Report. Cytokinin Controls Polarity of PIN1-Dependent Auxin Transport during Lateral Root Organogenesis

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1 Current Biology 24, 11 17, May 5, 214 ª214 Elsevier Ltd All rights reserved Cytokinin Controls Polarity of PIN1-Dependent Auxin Transport during Lateral Root Organogenesis Report Peter Marhavý, 1,2, Jérôme Duclercq, 1,2,7 Benjamin Weller, 4 Elena Feraru, 1,2,8 Agnieszka Bielach, 1,2 Remko Offringa, 5 Jirí Friml, 1,2, Claus Schwechheimer, 4 Angus Murphy, 6 and Eva Benková 1,2,, 1 Department of Plant Systems Biology, VIB, 952 Gent, Belgium 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, 952 Gent, Belgium Institute of Science and Technology Austria, 4 Klosterneuburg, Austria 4 Plant Systems Biology, Technische Universität München, 8554 Freising, Germany 5 Department of Molecular and Developmental Genetics, Institute Biology Leiden, Leiden University, 2 AL Leiden, the Netherlands 6 Plant Science and Landscape Architecture, University of Maryland, College Park, MD 2742, USA Summary The plant hormones auxin and cytokinin mutually coordinate their activities to control various aspects of development [1 9], and their crosstalk occurs at multiple levels [1, 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 enhances 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 phosphatases, desensitize PIN1 to cytokinin. Our results reveal conceptually novel, cytokinin-driven polarization mechanism that operates in developmental processes involving rapid auxin stream redirection, such as lateral root organogenesis, 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 7 Present address: Research Unit Ecology and Dynamics of Human- Influenced Systems (EDYSAN, FRE 498 CNRS), University of Picardie Jules Verne, 825 Amiens, France 8 Present address: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences (BOKU), 119 Vienna, Austria Correspondence: eva.benkova@ist.ac.at growth axis [6, 7] and of newly initiated organs [8, 9]. Lateral root organogenesis is a prominent example of a developmental 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 repolarization of the PIN-FORMED1 (PIN1) auxin transporter from the predominantly anticlinal orientation during the initial phases of lateral root primordium (LRP) organogenesis to the periclinal 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 trafficking. 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 degradation, 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 (.1 mm) concentrations rapidly depleted the 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 periclinal 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 concentrations, 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 periclinal 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 AHK 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

2 Current Biology Vol 24 No μM_ C nM_ B PIN1:: A PIN1:: an clinal membranes nM 4 E Rela ve fluorescence (%) Rela ve fluorescence (%) D nM PIN1:: an clinal membranes μM 4 G Rela ve fluorescence (%) Rela ve fluorescence (%) periclinal membranes 1 2 F PIN1:: 12 PIN1:: periclinal membranes μM Figure 1. PIN1 Depletion from Anticlinal LRP Membranes Stimulated by Cytokinin Real-time monitoring of at membranes in stage-iii LRPs untreated (A) and treated with cytokinin (B and C). Cytokinin sensitivity of was higher at the anticlinal (D and F) than at the periclinal (E and G) membranes. primordia were treated for hr, hr, and 7 hr with 5 nm (B, D, and E) and.1 mm (C, F, and G), respectively. Scale bars, mm. Error bars indicate the SEM compared to primordia at time hr (p <.5, p <.1, p <.1; n = 1 LRPs). White and yellow asterisks mark anticlinal and periclinal membranes, respectively. The insets show close-up views, with doubleheaded arrows indicating membranes at which signal was quantified., control medium;, cytokinin derivative N6-benzyladenine. See also Figure S1.

3 Cytokinin Controls Polarity of PIN1 1 need for the auxin flow redirection along the new primordia axis (Figure SH S1K). To examine the impact of the cytokinin perception on the PIN1 polarity, we analyzed cytokinin receptor mutant LRPs. Whereas the PIN1 polarity index did not change in the single cre1-12/ahk4 (Figures 2A, 2B, and 2D), it significantly shifted in favor of anticlinal PIN1 in multiple cre-12/ahk4,ahk and cre1-12/ahk4,ahk2 mutants (Figures 2A, 2C, and 2E 2H). PIN1 at anticlinal membranes of cre1-12/ ahk4,ahk2 LRPs was insensitive to cytokinin, supporting a role of cytokinin signaling in cytokinin-stimulated elimination of PIN1 from anticlinal membranes (Figures S1L and S1M). This PIN1 polarity index shift in cytokinin receptor mutants correlated with a defective LRP patterning, reflected by a smaller number of epidermal cells in stage-iv LRPs than in the control (Figures 2I and 2J). These results indicate that an intact cytokinin perception pathway contributes to proper PIN polarity establishment and redirection of auxin fluxes along new growth axes during primordia organogenesis. To examine the PIN1 sensitivity to cytokinin at different polar domains and cell types, we used root epidermal and cortical cells as a model system. Previously, hemagglutinin (HA)- tagged PIN1 or a 2 fusion, when produced ectopically in root epidermal cells (driven by PIN2 or 5S promoters), had been shown to exhibit an apolar and/or basal (rootward) membrane localization [1, 14]. Thus, the localization of PIN1 in epidermal cells differs from that of the innate epidermal PIN2, PIN2-GFP, or fusions, which are located at apical (shootward) membranes [1, 14]. In contrast, in cortex cells close to the meristem, both PIN1 and PIN2 predominantly localize to basal membranes [15]. This collection of transgenic lines with well-defined PIN localizations provided a convenient model system to test the cytokinin sensitivities at different polar domains. Short-term exposure to cytokinin significantly reduced the 2 membrane signal in epidermal cells (Figures A and D). In contrast, apically located PIN2-GFP and in root epidermal cells were fully insensitive to cytokinin (Figures B, C, E, and F). In cortex cells, in which all PIN variants are located at basal membranes, cytokinin significantly decreased the PIN abundance, regardless of the PIN variants (Figures A F). Immunolocalization of HA-tagged PIN1 enabled us to score the number of epidermal cells with nonpolar, basal, or apical PIN1 due to the partial cell separation in fixed root samples. Cytokinin preferentially decreased PIN1 at the basal, but not the apical, membranes, and the proportion of cells with only apically localized PIN1- HA was higher in cytokinin-treated roots (76.5% 6 8%; n = 29) than in untreated roots, with most cells exhibiting nonpolar (52% 6 11%, n = 11) or basal (24% 6 7%; n = 25) localizations (Figures G and H). These data support our observations that the PIN sensitivity to cytokinin depends primarily on the PIN polar localization rather than on cell type or developmental context. In root epidermal cells, the apically localized PIN2 mediates the shootward auxin stream, which is indispensable for gravity response [1, 2, 1, 16]. The ectopically expressed PIN1-HA and 2 fail to rescue the agravitropic root phenotype of pin2 due to their predominantly nonpolar or basal localizations [1, 16]. We hypothesized that cytokinin, through elimination of PIN1 at the basal, but not apical, membranes of the epidermal cells, might restore the shootward auxin flux and, consequently, the root gravitropism. To verify this assumption, we examined the auxin redistribution in epidermal cells with the DR5rev::GFP auxin reporter [8]. As expected, 4 hr of gravistimulation significantly enhanced the auxin response at the bottom (stimulated) side of control roots (Figures S2A and S2B). In gravistimulated PIN2::PIN1-HA/eir1-1, the auxin response on both sides of the roots was not statistically different (Figures S2A and S2B), but when PIN2::PIN1-HA/ eir1-1 seedlings were grown in the presence of cytokinin, gravistimulation resulted in an asymmetric DR5 response, indicating that cytokinin restored the shootward auxin transport (Figures S2A and S2B). Next, we tested whether cytokininmediated repair of the auxin redistribution in PIN2::PIN1-HA/ eir1-1 correlates with an improved root gravitropism. Cytokinin slightly delayed the gravitropism of wild-type roots and did not restore the agravitropic phenotype of eir1-1/pin2 (Figures S2C and S2D). In contrast, agravitropic PIN2::PIN1-HA as well as 5S::PIN1 roots were able to react to gravitropic stimulation when treated with cytokinin (Figures S2C and S2D). Measurement of auxin transport in roots revealed that the rootward transport in control, pin2, and PIN2::PIN1-HA lines was as strongly reduced by cytokinin as in the untreated pin1, consistent with observations that the PIN1-mediated rootward transport is primary cytokinin target (Figure S2E) [12]. In contrast, shootward transport was defective in pin2 and PIN2::PIN1-HA, but not in control and pin1 roots (Figure S2F) [1]. When control, pin1, and pin2 roots were treated with cytokinin, the shootward transport was slightly reduced, corroborating that the auxin flow mediated by PIN2 is modestly affected by cytokinin at the transcriptional [17], but not at the posttranslational, level [12]. Importantly, cytokinin rescued the shootward auxin transport in PIN2::PIN1-HA, but not in pin2, confirming that the cytokinin effect on PIN1 has a direct impact on the directional auxin transport (Figure S2F). In summary, these results demonstrate that cytokinin promotes the PIN1 degradation selectively from the basal membranes. Hence, this membrane-specific decrease in PIN1 at the basal side of epidermal cells in the presence of cytokinin restores the shootward auxin transport and, consequently, the gravitropic response. The protein phosphatase 2A (PP2A) and AGC kinases act antagonistically on the phosphorylation of PIN proteins and affect their apical-basal localization [18 21]. A PIN polarity shift toward the apical membrane is facilitated in pp2a mutants, by overexpression of the AGC kinases PINOID (PID), WAG1, and WAG2 or by phosphomimic mutations of PIN1. Conversely, the agc or PIN loss-of-phosphorylation mutations induce an apical-to-basal PIN polarity switch [18 22]. We argued that changed PIN1 localization in mutants defective in PP2A or AGC functions might result in PIN1- modulated cytokinin sensitivity. Cytokinin treatment reduced the PIN1 membrane signal in the pp2aa1 single mutant similarly as in the wild-type. However, in the pp2aa1,pp2aa2 and pp2aa1,pp2aa double mutants lacking two of the three regulatory A subunits of the PP2A complex, the cytokinin sensitivity was significantly reduced (Figures 4A and 4B). In roots overexpressing PID, the PIN1 sensitivity to cytokinin was much lower than that in control roots (Figures 4C and 4D). Thus, in lines with more phosphorylated and, hence, more apically localized PIN proteins, the PIN degradation is less sensitive to cytokinin. To examine the cytokinin sensitivity of a phosphorylated PIN1 subpopulation, we used antibodies raised against the PIN1 peptide carrying phosphorylated S21-P (PIN1-a- S21-P), previously shown to be targeted by PID [2]. Typically, cytokinin significantly reduced the PIN1 membrane signal in root meristem cells when immunodetected with standard anti-pin1 antibodies recognizing the hydrophilic loop of PIN1, but no decrease could be detected with

4 Current Biology Vol 24 No 9 14 A PIN1:: B C cre1/pin1:: cre1 ahk2/pin1:: D periclinal/an clinal Rela ve expression _ cre1-12_ E periclinal/an clinal Rela ve expression _ cre1-12 ahk2-2_.2 F PIN1:: G cre1 ahk/pin1:: H Rela ve expression periclinal/an clinal _ cre1-12 ahk-_.2 52 rel. intensity I PIN1:: cre1 ahk2/pin1:: cre1 ahk/pin1:: J Avegrage number of cells Col cre1/ahk2 cre1/ahk Figure 2. PIN Polarity Index in Cytokinin Receptor Mutants (A H) Shift of the polarity index of the membrane abundance in favor of the anticlinal LRP localization of cre1,ahk2 (C; quantified in E) and cre1,ahk (G; quantified in H), but not significant in cre1 (B; quantified in D) cytokinin receptor mutants. LRPs were monitored for 7 hr compared to wildtype (p <.5, p <.1; n = 1 LRPs). (I and J) Reduced numbers of epidermal cells at stage-iv LRPs when compared to control in both cre1,ahk and cre1,ahk2 (n= LRPs) (I; quantified in J). White and yellow asterisks mark anticlinal and periclinal membranes, respectively. A semi-quantitative color-coded heat-map of the GFP fluorescence intensity is provided. Scale bars, 2 mm. Error bars indicate the standard error of the mean., control medium. See also Figure S2. PIN1-a-S21-P antibodies (Figures 4E and 4F), indicating that a phosphorylated PIN1 subpopulation is recruited for degradation in response to cytokinin with reduced efficiency. The loss-of-phosphorylation mutations (Ala) and S1,A and the phosphomimic mutations PIN1- GFP(Asp) and S1,E were correlated with basal

5 Cytokinin Controls Polarity of PIN1 15 A B C PIN2::2 PIN2::PIN2-GFP PIN2:: ep c ep c D E F 12 PIN2::2 12 PIN2::PIN2-GFP Rela ve fluorescence (%) Rela ve fluorescence (%) ep c ep c Rela ve fluorescence (%) ep c ep c PIN2::PIN1GFP G ep cor ep H cor ep cor PIN2::PIN1-HA Polar cell localiza on (%) PIN2::PIN1- HA apical non-polar basal ep c ep c Figure. Basally Localized PIN Proteins from the Plasma Membranes Are Depleted by Cytokinin (A F) Cytokinin-depleted basally localized in cortex and epidermal cells (A; quantified in D), but not apically localized PIN2-GFP (B; quantified in E) and (C; quantified in F) in root epidermal cells. The PIN-GFP membrane signal was measured in root cortical (cor) and epidermal (ep) cells. Roots were treated for hr with.1 mm. Error bars indicate the SEM compared to untreated samples (p <.5; n = 1 roots, with 1 cells per root). (G and H) Cytokinin-depleted PIN1-HA from basal membranes of root epidermal cells and increased proportion of cells with only apically localized PIN1. The PIN1 membrane signal was detected by immunolocalization. Seedlings were grown 6 days on.6 mm (G; quantified in H). Scale bars, 15 mm. ; control medium;, cytokinin derivative N 6 -benzyladenine. and apical membrane localizations, respectively [2, 22]. We found that whereas the cytokinin sensitivity in (Ala) and S1,A was unchanged, the phosphomimic versions (Asp) and S1,E were considerably less sensitive to cytokinin (Figures 4G and 4H; Figures SA and SB). The phosphorylation status of PIN1 affected its cytokinin sensitivity also in developmentally more advanced LRPs. Unlike in control roots, in which cytokinin eliminated PIN1 from anticlinal, but less from periclinal, membranes (Figures 1D and 1G), it decreased significantly S1,A from both anticlinal and periclinal membranes (Figures SC and SD). In contrast, S1,E was insensitive to cytokinin treatment on both anticlinal and periclinal membranes (Figures SE and SF). Thus, phosphorylated PIN proteins are much less sensitive to cytokinin than dephosphorylated ones. Next, we tested the extent to which the phosphorylation status of PIN1 affects the cytokinin-controlled root development. Whereas under control conditions, 86% of the wildtype LRPs developed from stage I or II to stage III or IV (Figure SG), after cytokinin treatment 85% of the LRPs were fully arrested (Figures SG and S4A). LRP development was mildly affected in S1,(A) and S1,E lines, with % and % arrested primordia, respectively, probably due to a deficient PIN1 polarity establishment. In the PIN1- GFPS1,A mutants, cytokinin increased the proportion of arrested LRPs to 77%, whereas only % of the primordia expressing the S1,E were arrested (Figures SG and S4B), suggesting that modulations mimicking PIN1 phosphorylation reduce the cytokinin sensitivity of the PIN1 protein and, as a consequence LRP sensitivity to cytokinin might be attenuated.

6 Current Biology Vol 24 No 9 16 A Col pp2aa1 pp2aa1 pp2aa2 pp2aa1 pp2aa B expression (%) C D E Col PIN1 Col pp2aa1 pp2aa1 pp2aa2 pp2aa1 pp2aa Col Col PIN1-a-S21-P 5S::PID F Rela ve expression (%) expression (%) Col 5S::PID Col_ Col_ 2 PIN1 PIN1-a-S21-P G F I F (Ala) a) (Asp) (Asp) H Rela ve fluorescence (%) (Ala) (Asp) Figure 4. Reduced Cytokinin Sensitivity by Enhanced PIN1 Phosphorylation (A D) PIN1 cytokinin insensitivity in the endodermal (en) cells of pp2aa1 pp2aa and pp2aa1 pp2aa2 (A and B) and 5S::PID (C and D) roots compared to untreated samples (p <.5, p <.1; n = 1 roots, with 1 cells per root). The PIN1 signal was detected by immunolocalization and quantified in endodermal cells of 5-day-old root meristem hr after.1 mm treatment (B and D). Scale bars, mm (A) and 4 mm (C). (E and F) Cytokinin-reduced PIN1 membrane signal in root meristem cells by 8% 6 11% (n = 1), when immunodetected with antibodies recognizing the PIN1 hydrophilic loop, but not with PIN1-a-S21-P antibodies. (G and H) Cytokinin insensitivity of phosphomimic (Asp), but not of loss-of-phosphorylation (Ala) allele, compared to untreated samples (p <.5, p <.1; n = 1 LRPs). White arrowheads mark vacuoles with GFP accumulation. The membrane signal was measured in stage-i LRPs hr after treatment with.1 mm. Scale bars, 2 mm. Error bars indicate the SEM., control medium;, cytokinin derivative N 6 -benzyladenine. See also Figures S and S4. The auxin flow direction is largely defined by the membrane polarity of PIN transporters [1, 2]. The PIN polarity is highly dynamic, and its change during organogenesis or tropic responses [, 4, 8, 24, 25] is crucial for proper developmental output. However, the polarizing cues and mechanisms that underlie the rapid auxin flow redirection during the formation of new organs are still scarcely understood. Here, we demonstrate that cytokinin not only regulates the overall auxin flow

7 Cytokinin Controls Polarity of PIN1 17 capacity, but might also act as a polarizing cue and control the auxin stream directionality. In LRPs, because of the more efficient PIN1 elimination at anticlinal membranes, cytokinin alters the overall polarization in favor of periclinal PIN1, thereby directing the auxin stream toward the tips of newly formed primordia and, consequently, promoting LRP development. Similarly, by elimination of PIN1 from the basal membranes in the root epidermal cells, cytokinin can restore the gravitropic growth caused by the incorrect PIN polarity in roots that ectopically express PIN1. This selective cytokinin sensitivity of PIN1 depends on phosphorylation and it is reduced by an increased phosphorylation degree. In conclusion, this cytokinin-driven differential degradation represents a conceptually novel mechanism to fine-tune the PIN polarity that operates in a developmental program involving a rapid auxin transport redirection. Supplemental Information Supplemental Information includes four figures and Supplemental Experimental Procedures and can be found with this article online at doi.org/1.116/j.cub Acknowledgments We thank Candela Cuesta for technical assistance, Doron Scolnik for sharing expression and imaging results prior to publication, Jürgen Kleine-Vehn for discussions and critical reading of the manuscript, and Annick Bleys and Martine De Cock for help in preparing it. This work was supported by a European Research Council Starting Independent Research Grant (ERC-27-Stg-2762-HCPO to E.B.), the Division of Energy Biosciences, US Department of Energy (grant DE-FG2-6ER1584 to A.S.M.), and an EMBO for a postdoctoral fellowship (LTF to E.F.). Received: January 21, 214 Revised: March 25, 214 Accepted: April 1, 214 Published: April 24, 214 References and Notes 1. Müller, A., Guan, C., Gälweiler, L., Tänzler, P., Huijser, P., Marchant, A., Parry, G., Bennett, M., Wisman, E., and Palme, K. (1998). 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8 Current Biology, Volume 24 Supplemental Information Cytokinin Controls Polarity of PIN1-Dependent Auxin Transport during Lateral Root Organogenesis Peter Marhavý, Jérôme Duclercq, Benjamin Weller, Elena Feraru, Agnieszka Bielach, Remko Offringa, Jiří Friml, Claus Schwechheimer, Angus Murphy, and Eva Benková

9 A 2 Rela ve express ion _ 1 _5nM _.8 _.1µM.6.4 C PIN1:: E PIN1:: PIN1::.1µM_ 18h 18h 18h high_ D PIN1:: 5nM_ 18h 18h F Rela ve Increase of transversal length of LRP (%) _ 6 _5nM.1µM_ 4 2 G III_stage AHK2::GUS H I PIN1:: 45 J II_stage TCS:GFP 4 III_stage 5 _ 25 _ 2 cre ahk2_ 15 cre ahk2_ periclinal membranes 4 5 _ 25 _ 2 cre ahk2_ 15 cre ahk2_ 1 5 Figure S1 III_stage III_stage TCS:GFP M an clinal membranes III_stage AHK::GUS K II_stage PIN1:: Rela ve fluorescence CRE/AHK4::GUS Rela ve fluorescence L periclinal/an clinal Rela ve expression B periclinal/an clinal

10 DR5::GFP PIN2::PIN1-HA PIN2::PIN1-HA B 14 Ra o of DR5 -GFP fluorescence A DR5::GFP t/h t/4h 4 2 DR5-GFP D C DR5-GFP PIN2::PIN1-HA PIN2::PIN1-HA eir1-1 PIN2::PIN1-HA 5S::PIN1 Col Col_ Col_ eir1-1 _ 9 9 eir1-1_ 6 PIN2::PIN1 -HA _ E DPM. 1 seedlings -1 PIN2::PIN1 -HA _ 9 a 5S::PIN1_ 5S::PIN1_ Rootward IAA transport F control kin a a 1 8 b b b b b 6 4 a Shootward IAA transport a 1 a control kine n a a b 8 c b Col- Figure S DPM. 1 seedlings -1 9 pin1 pin2 PIN2::PIN1-HA Col- pin1 pin2 PIN2::PIN1-HA

11 A S1,A S1,E B Rela ve fluorescence S1,A S1,E C Rela ve fluorescence (%) PIN1:: S1,A _.1µM _ D Rela ve fluorescence (%) PIN1:: S1,A _periclinal.1µm _periclinal E Rela ve fluorescence (%) h h h 7 h PIN1:: S1,E _.1µM _ F Rela ve fluorescence (%) h h h 7 h PIN1:: S1,E _periclinal.1µm _periclinal G h h h 7 h h h h 7 h Stages I and II S1,A S1,E Control 2 µm Control 2 µm Control 2 µm Total LRP Developing LRP Arrested LRP Arrested/Total 6/4 4/47 11/7 24/1 1/ 8/2 Inhibition (%) , Fully arrested;, cytokinin derivative N 6 -benzyladenine. Figure S

12 A h h B (Asp) h (Asp) h Figure S4

13 Supplemental Figure Legends Figure S1- Related to Figure1.Cytokinin modulates PIN polarity s index and affect LRP growth (A, B) Real-time monitoring of membrane in stage-iii LRP. Periclinal/anticlinal polarity index of the membrane abundance is shifted in favor of the periclinal localization after cytokinin treatment when compared to the nontreated control (P<.5; P<.1; P<.1; n=1 LRP). Roots were treated for 7 h with low (5 nm) and high (.1 µm) cytokinin concentrations. (C to F) Shift of the periclinal/anticlinal polarity index of the membrane abundance after treatment with 5 nm, correlated with enhanced primordia growth (D and F). Growth of LRP treated with.1 µm is repressed, despite the changed polarity index, probably due to an overall PIN1 depletion from membranes (E and F) compared to primordia at time h (P<.5; P<.1; P<.1; n=2 LRP). LRP outgrowth was monitored for 18 h and growth increase is marked by a white arrow. Scale bars, 45 µm. (G) AHK2, AHK and AHK4/CRE1 expression in stage-iii LRP. Scale bars, µm. (H and J) signal at the plasma membrane in LRP stage-ii and III. (I and K) TCS:GFP expression in stage-ii and stage-iii LRP. Scale bars, µm. (L and M) Real-time monitoring of membrane in stage-iii LRP in wild type (Col-) and cre,ahk2. Cytokinin sensitivity of is higher at anticlinal (L) than at periclinal membranes (M), whereas PIN1 is insensitive in cre,ahk2 (L and M). Roots were treated for 7 h with (5 nm) cytokinin concentrations. Scale bars, µm. Error bars mark the standard error of the mean., control medium;, cytokinin derivative N 6 -benzyladenine.

14 Figure S2- Related to Figure2. Shootward auxin flux restored by cytokinin in the root epidermal cells that ectopically experss PIN1. (A and B) Enhanced DR5rev::GFP response at the bottom side of control roots, but not of gravistimulated PIN2::PIN1-HA,pin2/eir1 roots. Cytokinin treatment partially rescued the asymmetric auxin responses in PIN2::PIN1-HA,pin2/eir1 roots compared to time h (P<.5; P<.1; error bars indicate standard error of the mean, n=2 roots). Arrowheads mark gravistimulated root sides. (C and D) Gravitropic response of PIN2::PIN1-HA,pin2/eir1 and 5S:: roots rescued by cytokinin, but not of pin2/eir1. White and red arrows indicate change in gravity-stimulation. (E and F) H-indole--acetic acid rootward and shootward transport decreased and increased by kinetin application in PIN2::PIN1-HA, respectively (Analysis of variance, Dunnet s post-hoc, P<.5; error bars indicate standard deviation, n= pools of 1 seedlings for assays). Seven-day-old roots grown on medium with.1 µm (A and B) and.6 µm (C and D) or without cytokinin were exposed to a 24-h gravistimulus. Scale bars, µm (A),.5 cm (D)., control medium;, cytokinin derivative N 6 - benzyladenine. Figure S- Related to Figure4. Reduced cytokinin sensitivity by enhanced PIN1 phosphorylation. (A and B) cytokinin insensitivity of phosphomimic S1,E, but not of loss-ofphosphorylation S1,A alleles (P<.5; P<.1; n=1 LRP). White arrowheads indicate vacuoles with GFP accumulation. The membrane signal was measured in stage-i LRP h after treatment with.1 µm. (C to F) Real-time monitoring of at anticlinal and periclinal membranes in control and cytokinin-treated stage-iii LRP. Loss-ofphosphorylation S1,A at both anticlinal and periclinal membranes is cytokinin

15 sensitive (C and D), whereas the phosphomimic S1,E variant is insensitive (E and F) compared to primordia at time h (P<.5; P<.1; n=5 LRP). Error bars indicate the standard error of the mean. (G) Phosphorylation status of PIN1 modulates the cytokinincontrolled lateral root development. LRP development monitored for 12 hours., control medium;, cytokinin derivative N 6 -benzyladenine. Figure S4- Related to Figure 4. LRP sensitivity to cytokinin modulated by the PIN1 phosphorylation status. (A and B) Cytokinin treatment stimulating degradation and inhibiting LRP development (A) and cytokinin-insensitive (Asp) correlating with cytokinininsensitive LRP development (B). Representative real-time analysis of LRP development on control medium () and in the presence of.1 μm N 6 -benzyladenine () (n=1 primordia per line and treatment). Scale bars, 2 µm.

16 Supplemental Experimental Procedures Plant material The transgenic Arabidopsis thaliana (L.) Heynh. lines have been described elsewhere: PIN1:: and DR5rev::GFP [S1]; PIN2::PIN2-GFP [S2]; PIN2::PIN1-HA, eir1-1/pin2, PIN2::, and PIN2:: [S2]; PIN2::PIN1-HA/eir1/DR5::GFP [S]; 5S:: [S4]; 5::PID [S5]; pp2aa1, pp2aa1, pp2aa2, and pp2aa1,pp2aa [S6]; PIN1::(Asp) and PIN1::(Ala)[S7]; PIN1::S1.A and PIN1::S1.E [S8], eir1-1 [S9] ; pin1, and pin2 [S1] cre1-12,pin1::; cre1-12,ahk2,pin1::; cre1-12,ahk,pin1:: [S11]; TCS:GFP [S12]; CRE/AHK4::GUS, AHK2::GUS, AHK::GUS [S11]. Growth conditions Seeds of Arabidopsis (accession Columbia-) were plated on half-strength (.5) Murashige and Skoog (MS) medium (Duchefa) with 1% sucrose,.5 MS medium (Duchefa) and.8% agar (ph 5.7). For the root gravity assays,.5 % agar was used. The seeds were stratified for 2 days at 4 C. Seedlings were grown on vertically oriented plates in growth chambers under a 16-h light/8-h dark photoperiod at 18 C. Hormonal treatments Five- to 6-day-old seedlings were transferred onto solid MS media with or without the cytokinin derivative N 6 -benzyladenine () and incubated for h to 7 h in the dark at 22 C. The

17 concentrations used were 5 nm,.1 μm,.6 μm, and 2 μm as indicated for particular experiments. Real-time analyses of membrane protein dynamics and GFP signal quantification The membrane GFP signal was quantified on scans of stage-i LRP, stage-iii LRP, and primary root epidermal and cortical cells. Pictures were taken with a FV1 ASW confocal microscope (Olympus) and LSM7 (Zeiss) with a 2 or 6 (water immersion) objective. For all the experiments, the scans were done with identical microscopes and laser settings. At minimum, 1 stage-i LRP (two anticlinal plasma membranes) and five stage-iii LRP (two anticlinal and periclinal plasma membranes) were analyzed. Criteria for quantifications of the membrane signals in particular tissues and the microscope settings were as described [S11]. The fluorescence intensity of the membrane PIN-GFP signal was quantified with ImageJ (NIH; and Cellset software, as described [S1, S14]. The statistical significance was evaluated with Student's t-test. Images were processed in Adobe Illustrator. Immunolocalization Immunolocalizations were done on 5-day-old seedlings with an InSituPro robot (Intavis, Köln, Germany) according to the published protocol [S15]. Dilutions of antibodies used were: 1:1 for rabbit anti-pin1 [S16], 1:5 for mouse anti-ha (AbCam/HA.C5), and 1:6 for Cy antirabbit (Sigma). Signal intensities were evaluated with the quantification function of Image J 1.41 software ( and Cellset software. The rabbit PIN1-a-S21-P antibody was generated with the phosphorylated synthetic peptide LSATPRP-S(PO H 2 )-NLTNA followed by affinity purifications against the nonphosphorylated and phosphorylated peptide at

18 Eurogentec (Liège, Belgium). Col- seedlings were immunostained with rabbit PIN1-a-S21-P (dilution 1:). Analysis of LRP organogenesis and gravitropism Real-time LRP development was analyzed as described [S11]. For the LRP growth, the relative increase of the LRP length within 18 h was determined. At least 2 stage-v LRP were processed. For root gravitropism response analyses, seedlings were grown on.5% agar (ph 5.7) and.5 MS medium (Duchefa) on vertically oriented plates in growth chambers under a 16-h light/8-h dark photoperiod at 18 C. After 6 days, the plates were rotated by 9 degree for 24 h and the gravitropic responses were measured with the ImageJ software. The obtained values were processed in Excel 11.. software (Microsoft Corporation). Auxin transport measurements Auxin transport was measured as described [S17, S18]. Filter paper strips (2 mm) at the root apex were soaked with.25 MS (ph 5.5) ± kinetin (.5 μm) for a 2-h acclimation preceding the application of H-indole--acetic acid ( Ci mmol -1 ).

19 Supplemental References S1. Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D., Jürgens, G., and Friml, J. (2). Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115, S2. Wiśniewska, J., Xu, J., Seifertová, D., Brewer, P. B., Růžička, K., Blilou, I., Rouquié, D., Benková, E., Scheres, B., and Friml, J. (26). Polar PIN localization directs auxin flow in plants. Science 12, 88. S. Feraru, E., Feraru, M. I., Kleine-Vehn, J., Martinière, A., Mouille, G., Vanneste, S., Vernhettes, S., Runions, J., and Friml, J. (211). PIN polarity maintenance by the cell wall in Arabidopsis. Curr. Biol. 21, 8 4. S4. Růzicka, K., Ljung, K., Vanneste, S., Podhorská, R., Beeckman, T., Friml, J., and Benková, E. (27). Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution. Plant Cell 19, S5. Friml, J., Yang, X., Michniewicz, M., Weijers, D., Quint, A., Tietz, O., Benjamins, R., Ouwerkerk, P. B. F., Ljung, K., Sandberg, G., et al. (24). A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux. Science 6, S6. Michniewicz, M., Zago, M. K., Abas, L., Weijers, D., Schweighofer, A., Meskiene, I., Heisler, M. G., Ohno, C., Zhang, J., Huang, F., et al. (27). Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux. Cell 1, S7. Zhang, J., Nodzyński, T., Pĕnčík, A., Rolčík, J., and Friml, J. (21). PIN phosphorylation is sufficient to mediate PIN polarity and direct auxin transport. Proc. Natl. Acad. Sci. USA 17,

20 S8. Huang, F., Zago, M. K., Abas, L., van Marion, A., Galván-Ampudia, C. S., and Offringa, R. (21). Phosphorylation of conserved PIN motifs directs Arabidopsis PIN1 polarity and auxin transport. Plant Cell 22, S9. 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, S1. Friml, J., Vieten, A., Sauer, M., Weijers, D., Schwarz, H., Hamann, T., Offringa, R., and Jürgens, G. (2). Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426, S11. Marhavý, P., Bielach, A., Abas, L., Abuzeineh, A., Duclercq, J., Tanaka, H., Pařezová, M., Petrášek, J., Friml, J., Kleine-Vehn, J., et al. (211). Cytokinin modulates endocytic trafficking of PIN1 auxin efflux carrier to control plant organogenesis. Dev. Cell 21, S12. Bielach, A., Podlešáková, K., Marhavý, P., Duclercq, J., Cuesta, C., Müller, B., Grunewald, W., Tarkowski, P., and Benková, E. (212). Spatiotemporal regulation of lateral root organogenesis in Arabidopsis by cytokinin. Plant Cell 24, S1. Žádníková, P., Petrášek, J., Marhavý, P., Raz, V., Vandenbussche, F., Ding, Z., Schwarzerová, K., Morita, M. T., Tasaka, M., Hejátko, J., et al. (21). Role of PINmediated auxin efflux in apical hook development of Arabidopsis thaliana. Development 17, S14. Pound, M. P., French, A. P., Wells, D. M., Bennett, M. J., and Pridmore, T. P. (212). CellSeT: novel software to extract and analyze structured networks of plant cells from confocal images. Plant Cell 24,

21 S15. Sauer, M., Balla, J., Luschnig, C., Wiśniewska, J., Reinöhl, V., Friml, J., and Benková, E. (26). Canalization of auxin flow by Aux/IAA-ARF-dependent feedback regulation of PIN polarity. Genes Dev. 2, S16. Paciorek, T., Zazímalová, E., Ruthardt, N., Petrásek, J., Stierhof, Y.-D., Kleine-Vehn, J., Morris, D. A., Emans, N., Jürgens, G., Geldner, N., et al. (25). Auxin inhibits endocytosis and promotes its own efflux from cells. Nature 45, S17. Kubeš, M., Yang, H., Richter, G. L., Cheng, Y., Młodzińska, E., Wang, X., Blakeslee, J. J., Carraro, N., Petrášek, J., Žažímalová, E., et al. (212). The Arabidopsis concentrationdependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis. Plant J. 69, S18. Peer, W. A., and Murphy, A. S. (27). Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci. 12,