accción hormonal de las auxinas en plantas ¿por qué y cuándo se descubren las auxinas? ¿qué...
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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?
Darwin (1890s) studied phototropism – movement towards light
Darwin, C., and Darwin, F. (1881) The power of movement in plants. Appleton and Co., New York.; Photos courtesy of Dr. R.L. Nielsen
Darwin and others studied coleoptiles –tissues that protect monocot leaves during germination.
Teaching ToolsIN PLANT BIOLOGY
¿Por qué y cuándo se descubren las auxinas?
Cutting off or covering the coleoptile tip interferes with the response
These experiments showed that the light signal is perceived at the tip, although the bending occurs at the base.
Untreated coleoptile bends
Coleoptiles with tips shielded from light or removed do not bend
Teaching ToolsIN PLANT BIOLOGY
¿Por qué y cuándo se descubren las auxinas?
+ --
Darwin concluded that a signal moves from tip to base
Charles Darwin image courtesy of Patche99z
“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.”
Teaching ToolsIN PLANT BIOLOGY
¿Por qué y cuándo se descubren las auxinas?
Boysen-Jensen (1913) showed that the transmitted influence can move
through a gelatin block
Before
The signal cannot move through a solid block or butter, demonstrating that it is a water- soluble chemical.
After
Teaching ToolsIN PLANT BIOLOGY
¿Por qué y cuándo se descubren las auxinas?
Repositioning the tip can induce bending in uniform light
Teaching ToolsIN PLANT BIOLOGY
Tip removed and replaced to one side
BeforeControl
Tip removed
Tip removed and replaced
¿Por qué y cuándo se descubren las auxinas?
Paal (1919) showed that removing the tip and replacing it on one side of the base is sufficient to cause bending.
After
Asymmetric tip placement causes bending
In the 1930s, auxin was purified and shown to promote growth
Redrawn from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1: 162-182.
Frits Went collected auxin from shoot tips into agar blocks...
...and showed that the material collected in the agar blocks was the growth-promoting substance.
This bending assay for the growth-promoting effect of auxin was used as a basis for its purification.
Angle of curvature is proportional to amount of auxin in block
Teaching ToolsIN PLANT BIOLOGY
¿Por qué y cuándo se descubren las auxinas?
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?
Auxinas: estructura química
¿Qué metabolitos constituyen las auxinas?
Went FW (1928) Wushstoff und wachstum. Rec Trav Bot Neerl 25: 1–116
Auxinas sintéticasDerivadas de IAA NAA (naphtyl-acetic acid)
Derivadas del PAA 2,4-D, 2,4,5-T(otros efectos: herbicidas)
Antiauxinas: PCIB: 2-(p-chlorophenoxy)-2-isobutyric acid
NAA
Auxinas naturales(libres o conjugadas)IAAIBA (indole butyric acid)
4-Cl-IAA (4-cloroindole-3-acetic)
PAA (phenylacetic acid)Indole-3-acetic acid Phenyl-acetic acid
4-Cl-Indole-3-acetic acid
Cl
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?
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
Teaching ToolsIN PLANT BIOLOGY
¿Qué procesos son regulados por auxinas?
Auxin’s role in apical dominance was known in the 1930s
DecapitateReplace apex with agar block: without or with auxin.
Auxin suppresses bud outgrowth
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.
No auxin
Auxin
Bu
d L
eng
th
Teaching ToolsIN PLANT BIOLOGY
¿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
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.
A more recent experiment in which radish roots were dipped
into auxin and initiated lateral roots at a frequency proportional
to auxin concentration. µM IAA
Teaching ToolsIN PLANT BIOLOGY
Teaching ToolsIN PLANT BIOLOGY
¿Qué procesos son regulados por auxinas?
Gravitropism is a response to a change in orientation relative to gravity
Shoots are negatively
gravitropic – they grow away from the
center of gravity
Roots are positively gravitropic – they grow towards the center of gravity
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.
Teaching ToolsIN PLANT BIOLOGY
vertical
GRAVITY VECTOR
horizontal
time
length
Lower side
Upper side
¿Qué procesos son regulados por auxinas?
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?
Dissecting auxin action
TransportSynthesis IAA
Catabolism Conjugation
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.
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo abordar el estudio de la acción de las auxinas?
Synthesis IAA Transport Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism Conjugation
Synthesis IAA Transport Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism Conjugation
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. Teaching Tools
IN PLANT BIOLOGY
Gravity, Nutrient status, Ionic environment, Pathogens, Light: directionality, intensity, wavelength
Gravity, nutrient status, ionic environment, pathogens, Light: directionality, intensity, wavelength
Ethylene, brassinosteroids, cytokinin, gibberellic acid, jasmonic acid, strigolactones....
-positive and negative feedback(self-regulation)
-environmental factors-hormone interaction
Auxin response pathway regulation
¿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
Teaching ToolsIN PLANT BIOLOGY
¿Cómo abordar el estudio de la acción de las auxinas?
Auxin amount
Cell-specific auxin measurements
Using this method, a high resolution map has been produced of auxin concentration in the Arabidopsis root apex.
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.
The highest auxin concentration is in the quiescent center (QC) of the root apex.
QC
Teaching ToolsIN PLANT BIOLOGY
¿Cómo abordar el estudio de la acción de las auxinas?
Cell-specific auxin measurements
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.
Laser
Charged plates deflect cells into labeled and unlabeled pools.
Start with roots expressing GFP in defined cell populations
Dissociate cells and isolate protoplasts
Separate into single-cells
Recently the resolution of GC-MS measurements have been greatly refined.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo abordar el estudio de la acción de las auxinas?
Chemical analysis of auxin levels from plant extracts
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-3-acetic acid in plant tissues.Plant Physiol. 108: 1043-1047.
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo abordar el estudio de la acción de las auxinas?
Monitoring auxin response by reporter gene transcription
TGTCTC GUS (or GFP)
The DR5 reporter construct is widely used to monitor auxin response-level.
DR5 consists of seven repeats of an auxin-response element (AuxRE) which is a binding site for auxin-responsive transcription factors (ARFs).
AuxRE
Hours of staining
Auxin-response level increases with exogenous auxin (NAA)
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.
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¿Cómo abordar el estudio de la acción de las auxinas?
DR5 expression reveals high auxin activity in the root apex and QC
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo abordar el estudio de la acción de las auxinas?
Auxin can be detected by anti-auxin antibodies
DR5::GUSAnti-IAA antibody
+ auxin
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.
Auxin accumulation during lateral root initiation
Negative control
Anti-IAA antibody
QC
Primary root
Teaching ToolsIN PLANT BIOLOGY
¿Cómo abordar el estudio de la acción de las auxinas?
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?
Auxin biosynthesis pathways
cloned from bacteria
cloned from plants
SUR2
Indole-3-glycerolphosphate
tryptamine
tryptophan aminotransferase
N-hydroxyl tryptamineIndole-3-acetaldoximeIndole-3-propionic acidIndole-3-acetamide
1-aci-Nitro-2-IndolylethaneIndole-3-acetonitrileIndole-3-acetaldehyde
Indolic glucosinolates
FLOOZY(Petunia)
TAA1
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
flavin monooxygenase
yucca: un mutante de biosíntesis de IAAobtenido por “activation tagging”
Wt yucca Wt yucca
roots
light dark
yucca
Wt
Wt yucca
activationtagging
“yucca”: un fenotipo indicativo de
un mutante relacionado con auxinas
Zhao et al. (2001) Science 291:306
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
yucca es un mutante con niveles más altos de IAA
IPAR = isopenteniladenina ribósido
DR5-GUS = gendelator
de auxinas
iaaL = gende enzima que
conjuga IAA y Lys
Zhao et al. (2001) Science 291:307
Wt
yucca Wt yucca
Wt yucca
MS (4weeks) MS+IPAR
MS (7weeks) +IPAR
yucca-DR5-GUSDR5-GUS yucca yucca-iaaL iaaL
Supresión del fenotipo yucca
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Auxin synthesis is developmentally and environmentally controlled
Auxin biosynthesis is influenced by other hormones and environmental conditions
Sugawara, S., et al. (2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis. Proc. Natl. Acad. Sci. 106: 5430-5435.
Ethylene
Methyl Jasmonate
Red / Far-red light ratio
Temperature
Teaching ToolsIN PLANT BIOLOGY
¿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?
24-OCT-2011
¡¡¡ TAA1 y YUCCA constituyen una sola ruta !!!
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Mashiguchi et al.Won et al.
Mashiguchi et al (2011) PNAS, 24 oct, early ed
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
TAA1 and YUC1 act synergistically to enhance IAA biosynthesis in Arabidopsis.
Localized auxin synthesis produces developmentally important gradients
YUCCA1::GUS expression DR5::GFP expression (auxin-inducible promoter)
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.
The development of the Arabidopsis female gametophyte involves an auxin-gradient, which is generated in part by a gradient of auxin-biosynthesis.
Teaching ToolsIN PLANT BIOLOGY
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
Polar transport of auxin was recognized in the 1930s
Adapted from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1: 162-182.
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
This experiment, carried out by H.G. Van der Weij revealed that auxin in the shoot is translocated from tip to base.
When the segment is inverted, it is unable to transport auxin from base to tip.
Auxin transport – chemiosmotic model
Redrawn from Robert, H.S., and Friml, J. (2009) Auxin and other signals on the move in plants. Nat. Chem. Biol. 5: 325-332.
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-to-cell polar auxin transport
IAA- + H+IAAH
Cytoplasm pH 7
IAA-
IAAH
IAA- + H+
Cell wall pH 5.5
A proton ATPase maintains the differential pH gradient
H+
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
Auxin moves through efflux and influx carrier proteins
Cell-to-cell polar auxin transportThe 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.
The ABCB transporters contribute to auxin transport in a diverse ways. (These were formerly referred to as MDR or PGP proteins).
ABCB
ABCB
ABCB
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
Auxin transport: influx and efflux factors
- Influx carriers (4 members in Arabidopsis)
The most studied: AUX1Related 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 1-naphthylphthalamic acid blocks some aspect of the endocytic step in PIN protein cycling
TIBA 2, 3, 5-triiodobenzoic acid leads to an intracellular accumulation of PIN protein associatedwith 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
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.
AUX1/LAX
There are several genes encoding proteins like AUX1; these are the LAX proteins (Like AUX1).
Auxin uptake is disrupted in aux1
mutants.
Carrier-mediated
auxin uptake
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
Auxin transport is facilitated by AUX1/LAX proteins
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.
DAG = Days After Germination
In the aux1 mutant, auxin transport is disrupted, so auxin moves more slowly from shoot to root.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
The ABCB proteins are important for auxin transport
WT abcb19-1
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.
PGP
Loss-of-function of some ABCB genes causes diverse phenotypes including dwarfism and altered tropisms.
Arabidopsis encodes 21 ABCB proteins, many of which are necessary for proper auxin transport.
abcb1abcb19-1abcb19-2 abcb19-
2 – abcb1
WT
ABCB
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
The PIN proteins are named for the pin-formed mutant
pin-formed, which has a mutation in the PIN1 gene, makes some abnormal leaves and then a bare inflorescence.
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
Exon-intron structure of the eight AtPINsin Arabidopsis
Paponov et al (2005) TIPS 10:171
Transmembrane domains Translated exon sequence Chromosomal location and duplication events -recent- old
351 aa
647 aa
¿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
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
Immunolocalization of the AtPINs
Untreatedseedlings
4h NPA
(Usually PIN1)
e-f)More expressionNo change in localization
g-h)NPA affects PIN4 localizationDo PIN4 compensate de lossof function of PIN1 by NAPA?
i) At he bottom of each cellj) 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
a) Columnella cell layerb) + quiescent centrec) + quiescent centred) More diffuse
Columbia
¿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
Redrawn from Kleine-Vehn, J., et al. (2009). PIN auxin efflux carrier polarity is regulated by PINOID kinase-mediated recruitment into GNOM-independent trafficking in Arabidopsis. Plant Cell 21: 3839-3849.Teaching Tools
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¿Cómo se transportan a los tejidos diana?
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!
Teaching ToolsIN PLANT BIOLOGY
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.
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.
Auxin transport - summary
¿Cómo se transportan a los tejidos diana?
Auxin homeostasis - summary
Synthesis IAA
Catabolism ConjugationAuxin homeostasis is maintained by its
synthesiscatabolism conjugationtransport
All of these processes are tightly regulated
Transport
Teaching ToolsIN PLANT BIOLOGY
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
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?
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
Estrategia GENÉTICA: - generación de mutantes (de pérdida o ganancia de función)- selección por alteración de la respuesta hormonal- localización de la mutación en el genoma TIR1
At
ABPmaíz
Woo et al (2002) EMBO J 21:2877
¿Cómo se perciben las auxinas?
Auxin Perception: Two receptors?
1972 Hertel R, Thomson KS, Russo VEAIn vitro auxin binding to particulate cell fractions from corn coleoptilesPlanta 107: 325-340
IAA
Perception (receptor)
Perception (receptor)
ABP1
SCFTIR1
2005
Stefan Kepinski & Ottoline LeyserThe Arabidopsis F-box protein TIR1 is an auxin receptorNihal Dharmasiri, Sunethra Dharmasiri & Mark EstelleThe F-box protein TIR1 is an auxin receptorNature 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 92 104 117 151 163
GQRTPIHRHSCEE WDEDCFEAAKDEL
-COOHNH2-
- Problemas pendientes: localización y función
auxin-binding domain K-channel domain
Woo et al. (2002) EMBO J 21:2877-2885,
¿Cómo se perciben las auxinas?
AUXIN-BINDING Protein1 is necessary for cell division and expansion
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.
Wild-type control
EtOH-induced
ABP1-antisense
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 non-inductive conditions.
At day 12, ABP1 was silenced and plant growth was severely restricted.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las 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 eitheractivate 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)?
Elementos de la respuesta a auxinas
¿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
Inestabilidad Dimerización (con Aux/IAA y ARF)
I II III IV
Mutaciones en el dominio II las hacen “indegradales”
¿Cómo se perciben las auxinas?
This image shows the crystal structure of TIR1 complexed with auxin and the domain II region of an Aux/IAA protein.
TIR1 binds to the conserved degron in domain II
I II III IV
QVVGWPLVRSYRK
I II III IV
QVVGWPPVRSYRK
Wild-type degron
Mutant degron
I II III IV
I II III IV
Mutating the degron prevents the Aux/IAA protein from binding to TIR1 and being subsequently proteolyzed; the mutant proteins are more stable.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las auxinas?
Mutations in some Aux/IAA genes cause an auxin-resistant phenotype
Nagpal, P., et al. (2000) AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol, 123: 563-574.
Dominant, gain-of-function mutations in Aux/IAA genes all map to the conserved degron sequence in domain II
I II III IV
QVVGWPPVRSYRK
I II III IV
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las auxinas?
Aux/IAA genes are induced by auxin
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.
Control
Auxin depleted
Auxin treatment (mins)
15 30 60 90
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 auxin-induced genes is known as the Aux/IAA genes.
GmAux22
GmAux28
Teaching ToolsIN PLANT BIOLOGY
Auxin treatment (mins)
¿Cómo se perciben las auxinas?
Aux/IAA proteins are very short-lived nuclear proteins
PS-IAA6
Many of the Aux/IAA proteins have a very short half-life, suggesting a regulatory function.
Abel, S., Oeller, P.W., and Theologis, A. (1994). Early auxin-induced genes encode short-lived nuclear proteins. PNAS 91: 326-330.
A fusion protein with GUS shows
nuclear accumulation
Teaching ToolsIN PLANT BIOLOGY
¿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
Unión a DNA (Activación de la transcripción) Dimerización
III IV
- Se unen a los dominios TGTCTC AuxREs (específico de plantas)- Activan o reprimen la transcripción por unión
directa a los sitios TGTCTCcon 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
DNase footprint analysis showing ARF1 binding to an auxin-response element (AuxRE)
Increasing amounts of ARF1
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.
In DNase footprinting, the site at which a protein binds to DNA can be identified because that region is protected from DNase endonuclease digestion.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las auxinas?
Some ARFs are transcriptional activators and some repressors
Confirmed activators
ARF 2-4, 9 are confirmed
repressors
The Arabidopsis ARF protein family
Reprinted from Guilfoyle, T., and Hagen, G. (2007). Auxin response factors. Curr. Opin. Plant Biol. 10: 453 – 460 with permission from Elsevier.
We don’t know much about the ARF repressors yet.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las auxinas?
Aux/IAA proteins repress the activity of Auxin Response Factors (ARFs)
III IVDNA-binding domain
Activation / repression domain
I II IV
ARF
Aux/IAAIII
ARF and Aux/IAA proteins have sequence similarity in their C-terminal regions through which they can homodimerize.
ARF homodimer
ARF Aux/IAA heterodimer
Teaching ToolsIN PLANT BIOLOGY
¿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 levelAux/IAAs are degraded by SCFTIR1-mediated proteolysis, relieving repression of ARFs to activate or repress gene expression.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las auxinas?
The auxin signaling pathway is amazingly short!
Auxin
INPUT
OUTPUT
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las auxinas?
Perhaps the diversity of auxin responses is correlated with the
diversity in signaling proteins
Auxin
INPUT
OUTPUT
Arabidopsis has 29 Aux/IAA proteins
Arabidopsis has 23 ARF proteins
TIR1 is related to five other auxin- receptor proteins, AFB1 – AFB5.
The number of potential interactions between ARFs and Aux/IAA proteins is quite large, and has the potential to precisely control auxin-induced gene expression in a vast number of combinations.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se perciben las auxinas?
>700 in Arabidopsis
F-box-proteins
TIR1AFB1
AFB5
COI1
AUXIN SIGNALING F-BOX
(jasmónico)
}
¿Cómo se perciben las auxinas?
74 in humans53 in mice Dharmasiri et al. (2005) Developmental Cell 9:109–119
Ensayos con 3H-IAA
Binding to proteinsAuxin binds directly to SCF-TIR1positive signal in assays with GST-IAA7negative signal in assays with GST-AXR2-1(que no se une a SCF-TIR1)
benzoic acidL-tryptophan
IC50 (mM) 2,4-D, 1.4 1-NAA 1.3 IAA 0.12
Competición con auxinas frías
(valores obtenidos con extractos crudos)
¿Cómo se perciben las auxinas?
Inhibition of auxin signaling: terfestatin A
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
Yamazoe et al (2005) Plant Phys 139:779
¿Cómo se perciben las auxinas?
terfestatin A: effects on root growthYamazoe 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)
mock 5 mM IAA(2h)
5 mM IAA + 5 mM A
5 mM IAA + 10 mM HTA
5 mM IAA + 5 mM 2-FAA
BA3–GUS seedlings (GUS activity for 12 h)
¿Cómo se perciben las auxinas?
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?
Auxin action in whole-plant processes
Inhibit branching in the shoot
Promote branching in the root
Promote lateral organ initiation at the shoot
apical meristem
Maintain stem-cell fate at the
root apical meristem
Control patterning and vascular development
Response to nutrient distribution and abundance
Integrate growth signaling pathways
Integrate growth signaling pathways
Cell elongation
Light responses
Responses to pathogens
Responses to symbionts – nodule formation
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.
Auxin in action!
Teaching ToolsIN PLANT BIOLOGY
¿Qué queda por hacer?
How does auxin affect so many processes in plant growth and development?
Auxin-induced gene productsAux/IAA proteinsGH3 (auxin conjugation)ACS (ethylene synthesis)Jasmonic acid synthesisCKX (cytokinin degradation)Transcription factors
Auxin-repressed gene productsCell wall proteinsARRs (Cytokinin signaling)Transcription factors
Perception (receptor)
Target genes
Biological Functions
The answer is not immediately obvious.....
Some auxin-regulated genes are clearly connected with downstream functions, others are more difficult to interpret.
Teaching ToolsIN PLANT BIOLOGY
¿Qué queda por hacer?
Ongoing investigations
Synthesis IAA Transport Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism Conjugation
What regulates auxin synthesis?
What do all those transport proteins do? What controls their
activity and distribution??
Where does auxin elimination fit in?
What are the target genes, and what do they do?
What are the other TIR1 like proteins doing, and what does ABP1 do?
How do all these pieces fit together to make a functioning
plant????
Why so many ARFs and Aux/IAAs?
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.
Teaching ToolsIN PLANT BIOLOGY
¿Qué queda por hacer?
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).
Sugawara, S., et al. (2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis. Proc. Natl. Acad. Sci. 106: 5430-5435.
Mutations inYUCCA genes (circled) affect embryogenesis and in TAA1 genes affect environmental responses.
The IAOx pathway may be restricted to Arabidopsis and its close relatives.
Teaching ToolsIN PLANT BIOLOGY
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
La respuesta a auxinas: mecanismo de acción hormonal: proteolisis de elementos de respuesta
Aux/IAA
Aux/IAA
Aux/IAA
Aux/IAA
Aux/IAA
> Fosforilación de Aux/IAA> Ubiquitinación
> Proteolisis
> Desbloqueo de ARF
> Inducción de procesos> Inducción de Aux/IAA
Auxin
No hay fosforilación
¿Cómo se perciben las auxinas?
Auxin homeostasis is also controlled by conjugation and degradation
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.
IAA
(GH3 genes)
Overexpression of an auxin conjugating enzyme encoded by a GH3 gene reduces auxin levels in the plant and causes a dwarfed phenotype.
GH3.13 overexpression
Wild-type
GH3.13 overexpression
Wild-type
GH3 genes are auxin-induced Arabidopsis
Rice
Teaching ToolsIN PLANT BIOLOGY
¿Dónde se producen y a través de qué rutas se sintetizan las auxinas?
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).
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.
Teaching ToolsIN PLANT BIOLOGY
¿Cómo se transportan a los tejidos diana?
PIN1PIN1
PIN2PIN2
PIN3PIN3
PIN4PIN4
PIN7PIN7
AUX1AUX1
Auxin transport in the root tip
Petrasek et al (2009) Development 136: 2682
¿Cómo se transportan a los tejidos diana?
Auxin transport in developing lateral rootsPetrasek et al (2009) Development 136: 2682
¿Cómo se transportan a los tejidos diana?
Interacción > Aux/IAA con TIR1
Dos tipos de Aux/IAA> GST-IAA7 Estelle
> biotinylated 17 aa peptide domain II Leyser
TIR1> extracts from tir1-1 GVG-TIR1-myc seedlings
Detección: anti-myc antibody
Hay interacción si se forman complejos proteína (GST/biotina) que se detectan con anti-myc antibody
Detección de complejos TIR1-Aux/IAA: Pull-down experiments
¿Cómo se perciben las auxinas?