Auxinas

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Accción hormonal de las auxinas en plantas
¿Por qué y cuándo se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan y transportan?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
Accción hormonal de las auxinas en plantas
¿Por qué y cuándo se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan y transportan?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
¿Por qué y cuándo se descubren las auxinas?
Darwin (1890s) studied phototropism –
movement towards light
Darwin and others
studied coleoptiles –
tissues that protect
monocot leaves during
germination.
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Darwin, C., and Darwin, F. (1881) The power of movement in plants. Appleton and Co., New York.; Photos courtesy of Dr. R.L. Nielsen
¿Por qué y cuándo se descubren las auxinas?
Cutting off or covering the coleoptile tip
interferes with the response
+
Untreated
coleoptile
bends
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-
-
Coleoptiles with tips
shielded from light or
removed do not bend
These experiments showed
that the light signal is
perceived at the tip, although
the bending occurs at the
base.
¿Por qué y cuándo se descubren las auxinas?
Darwin concluded that a signal moves
from tip to base
“We must therefore conclude that when
seedlings are freely exposed to a lateral
light some influence is transmitted from
the upper to the lower part, causing the
latter to bend.”
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Charles Darwin image courtesy of Patche99z
¿Por qué y cuándo se descubren las auxinas?
Boysen-Jensen (1913) showed that the
transmitted influence can move
through a gelatin block
Before
After
The signal cannot
move through a
solid block or butter,
demonstrating that
it is a water- soluble
chemical.
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¿Por qué y cuándo se descubren las auxinas?
Repositioning the tip can induce
bending in uniform light
Tip removed and
replaced to one side
Tip
removed
Control
Before
Tip removed
and replaced
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Asymmetric tip placement
causes bending
Paal (1919) showed
that removing the tip
and replacing it on
one side of the base
is sufficient to cause
bending.
After
¿Por qué y cuándo se descubren las auxinas?
In the 1930s, auxin was purified and
shown to promote growth
Angle of
curvature is
proportional to
amount of
auxin in block
Frits Went collected auxin
from shoot tips into agar
blocks...
...and showed that
the material collected
in the agar blocks
was the growthpromoting substance.
This bending assay for the growthpromoting effect of auxin was used
as a basis for its purification.
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Redrawn from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1: 162-182.
Accción hormonal de las auxinas en plantas
¿Por qué y cuándo se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan y transportan?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
¿Qué metabolitos constituyen las auxinas?
Auxinas: estructura química
Went FW (1928) Wushstoff und wachstum. Rec Trav Bot Neerl 25: 1–116
Auxinas naturales
(libres o conjugadas)
IAA
IBA (indole butyric acid)
4-Cl-IAA (4-cloroindole-3-acetic)
PAA (phenylacetic acid)
Cl
4-Cl-Indole-3-acetic acid
Indole-3-acetic acid
Auxinas sintéticas
Derivadas de IAA
NAA (naphtyl-acetic acid)
Derivadas del PAA
2,4-D, 2,4,5-T
(otros efectos: herbicidas)
NAA
Antiauxinas: PCIB: 2-(p-chlorophenoxy)-2-isobutyric acid
Phenyl-acetic acid
Accción hormonal de las auxinas en plantas
¿Cuándo y por qué se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan y transportan?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
¿Qué procesos son regulados por auxinas?
Auxinas en plantas superiores
Procesos de Crecimiento y Desarrollo
- dominancia apical
- iniciación de raíces
- tropismos
- diferenciación de vasos
- fructificación
Fisiología Celular
- extrusión de protones
- división y elongación celular
- regulación de la expresión génica
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¿Qué procesos son regulados por auxinas?
Auxin’s role in apical dominance was
known in the 1930s
Bud Length
Decapitate
Replace apex
with agar
block: without
or with auxin.
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Auxin suppresses
bud outgrowth
No auxin
Auxin
Thimann, K.V., and Skoog, F. (1934). On the inhibition of bud development and other functions of growth substance in Vicia faba. Proceedings
of the Royal Society of London B. 114: 317-339 with permission; Went, F.W. and Thimann, K.V. (1937) Phytohormones. The Macmillan
Company, New York.
¿Qué procesos son regulados por auxinas?
Auxin’s root-promoting properties
were also known by the 1930s
Adventitious roots
are initiated from
grape stems treated
with auxin
A more recent experiment in
which radish roots were dipped
into auxin and initiated lateral
roots at a frequency proportional
to auxin concentration.
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µM
IAA
Thimann, K.V. (1938). Hormones and the analysis of growth. Plant Physiol. 13: 437-449.
Kerk, N.M., Jiang, K., and Feldman, L.J. (2000). Auxin metabolism in the root apical meristem. Plant Physiol. 122: 925-932.
¿Qué procesos son regulados por auxinas?
Gravitropism is a response to a change
in orientation relative to gravity
GRAVITY
VECTOR
Shoots are
negatively
gravitropic – they
grow away from the
center of gravity
vertical
Lower side
Roots are positively
gravitropic – they
grow towards the
center of gravity
horizontal
length
Upper side
time
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Luschnig, C., Gaxiola, R.A., Grisafi, P., and Fink, G.R. (1998) EIR1, a root-specific protein involved
in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev.12: 2175-2187.
Accción hormonal de las auxinas en plantas
¿Por qué y cuándo se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan y transportan?
¿Cómo se transportan a los tejidos diana?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
¿Cómo abordar el estudio de la acción de las auxinas?
Dissecting auxin action
Catabolism
Synthesis
IAA
Conjugation
Transport
Perception
(receptor)
TF activation/
inactivation
Target
genes
Biological
Functions
Auxin’s effects depend upon its synthesis, transport,
perception, signaling, and target gene responses. Most of
these functions are controlled by many genes with differing
cell specificities.
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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.
¿Cómo abordar el estudio de la acción de las auxinas?
Auxin response pathway regulation
-positive and negative feedback
(self-regulation)
-environmental factors
-hormone interaction
Conjugation
Catabolism
Synthesis
IAA
Transport
Perception
(receptor)
TF activation/
inactivation
Target
genes
Biological
Functions
Gravity, Nutrient
nutrient status, ionic
Ionic environment, pathogens,
Pathogens,
Light: directionality, intensity, wavelength
Ethylene, brassinosteroids, cytokinin, gibberellic acid, jasmonic acid, strigolactones....
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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.
¿Cómo abordar el estudio de la acción de las auxinas?
Tools in auxin research
•Auxin quantification from tissue or cell extracts
•Expression of auxin-induced genes
•In situ detection by auxin-specific antibodies
•Orientation of PIN auxin-transport proteins
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¿Cómo abordar el estudio de la acción de las auxinas?
Cell-specific auxin measurements
Auxin amount
The highest auxin
concentration is in
the quiescent
center (QC) of the
root apex.
Using this method, a high
resolution map has been
produced of auxin concentration
in the Arabidopsis root apex.
QC
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Petersson, S.V., Johansson, A.I., Kowalczyk, M., Makoveychuk, A., Wang, J.Y., Moritz, T., Grebe, M., Benfey, P.N., Sandberg, G., and
Ljung, K.(2009) An auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA
distribution and synthesis. Plant Cell 21:1659-1668.
¿Cómo abordar el estudio de la acción de las auxinas?
Cell-specific auxin measurements
Dissociate
cells and
isolate
protoplasts
Recently the resolution of
GC-MS measurements
have been greatly refined.
Separate into
single-cells
Laser
Charged plates deflect
cells into labeled and
unlabeled pools.
Start with roots
expressing GFP in
defined cell populations
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Petersson, S.V., Johansson, A.I., Kowalczyk, M., Makoveychuk, A., Wang, J.Y., Moritz, T., Grebe, M., Benfey, P.N., Sandberg, G., and
Ljung, K.(2009) An auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA
distribution and synthesis. Plant Cell 21:1659-1668.
¿Cómo abordar el estudio de la acción de las auxinas?
Chemical analysis of auxin levels from
plant extracts
Chemical analysis by gas
chromatography-mass
spectrometry (GC-MS) is an
important way to quantify auxin.
This experiment
shows an auxin
gradient from tip to
base in young
leaves but not
mature leaves.
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Edlund, A., Eklof, S., Sundberg, B., Moritz, T., and Sandberg, G. (1995) A microscale technique
for gas chromatography-mass spectrometry measurements of picogram amounts of indole-3acetic acid in plant tissues.Plant Physiol. 108: 1043-1047.
¿Cómo abordar el estudio de la acción de las auxinas?
Monitoring auxin response by reporter
gene transcription
GUS (or GFP)
TGTCTC
AuxRE
The DR5 reporter construct is
widely used to monitor auxin
response-level.
Hours of
staining
DR5 consists of seven repeats of
an auxin-response element
(AuxRE) which is a binding site
for auxin-responsive transcription
factors (ARFs).
Auxin-response level increases
with exogenous auxin (NAA)
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Ulmasov, T., Murfett, J., Hagen, G., and Guilfoyle, T.J. (1997). Aux/1AA proteins repress expression of reporter
genes containing natural and highly active synthetic auxin response elements. Plant Cell 9: 1963-1971.
¿Cómo abordar el estudio de la acción de las auxinas?
DR5 expression reveals high auxin
activity in the root apex and QC
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Reprinted by permission from Macmillan Publishers, Ltd. Blilou, I., et al. (2005) The PIN auxin
efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433: 39-44.
¿Cómo abordar el estudio de la acción de las auxinas?
Auxin can be detected by anti-auxin
antibodies
Auxin accumulation during lateral root initiation
DR5::GUS
Primary root
Anti-IAA antibody
Anti-IAA antibody
+ auxin
QC
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Negative
control
Reprinted from Benková, E. et al. (2003) Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell
115: 591-602, with permission from Elsevier.
Accción hormonal de las auxinas en plantas
¿Por qué y cuándo se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan
y transportan?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Auxin biosynthesis pathways
cloned from bacteria
cloned from plants
Indole-3-glycerolphosphate
tryptamine
FLOOZY
(Petunia)
TAA1
flavin
monooxygenase
tryptophan
aminotransferase
N-hydroxyl tryptamine
Indole-3-acetaldoxime
Indole-3-propionic acid
Indole-3-acetamide
SUR2
1-aci-Nitro-2-Indolylethane
Indole-3-acetonitrile
Indole-3-acetaldehyde
Indolic glucosinolates
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
yucca: un mutante de biosíntesis de IAA
obtenido por “activation tagging”
Zhao et al. (2001) Science 291:306
light
dark
roots
Wt
yucca
Wt
yucca
activation
tagging
yucca
“yucca”: un fenotipo indicativo de
un mutante relacionado con auxinas
Wt
Wt
yucca
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
yucca es un mutante con niveles más altos de IAA
Zhao et al. (2001) Science 291:307
MS (4weeks) MS+IPAR
IPAR = isopentenil
adenina ribósido
Wt
yucca
Wt
yucca
Supresión del fenotipo yucca
MS (7weeks) +IPAR
Wt
yucca
DR5-GUS = gen
delator
de auxinas
iaaL = gen
DR5-GUS
yucca-DR5-GUS
yucca
yucca-iaaL
iaaL
de enzima que
conjuga IAA y Lys
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Auxin synthesis is developmentally and
environmentally controlled
Methyl
Jasmonate
Ethylene
Red / Far-red light
ratio
Temperature
Auxin biosynthesis is
influenced by other hormones
and environmental conditions
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Sugawara, S., et al. (2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis.
Proc. Natl. Acad. Sci. 106: 5430-5435.
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
¿Funciones diferentes para las diferentes rutas?
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
¡¡¡ TAA1 y YUCCA constituyen una sola ruta !!!
24-OCT-2011
Mashiguchi et al.
Won et al.
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
TAA1 and YUC1 act synergistically
to enhance IAA biosynthesis in Arabidopsis.
Mashiguchi et al (2011) PNAS, 24 oct, early ed
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Localized auxin synthesis produces
developmentally important gradients
YUCCA1::GUS expression
DR5::GFP expression (auxininducible promoter)
The development of the Arabidopsis female gametophyte
involves an auxin-gradient, which is generated in part by
a gradient of auxin-biosynthesis.
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From Pagnussat, G.C., Alandete-Saez, M., Bowman, J.L., and Sundaresan, V. (2009) Auxin-dependent patterning and gamete specification
in the Arabidopsis female gametophyte.Science 324: 1684-1689. Reprinted with permission from AAAS.
¿Cómo se transportan a los tejidos diana?
Polar transport of auxin was
recognized in the 1930s
A segment cut from a
coleoptile can move
auxin from tip to base.
The upper agar block was
loaded with auxin, which
the segment translocated
to the lower block.
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When the segment is
inverted, it is unable to
transport auxin from
base to tip.
This experiment, carried out by
H.G. Van der Weij revealed
that auxin in the shoot is
translocated from tip to base.
Adapted from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1: 162-182.
¿Cómo se transportan a los tejidos diana?
Auxin transport – chemiosmotic model
Indole-3-acetic acid is a charged
anion (IAA-) in the cytoplasm (pH 7).
In the more acidic cell wall (pH 5.5)
some is uncharged (IAAH). The
uncharged form crosses the plasma
membrane into the cell where it is
deprotonated and unable to exit
other than through specific
transporters.
Cell wall
pH 5.5
A proton ATPase
maintains the
differential pH
gradient
Cytoplasm
pH 7
H+
IAA-
IAAH
IAA- + H+
Cell-to-cell
polar
auxin
transport
IAAH
IAA- + H+
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Redrawn from Robert, H.S., and Friml, J. (2009) Auxin and other signals on the move in plants. Nat. Chem. Biol. 5: 325-332.
¿Cómo se transportan a los tejidos diana?
Auxin moves through efflux and influx
carrier proteins
ABCB
ABCB
The AUX1/LAX
influx carriers
contribute to
movement of
IAAH into the
cytoplasm.
The PIN family of
proteins contributes to
directional movement
of auxin out of the cell.
Cell-to-cell
polar
auxin
transport
ABCB
The ABCB
transporters
contribute to auxin
transport in a diverse
ways. (These were formerly
referred to as MDR or PGP
proteins).
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Reprinted with permission from Macmillan Publishers, Ltd. Robert, H.S., and Friml, J. (2009)
Auxin and other signals on the move in plants. Nat. Chem. Biol. 5: 325-332.
¿Cómo se transportan a los tejidos diana?
Auxin transport: influx and efflux factors
- Influx carriers (4 members in Arabidopsis)
The most studied: AUX1
Related to a small subfamily of amino acid transporters,
the amino acid/auxin:proton symport permeases (AAAP),
which show homology to bacterial proteins of the
amino acid/polyamine/choline (APC) permease family,
- Efflux carriers
PIN family (8 members in Arabidopsis)
ABCB-type (ATP- binding casette subfamily B)
- Inhibitors
NPA
TIBA
1-naphthylphthalamic acid
blocks some aspect of the endocytic step in PIN protein cycling
2, 3, 5-triiodobenzoic acid
leads to an intracellular accumulation of PIN protein associated
with endosomes (Abas et al. 2006)
¿Cómo se transportan a los tejidos diana?
Auxin transport: influx and efflux
¿Cómo se transportan a los tejidos diana?
AUX1 takes up auxin into the cell
AUX1/LAX
Carriermediated
auxin
uptake
There are several
genes encoding
proteins like AUX1;
these are the LAX
proteins (Like AUX1).
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Auxin uptake is
disrupted in aux1
mutants.
Reprinted with permission from Macmillan Publishers, Ltd. Marchant, A. et al., (1999) AUX1 regulates root gravitropism in Arabidopsis by
facilitating auxin uptake within root apical tissues. EMBO J. 18: 2066–2073.
¿Cómo se transportan a los tejidos diana?
Auxin transport is facilitated by
AUX1/LAX proteins
In the aux1
mutant, auxin
transport is
disrupted, so
auxin moves
more slowly
from shoot to
root.
DAG = Days After Germination
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Marchant, A., Bhalerao, R., Casimiro, I., Eklof, J., Casero, P.J., Bennett, M., and Sandberg, G. (2002). AUX1 promotes lateral root formation by
facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling. Plant Cell 14: 589-597 Reprinted from
Muday, G.K., and DeLong, A. (2001). Polar auxin transport: Controlling where and how much. Trends Plant Sci. 6: 535–542, with permission
from Elsevier.
¿Cómo se transportan a los tejidos diana?
The ABCB proteins are important for
auxin transport
ABCB
PGP
WT
abcb19-2
abcb19abcb19-1 abcb1 2 –
abcb1
Arabidopsis encodes 21 ABCB
proteins, many of which are
necessary for proper auxin
transport.
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WT
abcb19-1
Loss-of-function of some ABCB
genes causes diverse
phenotypes including dwarfism
and altered tropisms.
Wu, G., Lewis, D.R., and Spalding, E.P. (2007) Mutations in Arabidopsis multidrug resistance-like ABC transporters separate the roles of
acropetal and basipetal auxin transport in lateral root development. Plant Cell 19: 1826–1837. Noh, B., Murphy, A.S., and Spalding, E.P. (2001)
Multidrug Resistance–like genes of Arabidopsis required for auxin transport and auxin-mediated development. Plant Cell 13: 2441–2454.
¿Cómo se transportan a los tejidos diana?
The PIN proteins are named for the pinformed mutant
pin-formed, which has a
mutation in the PIN1 gene,
makes some abnormal leaves
and then a bare inflorescence.
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Galweiler, L., Guan, C., Muller, A., Wisman, E., Mendgen, K., Yephremov, A., and Palme, K. (1998). Regulation of polar auxin transport by
AtPIN1 in Arabidopsis vascular tissue. Science 282: 2226 – 2230, reprinted with permission from AAAS.
¿Cómo se transportan a los tejidos diana?
Exon-intron structure of the eight AtPINs
in Arabidopsis
Transmembrane domains
Translated exon sequence
Chromosomal location and duplication events
-recent
- old
647 aa
351 aa
Paponov et al (2005) TIPS 10:171
¿Cómo se transportan a los tejidos diana?
Auxin gradients and transport during
embryogenesis
Petrasek et al (2009) Development 136: 2681
¿Cómo se transportan a los tejidos diana?
The distribution of PIN proteins
contributes to the auxin gradients
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Křeček , P., Skůpa , P., Libus, J., Naramoto, S., Tejos, R., Friml J., and Zažímalová, E. (2009) The
PIN-FORMED (PIN) protein family of auxin transporters. Genome Biology 10: 249.
¿Cómo se transportan a los tejidos diana?
Immunolocalization of the AtPINs
a) Columnella cell layer
b) + quiescent centre
c) + quiescent centre
d) More diffuse
Columbia
e-f)
More expression
No change in localization
g-h)
NPA affects PIN4 localization
Do PIN4 compensate de loss
of function of PIN1 by NAPA?
i) At he bottom of each cell
j) At the bottom in cortical cells
At the top in the epidermal layer
k-l-m) low level expression in
the epidermis
n-o-p) same pattern of expression
(Usually PIN1)
Untreated
seedlings
4h NPA
¿Cómo se transportan a los tejidos diana?
PIN proteins cycle between plasma membrane and
endosome --- Brefeldin A blocks endocytosis and
traps PIN proteins in endosomes
endosome
Plasma
membrane
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Redrawn from Kleine-Vehn, J., et al. (2009). PIN auxin efflux carrier polarity is regulated by PINOID kinasemediated recruitment into GNOM-independent trafficking in Arabidopsis. Plant Cell 21: 3839-3849.
¿Cómo se transportan a los tejidos diana?
Auxin transport - summary
Regulated movement of auxin is critical for
phototropism, gravitropism, embryonic
pattern formation and organogenesis.
Auxin transport proteins have been
identified. Factors that control their position
and activity are under investigation.
Arabidopsis has 6 AUX1/LAX
proteins, 21 ABCB proteins
and 8 PIN proteins - we don’t
yet understand all the
complexity of auxin transport!
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¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Auxin homeostasis - summary
Catabolism
Synthesis
IAA
Conjugation
Transport
Auxin homeostasis
is maintained by its
synthesis
catabolism
conjugation
transport
All of these processes
are tightly regulated
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Accción hormonal de las auxinas en plantas
¿Por qué y cuándo se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan y transportan?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
¿Cómo se perciben las auxinas?
Aproximaciones en la búsqueda de receptores
Estrategia BIOQUÍMICA:
- utilización de hormonas marcadas
- mezcla con extractos proteicos
- purificación de fracciones marcadas
- aislamiento del receptor
- Análisis de la secuencia N-terminal
- Construcción de oligonucleótidos
- Rastreo de una biblioteca de cDNA
- Secuenciación de clones positivos
ABP
maíz
Woo et al (2002) EMBO J 21:2877
Estrategia GENÉTICA:
- generación de mutantes (de pérdida o ganancia de función)
- selección por alteración de la respuesta hormonal
TIR1
- localización de la mutación en el genoma
At
¿Cómo se perciben las auxinas?
Auxin Perception: Two receptors?
1972
Perception
(receptor)
ABP1
Hertel R, Thomson KS, Russo VEA
In vitro auxin binding to particulate
cell fractions from corn coleoptiles
Planta 107: 325-340
IAA
2005
Perception
(receptor)
SCFTIR1
Stefan Kepinski & Ottoline Leyser
The Arabidopsis F-box protein TIR1
is an auxin receptor
Nihal Dharmasiri, Sunethra Dharmasiri & Mark Estelle
The F-box protein TIR1 is an auxin receptor
Nature 26 de mayo
¿Cómo se perciben las auxinas?
Aislamiento de la auxin-binding protein
(ABP) de maíz
-
Purificación: Mr: 40-45 kD (20 kD)
Análisis de la secuencia N-terminal
Construcción de oligonucleótidos
Rastreo de una biblioteca de cDNA
Secuenciación de clones positivos
Estructura primaria de la proteína
Análisis de la estructura
10
21
32
42
51
63
80
Woo et al. (2002) EMBO J 21:2877-2885,
92
104
117
151
163
NH2-
-COOH
GQRTPIHRHSCEE
auxin-binding domain
- Problemas pendientes: localización y función
WDEDCFEAAKDEL
K-channel domain
¿Cómo se perciben las auxinas?
AUXIN-BINDING Protein1 is necessary
for cell division and expansion
Wild-type
control
EtOHinduced
ABP1antisense
Loss-of-function of ABP1 is
embryo-lethal.
Plants were produced that
carry an inducible antisense
construct that silences the
ABP1 gene. They were
grown for 12 days in noninductive conditions.
At day 12, ABP1 was
silenced and plant growth
was severely restricted.
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Braun, N., et al. (2008). Conditional repression of AUXIN BINDING PROTEIN1
reveals that it coordinates cell division and cell expansion during postembryonic
shoot development in Arabidopsis and tobacco. Plant Cell 20: 2746-2762.
¿Cómo se perciben las auxinas?
Elementos de la respuesta a auxinas
Two families of transcription factors,
> Aux/IAA proteins, exert their effects by binding to the ARF proteins.
The effect of Aux/IAA binding is to repress transcription
> ARF (auxin response factors), bind DNA directly and either
activate or repress transcription depending on the ARF
Complejo SCF-TIR1: ubiquitinación de Aux/IAA (en presencia de IAA)
> SCF: SKP1, Cullin and F-box protein.
> TIR1 (F-box): Transport Inhibitor Response1 (tir no responden a auxinas)
La interacción SCF-Aux/IAA no se debe a etiquetado del sustrato
(fosforilación, glicosilación ...) sino a “modificación” en TIR1
¿Cómo promueven las auxinas la interacción SCF-Aux/IAA)?
¿Cómo se perciben las auxinas?
Aux/IAA genes
Aislamiento
- Inducción rápida tras tratamiento con IAA
- Similaridad de secuencia con Aux/IAA previos
- En ensayos de doble híbrido con proteínas Aux/IAA
Fuentes (probablemente sólo en plantas)
- dicots: guisante, soja, M truncatula, A thaliana (25), tomate, tabaco, algodón ...
- monocots: maíz, arroz ...
- árboles: pino ...
-Localización nuclear de la proteína
Estructura
- Dominios conservados
I
Mutaciones en el dominio II
las hacen “indegradales”
II
Inestabilidad
III
IV
Dimerización
(con Aux/IAA y ARF)
¿Cómo se perciben las auxinas?
TIR1 binds to the conserved degron in
domain II
I
II
III
QVVGWPLVRSYRK
I
II
III
QVVGWPPVRSYRK
Wild-type degron
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IV
Mutant degron
IV
Mutating the degron
prevents the Aux/IAA
protein from binding to
TIR1 and being
subsequently proteolyzed;
the mutant proteins are
more stable.
This image shows the crystal
structure of TIR1 complexed
with auxin and the domain II
region of an Aux/IAA protein.
¿Cómo se perciben las auxinas?
Mutations in some Aux/IAA genes cause
an auxin-resistant phenotype
Dominant, gain-of-function mutations in
Aux/IAA genes all map to the conserved
degron sequence in domain II
I
II
III
IV
QVVGWPPVRSYRK
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Nagpal, P., et al. (2000) AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol, 123: 563-574.
¿Cómo se perciben las auxinas?
Aux/IAA genes are induced by auxin
In the 1980’s, the new
techniques of molecular
biology allowed scientists to
isolate genes that were
strongly and rapidly induced
by auxin.
One family of these auxininduced genes is known as
the Aux/IAA genes.
GmAux22
GmAux28
Control
15
30
60
90
Auxin treatment (mins)
Auxin
depleted
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Walker, J.C. and Key, J.L. (1982) Isolation of cloned cDNAs to auxin-responsive poly(A)+RNAs of
elongating soybean hypocotyl. PNAS 79: 7185-7189.
¿Cómo se perciben las auxinas?
Aux/IAA proteins are very short-lived
nuclear proteins
Many of the
Aux/IAA proteins
have a very
short half-life,
suggesting a
regulatory
function.
A fusion
protein with
GUS shows
nuclear
accumulation
PS-IAA6
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IN PLANT BIOLOGY
Abel, S., Oeller, P.W., and Theologis, A. (1994). Early auxin-induced genes encode short-lived nuclear proteins. PNAS 91: 326-330.
¿Cómo se perciben las auxinas?
ARF (auxin response factors)
Características
- Familia de 23 genes en Arabidopsis
- Dominios C-terminales homógos al III y IV de las proteínasAux/IAA
III
Unión a DNA
(Activación de la transcripción)
IV
Dimerización
- Se unen a los dominios TGTCTC AuxREs (específico de plantas)
- Activan o reprimen la transcripción por unión
directa a los sitios TGTCTC
con otras ARFs o Aux/IAAA que no están unidas al DNA
¿Cómo se perciben las auxinas?
ARFs are transcription factors that
bind auxin-inducible promoters
Increasing amounts of
ARF1
DNase footprint
analysis showing
ARF1 binding to an
auxin-response
element (AuxRE)
In DNase footprinting, the
site at which a protein binds
to DNA can be identified
because that region is
protected from DNase
endonuclease digestion.
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Reprinted from Ulmasov, T., Hagen, G., and Guilfoyle, T. (1997). ARF1, a transcription factor that binds to auxin response elements.
Science 276: 1865 – 1868 with permission from AAAS.
¿Cómo se perciben las auxinas?
Some ARFs are transcriptional
activators and some repressors
The Arabidopsis
ARF protein family
Confirmed
activators
ARF 2-4, 9 are
confirmed
repressors
We don’t know much about
the ARF repressors yet.
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Reprinted from Guilfoyle, T., and Hagen, G. (2007). Auxin response factors.
Curr. Opin. Plant Biol. 10: 453 – 460 with permission from Elsevier.
¿Cómo se perciben las auxinas?
Aux/IAA proteins repress the activity of
Auxin Response Factors (ARFs)
ARF and Aux/IAA proteins have
sequence similarity in their Cterminal regions through which
they can homodimerize.
ARF
DNAbinding
domain
Activation /
repression
domain
ARF homodimer
ARF Aux/IAA heterodimer
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IV
Aux/IAA
I
Teaching Tools
III
II
III
IV
¿Cómo se perciben las auxinas?
Model for auxin-regulated
transcriptional control
Low auxin level
Some ARFs are
complexed with
Aux/IAA repressors,
interfering with their
action
High auxin level
Aux/IAAs are
degraded by SCFTIR1mediated proteolysis,
relieving repression of
ARFs to activate or
repress gene
expression.
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¿Cómo se perciben las auxinas?
The auxin signaling pathway is
amazingly short!
Auxin
INPUT
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OUTPUT
¿Cómo se perciben las auxinas?
Perhaps the diversity of auxin
responses is correlated with the
diversity in signaling proteins
Arabidopsis has 29
Aux/IAA proteins
Auxin
Arabidopsis has 23
ARF proteins
OUTPUT
INPUT
TIR1 is related to
five other auxinreceptor proteins,
AFB1 – AFB5.
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The number of potential
interactions between
ARFs and Aux/IAA
proteins is quite large,
and has the potential to
precisely control auxininduced gene
expression in a vast
number of
combinations.
¿Cómo se perciben las auxinas?
F-box-proteins
>700 in Arabidopsis
TIR1
AFB1
}
AUXIN
SIGNALING
F-BOX
AFB5
COI1
(jasmónico)
74 in humans
53 in mice
Dharmasiri et al. (2005) Developmental Cell 9:109–119
¿Cómo se perciben las auxinas?
Ensayos con 3H-IAA
Binding to proteins
Auxin binds directly to SCF-TIR1
positive signal in assays with GST-IAA7
negative signal in assays with GST-AXR2-1
(que no se une a SCF-TIR1)
Competición con auxinas frías
benzoic acid
L-tryptophan
IC50
2,4-D,
1-NAA
IAA
(mM)
1.4
1.3
0.12
(valores obtenidos con extractos crudos)
¿Cómo se perciben las auxinas?
Inhibition of auxin signaling: terfestatin A
Yamazoe et al (2005) Plant Phys 139:779
Terfestatin A (TrfA): terphenyl-b-glucoside
- isolated from Streptomyces sp. F40
screen for compounds that inhibit the expression of
auxin-inducible genes in Arabidopsis
- specifically inhibited the expression of primary auxin-inducible genes in roots,
did not affect abscisic acid and cytokinin related-genes.
- blocked the auxin-enhanced degradation of Aux/IAA repressor proteins
without affecting interaction between Aux/IAAs and TIR1.
- TrfA treatment antagonized auxin responses in roots
primary root inhibition,
lateral root initiation,
root hair promotion,
and root gravitropism,
- only limited effects on shoot auxin responses.
TrfA acts as a modulator of Aux/IAA stability
¿Cómo se perciben las auxinas?
terfestatin A: effects on root growth
Yamazoe et al (2005) Plant Phys 139:779
¿Cómo se perciben las auxinas?
2-FAA is a potent inhibitor of
auxin-mediated events
Sungur et al (2007) Plant Cell Physiol. 48: 1693
Compound A (A) = 2-hydroxy-N-(5-propyl-1,3,4-thiadiazol-2-yl) acetamide (HTA)
+ 2-furylacrylic acid (2-FAA)
BA3–GUS seedlings
(GUS activity for 12 h)
mock
5 mM IAA
(2h)
5 mM IAA
+ 5 mM A
5 mM IAA
+ 10 mM HTA
5 mM IAA
+ 5 mM 2-FAA
Accción hormonal de las auxinas en plantas
¿Por qué y cuándo se descubren las auxinas?
¿Qué metabolitos constituyen las auxinas?
¿Qué procesos son regulados por las auxinas?
¿Cómo abordar el estudio de la acción de las auxinas?
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
¿Cómo se transportan a los tejidos diana?
¿Cómo se perciben las auxinas?
¿Qué queda por hacer?
¿Qué queda por hacer?
Auxin action in whole-plant processes
Auxin in
action!
Promote lateral organ
initiation at the shoot
apical meristem
Light responses
Cell elongation
Responses to pathogens
Inhibit
branching in
the shoot
Integrate growth
signaling pathways
Control patterning and vascular
development
Maintain stemPromote
cell fate at the
branching
in the root
Integrate growth root apical
meristem
Response to nutrient
signaling pathways
distribution and
Responses to symbionts –
abundance
nodule formation
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IN PLANT BIOLOGY
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.
¿Qué queda por hacer?
How does auxin affect so many processes in
plant growth and development?
Perception
(receptor)
The answer is not immediately
obvious.....
Some auxin-regulated genes
are clearly connected with
downstream functions, others
are more difficult to interpret.
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Target
genes
Auxin-induced gene products
Aux/IAA proteins
GH3 (auxin conjugation)
ACS (ethylene synthesis)
Jasmonic acid synthesis
CKX (cytokinin degradation)
Transcription factors
Auxin-repressed gene
products
Cell wall proteins
ARRs (Cytokinin signaling)
Transcription factors
Biological
Functions
¿Qué queda por hacer?
Ongoing investigations
Where does auxin
elimination fit in?
Catabolism
Synthesis
Conjugation
IAA
What regulates
auxin synthesis?
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What do all those transport
proteins do? What controls their
activity and distribution??
Transport
Perception
(receptor)
What are the other
TIR1 like proteins
doing, and what
does ABP1 do?
TF activation/
inactivation
Why so many
ARFs and
Aux/IAAs?
What are the target
genes, and what do
they do?
Target
genes
Biological
Functions
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.
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Auxin biosynthesis: novel diagram
Tao et al (2008) Cell 133: 171
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Biosynthesis and homeostasis
IAA is produced from
tryptophan (Trp) via several
semi-independent pathways and
one Trp-independent pathway
(blue arrow).
Mutations inYUCCA
genes (circled) affect
embryogenesis and in
TAA1 genes affect
environmental
responses.
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IN PLANT BIOLOGY
The IAOx pathway
may be restricted
to Arabidopsis and
its close relatives.
Sugawara, S., et al. (2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis.
Proc. Natl. Acad. Sci. 106: 5430-5435.
¿Cómo se perciben las auxinas?
La respuesta a auxinas: mecanismo de acción
hormonal: proteolisis de elementos de respuesta
Aux/IAA
Aux/IAA
Auxin
No hay fosforilación
> Fosforilación
de Aux/IAA
> Ubiquitinación
Aux/IAA
Aux/IAA
> Proteolisis
Aux/IAA
> Desbloqueo de ARF
> Inducción de procesos
> Inducción de Aux/IAA
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Auxin homeostasis is also controlled by
conjugation and degradation
Overexpression of an auxin conjugating enzyme
encoded by a GH3 gene reduces auxin levels in
the plant and causes a dwarfed phenotype.
IAA
GH3 genes
are auxininduced
Rice
(GH3 genes)
Arabidopsis
Wild-type
GH3.13
overexpression
Wild-type
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GH3.13
overexpression
Zhang, S.-W., et al., (2009) Altered architecture and enhanced drought tolerance in rice via the cown-regulation of indole-3-acetic acid by
TLD1/OsGH3.13 activation. Plant Physiol. 151:1889-1901. Staswick, P.E., et al., (2005) Characterization of an Arabidopsis enzyme family that
conjugates amino acids to indole-3-acetic acid. Plant Cell 17: 616-627.
¿Cómo se transportan a los tejidos diana?
Polar Auxin Transport
Auxin moves long distances
through the phloem.
Auxin also moves via auxin
transport proteins.
Auxin normally moves from the tip
of the shoot towards the tip of the
root. At the root tip, auxin changes
direction and moves short
distances up the root again
(basipetally).
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Reprinted with permission from Macmillan Publishers, Ltd. Robert, H.S., and Friml, J. (2009) Auxin and other signals on the move in plants.
Nat. Chem. Biol. 5: 325-332. Reprinted from Muday, G.K., and DeLong, A. (2001). Polar auxin transport: Controlling where and how much.
Trends Plant Sci. 6: 535–542, with permission from Elsevier.
¿Cómo se transportan a los tejidos diana?
Auxin transport in the root tip
AUX1
Petrasek et al (2009) Development 136: 2682
PIN1
PIN2
PIN4
PIN3 PIN7
¿Cómo se transportan a los tejidos diana?
Auxin transport in developing lateral roots
Petrasek et al (2009) Development 136: 2682
¿Cómo se perciben las auxinas?
Detección de complejos TIR1-Aux/IAA:
Pull-down experiments
Interacción
> Aux/IAA con TIR1
Dos tipos de Aux/IAA
> GST-IAA7
> biotinylated 17 aa peptide domain II
TIR1
Detección:
Estelle
Leyser
> extracts from tir1-1 GVG-TIR1-myc seedlings
anti-myc antibody
Hay interacción si se forman complejos proteína (GST/biotina)
que se detectan con anti-myc antibody
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