The Story of Auxin – 1890 to present Darwin and others studied coleoptiles –tissues that protect monocot leaves during germination Charles 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

What is the signal that carries information from tip to base? Cutting off or covering the coleoptile tip interferes with the response Coleoptiles with tips shielded from light or removed do not bend “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.” Untreated coleoptile bends signal What is the signal that carries information from tip to base? These experiments showed that the light signal is perceived at the tip, although the bending occurs at the base – some signal must move from tip to base

Boysen-Jensen (1913) showed that the transmitted influence can move through a gelatin block Before After Left to right – solid, butter, gelatin, control. The base of the coleoptile was shaded. The signal cannot move through a solid block or butter, demonstrating that it is a water- soluble chemical. Left to right – solid, butter, gelatin, control (no cutting). By shading the base, he ensured that the signal had to move from the site of perception above the cut surface through the butter or block and stimulate bending in the base.

Repositioning the tip can induce bending in uniform light Tip removed and replaced to one side 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. Leftmost coleoptile is the uncut control, second is cut coleoptile, third is coleoptile with tip replaced symmetrically, and fourth is coleoptile with tip replaced to the side, which initiates bending. Note that these experiments are done in uniform, not unidirectional, light. These experiments suggest that the signal from the tip promotes growth and that the light changes its distribution. Tip removed Before After Control Tip removed and replaced

In the 1930s, auxin was purified and shown to promote growth 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 Indole-3-acetic acid, IAA Redrawn from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1: 162-182.

Auxin: a 21st century perspective Auxin homeostasis Tools in auxin research Polar auxin transport Perception and signaling Auxin action in whole-plant processes Interactions with other signals

Auxin synthesis and homeostasis, transport and signaling IAA Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions Catabolism Conjugation ABP1 Cell surface proteins Synthesis IAA Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions Catabolism Conjugation 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. Curr. Opin. Plant Biol.13: 12-20.

Auxin is produced through several pathways Indole Trp IAA IPA TAM IAAld IAM IAOx IAN Trp-independent pathway IAOx pathway IAM pathway TAM pathway IAA is produced from tryptophan (Trp) via several pathways, and one Trp-independent pathway The IAOx pathway may be restricted to Arabidopsis and its close relatives Trp-dependent pathways Catabolism Conjugation The pool of active auxin is also regulated by catabolism and conjugation Light, nutrients, and other hormones all regulate auxin synthesis Adapted from Rosquete M.R., Barbez, E. and Kleine-Vehn, J. (2011) Cellular auxin homeostasis: gatekeeping is housekeeping. Mol. Plant 5: 772–786. See also Mashiguchi, K.,et al. (2011) The main auxin biosynthesis pathway in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 108: 18512–18517. .

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.

The Cholodny-Went theory of tropic curvature The Cholodny-Went theory of tropic curvature states that perception of a stimulus (e.g. asymmetric light or gravity) initiates a lateral relocation of auxin, resulting in differential growth.

Coleoptiles or shoots move auxin to the shaded side Cell length Auxin concentration DR5::GUS IAA accumulates on the shaded side of phototropically stimulated Brassica oleracea hypocotyls. Auxin-induced transcription of an auxin-sensitive promoter is increased on the dark side of this phototropically-stimulated Arabidopsis hypocotyl. Increased auxin promotes cell elongation on the shaded side, causing bending toward the light. Esmon, C.A. et al. (2006) A gradient of auxin and auxin-dependent transcription precedes tropic growth responses. Proc. Natl. Acad. Sci. USA 103: 236–241. Reprinted by permission from Macmillan Publishers, Ltd: Friml, J., Wisniewska, J., Benkova, E., Mendgen, K., and Palme, K. (2002) Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 415: 806-809.

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-to-cell polar auxin transport IAA- + H+ IAAH Cytoplasm pH 7 IAA- Cell wall pH 5.5 A proton ATPase maintains the differential pH gradient H+ Redrawn from Robert, H.S., and Friml, J. (2009) Auxin and other signals on the move in plants. Nat. Chem. Biol. 5: 325-332.

Auxin moves through efflux and influx carrier proteins Cell-to-cell polar auxin transport 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. The ABCB transporters contribute to auxin transport in a diverse ways. (These were formerly referred to as MDR or PGP proteins). 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.

PIN proteins orient asymmetrically in plant cells PIN1 localizes to the lower surface of root cortex cells Root Apex Root Base PIN1 is responsible for auxin flow from shoot apex to root apex. Reprinted by permission from Macmillan Publishers, Ltd. Dhonukshe, P., et al. (2008). Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions. Nature 456: 962-966. Reproduced with permssion from Dolan, L., et al. (1993). Cellular organisation of the Arabidopsis thaliana root. Development 119: 71-84.

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.

Auxin’s effects are mediated through at least two types of receptor IAA Perception (receptor) ABP1 SCFTIR1 ABP1 was first identified in the 1970s, but only recently are its functions becoming clear…

ABP1 mediates rapid auxin responses at the plasma membrane Perception of auxin by ABP1 at the outer face of the plasma membrane initiates signals that lead to proton-pump activation, wall acidification and wall loosening. Reprinted from Tromas, A., Paponov, I., and Perrot-Rechenmann, C. (2010). AUXIN BINDING PROTEIN 1: functional and evolutionary aspects. Trends Plant Sci. 15: 436–446 with permission from Elsevier.

Auxin acts like a molecular glue that holds coreceptor proteins together TIR1 IAA Aux/IAA TIR1 is an F-box protein, part of the SCFTIR1 ubiquitin ligase complex It forms a coreceptor complex with Aux/IAA proteins SCFTIR1 complex Tan, X., Calderon-Villalobos, L.I.A., Sharon, M., Zheng, C., Robinson, C.V., Estelle, M. and Zheng, N. (2007). Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature. 446: 640-645. Jena Library Jena Library

The auxin signaling pathway Auxin promotes the association of Aux/IAA proteins and the SCFTIR1 ubiquitin ligase complex ARF Aux/IAA LOW AUXIN LEVELS SCFTIR1 IAA Aux/IAA HIGH AUXIN LEVELS …leading to proteolysis of Aux/IAA IAA At low auxin levels, Aux/IAA proteins and ARF proteins associate and interfere with ARF action ARF Elimination of Aux/IAA proteins stops them from interfering with ARF proteins, which can activate or repress transcription

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 Cell elongation Light responses Responses to pathogens Responses to symbionts – nodule formation Auxin in action! 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.

Patterns of auxin accumulation contribute to developmental patterning It has recently become clear that one of auxin’s key roles involves establishing and conveying positional information, and that it can act both as a morphogen and developmental trigger. Reprinted by permission from Macmillan Publishers, Ltd. Sorefan, K. et al. (2009) A regulated auxin minimum is required for seed dispersal in Arabidopsis. Nature 459: 583-586. Dubrovsky, J.G., et al., (2008) Auxin acts as a local morphogenetic trigger to specify lateral root founder cells. Proc. Natl. Acad. Sci. 105: 8790–8794, copyright © by the National Academy of Sciences. Petersson, S.V., et al. (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. Reprinted from Heisler, M.G., et al. (2005). Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem. Curr. Biol. 15: 1899–1911, with permission from Elsevier.Reproduced with permission from Petrášek, J., and Friml, J. (2009) Auxin transport routes in plant development. Development 136: 2675-2688.

Auxin acts like a morphogen at the root apex Auxin amount Quiescent center Cells at the quiescent center experience the highest auxin concentrations and remain quiescent. Cells near the tip experience intermediate auxin levels and divide frequently Cells experiencing a low auxin level elongate and differentiate 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.

Auxin also acts like a trigger, initiating developmental events The experimental increase of auxin synthesis in one cell is sufficient to trigger a lateral root formation. A localized auxin maximum (or minimum) can be sufficient to initiate a developmental event. Here the red indicates cells triggered to differentiate by elevated auxin levels. Dubrovsky, J.G., et al., (2008) Auxin acts as a local morphogenetic trigger to specify lateral root founder cells. Proc. Natl. Acad. Sci. 105: 8790–8794, copyright © by the National Academy of Sciences.

Perception (receptor) TF activation/ inactivation Summary Synthesis IAA Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions Catabolism Conjugation ABP1 Cell surface proteins In the past 30 years we have identified many of the molecular characters in the auxin story, and have a pretty good idea of its major themes, but the story is far from complete. 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.