Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis in Dictyostelium discoideum

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.

An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato

Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases β-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes β-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene β-cyclase, an enzyme that converts lycopene to β-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that β-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.