Role of cytokinin in plant development Cytokinin – substance crucial for sustain of cell proliferation Cytokinins CYTOKININ - Metabolism Synthesis – IPT genes Degradation – CK-oxidaseDegradation – CK-oxidase Conjugation Cytokinin biosynthesis Agrobacterium tumefaciens – IPT gene for cytokinin biosynthesis Isolation of Arabidopsis IPTs Takei et al., 2001 Arabidopsis IPTs (8) Differential expression patterns Differently responsive promotors (cytokinin, auxin, nitrate, combinations) Kakimoto 2004 (cytokinin, auxin, nitrate, combinations) mutants, overexpression ??? Cytokinin – role in plant development Auxin and cytokinin roots Skoog et Miller, 1957 shoots Isolation of CK-oxidase (AtCKX) Werner et al., 2001 AtCKXs overexpression in tobacco Werner et al., 2001 Effect of AtCKX on tobacco root Werner et al., 2001 Morphology of AtCKX tobacco plants CK in shoot – positive regulator CK in root – negative regulator Werner et al., 2001 Cytokinin – root meristem development Dello Ooio et al., 2007 Cytokinin - lateral root organogenesis Laplaze et al., 2007 Cytokinin effect is stage specific Laplaze et al., 2007 Cytokinin –signal perception and transduction Isolation of CK independent (cki1) mutant Kakimoto et al., 1996 Identification of CKI1 gene Kakimoto et al., 1996 Verification - 35S::CKI1 transgene +CK -CK Kakimoto et al., 1996 Next strike - CK response mutant (cre1) Wt pGTV pGTV-CRE1 Inoue et al., 2001 Wt cre1 His kinase transduction pathway Piece of genius - complementation Hog1 kinase hyperactivation Inoue et al., 2001 Cytokinin receptors – what else? 3 CRE1 homologous proteins (AHKs) multiple mutant phenotypes – additive (not lethal)(not lethal) What does CKI1? His kinase pathway components in Arabidopsis Response Regulators in Arabidopsis CK responsive genes – ARR type A Brandstatter and Kieber, 1999 Response regulator ARR type Anegative regulators of cytokinin signalling To et al., 2004 Phenotypes of arr type B positive regulator of CK signalling wt 35S::ARR1 arr1-1 Sakai et al., 2001 AHPs mediate transfer of cytokinin signal between cytoplasm and nucleus Hutchison et al.,2006 Games with protoplasts Hwang Opposite effects of two classes of ARRs on CK signalling AHPs – signal to nucleus Hextuple of type A ARRs confirms role as negative regulators e.g. Root elongation assay Transcritpion Cell division Shoot formation Delayed senescence Vascular develppment Cytokinin – root meristem development Dello Ioio et al., 2007 Auxin related mutants with short meristem Blilou et al.,2005 Auxin transport mutantsMS cytokinin Cytokinin modulates auxin transport MS BA PIN2:GFP (48 h) PIN7:GFP (24h)PIN3:GFP (24h) PIN1:GFP (24 h) MS BA 40 60 80 100 120 5 10 15 25 30 hours MS BA 45 50 MS BA 90 100 MS BA 60 70 80 90 100 110 120 5 10 15 24 28 48 hours MS BA* * * * * * * MS BA MS BA 20 25 30 35 40 45 5 10 24 48 hours 50 60 70 80 90 5 10 15 24 28 48 hours * * * * * * * 0 50 100 150 200 250 300 PIN1 PIN2 PIN3 PIN7 Relativeexpression(%) MS 1h 2h 3h 4h 5h 6h Q-RT PCR Ruzicka et al., 2009 MS BA 0 20 40 60 80 100 120 140 0 0.1 1 10 µM BA * * * Cytokinin reduces auxin efflux in tobacco BY2 cells PIN1:RFP 0,00 0,50 1,00 1,50 0 5 10 15 20 25 time (min) M S BA Ruzicka et al., 2009 CK – auxin signalling pathways interaction to modulate auxin transport CK receptor CK response regulator AUX/IAA negative regulator Expression of IAA3/SHY2 is CK regulated Dello Ioio et al., 2008 Model of cytokinin and auxin interaction in root meristem Dello Ioio et al., 2008 Cytokinin signalling regulates vascular development Mahonen et al., 2006 ahp6 suppress wol insensitivity to cytokinin AHP6 codes for histidine phosphotransfer protein 6 AHP6 is negative regulator of cytokinin signalling