RESUMO
Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6â³-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6â³RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6â³RhaT enzyme expressed in Escherichia coli exhibited 6â³-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6â³RhaT converted the administered quercetin into rutin, suggesting that FeF3G6â³RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6â³RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6â³RhaT is involved in rutin biosynthesis in common buckwheat.
Assuntos
Fagopyrum/metabolismo , Hexosiltransferases/metabolismo , Rutina/biossíntese , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Fagopyrum/enzimologia , Hexosiltransferases/genética , Hexosiltransferases/isolamento & purificação , Fenóis/metabolismo , Reação em Cadeia da Polimerase , Plântula/enzimologia , Análise de Sequência de RNA , Especificidade por SubstratoRESUMO
Peptides released from eggs of marine invertebrates play a central role in fertilization. About 80 different peptides from various phyla have been isolated, however, with one exception, their respective receptors on the sperm surface have not been unequivocally identified and the pertinent signaling pathways remain ill defined. Using rapid mixing techniques and novel membrane-permeable caged compounds of cyclic nucleotides, we show that the sperm-activating peptide asterosap evokes a fast and transient increase of the cGMP concentration in sperm of the starfish Asterias amurensis, followed by a transient cGMP-stimulated increase in the Ca(2+) concentration. In contrast, cAMP levels did not change significantly and the Ca(2+) response evoked by photolysis of caged cAMP was significantly smaller than that using caged cGMP. By cloning of cDNA and chemical crosslinking, we identified a receptor-type guanylyl cyclase in the sperm flagellum as the asterosap-binding protein. Sperm respond exquisitely sensitive to picomolar concentrations of asterosap, suggesting that the peptide serves a chemosensory function like resact, a peptide involved in chemotaxis of sperm of the sea urchin Arbacia punctulata. A unifying principle emerges that chemosensory transduction in sperm of marine invertebrates uses cGMP as the primary messenger, although there may be variations in the detail.
