (Dogbo et al., 1988). The enzyme phytoene synthase has been purified to homogeneity phytoene synthase from this source is a monomer wirti a molecular weight of 47,500 (Dogbo et al., 1988). 

In addition Vick and Beevers (1978) isolated proplastids from endosperm of germinating castor beans and demonstrated that these organelles were sites of fatty acid synthesis. Kleinig and Liedvogel (1978) have obtained chromoplasts from daffodO petals and have demonstrated extensive incorporation of [ C]acetate into palmitic acid. Nothelfer ei al. (1977) have also shown in soybean tissue culture suspension cells that proplastids are capable of synthesizing fatty acids. 

The chloroplasts and chromoplasts were isolated after immersion of fruit pericarp in sucrose solution containing p-mecraptoethanol, EDTA, Tris and ascorbic acid. 

Beyer, P., Kreuz, K., Kleinig, H. (1980) 6-carotene synthesis in isolated chromoplasts from Narcissus pseudonarcissus. Plants 130, 435-438. 

---