1. Petr GALUSZKA Centre of the Region Haná for Biotechnological and Agricultural Research Palacký University in Olomouc FROM CYTOKININ METABOLISM TO AGRICULTURE

2. Gajdošová et al., JXB 2011 CK qualitative content analysis barleymaizeCK-typeArabidopsis pmol/g of leaf fresh weight 15.12.1iP22.2 36.41.4tZ60.5 206.7164.3cZ17.5 7.51.7DHZ4.2

3. CK qualitative analysis during maize development pmol/g of leaf fresh weight kernel germination shoot developmentroot development 20th day 20th day isopentenyladenine trans-zeatin cis-zeatin dihydrozeatin

4. CYTOKININ SENSING in Zea mays Histidine kinase – CK receptor bound to membranes Apparent K D (nM) CKZmHK1ZmHK2ZmHK3 isopentenyladenine2.30.62.7 trans-zeatin240.47.2 cis-zeatin268.421 dihydrozeatin6302232 tZR300070280 iPRnot detectable400 tZOGnot detectable S. Lomin et al., JXB 2011 Contrary to Arabidopsis receptors, there is higher sensitivity to cZ and DHZ at least on some maize receptors

5. iP ? cZ DHZ tZ zeatin metabolism in planta is rather obscure de novo biosynthesis tRNA degradation CYP450 zeatin isomerase IPT LOG zeatin reductase ? proved in planta gene is known proved in vitro activity detected hypothesized

6. Cytokinin dehydrogenases have different preferences CKXiPtZcZDHZ AtCKX1100721160 AtCKX21007820 AtCKX31002000 AtCKX41008940 AtCKX510068 0 AtCKX610013370 AtCKX7100301160 ZmCKX11001221380 ZmCKX101001541650 HvCKX210019000 CKX relative activity CKX gene families: Arabidopsis thaliana7 genes Zea mays13 genes Oryza sativa11 genes Hordeum vulgare11 genes

7. CKX as a transgene Constitutive overexpression of CKX causes cytokinin-deficient phenotype Main function of CKs on plant differentiation proved together with opposite role on root and shoot development Cytokinin content 30–50% of WT Construct: 35S::HvCKX2 pmol/g fresh w CKWTHvCKX2 seedlings iP115.831.5 tZ21.710.4

8. Transformation of Barley with CKX genes Ubiqutin::ZmCKX1 Ubiqutin::HvCKX2 WT trsg WT Z2 Z4 Z7 Z8 Z10 WTZ10 Constitutive CKX overexpression in cereal: prematured senescence no onset of flowering (sterility) enlarged root system (dependent of transgene) pmol/g fresh w CKWTtrsg root iP22.216.1 tZ87.3122 cZ42.8188 DHZ2.87.5 shoot iP14.010.8 tZ37.612.2 cZ161101 DHZ2.62.9

9. CKX and IPT gene families in barley 11 CKX genes 2 tRNA IPT genes 5 de novo IPT genes

10. wild type situation our transgenic approach