Plant Physiology Preview. Published on January 13, 2010, as DOI: /pp

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1 Plant Physiology Preview. Published on January 13, 2010, as DOI: /pp Scientific Correspondence Running Head: F-box receptors and rapid auxin-induced growth Author to whom correspondence should be adresse: Hartwig Lüthen, Biozentrum Flottbek, University of Hamburg, Ohnhorststr. 18, Hamburg, Germany, Tel Copyright 2010 by the American Society of Plant Biologists

2 Title: Rapid auxin-induced cell expansion and gene expression: A four-decade old question revisited Authors Daniel Schenck Biozentrum Flottbek, University of Hamburg, Ohnhorststr. 18, Hamburg, Germany, Tel Tel , May Christian, Biozentrum Flottbek, University of Hamburg, Ohnhorststr. 18, Hamburg, Germany, Tel Tel , Alan Jones Departments of Biology and Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 2759, alan_jones@unc.edu, Tel: (919) Hartwig Lüthen Biozentrum Flottbek, University of Hamburg, Ohnhorststr. 18, Hamburg, Germany, h.luthen@botanik.uni-hamburg.de, Tel Footnotes: Funded by the NIGMS, DOE, and NSF (A.M.J), Graduiertenförderung Uni Hamburg (D.S.) and a Feodor Lynen Research Fellowship (M.C and A.M.J.). Corresponding author: Hartwig Lüthen, Biozentrum Flottbek, University of Hamburg, Ohnhorststr. 18, Hamburg, Germany, h.luthen@botanik.uni-hamburg.de. Research Area: Signal Transduction and Hormone Action (Scientific Correspondence) 2

3 Footnotes: Funded by the NIGMS, DOE, and NSF (A.M.J.), Graduietenförderung University of Hamburg (D.S. and H.L.) and Feodor Lynen Research Fellowship (M.C. and A.N.J.) We are most grateful to M. Estelle, R. Hertel, O. Leyser, A. Theologis, and L. Vanderhoef for their helpful comments and for sharing their perspectives and reagents. 3

4 The classical effect of the plant hormone auxin is very rapid stimulation of cell expansion followed by sustained growth over a longer time period. However, auxins are also important in other responses such as cell division and differentiation. Recently, the TRANSPORT INHIBITOR RESPONSE1/AUXIN BINDING F-BOX PROTEIN (TIR1/AFB) family of auxin receptors regulating expression of auxin induced genes has garnered much attention regarding the precise role that TIR1/AFBs serve in auxin responses. Here, we show that major changes in gene expression mediated by these important receptors do not play the major role in triggering the very rapid phase of cell elongation. Fifty years after Charles and Francis Darwin predicted its existence, the plant hormone auxin was discovered in the 1930 s to be a key molecule regulating rapid plant cell expansion (5-10 minutes). Soon thereafter, synthetic auxins such as 2,4 dichlorophenoxyacetic acid (2,4D) were discovered and these became economically-important growth regulators for agriculture. It was learned that auxin also affects plant development such as organ formation. A remarkable feature about the mode of action of auxin is that it induces changes in global gene expression within 5-15 minutes of application (Abel et al., 1994); this remains one of the fastest regulation of gene transcription known. Changes in protein level are detectable around minutes after auxin application (Oeller and Theologis, 1995). By the 1960 s, the central question in the auxin field was whether the rapid auxin-induced change in instantaneous growth strictly requires the immediate synthesis of growth-limiting proteins through auxin-induced gene expression (Cleland, 1971), or rather is triggered by a direct stimulation of growth-limiting processes (Hager et al., 1971). It has been proposed that sustained growth involves both gene expression and stimulation of growth-limiting processes (Vanderhoef et al., 1976). This longstanding question remains unanswered. However, with the recent discovery of a family of nuclear, auxin-binding, F-box (AFB) proteins (Dhamarsiri et al., 2005a, Kepinsky and Leyser, 2005), including the prototype TIR1, as well as AFB1, AFB2, AFB3, AFB4 and AFB5 (Dhamarsiri et al. 2005b) and a means to measure auxin-induced gene expression and growth at high resolution, this question can be revisited using three new tools. These tools are: i) a set of loss-of-function mutations in the TIR1/AFB gene family. It is known that the AFB proteins activate the expression of auxin-induced, primary-response genes by affecting the steady-state-level of a large family of transcription factors that directly or indirectly mediate hundreds of auxin-regulated genes, including genes encoding proteins that metabolize and transport auxin. Single and combined mutations in the AFB gene family confer auxin-deficient phenotypes. ii) The synthetic auxin responsive promoter driving green fluorescent protein (GFP) expression (DR5rev::GFP) which enables one to measure rapidly and in situ at the cellular activity of this large set of auxin-induced genes. Although indirect, this method has become one of the accepted techniques to assess auxin-inducible gene expression (Heisler et al., 2005). Finally, iii), growth measurements were adopted to Arabidopsis that enabled us to capture instantaneous growth kinetics at high resolution (Christian and Lüthen, 2000). Combining all of these, we monitored auxin-induced gene expression and growth of the hypocotyl at a high temporal resolution. Exogenously-applied auxin induces changes in global gene expression ~20-fold in wild type hypocotyls as reported by DR5rev::GFP (Fig. 1A). In contrast to wild type hypocotyls, auxininduced gene expression was strongly attenuated in auxin-treated hypocotyls of plants lacking 3 of 6 known F-box auxin receptors (Fig. 1A, lower row). The assay time was extended to 24 hours to emphasize that auxin-induced gene expression via the DR5rev::GFP reporter was barely detectable in the afb mutant (Fig. 1A). Despite great attenuation of gene expression in 4

5 the afb mutant, normalized auxin-induced growth was essentially unchanged compared to wild type hypocotyls (Fig. 1B). Auxin sensitivity, auxin uptake, and rapid auxin-induced growth The tir/afb mutants did not alter the sensitivity of the rapid growth reponse to the natural auxin, indole acetic acid (IAA) (Fig. 2A). However, growth induced by the synthetic auxin 2,4-D was strongly affected (Fig. 2B). We hypothesized that a primary reason for the impaired 2,4-D activity and for the residual delayed response to the endogenous auxin IAA in the mutant is altered uptake and transport of the hormone in the mutant tissue. To overcome this possible complication, we used 2,4-D methyl ester, a synthetic, lipophilic pro-auxin (Christian et al. 2008, Salvidi-Goldstein et al., 2008,) that rapidly enters plants cells by an unfacilitated pathway and then becomes cleaved intracellularly by esterases to yield active free 2,4-D. As shown in Fig. 2C, 2,4-D methyl ester induces the rapid change in growth in both wild type and mutant hypocotyls similarly. This is consistent with the idea that one of the reasons for the developmental phenotypes in the afb mutants is due to altered auxin uptake or transport. It also raises a caution to those trying to interpret these phenotypes in the context of AFB mechanism of action since many basic processes such as auxin transport and metabolism are disrupted in the afb mutants in addition to auxin signaling as shown by Dhamasiri, et al. (2005b). It is possible that DR5rev::GFP does not report all auxin-regulated genes and that there remains a small subset of growth-limiting genes regulated by some other auxin-binding protein than TIR1, AFB1, AFB2 or AFB3 in the tir1/afb mutants. However, we can likely exclude AFB4 and AFB5. AFB5 binds auxins that are structurally different than IAA (Walsh et al., 2006) and AFB4 is not expressed in the hypocotyl based on public expression data. We conclude that the near-instantaneous induction of auxin-induced growth is not strictly dependent on the global change in auxin-induced genes via the TIR1/AFB pathway. This view is strengthened by the observation that the auxin resistant mutant axr1-12 displays a normal onset of the auxin growth response (Fig. S2),We speculate that auxin-induced gene expression is undoubtably requisite for sustained auxin-induced cell expansion and long-term development (e.g. the auxin effect on root formation) but auxin s regulation at the level of changes in global gene expression may not constitute the rate-limiting step for the near instantaneous cell expansion. Feedback mechanisms between the TIR1/AFB-dependent and - independent pathways may well exist and are promising targets for investigations using physiological and molecular tools. It is well established that ACC synthase is coded by a gene inducible through the auxin- TIR1/AFB pathway (Abel et al. 1994, Wang et al. 2005), and there is evidence harking back to the 1960s that auxin transport is regulated by auxin (Apfelbaum and Burg 1972). Crosstalk between the TIR1 pathway and independent pathways has recently been shown for the control of root elongation growth (Tromos et al. 2009). 5

6 Literature cited Abel S, Oeller PW, Theologis A. (1994) Early auxin-induced genes encode short-lived nuclear proteins. Proc Natl Acad Sci USA 91: Abel S, Nguyen MD, Chow W, Theologis A ACS4, a primary Indoleacetic acidresponsive gene encoding 1-aminocyclopropane- 1-carboxylatesynthase in Arabidopsis thaliana. Structural characterization, expression in Escherichia coli, and expression characteristics in response to auxin. J Biol Chem 270: Christian M, Lüthen H (2000) New methods to analyse auxin-induced growth I: Classical auxinology goes Arabidopsis. Plant Growth Regul. 32: Christian, M, Hannah, WB, Lüthen, H, Jones, AM (2008) New auxins from a chemical genomics approach. J. Expt. Bot 59: Cleland R (1971) Instability of the growth-limiting proteins of the Avena coleoptile and their pool size in relation to auxin. Planta 99: 1-11 Dharmasiri N, Dharmarsiri S, Weijers D, Lechner E, Yamada M, Hobbie L, Ehrismann JS, Jürgens G, Estelle M (2005b) Plant development is regulated by a family of auxin receptor F Box proteins. Dev Cell 9: Dharmasiri N, Dharmasiri S, Estelle M (2005a) The F-box protein TIR1 is an auxin receptor. Nature 435: Hager A, Menzel H, Krauss A (1971) Versuche und Hypothese zur Primärwirkung des Auxins beim Streckungswachstum. Planta 100: Heisler MG, Ohno C, Das P, Sieber P, Reddy GV, Long JA, Meyerowitz EM (2005) Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem. Curr Biol 15: Kepinsky S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435: Oeller PW, Theologis A (1995) Induction kinetics of the nuclear proteins encoded by the early indoleacetic acid-inducible genes, PS-IAA4/5 and PS-IAA6 in pea (Pisum sativum L.). Plant J. 7: Savaldi-Goldstein S, Baiga TG, Pojer P, Dabi T, Butterfield C, Parry G, Santner A, Dharmasiri N, Tao Y, Estelle M, Noel JP, Chory J (2008) New auxin analogs with growth promoting effects in intact plants reveal a chemical strategy to improve hormone delivery. Proc Natl Acad Sci USA 105: Vanderhoef LN, Stahl CA, Lu T-YS (1976) Two elongation responses to auxin respond differently to protein synthesis inhibition. Plant Physiol 58: Tromas A, Braun N, Muller P,Khodus T, Paponov IA, Palme K, Ljung K, Lee J-Y, Benfey P, Murray JAH, Scheres B, Perrot-Rechenmann C (2009) The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth. PLOS One 4: e6648 6

7 Walsh TA, Neal R, Merlo AO, Honma M, Hicks GR, Wolff K, Matsumura W, Davies JP (2006) Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis. Plant Physiol 142: Wang NN, Shih M-C, Li N (2004) The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC-synthase genes AtACS4, AtACS5 and AtACS7 induced by hormones and stresses. J Exp Bot 56:

8 Figure Legends Figure 1 A, Hypocotyls of Arabidopsis, expressing GFP under the control of the auxin-inducible DR5 promotor. Upper row: Wild type (DR5rev::GFP Col-0), lower row: afb mutant (DR5rev::GFP tir1-1 afb2-3 afb3-4) in Col- 0/Ws background. The times after addition of 10-5 M IAA and change of fluorescence due to auxin-dependent GFP synthesis are indicated. Relative values of the fluorescent signal determined through CCD-fluorimetry are shown for the corresponding hypocotyls. Corrected fluorescence at t=0 was set as 100%. B, Elongation growth induced by 10-5 M IAA in wild type (closed squares) and the afb (tir1-1 afb1-3 afb2-3 afb 3-4) mutant (circles). Auxin was added at the time indicated by the arrow. Kinetic data was normalized by the ability to respond to the fungal toxin, fussicoccin. For details on the normalization and the nature of slight delay in mutant growth refer to the supplemental Figure S1. A wild type control without auxin addition is also shown (open squares). Figure 2 A, Dose-response curves for amplitude of IAA-induced growth, measured 150 minutes after the application of the hormone, in the wild type and in the receptor mutants. B, Same as above for the synthetic auxin 2,4-D. Note the strong apparent effect of the receptor mutation on 2,4-D sensitivity C, Time course of growth induced by the membrane permeable synthetic auxin 2,4-D methyl ester and 2,4-D. Mutant curves were normalized for the fusicoccin lag phase as described in the supplemental data. Note that wild type and mutant responses to 2,4-D ME are virtually identical in both amplitude and lag phases while the mutation abolishes 2,4-D induced growth in the mutation. 8

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