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.

Root Engineering in Barley: Increasing Cytokinin Degradation Produces a Larger Root System, Mineral Enrichment in the Shoot and Improved Drought Tolerance

“…The barley root consists of several seminal roots derived from the embryo and a nodal root system derived from the nodes at the base of the main stem or of the tillers. Previous attempts to manipulate the barley root system by the ectopic expression of CKX genes have proven difficult, as strong detrimental effects on shoot development and reproduction could not be avoided (Mrízová et al, 2013;Pospíšilová et al, 2016). This indicated that a successful approach may depend on restricting the manipulation of CKX gene expression to roots.…”

Silencing ofTaCKX1mediates expression of otherTaCKXgenes to increase grain yield in wheat

“…The lower plant height and higher root weight observed in the group of silenced plants of wheat is in agreement with opposed regulation of these traits by CKs and IAA mentioned above (Brenner and Schmulling 2012). CKs are considered as negative regulators of root growth, so their reduction causes enhanced root growth (Werner et al, 2010; Mrizova et al, 2013), while excess of CKs inhibits primary root growth in Arabidopsis (Riefler et al, 2006; Dello Ioio et al, 2012). It was also demonstrated that long-distance, basipetal transport of cytokinin controls polar auxin transport and maintains the vascular pattern in the root meristem (Bishopp et al, 2011).…”

De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea

“…data). Genes for RR, HK and LOG were numbered based on the closest rice orthologues (Ito and Kurata, ; Kurakawa et al ., ) while those for CKX, IPT, b‐actin and elongation factor 1 were based on barley orthologues (Mrízová et al ., ). Predicted sequences were deposited in the NCBI database (http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12838/suppinfo).…”