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

Christian, May

Jones, Alan

Lüthen, Hartwig

Schenck, Daniel

PubMed

Schenck, D.

Christian, M.

Jones, A.

Luthen, H.

Carolina Digital Repository

Scientific CorrespondenceRapid Auxin-Induced Cell Expansion and GeneExpression: A Four-Decade-Old Question Revisited1[C][W]Daniel Schenck, May Christian, Alan Jones, and Hartwig Lüthen*Biozentrum Flottbek, University of Hamburg, 22609 Hamburg, Germany (D.S., M.C., H.L.); and Departmentsof Biology and Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599–3280 (A.J.)The classical effect of the plant hormone auxin isvery rapid stimulation of cell expansion followed bysustained growth over a longer time period. However,auxins are also important in other responses, such ascell division and differentiation. Recently, the TRANS-PORT INHIBITOR RESPONSE1/AUXIN-BINDINGF-BOX PROTEIN (TIR1/AFB) family of auxin recep-tors regulating the expression of auxin-induced geneshas garnered much attention regarding the preciserole that TIR1/AFBs serve in auxin responses. Here,we show that major changes in gene expression me-diated by these important receptors do not play themajor role in triggering the very rapid phase of cellelongation.Fifty years after Charles and Francis Darwin pre-dicted its existence, the plant hormone auxin was dis-covered in the 1930s to be a key molecule regulatingrapid plant cell expansion (5–10 min). Soon thereafter,synthetic auxins such as 2,4-dichlorophenoxyacetic acid(2,4-D) were discovered, and these became economi-cally important growth regulators for agriculture. Itwas learned that auxin also affects plant developmentsuch as organ formation. A remarkable feature aboutthe mode of action of auxin is that it induces changesin global gene expression within 5 to 15 min ofapplication (Abel et al., 1994); this remains one of thefastest regulations of gene transcription known.Changes in protein level are detectable around 15 to30 min after auxin application (Oeller and Theologis,1995). By the 1960s, the central question in the auxinfield was whether the rapid auxin-induced change ininstantaneous growth strictly requires the immediatesynthesis of growth-limiting proteins through auxin-induced gene expression (Cleland, 1971) or rather istriggered by a direct stimulation of growth-limitingprocesses (Hager et al., 1971). It has been proposed thatsustained growth involves both gene expression andstimulation of growth-limiting processes (Vanderhoefet al., 1976). This long-standing question remainsunanswered.However, with the recent discovery of a family ofnuclearAFB proteins (Dharmasiri et al., 2005a; Kepinskyand Leyser, 2005), including the prototype TIR1 as wellas AFB1, AFB2, AFB3, AFB4, and AFB5 (Dharmasiriet al., 2005b) and a means to measure auxin-inducedgene expression and growth at high resolution, thisquestion can be revisited using three new tools. (1) Aset of loss-of-function mutations in the TIR1/AFB genefamily. It is known that the AFB proteins activate theexpression of auxin-induced, primary-response genesby affecting the steady-state level of a large family oftranscription factors that directly or indirectly mediatehundreds of auxin-regulated genes, including genesencoding proteins that metabolize and transportauxin. Single and combined mutations in the AFBgene family confer auxin-deficient phenotypes. (2) Thesynthetic auxin-responsive promoter driving GFP ex-pression (DR5rev::GFP), which enables one to measurerapidly and in situ the cellular activity of this largeset of auxin-induced genes. Although indirect, thismethod has become one of the accepted techniques toassess auxin-inducible gene expression (Heisler et al.,2005). (3) Growth measurements were adopted toArabidopsis (Arabidopsis thaliana) that enabled usto capture instantaneous growth kinetics at high res-olution (Christian and Lüthen, 2000). Combining allof these, we monitored auxin-induced gene expres-sion and growth of the hypocotyl at a high temporalresolution.Exogenously applied auxin induces changes inglobal gene expression approximately 20-fold inwild-type hypocotyls, as reported with DR5rev::GFP(Fig. 1A). In contrast to wild-type hypocotyls, auxin-induced gene expression was strongly attenuated inauxin-treated hypocotyls of plants lacking three of sixknown F-box auxin receptors (Fig. 1A, bottom row).The assay time was extended to 24 h to emphasize thatauxin-induced gene expression via the DR5rev::GFPreporter was barely detectable in the afb mutant (Fig.1A). Despite great attenuation of gene expression in1 This work was supported by the National Institute of GeneralMedical Sciences, the Department of Energy, and the NationalScience Foundation (A.J.), the Graduietenförderung University ofHamburg (D.S. and H.L.), and the Feodor Lynen Research Fellow-ship (M.C. and A.J.).* Corresponding author; e-mail h.luthen@botanik.uni-hamburg.de.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Hartwig Lüthen (h.luthen@botanik.uni-hamburg.de).[C] Some figures in this article are displayed in color online but inblack and white in the print edition.[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.109.149591Plant Physiology, March 2010, Vol. 152, pp. 1183–1185, www.plantphysiol.org  2010 American Society of Plant Biologists 1183 www.plantphysiol.orgon July 13, 2020 - Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved.the afbmutant, normalized auxin-induced growth wasessentially unchanged compared with that in wild-type hypocotyls (Fig. 1B).AUXIN SENSITIVITY, AUXIN UPTAKE, AND RAPIDAUXIN-INDUCED GROWTHThe tir/afbmutants did not alter the sensitivity of therapid growth response to the natural auxin, indoleacetic acid (IAA; Fig. 2A). However, growth inducedby the synthetic auxin 2,4-D was strongly affected (Fig.2B). We hypothesized that a primary reason for theimpaired 2,4-D activity and for the residual delayedresponse to the endogenous auxin IAA in the mutantis altered uptake and transport of the hormone in themutant tissue. To overcome this possible complication,we used 2,4-D methyl ester, a synthetic, lipophilicproauxin (Christian et al., 2008; Savaldi-Goldsteinet al., 2008) that rapidly enters plant cells by anunfacilitated pathway and then becomes cleaved in-tracellularly by esterases to yield active free 2,4-D. AsFigure 1. A, Hypocotyls of Arabidopsis expressing GFP under thecontrol of the auxin-inducible DR5 promoter. The top row shows thewild type (DR5rev::GFP, ecotype Columbia), and the bottom rowshows the afb mutant (DR5rev::GFP tir1-1 afb2-3 afb3-4) in theColumbia/Wassilewskija background. The times after addition of1025 M IAA and change of fluorescence due to auxin-dependent GFPsynthesis are indicated. Relative values of the fluorescent signal deter-mined through CCD fluorimetry are shown for the correspondinghypocotyls. Corrected fluorescence at time 0 was set as 100%. B,Elongation growth induced by 1025 M IAA in the wild type (blacksquares) and the afbmutant (tir1-1 afb1-3 afb2-3 afb 3-4; circles). Auxinwas added at the time indicated by the arrow. Kinetic data werenormalized by the ability to respond to the fungal toxin fusicoccin. Fordetails on the normalization and the nature of the slight delay in mutantgrowth, see Supplemental Figure S1. Awild-type control without auxinaddition is also shown (white squares). [See online article for colorversion of this figure.]Figure 2. A, Dose-response curves for the amplitude of IAA-inducedgrowth, measured 150 min after the application of the hormone, in thewild type (ecotype Columbia [Col-0]) and the receptor mutants. B,Same as A for the synthetic auxin 2,4-D. Note the strong apparent effectof the receptor mutation on 2,4-D sensitivity. C, Time course of growthinduced by the membrane-permeable synthetic auxin 2,4-D methylester and 2,4-D. Mutant curves were normalized for the fusicoccin lagphase as described in Supplemental Materials and Methods S1. Notethat wild-type and mutant responses to 2,4-D methyl ester are virtuallyidentical in both amplitude and lag phases, while the mutationabolishes 2,4-D-induced growth in the mutation. [See online articlefor color version of this figure.]Schenck et al.1184 Plant Physiol. Vol. 152, 2010 www.plantphysiol.orgon July 13, 2020 - Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved.shown in Figure 2C, 2,4-D methyl ester induces therapid change in growth in both wild-type and mutanthypocotyls similarly. This is consistent with the ideathat one of the reasons for the developmental pheno-types in the afb mutants is altered auxin uptake ortransport. It also raises a caution to those trying tointerpret these phenotypes in the context of AFBmechanism of action, since many basic processessuch as auxin transport and metabolism are disruptedin the afb mutants in addition to auxin signaling, asshown by Dharmasiri et al. (2005b).It is possible that DR5rev::GFP does not report allauxin-regulated genes and that there remains a smallsubset of growth-limiting genes regulated by someother auxin-binding protein than TIR1, AFB1, AFB2, orAFB3 in the tir1/afb mutants. However, we can likelyexclude AFB4 and AFB5. AFB5 binds auxins that arestructurally different from IAA (Walsh et al., 2006),and AFB4 is not expressed in the hypocotyl based onpublic expression data. We conclude that the near-instantaneous induction of auxin-induced growth isnot strictly dependent on the global change in auxin-induced genes via the TIR1/AFB pathway. This viewis strengthened by the observation that the auxin-resistant mutant axr1-12 displays a normal onset of theauxin growth response (Supplemental Fig. S2). Wespeculate that auxin-induced gene expression is un-doubtedly requisite for sustained auxin-induced cellexpansion and long-term development (e.g. the auxineffect on root formation), but auxin’s regulation at thelevel of changes in global gene expression may not con-stitute the rate-limiting step for the near-instantaneouscell expansion. Feedback mechanisms between theTIR1/AFB-dependent and -independent pathwaysmay well exist and are promising targets for investi-gations using physiological and molecular tools. It iswell established that 1-aminocyclopropane-1-carboxylicacid synthase is coded by a gene inducible through theauxin-TIR1/AFB pathway (Abel et al., 1995; Wanget al., 2005), and there is evidence harking back to the1960s that auxin transport is regulated by auxin(Apfelbaum and Burg, 1972). Cross talk between theTIR1 pathway and independent pathways has recentlybeen shown for the control of root elongation growth(Tromas et al., 2009).Supplemental DataThe following materials are available in the online version of this article.Supplemental Figure S1. Lag phase of fusicoccin-induced growth the afbmutant.Supplemental Figure S2. Auxin-induced growth in the axr1 mutant.Supplemental Materials and Methods S1. Supplemental materials andmethods and additional data.ACKNOWLEDGMENTSWe are most grateful to M. Estelle, R. Hertel, O. Leyser, A. Theologis, and L.Vanderhoef for their helpful comments and for sharing their perspectives andreagents.Received October 19, 2009; accepted January 6, 2010; published January 13,2010.LITERATURE CITEDAbel S, Nguyen MD, Chow W, Theologis A (1995) ACS4, a primaryindoleacetic acid-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis thaliana: structural characterization,expression in Escherichia coli, and expression characteristics in responseto auxin. J Biol Chem 270: 19093–19099Abel S, Oeller PW, Theologis A (1994) Early auxin-induced genes encodeshort-lived nuclear proteins. Proc Natl Acad Sci USA 91: 326–330ApfelbaumA, Burg SP (1972) Effects of ethylene and 2,4-dichlorophenoxy-acetic acid on cellular expansion in Pisum sativum. Plant Physiol 50: 125–131Christian M, Hannah WB, Lüthen H, Jones AM (2008) New auxins from achemical genomics approach. J Exp Bot 59: 2757–2767Christian M, Lüthen H (2000) New methods to analyse auxin-inducedgrowth. I. Classical auxinology goes Arabidopsis. Plant Growth Regul32: 107–114Cleland R (1971) Instability of the growth-limiting proteins of the Avenacoleoptile and their pool size in relation to auxin. Planta 99: 1–11Dharmasiri N, Dharmasiri S, Estelle M (2005a) The F-box protein TIR1 isan auxin receptor. Nature 435: 441–445Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L,Ehrismann JS, Jürgens G, Estelle M (2005b) Plant development isregulated by a family of auxin receptor F box proteins. Dev Cell 9:109–119Hager A, Menzel H, Krauss A (1971) Versuche und Hypothese zurPrimärwirkung des Auxins beim Streckungswachstum. Planta 100:47–75Heisler MG, Ohno C, Das P, Sieber P, Reddy GV, Long JA, MeyerowitzEM (2005) Patterns of auxin transport and gene expression duringprimordium development revealed by live imaging of the Arabidopsisinflorescence meristem. Curr Biol 15: 1899–1911Kepinsky S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an auxinreceptor. Nature 435: 446–451Oeller PW, Theologis A (1995) Induction kinetics of the nuclear proteinsencoded by the early indoleacetic acid-inducible genes, PS-IAA4/5 andPS-IAA6 in pea (Pisum sativum L.). Plant J 7: 37–48Savaldi-Goldstein S, Baiga TG, Pojer P, Dabi T, Butterfield C, Parry G,Santner A, Dharmasiri N, Tao Y, Estelle M, et al (2008) New auxinanalogs with growth promoting effects in intact plants reveal a chemicalstrategy to improve hormone delivery. Proc Natl Acad Sci USA 105:15190–15195Tromas A, Braun N, Muller P, Khodus T, Paponov IA, Palme K, Ljung K,Lee JY, Benfey P, Murray JAH, et al (2009) The AUXIN BINDINGPROTEIN 1 is required for differential auxin responses mediating rootgrowth. PLoS One 4: e6648Vanderhoef LN, Stahl CA, Lu TYS (1976) Two elongation responses toauxin respond differently to protein synthesis inhibition. Plant Physiol58: 402–404Walsh TA, Neal R, Merlo AO, Honma M, Hicks GR, Wolff K, MatsumuraW, Davies JP (2006) Mutations in an auxin receptor homolog AFB5 andin SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis. PlantPhysiol 142: 542–552Wang NN, Shih MC, Li N (2005) The GUS reporter-aided analysis ofthe promoter activities of Arabidopsis ACC-synthase genes AtACS4,AtACS5 and AtACS7 induced by hormones and stresses. J Exp Bot 56:909–920Auxin Receptor and Elongation GrowthPlant Physiol. Vol. 152, 2010 1185 www.plantphysiol.orgon July 13, 2020 - Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved.

Rapid Auxin-Induced Cell Expansion and Gene Expression: A Four-Decade-Old Question Revisited

Daniel Schenck

May Christian

Alan Jones

Hartwig Lüthen

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Rapid Auxin-Induced Cell Expansion and Gene Expression: A Four-Decade-Old Question Revisited1[C][W]

Rapid Auxin-Induced Cell Expansion and Gene Expression: A Four-Decade-Old Question Revisited1[C][W]

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