Functional Characterization of CsBGlu12, a β-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols.

Glycosylation and deglycosylation are impressive mechanisms that allow plants to regulate the biological activity of an array of secondary metabolites. Although glycosylation improves solubility and renders the metabolites suitable for transport and sequestration, deglycosylation activates them to carry out biological functions. Herein, we report the functional characterization of CsBGlu12, a β-glucosidase from Crocus sativus. CsBGlu12 has a characteristic glucoside hydrolase 1 family (α/β)8 triose-phosphate isomerase (TIM) barrel structure with a highly conserved active site. In vitro enzyme activity revealed that CsBGlu12 catalyzes the hydrolysis of flavonol β-glucosides and cello-oligosaccharides. Site-directed mutagenesis of any of the two conserved catalytic glutamic acid residues (Glu200 and Glu414) of the active site completely abolishes the β-glucosidase activity. Transcript analysis revealed that Csbglu12 is highly induced in response to UV-B, dehydration, NaCl, methyl jasmonate, and abscisic acid treatments indicating its possible role in plant stress response. Transient overexpression of CsBGlu12 leads to the accumulation of antioxidant flavonols in Nicotiana benthamiana and confers tolerance to abiotic stresses. Antioxidant assays indicated that accumulation of flavonols alleviated the accretion of reactive oxygen species during abiotic stress conditions. β-Glucosidases are known to play a role in abiotic stresses, particularly dehydration through abscisic acid; however, their role through accumulation of reactive oxygen species (ROS) scavenging flavonols has not been established. Furthermore, only one β-glucosidase 12 homolog has been characterized so far. Therefore, this work presents an important report on characterization of CsBGlu12 and its role in abiotic stress through ROS scavenging.