title: Biochemical characterization of the maize cytokinin dehydrogenase family and cytokinin profiling in developing maize plantlets in relation to the expression of cytokinin dehydrogenase genes.
abstract: The cytokinin dehydrogenases (CKX; EC 1.5.99.12) are a protein family that maintains the endogenous levels of cytokinins in plants by catalyzing their oxidative degradation. The CKX family in maize (Zea mays L.) has thirteen members, only two of which--ZmCKX1 and ZmCKX10--have previously been characterized in detail. In this study, nine further maize CKX isoforms were heterologously expressed in Escherichia coli, purified by affinity and ion-exchange chromatography and biochemically characterized. ZmCKX6 and ZmCKX9 could only be expressed successfully after the removal of putative sequence-specific vacuolar sorting signals (LLPT and LPTS, respectively), suggesting that these proteins are localized to the vacuole. Substrate specificity analyses revealed that the CKX isoforms can be grouped into two subfamilies: members of the first strongly prefer cytokinin free bases while members of the second degrade a broad range of substrates. The most active isoform was found to be ZmCKX1. One of the studied isoforms, ZmCKX6, seemed to encode a nonfunctional enzyme due to a mutation in a conserved HFG protein domain at the C-terminus. Site-directed mutagenesis experiments revealed that this domain is essential for CKX activity. The roles of the maize CKX enzymes in the development of maize seedlings during the two weeks immediately after radicle emergence were also investigated. It appears that ZmCKX1 is a key regulator of active cytokinin levels in developing maize roots. However, the expression of individual CKX isoforms in the shoots varied and none of them seemed to have strong effects on the cytokinin pool.
authors: Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Molecular Biology, Šlechtitelů 11, Olomouc 783 71, Czech Republic. Electronic address: david.zalabak@upol.cz.
authors: David
authors: D
authors: Zalabák
authors: Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Molecular Biology, Šlechtitelů 11, Olomouc 783 71, Czech Republic. Electronic address: petr.galuszka@upol.cz.
authors: Petr
authors: P
authors: Galuszka
authors: Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Molecular Biology, Šlechtitelů 11, Olomouc 783 71, Czech Republic. Electronic address: katarina.mrizova@gmail.com.
authors: Katarina
authors: K
authors: Mrízová
authors: Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Chemical Biology and Genetics, Šlechtitelů 11, Olomouc 783 71, Czech Republic. Electronic address: katka.vaclavik@seznam.cz.
authors: Kateřina
authors: K
authors: Podlešáková
authors: Key Lab of Plant Nutrition, MOA, College of Resources and Environmental Science, China Agricultural University, 100193 Beijing, China. Electronic address: rilianggu@cau.edu.cn.
authors: Riliang
authors: R
authors: Gu
authors: Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Chemical Biology and Genetics, Šlechtitelů 11, Olomouc 783 71, Czech Republic. Electronic address: jitka.frebortova@upol.cz.
authors: Jitka
authors: J
authors: Frébortová
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003583:Cytokinins / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism
chemical_list: D003583:Cytokinins; D017931:DNA Primers; D010088:Oxidoreductases; C019984:cytokinin oxidase
keywords: 2,6-dichlorophenolindophenol; AtCKX; CKX; Cytokinin; Cytokinin dehydrogenase; DCPIP; DHZ; DMAPP; EDTA; Escherichia coli IMPACT expression system; IPT; N(6)-(Δ(2)-isopentenyl)adenine; N(6)-(Δ(2)-isopentenyl)adenine-N9-glucoside; N(6)-(Δ(2)-isopentenyl)adenosine; N(6)-(Δ(2)-isopentenyl)adenosine-5′-monophosphate; Substrate preference; Zea mays L.; ZmCKX; cZ; cZ9G; cZRMP; cis-zeatin; cis-zeatin riboside-5′-monophosphate; cis-zeatin-N9-glucoside; cytokinin dehydrogenase; cytokinin dehydrogenase from Arabidopsis thaliana; cytokinin dehydrogenase from Zea mays; dihydrozeatin; dimethylallyl diphosphate; ethylenediaminetetraacetic acid; iP; iP9G; iPR; iPRMP; isopentenyl transferase; tZ; tZ9G; tZR; tZRMP; trans-zeatin; trans-zeatin riboside; trans-zeatin riboside-5′-monophosphate; trans-zeatin-N9-glucoside