Kaempferol rhamnoside catabolism in rosette leaves of senescing Arabidopsis and postharvest stored radish.

Flavonol rhamnosides including kaempferitrin (i.e., kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside) occur throughout the plant kingdom. Mechanisms governing flavonol rhamnoside biosynthesis are established, whereas degradative processes occurring in plants are relatively unknown. Here, we investigated the catabolic events affecting kaempferitrin status in the rosette leaves of Arabidopsis thaliana L. Heynh. (Arabidopsis) and Raphanus sativus L. (radish), respectively, in response to developmental senescence and postharvest handling. On a per plant basis, losses of several kaempferol rhamnosides including kaempferitrin were apparent in senescing leaves of Arabidopsis during development and postharvest radish stored at 5 °C. Conversely, small pools of kaempferol 7-O-α-rhamnoside (K7R), kaempferol 3-O-α-rhamnoside (K3R), and kaempferol built up in senescing leaves of both species. Evidence is provided for ⍺-rhamnosidase activities targeting the 7-O-α-rhamnoside of kaempferitrin and K7R in rosette leaves of both species. An HPLC analysis of in vitro assays of clarified leaf extracts prepared from developing Arabidopsis and postharvest radish determined that these metabolic shifts were coincident with respective 237% and 645% increases in kaempferitrin 7-O-⍺-rhamnosidase activity. Lower activity rates were apparent when these ⍺-rhamnosidase assays were performed with K7R. A radish ⍺-rhamnosidase containing peak eluting from a DEAE-Sepharose Fast Flow column hydrolyzed various 7-O-rhamnosylated flavonols, as well as kaempferol 3-O-ß-glucoside. Together it is apparent that the catabolism of 7-O-α-rhamnosylated kaempferol metabolites in senescing plant leaves is associated with a flavonol 7-O-α-rhamnoside-utilizing α-rhamnosidase.

An Apoplastic ß-Glucosidase is Essential for the Degradation of Flavonol 3-O-ß-Glucoside-7-O-α-Rhamnosides in Arabidopsis.

Flavonol bisglycosides accumulate in plant vegetative tissues in response to abiotic stress, including simultaneous environmental perturbations (i.e. nitrogen deficiency and low temperature, NDLT), but disappear with recovery from NDLT. Previously, we determined that a recombinant Arabidopsis ß-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-glucosides, forming flavonol 7-O-α-rhamnosides and flavonol aglycones, respectively. In this study, the transient expression of a BGLU15-Cherry fusion protein in onion epidermal cells demonstrated that BGLU15 was localized to the apoplast. Analysis of BGLU15 T-DNA insertional inactivation lines (bglu15-1 and bglu15-2) revealed negligible levels of BGLU15 transcripts, whereas its paralogs BGLU12 and BGLU16 were expressed in wild-type and bglu15 plants. The recombinant BGLU16 did not hydrolyze quercetin 3-O-ß-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-ß-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-ß-glucoside-7-O-α-rhamnoside hydrolase activity, whereas this activity increased by 223% within 2 d of NDLT recovery in wild-type plants. The levels of flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and quercetin 3-O-ß-glucoside were high and relatively unchanged in shoots of bglu15 mutants during recovery from NDLT, whereas rapid losses were apparent in wild-type shoots. Moreover, losses of two flavonol 3-O-ß-neohesperidoside-7-O-α-rhamnosides and kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside were evident during recovery from NDLT, regardless of whether BGLU15 was present. A spike in a kaempferol 7-O-α-rhamnoside occurred with stress recovery, regardless of germplasm, suggesting a contribution from hydrolysis of kaempferol 3-O-ß-neohesperidoside-7-O-α-rhamnosides and/or kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside by hitherto unknown mechanisms. Thus, BGLU15 is essential for catabolism of flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-glucosides in Arabidopsis.