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β-Glucosidases are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. Plant β-glucosidases are involved in many physiological processes like cell wall formation, phytohormone release, and activation of defense compounds. Activation of secondary metabolites by de-glycosylation is a widespread anti-herbivore defense strategy that allows plants to store harmless glycosides and hydrolyze them to toxic products upon attack. The main insect resistance factors in maize plants are 1,4-benzoxazin-3-one derivates (BXDs), such as DIMBOA-Glc and DIBOA-Glc, which are stored as glucosides and activated by plant β-glucosidases. So far, two β-glucosidases (ZmGlu1 & ZmGlu2) are known to hydrolyze DIMBOA-Glc and DIBOA-Glu. Of the 26 GH1 genes found in the genome of the maize inbred line B73, ZmGlu1 and ZmGlu2 form a distinct gene subfamily with 6 other ZmGlu genes (ZmGlu3-8). Since ZmGlu3-8 share 70-80% sequence similarity with ZmGlu1 & 2, it is likely that they have similar catalytic properties and might also accept BXDs as substrates. In one part of this project I am going to characterize the expression levels of ZmGlu1-8 in different maize organs and after different biotic stresses using quantitative real time PCR (qRT-PCR). To gain first insights into the specific biochemical activity of the uncharacterized ZmGlu enzymes, the B73 alleles of ZmGlu3-8 will be amplified from pooled cDNAs and cloned into the vector pASK-IBA37plus. The substrat specifities of the recombinant enzymes will be determined in assays containing different BXDs.
Another subproject is the characterization of β-glucosidase-aggregating factors. Some maize lines (“null” lines) possess only marginal soluble β-glucosidase activity after tissue disruption. In these lines, β-glucosidases are bound to a protein called β-glucosidase-aggregating factor (BGAF) and form insoluble protein complexes. BGAF-β-glucosidase aggregation has been suggested to protect β-glucosidases from proteolytic degradation in the insect gut, however, the exact function of BGAFs, even in the context of β-glucosidase stabilization, is yet unclear. The interactions of purified recombinant BGAF proteins with recombinant ZmGlu proteins will be tested with pull-down and gel shift assays. Furthermore, we will investigate the cellular localization of ZmGlu proteins and BGAFs in crown roots by using specific antibodies.
last updated on 2017-09-08