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

Abstract

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 β-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-β-glucoside-7-O-α-rhamnosides and flavonol 3-O-β-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-β-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-β-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-β-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-β-glucoside-7-O-α-rhamnosides and quercetin 3-O-β-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-β-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-β-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-β-glucoside-7-O-α-rhamnosides and flavonol 3-O-β-glucosides in Arabidopsis.

Genome-Wide Analysis of Glycoside Hydrolase Family 1 β-glucosidase Genes in Brassica rapa and Their Potential Role in Pollen Development

“…Phylogenetic analysis generated an interesting finding, that the clustering or groupings of genes were related to the chromosomal locus or function. Based on the functions of the AtBGLUs , flavonol accumulation ( AtBGLU1 - 6 ) and anthocyanin glucosyltransferase ( AtBGLU7 - 11 )-related genes were highlighted by subgroup GH1-a [10,11,12]; flavonoid utilization-related AtBGLUs ( AtBGLU12 - 17 ) were represented by the GH1-e subgroup [10,13]; myrosinase-encoding AtBGLUs ( AtBGLU34 - 39 ) belonged to the GH1-d subgroup [14,15,16], and scopolin hydrolysis-related AtBGLUs ( AtBGLU21 -23) were grouped into GH1-i [21,22]. Most of the genes within the same clusters on a chromosome were grouped into the same subfamily, which is similar to the findings using Arabidopsis , i.e., BrBGLU5 / 6 , BrBGLU11 / 12 , BrBGLU31 / 32 / 33 , BrBGLU40 / 41 , BrBGLU58 / 59 , and BrBGLU61 / 62 .…”

“…On the basis of Arabidopsis study, most subfamilies of BGLUs in Figure 2 may be associated with specific functions: GH1-a for flavonoid and anthocyanin metabolism, GH1-e for flavonoid utilization, GH1-d for myrosinases, and GH1-i for scopolin hydrolysis. At least 12 genes are known to be involved in flavonoid metabolism in GH1-a: AtBGLU1-6 for flavonol accumulation [10,11], AtBGLU7-11 as anthocyanin glucosyltransferases [10,11,12], and AtBGLU15 for flavonol bisglycoside catabolism under abiotic stress [13]. AtBGLU12 - 17 in the GH1-e subgroup code for flavonoid-utilizing BGLUs in legumes [10].…”

“…In addition to classifications based on genomic DNA organization, Arabidopsis BGLUs ( AtBGLU s) could be classified in relation to their known functions, which shows that genes within the same subfamily may function in similar processes. A large number of AtBGLU s are involved in flavonoid metabolism: AtBGLU1-6 for flavonol accumulation [10,11], AtBGLU7-11 for anthocyanin glucosyltransferase [11,12], and AtBGLU12-17 for flavonoid utilization [10,13]. Seven genes ( AtBGLU26 , AtBGLU34-39 ) function as myrosinases for chemical defense against herbivores and pathogen attacks [14,15,16].…”

Sclerotinia sclerotiorum Circumvents Flavonoid Defenses by Catabolizing Flavonol Glycosides and Aglycones

“…Plants also contain numerous glycoside hydrolases, which cleave flavonol glycosides during plant recovery from synergistic abiotic stresses, such as nitrogen deficiency and low temperature (Xu et al, 2004;Roepke and Bozzo, 2015;Roepke et al, 2017). The b-glucosidase BGLU15 characterized from Arabidopsis cleaves the 3-O-b-glucosides from K3G7R to produce kaempferol 7-O-rhamnoside (Roepke et al, 2017). Kaempferol 7-Orhamnoside was, however, not found among the fungal degradation products of flavonol glycoside extracts in our study, suggesting a different hydrolysis pathway of glycosidic linkages in fungi.…”

Comprehensive identification and characterization of abiotic stress and hormone responsive glycosyl hydrolase family 1 genes in Medicago truncatula

“…The remaining six genes (except MtBGLU19) in cluster I have phylogenetically closest relationship with BGLUs of other plants. Their close related gene AtBGLU15 of Arabidopsis is essential for the degradation of flavonol glucosides during the process of recovering from nitrogen deficiency and low temperature[41]. In rice, their closest homologous gene Os4BGlu12, is a wound-induced and functions in hydrolyzing cell-wall derived disaccharides and glycosides[42].…”