RESUMO
The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Although the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that a member of the nitrate/peptide transporter family is required for the accumulation and transport of pollen-specific flavonol 3-o-sophorosides, characterized by a glycosidic ß-1,2-linkage, to the pollen surface of Arabidopsis (Arabidopsis thaliana). Ectopic, transient expression in Nicotiana benthamiana epidermal leaf cells demonstrated localization of this flavonol sophoroside transporter (FST1) at the plasmalemma when fused to green fluorescent protein (GFP). We also confirmed the tapetum-specific expression of FST1 by GFP reporter lines driven by the FST1 promoter. In vitro characterization of FST1 activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1 insertion mutant by complementation with an FST1 genomic fragment restored the accumulation of flavonol glycosides in pollen grains to wild-type levels, corroborating the requirement of FST1 for transport of flavonol-3-o-sophorosides from the tapetum to the pollen surface.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flavonóis/metabolismo , Glicosídeos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Pólen/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Mutação/genética , Filogenia , Epiderme Vegetal/citologia , Extratos Vegetais/química , Pólen/ultraestrutura , Regiões Promotoras Genéticas/genética , Propanóis/química , Propanóis/metabolismo , Frações Subcelulares/metabolismo , Sobrevivência de Tecidos , Transcrição Gênica , Raios UltravioletaRESUMO
A change regarding the extent of adhesion - hereafter referred to as adhesion plasticity - between adhesive and less-adhesive states of mammalian cells is important for their behavior. To investigate adhesion plasticity, we have selected a stable isogenic subpopulation of human MDA-MB-468 breast carcinoma cells growing in suspension. These suspension cells are unable to re-adhere to various matrices or to contract three-dimensional collagen lattices. By using transcriptome analysis, we identified the focal adhesion protein tensin3 (Tns3) as a determinant of adhesion plasticity. Tns3 is strongly reduced at mRNA and protein levels in suspension cells. Furthermore, by transiently challenging breast cancer cells to grow under non-adherent conditions markedly reduces Tns3 protein expression, which is regained upon re-adhesion. Stable knockdown of Tns3 in parental MDA-MB-468 cells results in defective adhesion, spreading and migration. Tns3-knockdown cells display impaired structure and dynamics of focal adhesion complexes as determined by immunostaining. Restoration of Tns3 protein expression in suspension cells partially rescues adhesion and focal contact composition. Our work identifies Tns3 as a crucial focal adhesion component regulated by, and functionally contributing to, the switch between adhesive and non-adhesive states in MDA-MB-468 cancer cells.
