Biochemical Characterization of Flavonol Rhamnoside Catabolism in Arabidopsis thaliana and Raphanus sativus Leaves

Abstract

Flavonol rhamnosides such as kaempferitrin are plant secondary metabolites, whose biosynthetic mechanisms are well known. The concentrations of flavonol rhamnosides in plants decrease with abiotic stress recovery, leaf senescence, fruit maturation, and postharvest handling. Despite this, the catabolic processes promoting flavonol rhamnoside losses in plants are unknown. This thesis explores the catabolic events affecting kaempferitrin levels in the leaves of Arabidopsis thaliana L. Heynh. (Arabidopsis) and Raphanus sativus L. (radish), in response to developmental senescence and postharvest handling. Losses of several kaempferol rhamnosides including kaempferitrin were evident in the leaves of both plants. By contrast, the levels of kaempferitrin catabolites kaempferol 7-O-α-rhamnoside, kaempferol 3-O-α-rhamnoside, and kaempferol increased in these same tissues. Furthermore, evidence is provided for kaempferitrin and kaempferol-7-O-α-rhamnoside 7-O-α-rhamnosidase hydrolysis activities in leaves of both species. Kaempferitrin degradation in leaves sampled from developing Arabidopsis and postharvest radish overlapped with the marked induction in kaempferitrin 7-O-α-rhamnosidase activity, and smaller increases in the hydrolysis of kaempferol 7-O-α-rhamnoside. A flavonol 7-O-α-rhamnoside α-rhamnosidase was purified to homogeneity from postharvest stored radish leaves. Analysis of the final preparation by SDS-PAGE revealed two polypeptides of 36 kDa and 37 kDa. Moreover, size-exclusion chromatography and non-denaturing PAGE revealed the α-rhamnosidase exists as a tetramer. Mass spectrometry analysis of peptide sequences from the final enzyme preparation matched the in silico polypeptide translation of a radish fructose 1,6-bisphosphate aldolase 2 gene. The purified α-rhamnosidase had little capacity to cleave fructose 1,6-bisphosphate, a hallmark activity of fructose 1,6-bisphosphate aldolases. In vitro biochemical analyses revealed the radish enzyme is likely a glycosidase as it hydrolyzed kaempferitrin but also other rhamnosylated and glucosylated flavonols. Moreover, this hydrolysis activity was inhibited by known and hypothetical reactions products such as rhamnose, and kaempferol. On the whole, these findings indicate that the hydrolysis of 7-O-α-rhamnosylated kaempferol metabolites by a flavonol 7-O-α-rhamnoside-utilizing α-rhamnosidase is a metabolic phenomenon of the Brassicaceae plants Arabidopsis and radish.