CYTOKININ - Metabolism
Synthesis – IPT genes
Degradation – CK-oxidase
Conjugation
Cytokinin – substance crucial
for sustain of cell proliferation
Auxin and cytokinin
Skoog et Miller, 1957
roots
shoots
Cytokinins
Agrobacterium tumefaciens – IPT (isopentenyltransferase)
gene for cytokinin biosynthesis
Cytokinin biosynthesis
Takei et al., 2001
Isolation of Arabidopsis IPTs
Kakimoto 2004
Arabidopsis IPTs (8)
Differential expression patterns
Differently responsive promotors
(cytokinin, auxin, nitrate, combinations)
mutants, overexpression ???
Werner et al., 2001
Isolation of CK-oxidase (AtCKX)
AtCKXs overexpression in tobacco
Werner et al., 2001
Effect of AtCKX on tobacco root
Werner et al., 2001
Cytokinin
–signal perception and transduction
Kakimoto et al., 1996
Isolation of CK independent (cki1) mutant
Identification of CKI1 gene
Kakimoto et al., 1996
Verification - 35S::CKI1 transgene
+CK
-CK
Kakimoto et al., 1996
Inoue et al., 2001
Next strike
- CK response
mutant (cre1)
Wt
cre1
pGTV pGTV-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)
What does CKI1?
His kinase transduction pathway
His kinase pathway
components
in Arabidopsis
Response Regulators in Arabidopsis
Brandstatter and Kieber, 1999
CK responsive genes –
ARR type A
Response regulator ARR type Anegative
regulators of cytokinin signalling
To et al., 2004
Hutchison et al.,2006
AHPs mediate transfer of cytokinin signal between
cytoplasm and nucleus
Phenotypes of arr type B positive regulator
of CK signalling
wt
35S::ARR1
arr1-1
Sakai et al., 2001
Hwang
Games with protoplasts
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
Regulation of plant development
by ethylene
Mutant screens for ethylene pathway genes
airC2H4 air C2H4
Wt mutants
*
Roman et al., 1994
* phenotype rescued by inhibitor of ethylene biosynthesis
Screen for ethylene mutants
I. Genes involved in regulation of biosynthesis
eto mutants – constitutive triple response
WT eto2
-5
0
5
10
15
20
25
30
35
40
WT eto2-1
ethyleneproductionpl.mg-1
.h-1
eto2 eto3
- overproduction of ethylene
* *
* * *
Screen for ethylene mutants
II. Genes invoved in signalling pathway
Roman et al., 1994
Roman et al., 1994
Roman et al., 1994
ETR1 codes for histidine kinase
Bleecker et al.,1988 Hua et al., 1998 Sakai et al., 1998
etr1 ein4 ers2 etr2
His-kinases in Arabidopsis
Ethylene signalling
– homology to two component system ?
Ctr1 – codes for protein kinase of Raf family
ETR1 interacts with CTR1
Clark et al., 1998
CTR1 ctr1-1
CTR1 has protein kinase activity
-ethylene receptor dominant mutation > ethylene insensitivity
-ethylene receptor loss of function mutation > constitutive ethylene response
-ctr1 loss of function mutation > constitutive ethylene response
ctr1
Ethylene signal transduction pathway
Guo and Ecker, 2003
MODELS