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Using a polyclonal antibody (P23) generated against the human platelet integrin αIIbβ3 and a FITC-conjugate secondary antibody, fluorescence is observed at the surface of protoplasts isolated from Arabidopsis thaliana and Rubus fruticosus. Arabidopsis thaliana cells grown in suspension culture containing P23 and glycylarginylglycylaspartylserine (GRGDS), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins demonstrated aberrant cell wall/plasma membrane interactions and organization. When glycoproteins from these plants, purified on a concanavalin A Sepharose 4B, were subjected to SDS/PAGE and Western blotting, under reduced and non-reduced conditions, immunoblots probed with P23 revealed bands in both species. A shift in electrophoretic mobility is observed to different apparent molecular mass when no reducing agent is present. When purified by immunoaffinity chromatography on anti-αIIbβ3 Sepharose or Sepharose linked to the synthetic peptide D-Arg-Gly-Asp-Trp, the major antigenic components detected migrate at 30 kDa and 60 kDa in the first experiment and 60 kDa in the second one. Only the 60-kDa component is immunodetected with antibodies specific for either the β3 platelet chain or the αIIb polypeptide, suggesting the presence of two polypeptides co-migrating. To address more precisely the structure of this complex in plants, competition assays were performed. A significant inhibition is observed with CS3 a monoclonal antibody that interacts with the complexed form αIIbβ3 but not the dissociated subunits. Further structural similarities with the animal αIIbβ3 complex is demonstrated with Western blotting detection after plant glycoproteins immunoprecipitation with CS3 in absence or presence of 5 mM EDTA to dissociate the complex. We also present data on the characterization of a polyclonal antibody, named AcAt2, raised against Arabidopsis glycocoproteins... [ABSTRACT FROM AUTHOR]

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.