Arabidopsis 2010: Collaborative Project on the Functional Genomics of Arabidopsis beta-Glucosidase and beta-Galactosidase Gene Families

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

Widely distributed in animals, plants and microbes, O-glycoside hydrolases (EC 3.2.1.-) are enzymes that catalyze the cleavage of chemical bonds between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Responding to the challenge of the 2010 Project to identify the function of all Arabidopsis thaliana genes within the next decade, this collaborative research will focus on approximately 75 members of two related families of glycoside hydrolases (http://www.biol.vt.edu/faculty/esen/glycosidase.lab.html and http://afmb.cnrs-mrs.fr/~pedro/CAZY/). Family 1 includes beta -glucosidases (EC 3.2.1.21) and myrosinases (EC 3.2.3.1), which function in higher plants in chemical defense against herbivores and pathogens, lignin biosynthesis, and plant growth and development. Family 35 contains the beta -galactosidases (EC 3.2.1.23), which play key roles in fruit ripening, flower senescence, mobilization of carbohydrate reserves, and galactolipid turnover. To date, the precise biochemical roles of only three of these Arabidopsis hydrolases are known with certainty. The purpose of this multidisciplinary collaborative research is to assign biological functions to beta-glucosidases and beta-galactosidases encoded by the Arabidopsis genome. After using phylogenetic analysis to identify subfamilies that contain closely related enzymes, cDNAs encoding each target hydrolase will be obtained. Each hydrolase will then be overexpressed in foreign host (e.g. yeast and bacteria) cells and purified to ascertain its biological function by measuring its enzymatic activity toward a wide range of natural glycosidic substrates isolated primarily from Arabidopsis and related crucifers for this purpose. In parallel studies, three-dimensional structures of selected subfamily representatives will be determined by homology modeling and x-ray diffraction, providing novel insights into how these hydrolases recognize and bind their substrates. This information will be of paramount importance in future research to alter the substrate specificity of Family 1 and Family 35 hydrolases for biotechnological purposes, including biomass conversion and improvements in anti-herbivore defenses and fruit ripening.