title: Overexpression of cytokinin dehydrogenase genes in barley (Hordeum vulgare cv. Golden Promise) fundamentally affects morphology and fertility.
abstract: Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, Czech Republic.
authors.forename: Katarína
authors.initials: K
authors.lastname: Mrízová
authors.forename: Eva
authors.initials: E
authors.lastname: Jiskrová
authors.forename: Šárka
authors.initials: Š
authors.lastname: Vyroubalová
authors.forename: Ondřej
authors.initials: O
authors.lastname: Novák
authors.forename: Ludmila
authors.initials: L
authors.lastname: Ohnoutková
authors.forename: Hana
authors.initials: H
authors.lastname: Pospíšilová
authors.forename: Ivo
authors.initials: I
authors.lastname: Frébort
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Harwood
authors.forename: Petr
authors.initials: P
authors.lastname: Galuszka
mesh_terms: D016960:Agrobacterium tumefaciens; D003583:Cytokinins / Q000096:biosynthesis / Q000235:genetics; D005298:Fertility / Q000235:genetics; D015870:Gene Expression; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics; D018517:Plant Roots / Q000196:embryology* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics
chemical_list: D003583:Cytokinins; D010940:Plant Proteins; D010088:Oxidoreductases; C019984:cytokinin oxidase