Activity profiling of αlpha-glycosidases in plants

ABSTRACT: With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity profiling of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis, these probes label members of the GH31 family of Glycosyl Hydrolases, including ER-resident α-glucosidase-II RSW3/PSL5 and Golgi-resident α-mannosidase-II HGL1, which both trim N-glycans on glycoproteins. We also detect the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucan in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. α-glycosidase labelling causes specific signals at 100-130 kDa that are pH-dependent and can be supressed by α-glycosidase inhibitors miglitol and acarbose. The α-glycosidase probes display similar miglitol-sensitive signals in leaf extracts of in a broad range of plant species. To show its use on a non-model plant, we applied glycosidase activity profiling on Crocus sativa, a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 70 active glycosidases in stigma stages 1 and 4 of saffron (Crocus sativa L.), ten of which are differentially active. We also uncover massive changes in hydrolase activities in corms upon infection with Fusarium oxysporum using multiplex fluorescent labelling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease by which active α-glycosidases and other hydrolases can be displayed in non-model plants.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Crocus Sativus Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Stigma, Leaf

SUBMITTER: Farnusch Kaschani  

LAB HEAD: Farnusch Kaschani

PROVIDER: PXD009014 | Pride | 2018-05-17

REPOSITORIES: Pride