Cytokinin oxidase/dehydrogenase genes in barley and wheat: cloning and heterologous expression.
The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinin-deficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found to be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented.
Galuszka
Petr
P
Division of Molecular Biology, Department of Biochemistry, Faculty of Science, Palacký Univesity, Olomouc, Czech Republic. galuszka@prfholnt.upol.cz
Frébortová
Jitka
J
Werner
Tomás
T
Yamada
Mamoru
M
Strnad
Miroslav
M
Schmülling
Thomas
T
Frébort
Ivo
I
D015086:2,6-Dichloroindophenol / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D003583:Cytokinins / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
D016227:Benzoquinones; D003583:Cytokinins; D011994:Recombinant Proteins; C109863:ubiquinone-O; D015086:2,6-Dichloroindophenol; D010088:Oxidoreductases; C019984:cytokinin oxidase