Accción hormonal de las auxinas en plantas ¿Por qué y cuándo se descubren las auxinas? ¿Qué...

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


http://extension.entm.purdue.edu/pestcrop/2004/issue8/index.html

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



+ --

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.”



http://commons.wikimedia.org/wiki/User:Patche99z

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



Repositioning the tip can induce bending in uniform light


Tip removed and replaced to one side

BeforeControl

Tip removed

Tip removed and replaced


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



http://www.jstor.org/stable/pdfplus/4353103.pdf

Auxinas: estructura química


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


¿Cuándo y por qué se descubren las 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


¿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



http://rspb.royalsocietypublishing.org/content/114/789/317.full.pdf+html

http://www.archive.org/stream/phytohormones00went#page/62/mode/2up

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




http://www.plantphysiol.org/cgi/reprint/13/3/437

http://www.plantphysiol.org/cgi/content/abstract/122/3/925

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.


vertical

GRAVITY VECTOR

horizontal

time

length

Lower side

Upper side








¿Cómo se transportan a los tejidos diana?



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.



http://dx.doi.org/doi:10.1016/j.pbi.2009.10.006

Synthesis IAA Transport Perception (receptor)


Target genes





Target genes



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


http://dx.doi.org/doi:10.1016/j.pbi.2009.10.006

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



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



http://www.plantcell.org/cgi/content/full/21/6/1659

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.



http://www.plantcell.org/cgi/content/full/21/6/1659

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.



http://www.plantphysiol.org/cgi/reprint/108/3/1043

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.



http://www.plantcell.org/cgi/reprint/9/11/1963

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.



http://www.nature.com/nature/journal/v433/n7021/full/nature03184.html

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



http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WSN-4C5HP45-B-1&_cdi=7051&_user=121723&_pii=S0092867403009243&_orig=browse&_coverDate=11%2F26%2F2003&_sk=998849994&view=c&wchp=dGLbVzz-zSkWb&md5=fd57c0519e2f4f99aa4fb011c1b0d16f&ie=/sdarticle.pdf

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


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


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



¿Funciones diferentes para las diferentes rutas?


24-OCT-2011

¡¡¡ TAA1 y YUCCA constituyen una sola ruta !!!


Mashiguchi et al.Won et al.

Mashiguchi et al (2011) PNAS, 24 oct, early ed


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.



http://www.sciencemag.org/cgi/reprint/324/5935/1684.pdf

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.



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.

http://www.jstor.org/stable/pdfplus/4353103.pdf

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+



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.



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)


Auxin transport: influx and efflux


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



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.



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



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.



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


Auxin gradients and transport during embryogenesis

Petrasek et al (2009) Development 136: 2681


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.



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


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

IN 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.

Arabidopsis has 6 AUX1/LAX proteins, 21 ABCB proteins and 8 PIN proteins - we don’t yet understand all the complexity of auxin transport!




Auxin transport - summary


Auxin homeostasis - summary

Synthesis IAA

Catabolism ConjugationAuxin homeostasis is maintained by its

synthesiscatabolism conjugationtransport

All of these processes are tightly regulated

Transport



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


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



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


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,


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.



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


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”


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.



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



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


Auxin treatment (mins)


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



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


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.



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.



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



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.



The auxin signaling pathway is amazingly short!

Auxin

INPUT

OUTPUT



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.



>700 in Arabidopsis

F-box-proteins

TIR1AFB1

AFB5

COI1

AUXIN SIGNALING F-BOX

(jasmónico)

}


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)


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


terfestatin A: effects on root growthYamazoe et al (2005) Plant Phys 139:779


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)


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!



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


Target genes


The answer is not immediately obvious.....

Some auxin-regulated genes are clearly connected with downstream functions, others are more difficult to interpret.



Ongoing investigations



Target genes



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.



Auxin biosynthesis: novel diagram

Tao et al (2008) Cell 133: 171


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.



http://www.pnas.org/content/106/13/5430.full

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


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



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.



PIN1PIN1

PIN2PIN2

PIN3PIN3

PIN4PIN4

PIN7PIN7

AUX1AUX1

Auxin transport in the root tip

Petrasek et al (2009) Development 136: 2682


Auxin transport in developing lateral rootsPetrasek et al (2009) Development 136: 2682


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


Accción hormonal de las auxinas en plantas ¿Por qué y cuándo se descubren las auxinas? ¿Qué...

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