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Current Biology 24, 1031–1037, May 5, 2014 ª2014 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.04.002 Report Cytokinin Controls Polarity of PIN1-Dependent Auxin Transport during Lateral Root Organogenesis Peter Marhavy ´, 1,2,3 Je ´ro ˆ me Duclercq, 1,2,7 Benjamin Weller, 4 Elena Feraru, 1,2,8 Agnieszka Bielach, 1,2 Remko Offringa, 5 Ji ´ Friml, 1,2,3 Claus Schwechheimer, 4 Angus Murphy, 6 and Eva Benkova ´ 1,2,3, * 1 Department of Plant Systems Biology, VIB, 9052 Gent, Belgium 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium 3 Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria 4 Plant Systems Biology, Technische Universita ¨ t Mu ¨ nchen, 85354 Freising, Germany 5 Department of Molecular and Developmental Genetics, Institute Biology Leiden, Leiden University, 2333 AL Leiden, the Netherlands 6 Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA Summary The plant hormones auxin and cytokinin mutually coordi- nate their activities to control various aspects of develop- ment [1–9], and their crosstalk occurs at multiple levels [10, 11]. Cytokinin-mediated modulation of auxin transport provides an efficient means to regulate auxin distribution in plant organs. Here, we demonstrate that cytokinin does not merely control the overall auxin flow capacity, but might also act as a polarizing cue and control the auxin stream directionality during plant organogenesis. Cytokinin en- hances the PIN-FORMED1 (PIN1) auxin transporter depletion at specific polar domains, thus rearranging the cellular PIN polarities and directly regulating the auxin flow direction. This selective cytokinin sensitivity correlates with the PIN protein phosphorylation degree. PIN1 phosphomimicking mutations, as well as enhanced phosphorylation in plants with modulated activities of PIN-specific kinases and phos- phatases, desensitize PIN1 to cytokinin. Our results reveal conceptually novel, cytokinin-driven polarization mecha- nism that operates in developmental processes involving rapid auxin stream redirection, such as lateral root organo- genesis, in which a gradual PIN polarity switch defines the growth axis of the newly formed organ. Results and Discussion Flexible modulation of directional auxin flow is at the core of plant developmental plasticity and adaptation to fluctuating environmental conditions. Redirection of auxin fluxes enables tropic responses [1–5], as well as specification of the embryo growth axis [6, 7] and of newly initiated organs [8, 9]. Lateral root organogenesis is a prominent example of a develop- mental process during which redirection of auxin flow occurs, thereby defining the growth axis of developing lateral roots [8]. This auxin flow redirection depends on the gradual repolariza- tion of the PIN-FORMED1 (PIN1) auxin transporter from the predominantly anticlinal orientation during the initial phases of lateral root primordium (LRP) organogenesis to the pericli- nal cell membranes, thus directing the auxin transport stream toward the primordia tips. However, the polarizing cues and underlying mechanisms that direct the auxin flow during primordia formation are still unknown. Developmental regulation of the root system by cytokinin is partially mediated by interference with the endocytic traf- ficking. The cytokinin-stimulated PIN1 lytic degradation from stage-I LRP cell membranes correlates with repression of primordia organogenesis [12]. To get a broader view on the developmental role of the cytokinin-controlled PIN1 degrada- tion, we characterized the cytokinin effects on the LRPs at the more advanced developmental stage III, when distinct PIN1 polarities at either the anticlinal or periclinal membranes (transversal and longitudinal to the primary root growth axis, respectively) are established (Figure 1A). Cytokinin at low (5 nM) or high (0.1 mM) concentrations rapidly depleted the PIN1-GFP fusion signal from anticlinal membranes when compared to untreated primordia (Figure 1A–1D and 1F), but had no or a weaker impact at periclinal membranes (Figures 1A–1C, 1E, and 1G). The differential cytokinin sensitivity of PIN1 at anticlinal versus periclinal LRP membranes implies that cytokinin might modulate the PIN polarity index (the PIN1 ratio at periclinal versus anticlinal membranes) in favor of the periclinal location and, consequently, enhance the auxin flow toward the primordia tips to promote their development. Evaluation of the PIN1 polarity index revealed a clear shift toward the pericli- nal membranes of LRPs treated with cytokinin (Figures S1A and S1B available online). Importantly, application of 5 nM cytokinin on stage-V LRPs onward significantly stimulated the primordia outgrowth when compared to control primordia (Figures S1C, S1D, and S1F). In contrast, high cytokinin con- centrations, despite the modified PIN1 polarity index, retarded primordia outgrowth, probably due to high overall decrease in PIN1 abundance (Figures S1C, S1E, and S1F; Figures 1F and 1G). These results indicate that cytokinin differentially targets PIN1 at anticlinal or periclinal LRP membranes. Consequently, because the PIN1 polarization index is altered in favor of peri- clinal PIN1, the auxin flow toward the primordia tips might be enhanced and promote primordia development. Cytokinin signaling mediated through the ARABIDOPSIS HISTIDINE KINASE4 (AHK4)/CYTOKININ RESISTANT 1 (CRE1) cytokinin receptor was found to contribute to this regulatory pathway [12]. Expression of cytokinin receptors AHK4, AHK2, and AHK3 and the TCS::GFP cytokinin reporter was observed in the inner layers of LRPs (Figures S1G and S1K). However, whereas expression of cytokinin receptors could be detected from developmental stage I on (Figure S1G) [12], the cytokinin response was significantly activated from stage III on, correlating with the PIN1 polarization time and 7 Present address: Research Unit Ecology and Dynamics of Human- Influenced Systems (EDYSAN, FRE 3498 CNRS), University of Picardie Jules Verne, 80025 Amiens, France 8 Present address: Department of Applied Genetics and Cell Biology, Univer- sity of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria *Correspondence: [email protected]

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  • Current Biology 24, 10311037, May 5, 2014 2014 Elsevier Ltd All rights rese

    Cytokinin Controls Polarity

    e

    Jir Friml, Claus Schwechheimer, Angus Murphy,and Eva Benkova1,2,3,*

    Flexible modulation of directional auxin flow is at the core of enhanced and promote primordia development.7the cytokinin response was significantly activated fromsity of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria

    stage III on, correlating with the PIN1 polarization time and*Correspondence: [email protected] developmental plasticity and adaptation to fluctuatingenvironmental conditions. Redirection of auxin fluxes enablestropic responses [15], as well as specification of the embryo

    Cytokinin signaling mediated through the ARABIDOPSISHISTIDINE KINASE4 (AHK4)/CYTOKININ RESISTANT 1 (CRE1)cytokinin receptor was found to contribute to this regulatorypathway [12]. Expression of cytokinin receptors AHK4,AHK2, and AHK3 and the TCS::GFP cytokinin reporter wasobserved in the inner layers of LRPs (Figures S1G and S1K).However, whereas expression of cytokinin receptors couldbe detected from developmental stage I on (Figure S1G) [12],

    Present address: Research Unit Ecology and Dynamics of Human-

    Influenced Systems (EDYSAN, FRE 3498CNRS), University of Picardie Jules

    Verne, 80025 Amiens, France8Present address: Department of Applied Genetics andCell Biology, Univer-1Department of Plant Systems Biology, VIB, 9052 Gent,Belgium2Department of Plant Biotechnology and Bioinformatics,Ghent University, 9052 Gent, Belgium3Institute of Science and Technology Austria, 3400Klosterneuburg, Austria4Plant Systems Biology, Technische Universitat Munchen,85354 Freising, Germany5Department of Molecular and Developmental Genetics,Institute Biology Leiden, Leiden University, 2333 AL Leiden,the Netherlands6Plant Science and Landscape Architecture, University ofMaryland, College Park, MD 20742, USA

    Summary

    The plant hormones auxin and cytokinin mutually coordi-nate their activities to control various aspects of develop-

    ment [19], and their crosstalk occurs at multiple levels[10, 11]. Cytokinin-mediated modulation of auxin transport

    provides an efficient means to regulate auxin distributionin plant organs. Here, we demonstrate that cytokinin does

    not merely control the overall auxin flow capacity, but might

    also act as a polarizing cue and control the auxin streamdirectionality during plant organogenesis. Cytokinin en-

    hances the PIN-FORMED1 (PIN1) auxin transporter depletionat specific polar domains, thus rearranging the cellular PIN

    polarities and directly regulating the auxin flow direction.This selective cytokinin sensitivity correlates with the PIN

    protein phosphorylation degree. PIN1 phosphomimickingmutations, as well as enhanced phosphorylation in plants

    with modulated activities of PIN-specific kinases and phos-phatases, desensitize PIN1 to cytokinin. Our results reveal

    conceptually novel, cytokinin-driven polarization mecha-nism that operates in developmental processes involving

    rapid auxin stream redirection, such as lateral root organo-genesis, in which a gradual PIN polarity switch defines the

    growth axis of the newly formed organ.

    Results and Discussionof PIN1-Dependent Auxin Traduring Lateral Root Organog

    Peter Marhavy,1,2,3 Jerome Duclercq,1,2,7 Benjamin Weller,4

    Elena Feraru,1,2,8 Agnieszka Bielach,1,2 Remko Offringa,51,2,3 4 6rved http://dx.doi.org/10.1016/j.cub.2014.04.002

    Report

    nsportnesis

    growth axis [6, 7] and of newly initiated organs [8, 9]. Lateralroot organogenesis is a prominent example of a develop-mental process during which redirection of auxin flow occurs,thereby defining the growth axis of developing lateral roots [8].This auxin flow redirection depends on the gradual repolariza-tion of the PIN-FORMED1 (PIN1) auxin transporter from thepredominantly anticlinal orientation during the initial phasesof lateral root primordium (LRP) organogenesis to the pericli-nal cell membranes, thus directing the auxin transport streamtoward the primordia tips. However, the polarizing cues andunderlying mechanisms that direct the auxin flow duringprimordia formation are still unknown.Developmental regulation of the root system by cytokinin is

    partially mediated by interference with the endocytic traf-ficking. The cytokinin-stimulated PIN1 lytic degradation fromstage-I LRP cell membranes correlates with repression ofprimordia organogenesis [12]. To get a broader view on thedevelopmental role of the cytokinin-controlled PIN1 degrada-tion, we characterized the cytokinin effects on the LRPs at themore advanced developmental stage III, when distinct PIN1polarities at either the anticlinal or periclinal membranes(transversal and longitudinal to the primary root growth axis,respectively) are established (Figure 1A). Cytokinin at low(5 nM) or high (0.1 mM) concentrations rapidly depleted thePIN1-GFP fusion signal from anticlinal membranes whencompared to untreated primordia (Figure 1A1D and 1F), buthad no or a weaker impact at periclinal membranes (Figures1A1C, 1E, and 1G).The differential cytokinin sensitivity of PIN1 at anticlinal

    versus periclinal LRP membranes implies that cytokinin mightmodulate the PIN polarity index (the PIN1 ratio at periclinalversus anticlinal membranes) in favor of the periclinal locationand, consequently, enhance the auxin flow toward theprimordia tips to promote their development. Evaluation ofthe PIN1 polarity index revealed a clear shift toward the pericli-nal membranes of LRPs treated with cytokinin (Figures S1Aand S1B available online). Importantly, application of 5 nMcytokinin on stage-V LRPs onward significantly stimulatedthe primordia outgrowth when compared to control primordia(Figures S1C, S1D, and S1F). In contrast, high cytokinin con-centrations, despite the modified PIN1 polarity index, retardedprimordia outgrowth, probably due to high overall decrease inPIN1 abundance (Figures S1C, S1E, and S1F; Figures 1F and1G). These results indicate that cytokinin differentially targetsPIN1 at anticlinal or periclinal LRPmembranes. Consequently,because the PIN1 polarization index is altered in favor of peri-clinal PIN1, the auxin flow toward the primordia tips might be

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    Figure 1. PIN1 Depletion from Anticlinal LRP Membranes Stimulated by Cytokinin

    Real-timemonitoring of PIN1-GFP at membranes in stage-III LRPs untreated (A) and treated with cytokinin (B and C). Cytokinin sensitivity of PIN1-GFP was

    higher at the anticlinal (D and F) than at the periclinal (E and G)membranes. PIN1-GFP primordia were treated for 1.5 hr, 3 hr, and 7 hr with 5 nMBA (B, D, and

    E) and 0.1 mMBA (C, F, and G), respectively. Scale bars, 30 mm. Error bars indicate the SEM compared to primordia at time 0 hr (*p < 0.05, ***p < 0.001, ****p