title: Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.
abstract: Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Haibin
authors.initials: H
authors.lastname: Lu
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Balakumaran
authors.initials: B
authors.lastname: Chandrasekar
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Julian
authors.initials: J
authors.lastname: Oeljeklaus
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Johana C
authors.initials: JC
authors.lastname: Misas-Villamil
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Zheming
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shindo
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
authors.forename: Markus
authors.initials: M
authors.lastname: Kaiser
authors.affiliation: Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.) renier.vanderhoorn@plants.ox.ac.uk.
authors.forename: Renier A L
authors.initials: RA
authors.lastname: van der Hoorn
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D057056:Cysteine Proteases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D018525:Germination / Q000235:genetics; D008956:Models, Chemical; D015394:Molecular Structure; D009154:Mutation; D010802:Phylogeny; D010940:Plant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D012639:Seeds / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010940:Plant Proteins; D057056:Cysteine Proteases; D003546:Cysteine Endopeptidases; C089278:vacuolar processing enzyme