title: Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles.
abstract: The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX-GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants.
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic.
authors.forename: David
authors.initials: D
authors.lastname: Zalabák
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic.
authors.forename: Patricie
authors.initials: P
authors.lastname: Johnová
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Plíhal
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic.
authors.forename: Karolina
authors.initials: K
authors.lastname: Šenková
authors.affiliation: Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic.
authors.forename: Olga
authors.initials: O
authors.lastname: Šamajová
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Jiskrová
authors.affiliation: Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Novák
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
authors.forename: David
authors.initials: D
authors.lastname: Jackson
authors.affiliation: DuPont Pioneer Ag Biotech, DuPont Knowledge Centre, Turkapally Village, Hyderabad 500078, Telangana, India.
authors.forename: Amitabh
authors.initials: A
authors.lastname: Mohanty
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, Olomouc 783 71, Czech Republic. Electronic address: petr.galuszka@upol.cz.
authors.forename: Petr
authors.initials: P
authors.lastname: Galuszka
mesh_terms: D017360:Arabidopsis / Q000166:cytology; D003198:Computer Simulation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D059828:Plant Cells / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals; D021381:Protein Transport; D011523:Protoplasts / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013535:Suspensions; D014617:Vacuoles / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013535:Suspensions; D049452:Green Fluorescent Proteins; D010088:Oxidoreductases; C019984:cytokinin oxidase
keywords: Cytokinin; Cytokinin dehydrogenase; Subcellular localization; Vacuole; Zea mays L.