Download Synthesis CK Transport Perception (receptor)

Cytokinins
Cytokinins (CKs):
•promote cell division in the shoot
•delay leaf senescence
•regulate nutrient allocation
•promote root nodule development
•contribute to environmental signaling
and pathogen responses
•regulate auxin action and distribution
+ CK
+ auxin
+ auxin
and CK
© 2013 American Society of Plant Biologists
Structure of major cytokinins
adenine
Cytokinins are N6substituted adeninerelated compounds.
Trans-zeatin and
isopentenyl-adenine
are the most active and
abundant CKs
trans-zeatin (tZ)
Isopentenyladenine (iP)
© 2013 American Society of Plant Biologists
Agrobacterium tumefaciens induces
hormone-based tumors
Agrobacterium tumefaciens is a
natural plant pathogen. It causes
crown gall disease and tumorlike growths by inducing the plant
to produce auxin and cytokinin
Plant Cell
Nucleus
DNA
Agrobacterium
tumefaciens
Agrobacterium
tumefaciens on the
surface of a plant cell
Agrobacterium tumefaciens carries
tumor-inducing (Ti) plasmids. A
subset of the plasmid DNA called
transfer-DNA (T-DNA) is mobilized
into the plant nucleus
SEM courtesy of Martha Hawes, University of Arizona; grown gall by C-M
© 2013 American Society of Plant Biologists
T-DNA includes genes for
biosynthesis of auxin and cytokinin
Normal T-DNA
Ti
plasmid
Auxin and CK
produced
normal tumor
Auxin produced
These studies showed
that the Agrobacterium
tmr gene encodes a
cytokinin biosynthetic
enzyme
rooty tumor
No tmr gene
CK produced
shooty tumor
No tms gene
© 2013 American Society of Plant Biologists
The tmr gene encodes isopentenyltransferase, a key enzyme in CK
synthesis
Isopentenyltransferase (IPT)
AMP
DMAPP –
dimethylallyldiphosphate
iPRMP
isopentenyladenine (iP)
riboside phosphate
The bacterial IPT gene uses AMP
exclusively as a substrate whereas plant
IPT genes prefer ADP or ATP
Haberer, G. and Kieber, J.J. (2002) Cytokinins. New insights into a classic phytohormone. Plant Physiol. 128: 354-362.
© 2013 American Society of Plant Biologists
IPT overexpression causes reduced
apical dominance, reduced root
growth and delayed leaf senescence
Elevated CK
promotes shoot
bud outgrowth
Elevated CK
promotes shoot
growth and restricts
root growth
Wild type
IPT overexpression
Medford, J.I., et al. (1989) Alterations of endogenous cytokinins in plants using a
chimeric isopentenyl transferase gene Plant Cell1: 403-413.
© 2013 American Society of Plant Biologists
Cytokinins can be inactivated by
conjugation or degradation
The CKX genes are
important regulators of
active cytokinin levels
Adenine
Phosphoribosyl
A
Transferase 1
(APT1)
O-glycosylation
or O-acetylation
Glucosylation site
Irreversible
Degradation
Cytokinin
oxidase
(CKX)
Reversible
Conjugation
Adenylation
LOG
ACTIVE
FORM
CKX genes are
CK-induced
Kieber, J.J. (2002) Cytokinins: March 27, 2002. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists. doi: 10.1199/tab.0063 See also
Bajguz, A. and Piotrowska, A. (2009) Conjugates of auxin and cytokinin. Phytochemistry 70: 957–969.
© 2013 American Society of Plant Biologists
Cytokinin signaling takes place
through a phosphorelay system
Type-C
ARR
ARR22
ARR24
CK binding to the
membrane-bound
receptor histidine
kinases leads them to
autophosphorylate
The receptor HKs
transfer the
phosphoryl group to
histidine
phosphotransfer
proteins (HPTs)
The HPTs transfer
the phosphoryl
group to response
regulators
Adapted from Schaller, G.E., Kieber, J.J., and Shiu, S (2008) Two-component signaling elements and histidyl-aspartyl phosphorelays. The Arabidopsis Book: ASPB.
© 2013 American Society of Plant Biologists
Arabidopsis has three CK receptors,
AHK2, 3 and 4
Cytokinin receptor (AHK2)
Cytokinin receptor (AHK3)
wol
WT
AHK4 was identified as a wooden-leg mutant
(wol) and a cytokinin response mutant (cre1)
The wol root is
truncated due to
impaired differentiation
in central cylinder. The
cre1 mutants do not
produce shoots in
tissue culture
Wild-type
cre1
Reproduced with permission from Scheres, B. et al. (1995) Mutations affecting the radial organisation of the Arabidopsis root display specific defects throughout the
embryonic axis. Development 121: 53-62. Reprinted by permission from Macmillan Publishers Ltd.: Nature. Inoue, T., et al. (2001). Identification of CRE1 as a cytokinin
receptor from Arabidopsis. Nature 409: 1060-1063.
© 2013 American Society of Plant Biologists
Downstream of the receptors: HPTs
and RRs
The receptors pass the
phosphoryl groups to a histidine
phosphotransfer protein (HPT or
AHP*) which passes it to a
response regulator (RR or ARR*).
Type-B ARRs are transcription
factors, whereas Type- A ARRs
are inhibitors of CK signaling
*AHP and ARR refer to the Arabidopsis proteins
Reproduced with permission from El-Showk, S., Ruonala, R. and Helariutta, Y. (2013) Crossing paths: cytokinin signalling and crosstalk. Development 140: 1373–1383.
© 2013 American Society of Plant Biologists
Receptors relay phosphoryl groups
to His phosphotransfer proteins
Type-C
ARR
ARR22
ARR24
Three CK
receptors
Five histidine
phosphotransfer
proteins (HPTs) - In
Arabidopsis known
as AHPs
23 response
regulators (RRs) of
three types
– In Arabidopsis
known as ARRs
Adapted from Schaller, G.E., Kieber, J.J., and Shiu, S (2008) Two-component signaling elements and histidyl-aspartyl phosphorelays. The Arabidopsis Book: ASPB.
© 2013 American Society of Plant Biologists
There are three types of response
regulators
Receiver
domain
D
Arabidopsis: 10 Type-A ARRs
D
Arabidopsis: 2 Type-C ARRs
D
Arabidopsis: 11 Type-B ARRs
DNA-binding
domain
E
Negative
regulators
Type-B ARRs are transcriptional activators
with C-terminal DNA- binding domains
Pseudoresponse regulators (PRRs) are related
proteins but not involved in CK signaling
PRRs usually lack the conserved
Asp residue in the receiver domain
© 2013 American Society of Plant Biologists
ARR1, a type-B ARR, is a positive
regulator of CK signaling
ARR1 overexpression
Overexpression of
ARR1 makes tissues
more sensitive to CK
Wild-type
arr1 loss-of-function
The concentration of CK
needed to produce green
tissues is a good measure
of CK sensitivity
Loss-of-function arr1
mutants are less
sensitive to CK
From Sakai, H., Honma, T., Aoyama, T., Sato, S., Kato, T., Tabata, S., and Oka, A. (2001). ARR1, a transcription factor
for genes immediately responsive to cytokinins. Science 294: 1519-1521; reprinted with permission from AAAS.
© 2013 American Society of Plant Biologists
Type A ARRs are negative regulators
of CK signaling
Type-A ARR
D
CK-inducible
ARR6 promoter
Expression of
type-A ARRs
interferes with
CK-induced
transcription in
protoplasts
LUC
Reprinted by permission from Macmillan Publishers Ltd.: Nature . Hwang, I., and Sheen, J. (2001). Two-component
circuitry in Arabidopsis cytokinin signal transduction. Nature 413: 383-389, copyright 2001,
© 2013 American Society of Plant Biologists
Type-C ARRs may remove
phosphoryl groups from the system
Histidine kinase
activity adds
phosphoryl groups
to system
Phosphorelay system
Type-A ARRs
Putative competition
between type-A and
type-B ARRs
?
Type-C ARRs remove
phosphoryl groups from the
system via histidine
phosphatase activity
Type-B ARRs – CKinduced transcription
© 2013 American Society of Plant Biologists
CK action in whole-plant processes
CKs regulate
•Root vascular tissue
development
•Shoot and root developmental
patterning
•Nutrient uptake and allocation
•Leaf senescence
•Many other processes
Catabolism
Synthesis
CK
Cytokinin’s roles in wholeplant processes
Conjugation
Transport
Perception
(receptor)
TF activation/
inactivation
Target
genes
Biological
Functions
© 2013 American Society of Plant Biologists
Cytokinins contribute to developmental
patterning and meristem functions
CK promotes
cell division and
stem cell fate at
the shoot apical
meristem
CK inhibits root
meristem size
and cell division
and promotes
cell differentiation
at the root apical
meristem
Reprinted by permission from Macmillan Publishers, Ltd: NATURE Wolters, H., and Jürgens, G. (2009). Survival of the flexible:
Hormonal growth control and adaptation in plant development. Nat. Rev. Genet. 10: 305–317. Copyright 2009.
© 2013 American Society of Plant Biologists
Cytokinin and auxin regulate
organogenesis in tissue culture
+ CK
+ auxin
Tobacco leaf discs are placed
into sterile culture dishes on
medium containing various
hormones
+ auxin
and CK
TIME
Images courtesy of Richard Amasino.
© 2013 American Society of Plant Biologists
CKs contribute to nutrient uptake
and allocation
Sink
Source
CO2
Sink
Shoot systems are a
source of sugars and
primary metabolites that
are distributed to
nutrient sinks including
flowers and fruits, roots,
and young leaves
Source
Root systems take
up mineral nutrients
such as nitrate,
sulfate and
phosphate
Sink
NO3SO42PO43-
Elevated CK levels
increase expression of
photosynthetic enzymes
and delay leaf
senescence
© 2013 American Society of Plant Biologists
CKs contribute to nutrient uptake
and allocation
Elevated levels of nitrate
or phosphate increase the
rate of CK synthesis,
which ultimately
decreases root growth
rate. In turn, elevated CK
represses nutrient uptake
Model showing the role of
CK and other hormones on
nitrogen acquisition
Reprinted from Kiba, T., Kudo, T., Kojima, M. and Sakakibara, H. (2011). Hormonal control of nitrogen acquisition:
roles of auxin, abscisic acid, and cytokinin. J. Exp. Bot. 62: 1399-1409 by permission from Oxford University Press.
© 2013 American Society of Plant Biologists
Cytokinins delay leaf senescence
SAG:IPT Control
Plants that express IPT
under the regulation of a
senescence-induced
promoter (SAG) have
significantly delayed leaf
senescence
SAG:IPT Control
From Gan, S., and Amasino, R.M. (1995) Inhibition of leaf senescence by autoregulated production of
cytokinin Science 270: 1986-1988. Reprinted with permission from AAAS.
© 2013 American Society of Plant Biologists
CKs can negatively affect stress
tolerance
Loss-of-function
mutants affecting CK
synthesis (atipt) or
signaling (ahp) are
drought tolerant
ahp2,3,5
C
CK also reduces the
plant’s sensitivity to
ABA, so it acts through
ABA-dependent and
independent pathways
Stress
Wild type
Three-week-old plants exposed to
drought stress for13 or 14 days and
then re-watered for three days
Drought
tolerance is
conferred in
part by an
increase in
membrane
stability
CK
ABA
Stress
tolerance
Nishiyama, R., et al., (2011) Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid
biosynthesis. Plant Cell. 23 : 2169-2183. Nishiyama, R., Watanabe, Y., Leyva-Gonzalez, M.A., Ha, C.V., Fujita, Y., Tanaka, M., Seki, M., Yamaguchi-Shinozaki, K., Shinozaki, K., Herrera-Estrella, L., Tran, L.S.
(2013) Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response. Proc. Natl. Acad. Sci. USA. 2013 110: 4840–4845.
© 2013 American Society of Plant Biologists
CKmediated
processes
There are many other
processes mediated by
CK. Identifying the
specific genes that
contribute to each of
these will help us to
understand the myriad
roles that CK plays in
coordinating plant
growth
Reprinted from Werner, T., and Schmülling, T. (2009). Cytokinin
action in plant development. Current Opinion in Plant Biology 12:
527-538, with permission from Elsevier copyright 2009.
© 2013 American Society of Plant Biologists
Cytokinin action - summary
CKs have diverse roles – from regulating vascular
differentiation and meristem function to regulation
of nutrient allocation and leaf senescence
We are beginning to correlate specific genes with
specific functions but there are still many
unresolved questions
CKs provide many unexploited opportunities for
improving agricultural yields through increased
stress tolerance and seed yields
© 2013 American Society of Plant Biologists
Ongoing investigations
How do catabolism and
conjugation contribute to
in vivo functions?
Catabolism
Synthesis
What signals are carried by
xylem-borne tZ versus phloemborne iP?
Conjugation
CK
Why is localized
CK synthesis
sometimes
critical and
sometimes not?
Transport
Perception
(receptor)
Are signals from
the three receptors
integrated or kept
separate?
What are the target
genes, and what do
they do?
TF activation/
inactivation
Target
genes
Biological
Functions
How do the type-A
and type-C ARRs
work? What is the
relationship with
CRFs?
How do all these
pieces fit together to
make a functioning
plant????
Adapted from Kieffer, M., Neve, J., and Kepinski, S. (2010). Defining auxin response contexts in plant development. Current Opinion in Plant Biology 13: 12-20.
© 2013 American Society of Plant Biologists