Poster: Secondary metabolism

Abs # 260: Identification of Arabidopsis thaliana β-glucosidases that hydrolyse monolignol glucosides.

Presenter:	Escamilla-Trevino, Luis L, l-escamilla@uiowa.edu
Authors	Escamilla-Trevino, Luis L (A)   Laust, Amanda K (A)   Netland, Jason M (A)   Carroll, Bradley R (A)   Shih, Ming-Che  (A)   Cheng, Chi-Lien  (A)   Poulton, Jonathan E (A)
Affiliations:	(A): Department of Biological Sciences, The University of Iowa
Web Site:	http://www.biology.uiowa.edu/arabidopsis/

In higher plants, Glycoside Hydrolase (GH) Family 1 β-glucosidases have been implicated in several fundamental processes including anti-herbivore defense, lignification, and hydrolysis of phytohormone glycoside conjugates. Phylogenetic analysis of Arabidopsis GH Family 1 has revealed that three members, namely At1g61810 (BGLU45), At1g61820 (BGLU46), and At4g21760 (BGLU47), cluster with the gene encoding Pinus contorta coniferin β-glucosidase, leading to our hypothesis that they may be involved in lignification. To test this hypothesis, BGLU45 and BGLU46 proteins have been expressed in the yeast Pichia pastoris and assayed for their ability to hydrolyse monolignol glucosides. After purification to homogeneity by ammonium sulfate fractionation and hydrophobic interaction chromatography, BGLU46 exhibited activity toward salicin (100%), p-coumaryl alcohol glucoside (77%), phenyl β-glucoside (65%), coniferin (5%), and syringin (3%). Among β-linked p-nitrophenyl (PNP)-glycosides, it cleaved PNP-glucoside (100 %), PNP-galactoside (21%), and PNP-xyloside (2%) but was inactive toward PNP-mannoside, PNP-fucoside, and PNP-arabinoside. In induced culture supernatants, recombinant BGLU45 showed activity toward coniferin and syringin but not toward PNP-glucoside. In addition to BGLU45 purification and characterization, efforts are underway to determine the expression patterns of BGLU45 and BGLU46 by relative RT-PCR, promoter-GUS fusions, and in situ hybridization. (Supported by NSF grant MCB-0114666).