Lecture 11: Cytokinins

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Lecture 11 Cytokinins
Discovery of gibberellins Biosynthesis Physiolog
ical effects Signal transduction
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Differentiated plant cells can resume division
Examples - mature cells of cortex/phloem resume
division to form secondary meristems -gt
vascular cambium, cork cambium - wounding of
plant cells initiates cell divisions at the wound
site (even highly specialized cells, i.e.
phloem, guard cells) - but wound-induced
mitotic activity is self-limiting (cells
redifferentiate forming a protective layer of
cork cells)
Infection with Agrobacterium tumefaciens at the
wound site causes tumor-forming disease
crown-gall (unorganized mass of tumor-like
tissue) ? natural evidence of the mitotic
potential of mature plant cells Agrobacterium
makes them tumor-like
G. Haberlandt 1913 vascular tissue contains a
water-soluble substance that stimulates division
of wounded potato tuber tissue ? discovery of
cytokinins in 1950s
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Discovery, identification, properties
Philip White nutrient medium, supplemented with
auxin and 10-20 coconut milk supports cell
division of mature, differentiated cells from a
variety of tissues and species, leading to
formation of callus tissue. ? Coconut milk
contains substance/s that stimulate/s mature
cells to enter and remain in cell division
cycle. Coconut milk was shown to contain the
cytokinin zeatin. The very first cytokinin
discovered was the synthetic analog kinetin.
Kinetin N-(2-furanylmethyl)-1H-purin-6-amine
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Zeatin is the most abundant natural cytokinin
Extracts of immature endosperm of corn (Zea mays)
contain a substance with the same biological
effect as kinetin. This substance stimulates
mature plant cells to divide when added to
culture medium along with auxin. Letham (1973)
isolated that substance, called zeatin.
Kinetin
6-(4-Hydroxy-3-methylbut-2-enylamino)purine
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Cytokinins occur in both free and bound forms
Hormonally active cytokinins are free molecules
(not covalently bound to anything)
Transfer RNA (tRNA) contains a zeatin as a
hypermodified base Example this yeast tRNA for
tyrosine contains IP isopentenyladenosine
Other forms of conjugation Alanine
(irreversible) Glucose (cytokinin glucosides, may
be a storage form reversible by glucosidase)
? pseudouridine UH2 dihydrouridine Me
methylated base
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Some plant pathogenic bacteria, insects, and
nematodes secrete free cytokinins
Many microorganisms produce or cause plants to
synthesize cytokinins, e.g. ribosylzeatin and IP
Isopentenyl adenine (iP)
Infection of the plant tissue by these
microorganisms causes tissue to divide and form
special structures in some cases (insect galls
used as feeding sites)
Witches broom on balsam fir caused by
Corynebacterium fascians Lateral buds (normally
dormant) stimulated by bacterial cytokinin to
grow
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Crown gall cells have acquired a gene for
cytokinin synthesis
Tumor induction by Agrobacterium tumefaciens
T-DNA carries genes for biosynthesis of -
trans-zeatin (isopentenyll transferase) and auxin
(2 genes Trp monooxygenase and IAM
hydrolase) - unusual amino acid derivatives,
opines, which the bacterium but not the plant
utilizes as a nitrogen source
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Cytokinin biosynthesis
(Dimethylallyl diphosphate)
IPT isopentenyl transferase (8 genes in
Arabidopsis)
Unidentified hydroxylase
Root apical meristems major site of free
cytokinins in whole plant
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Cytokinin transport and metabolism
Transport of cytokinins from roots to shoot via
xylem -gt evidence from analysis of xylem
exudates - cytokinins in xylem exudates are
mainly in form of zeatin ribosides - transport
from root to shoot is regulated by unidentified
signals (grafting experiments)
Cytokinins are rapidly metabolized by cytokinin
oxidase, thereby inactivating cytokinins.
Activity of cytokinin oxidase induced by high
cytokinin concentrations.
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Biological roles of cytokinins
Tobacco plants overexpressing the gene for
cytokinin oxidase ? reduction in endogenous
cytokinin levels ? Inhibition of shoot growth due
to reduction in rate of cell proliferation in
SAM ? Cytokinins regulate cell division in vivo
AtCKX2-OX
AtCKX1-OX
AtCKX1 and AtCKX2 Arabidopsis cytokinin
oxidase
WT
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Cytokinin is required for normal growth of SAM
SAM of wild-type tobacco SAM transgenic tobacco
overexpressing AtCKX1 ? cytokinin deficiency
? reduced size of SAM
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Cytokinin suppresses growth of roots
Roots stained with fluorescent dye to stain
nucleus
WT tobacco
AtCKX1-OX Cytokinin-deficient
Enhancement of root growth by increasing size of
RAM Cytokinins have opposite roles
in regulating cell proliferation in RAM and
SAM
WT tobacco
AtCKX1-OX Cytokinin-deficient
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Cytokinins regulate specific components of the
cell cycle
Cyclin-dependent protein kinases (CDKs) together
with cyclins (small proteins of 38 KDa)
regulate eukaryotic cell cycle (see Chapter 1 for
recap) - major CDK, CDC2 (CELL DIVISION CYCLE
2) is regulated by auxin - auxin-induced CDK is
inactive high levels of CDK are not sufficient
for cell division - cytokinin activates
phosphatase to remove one (the inhibitory)
phosphate group from CDC2 (is inactive with
two phosphate groups bound) - cytokinin
elevates expression of CYDC3, encoding D-type
cyclin
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Cytokinins regulate specific components of the
cell cycle

• cytokinin activates phosphatase
• to remove one (the inhibitory)
• phosphate group from CDC2
• cytokinin elevates expression of
• CYDC3, encoding D-type cyclin,
• which are regulated in animals
• by variety of growth factors in G1
• phase

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The auxincytokinin ratio regulates morphogenesis
in cultured tissues
Shoots
No growth
Callus
Plant tissue
Roots
Nutrient agar
Auxin Kinetin
Ratio 15 150 0.003
High auxincytokinin ratio root Low
auxin/cytokinin ratio shoot Intermediate levels
callus
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The auxincytokinin ratio regulates morphogenesis
in cultured tissues
Map of Agrobacterium T-DNA in Ti plasmid showing
effects of T-DNA mutations on crown gall tumor
formation
Mutations or deletions of these regions result in
Ti plasmid that initiate tumors with specific
characteristics
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Cytokinins modify apical dominance and promote
lateral bud growth
Apical dominance by auxin, determines plant
form e.g. maize strong apical dominance, few
lateral branches shrubby plants many
lateral buds Cytokinin initiates growth of
lateral buds Well-studied example Stages of bud
development in Funaria moss (the bud
goes on to produce the leafy gametophyte stage of
the moss)
Protonema filament derived from germinating
moss spores
Bud formation of Funaria moss dependent on red
light cytokinin added to medium can substitute
for light.
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Cytokinins delay leaf senescence
Senescence programmed aging process
leading to death
Leaf senescence is retarded in transgenic tobacco
containing cytokinin biosynthesis gene IPT. IPT
is expressed in response to signals that induce
senescence (e.g. shorter days of fall, drought,
or the lack of nutrient causes less production of
auxin and this begins senescence).
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Cytokinins promote chloroplast development
Plastids develop as etioplasts in untreated, dark
grown control
Cytokinin treatment resulted in thylakoid
formation in plastids of dark-grown seedlings
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A cytokinin receptor related to bacterial
two-component receptors has been identified
Discovery of CKI1 cell line overexpressing CKI1
grows in culture in absence of added
cytokinin encodes protein similar in sequence
to bacterial two-component sensor histidine
kinases (ubiquitous receptors in prokaryotes)
Example CRE1 cytokinin receptor cre1 mutants
fail to develop shoots from un- differentiated
culture cells in response to cytokinin
Hpt histidine phosphotransfer protein, AHP in
Arabidopsis (5 genes present)
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Induction of type-A ARR genes in response to
cytokinin
Time following cytokinin treatment (min) 0
2 5 10 15 25 30 40
60 120 180
Probe ARR4 ARR5 ARR6 ARR7 ARR16 Tubuli
n
Northern blot probed with individual type-A
genes. Each lane contains RNA from Arabidopsis
seedlings treated with cytokinins for the
indicated times.
ARR5 is expressed in apical meristems of shoots
and roots.
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Cytokinin induces the transient movement of some
AHP proteins into the nucleus
Arabidopsis protoplasts expressing various AHP
genes fused to GFP and treated with zeatin and
monitored for 1.5 hrs.
- Zeatin Zeatin, 0.5 h Zeatin, 1.5 h
AHP1-GFP
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Model of cytokinin signaling
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Model of cytokinin signaling
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Model of cytokinin signaling