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Biochemical Characterization of β-Glucosidase-mediated Catabolism of Flavonol Bisglycosides in Arabidopsis thaliana

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Title: Biochemical Characterization of β-Glucosidase-mediated Catabolism of Flavonol Bisglycosides in Arabidopsis thaliana
Author: Roepke, Jonathon
Department: Department of Plant Agriculture
Program: Plant Agriculture
Advisor: Bozzo, Gale G.
Abstract: Kaempferol- and quercetin 3-O-β-glucoside-7-O-α-rhamnoside (K3G7R and Q3G7R, respectively) are major flavonol bisglycosides that accumulate in vegetative organs of Arabidopsis thaliana in response to dual abiotic stresses (i.e., nitrogen deficiency and low temperature, NDLT). However, these metabolites are lost during a 5 day recovery from NDLT. Typically, catabolism of related chemicals relies on β-glucosidase (BGLU) action. Major losses of K3G7R, Q3G7R, flavonol 3-O-β-rutinoside-7-O-α-rhamnosides and flavonol bisrhamnosides coincided with an approximate 250% spike in extractable Q3G7R BGLU activity within 2 days of NDLT recovery. QTOF-MS/MS analysis identified quercetin 7-O-α-rhamnoside as the Q3G7R hydrolysate. A phylogenetic analysis of all 47 Arabidopsis glycoside hydrolase family 1 members identified a clade consisting of BGLU12 -17, inclusive, that were most similar to BGLUs from other plant species known to utilize isoflavonoid β-O-glucosides, chemicals that are structurally similar to flavonol bisglycosides. A 300% higher level of BGLU15 transcripts was apparent within 1 day of recovery from NDLT; lower or negligible changes in expression were evident for the remaining BGLUs. Recombinant thioredoxin-His6-tagged mature BGLU15 was expressed in Escherichia coli and purified to apparent homogeneity. A comparison of a wide spectrum of O-linked β-glucosides revealed that recombinant BGLU15 preferentially hydrolysed the 3-O-β-glucoside of Q3G7R and K3G7R; flavonol 3-O-β-glucosides were also used, albeit with lower efficiency. Real time quantitative PCR revealed that T-DNA inactivation lines bglu15-1 and bglu15-2 contained negligible BGLU15 transcripts and cell-free leaf extracts of the bglu15 mutants had severely reduced Q3G7R hydrolysis relative to wild-type (WT) plants. UHPLC-DAD-MSn revealed that levels of K3G7R, Q3G7R and quercetin 3-O-β-glucoside remained elevated during the recovery period in leaves of bglu15 relative to WT plants. Moreover, losses of flavonol 3-O-β-rutinoside-7-O-α-rhamnosides and kaempferol bisrhamnoside were evident during the recovery phase, regardless of whether BGLU15 was present. With respect to catabolite production, a transient increase in kaempferol 7-O-α-rhamnoside was apparent with recovery, but this change was slightly reduced in bglu15 mutants. Together, all biochemical data indicate that BGLU15 is essential for flavonol- 3-O-β-glucoside and 3-O-β-glucoside-7-O-α-rhamnoside catabolism in Arabidopsis. This information is important for genetic engineering or traditional breeding strategies aimed at limiting flavonol losses in commercially important horticultural and agricultural commodities.
Date: 2015-09
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