Barley is one of the most important cereals, which is used for breweries, animal and human feeds. Genetic manipulation of plant hormone cytokinins may influence several physiological processes, besides others stress tolerance, root formation and crop yield. In planta, endogenous cytokinin status is finely regulated by the enzyme cytokinin dehydrogenase (EC 1.5.99.12; CKX), that irreversible degrades the side chain of adenine-derived isoprenoid cytokinins. Increasing grain yield by mean of manipulation of endogenous cytokinin content was assayed by the silencing of the HvCKX1 gene. Moreover, to elucidate the putative role of HvCKX1 gene on grain production, knocked-out Hvckx1 mutant plants were generated using the RNA-guided Cas9 system. Homozygote transgenic plants with silenced HvCKX1 gene and azygous knock-out Hvckx1 mutants have been selected and analyzed. Both reduced expression of HvCKX1 gene and CKX activity were measured in different stages of barley grain development. Phenotyping of the transgenic lines revealed reduced root growth, however, plants produced more tillers and grains than azygous wild-type controls and the total yield was increased up to 15 per cent. Although plant productivity was increased, total grain biomass was decreased to 80% of WT grains. RNA-seq analysis of knock-down transgenic lines revealed that several important macronutrient transporters were downregulated in the stage of massive starch accumulation. It suggests that local accumulation of cytokinins negatively affected nutrients flow resulting in reduced grain biomass. Obtained results confirmed the key role of HvCKX1 for regulation of cytokinin content in barley.

Central Laboratories and Research Support Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc Czechia

Department of Genetic Resources for Vegetables Medicinal and Special Plants Crop Research Institute Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc Czechia

Department of Molecular Biology Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc Czechia

Institute of Molecular and Translation Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Czechia

Plant Reproductive Biology Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben Gatersleben Germany

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Front Plant Sci. 2019 Feb 04;9:1996. doi: 10.3389/fpls.2018.01996. PubMed

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