Chromoplast development in ripening bell pepper fruits is characterized by a massive synthesis of carotenoid pigments, resulting in their distinctive red color. We have shown that 95% of these pigments accumulate in chromoplasts in specific lipoprotein fibrils. In addition to carotenoids, purified fibrils contain galactolipids, phospholipids, and a single, 32-kD protein, designated fibrillin, which has antigenically related counterparts in other species. Fibrils were reconstituted in vitro when purified fibrillin was combined with carotenoids and polar lipids in the same stoichiometric ratio found in fibrils in vivo. Antibodies directed against fibrillin were used to isolate a fibrillin cDNA clone and, in immunological studies, to follow its accumulation during the chloroplast-to-chromoplast transition under different conditions. A model for fibril architecture is proposed wherein carotenoids accumulate in the center of the fibrils and are surrounded by a layer of polar lipids, which in turn are surrounded by an outer layer of fibrillin. Topological analysis of purified fibrils verified this structure. Collectively, these results suggest that the process of fibril self-assembly in chromoplasts is an example of a general phenomenon shared among cells that target excess membrane lipids into deposit structures to avoid their destabilizing or toxic effects. In addition, we have shown that abscisic acid stimulates this phenomenon in chromoplasts, whereas gibberellic acid and auxin delay it.

The addition of phytyl side chain to chlorophylls, tocopherols and phylloquinone is prerequisite to their integration into plastid membranes. We have cloned a cDNA encoding a pre-geranylgeranyl reductase from Arabidopsis thaliana. The deduced primary structure predicts a mature size with a molecular mass of 47 kDa and displays a characteristic dinucleotide binding domain. Geranylgeranyl reductase expressed in Escherichia coli sequentially catalyzes the reduction of geranylgeranyl-chlorophyll a into phytyl-chlorophyll a as well as the reduction of free geranylgeranyl diphosphate into phytyl diphosphate. Due to its multifunctionality and weak hydrophobicity, we suggest that in plastid the same geranylgeranyl reductase is recruited into the chlorophyll, the tocopherol and the phylloquinone pathways. The geranylgeranyl reductase gene is up-regulated during etioplast to chloroplast and chloroplast to chromoplast development.

SummaryThe nature of isoprenoids synthesized in plants is primarily determined by the speci®city of prenyltransferases. Several of these enzymes have been characterized at the molecular level. The compartmentation and molecular regulation of geranyl diphosphate (GPP), the carbon skeleton that is the backbone of myriad monoterpene constituents involved in plant defence, allelopathic interactions and pollination, is poorly understood. We describe here the cloning and functional expression of a GPP synthase (GPPS) from Arabidopsis thaliana. Immunohistological analyses of diverse non-secretory and secretory plant tissues reveal that GPPS and its congeners, monoterpene synthase, deoxy-xylulose phosphate synthase and geranylgeranyl diphosphate synthase, are equally compartmentalized and distributed in non-green plastids as well in chloroplasts of photosynthetic cells. This argues that monoterpene synthesis is not solely restricted to specialized secretory structures but can also occur in photosynthetic parenchyma. These data provide new information as to how monoterpene biosynthesis is compartmentalized and induced de novo in response to biotic and abiotic stress in diverse plants.

SummaryThe late steps of carotenoid biosynthesis in plants involve the formation of xanthophyUs. Little is known about the enzymology of these steps. This paper reports the purification to homogeneity of a xanthophyll biosynthetic enzyme from Capsicum annuum chromoplasts, which catalyzes the conversion of the ubiquitous 5,6.epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Owing to its bifunctionallty, the name capsanthin-capsorubin synthase is proposed for this new enzyme. The purified enzyme is a monomer with a molecular mass of 50 kDa. Antibodies raised against this enzyme allowed the isolation of a fulllength cDNA clone encoding a capsanthin capserubin synthase high molecular weight precursor. The primary deduced structure reveals the presence of a consensus nucleotide binding site. The capeanthincapsorubin synthase gene is specifically expressed during chromoplast development in fruits accumulating ketocarotenoids, but not in mutants impaired in this biosynthetic step.

Chromoplast development in ripening bell pepper fruits is characterized by a massive synthesis of carotenoid pigments, resulting in their distinctlve red color. We have shown that 95% of these pigments accumulate in chromoplasts in speclfic lipoprotein fibrils. In addltion to carotenoids, purified fibrils contain galactolipids, phospholipids, and a single, 32-kD protein, designated fibrillin, which has antigenically related counterparts in other species. Fibrlls were reconstltuted in vitro when purified fibrillin was combined with carotenoids and polar llpids in the same stoichlometrlc ratlo found in fibrils in vivo. Antibodies dlrected against fibrillin were used to isolate a fibrillin cDNA clone and, in immunological studies, to follow its accumulation during the chloroplast-tothromoplast transltlon under different condltions. A model for fibril amhitecture is proposed wherein carotenoids accumulate in the center of the fibrils and are surrounded by a layer of polar lipids, which in turn are surrounded by an outer layer of fibrillin. Topological analysis of purlfied fibrils verified this structure. Collectively, these results suggest that the process of fibrll self-assembly in chromoplasts 1s an example of a general phenomenon shared among cells that target excess membrane llpids into deposit structures to avoid their destabilizing or toxic effects. In addition, we have shown that abscisic acid stimulates this phenomenon in chromoplasts, whereas gibberellic acid and auxin delay it.