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

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1 Current Biology,, May, ª Elsevier Ltd All rights reserved Cytokinin Controls Polarity of PIN-Dendent Auxin Transport during Lateral Root Organogenesis Rort Peter Marhavý,,, Jérôme Dulerq,,, Benjamin Weller, Elena eraru,,,8 Agnieszka Bielah,, Remko Offringa, Jirí riml,,, Claus Shwehheimer, Angus Murphy, 6 and Eva Benková,,, Dartment of Plant Systems Biology, VIB, 9 ent, Belgium Dartment of Plant Biotehnology and Bioinformatis, hent University, 9 ent, Belgium Institute of Siene and Tehnology Austria, Klosterneuburg, Austria Plant Systems Biology, Tehnishe Universität Münhen, 8 reising, ermany Dartment of Moleular and Developmental enetis, Institute Biology Leiden, Leiden University, AL Leiden, the Netherlands 6 Plant Siene and Landsape Arhiteture, University of Maryland, lege Park, MD, USA Summary The plant hormones auxin and ytokinin mutually oordinate their ativities to ontrol various aspets of development [ 9], and their rosstalk ours at multiple levels [, ]. Cytokinin-mediated modulation of auxin transport provides an effiient means to regulate auxin distribution in plant organs. Here, we demonstrate that ytokinin does not merely ontrol the overall auxin flow apaity, but might also at as a polarizing ue and ontrol the auxin stream diretionality during plant organogenesis. Cytokinin enhanes the PIN-ORMED (PIN) auxin transporter dletion at speifi polar domains, thus rearranging the ellular PIN polarities and diretly regulating the auxin flow diretion. This seletive ytokinin sensitivity orrelates with the PIN protein phosphorylation degree. PIN phosphomimiking mutations, as well as enhaned phosphorylation in plants with modulated ativities of PIN-speifi kinases and phosphatases, desensitize PIN to ytokinin. Our results reveal ontually novel, ytokinin-driven polarization mehanism that operates in developmental proesses involving rapid auxin stream rediretion, suh as lateral root organogenesis, in whih a gradual PIN polarity swith defines the growth axis of the newly formed organ. Results and Disussion lexible modulation of diretional auxin flow is at the ore of plant developmental plastiity and adaptation to flutuating environmental onditions. Rediretion of auxin fluxes enables tropi responses [ ], as well as speifiation of the embryo Present address: Researh Unit Eology and Dynamis of Human- Influened Systems (EDYSAN, RE 98 CNRS), University of Piardie Jules Verne, 8 Amiens, rane 8 Present address: Dartment of Applied enetis and Cell Biology, University of Natural Resoures and Life Sienes (BOKU), 9 Vienna, Austria Correspondene: eva.benkova@ist.a.at growth axis [6, ] and of newly initiated organs [8, 9]. Lateral root organogenesis is a prominent example of a developmental proess during whih rediretion of auxin flow ours, thereby defining the growth axis of developing lateral roots [8]. This auxin flow rediretion dends on the gradual rolarization of the PIN-ORMED (PIN) auxin transporter from the predominantly antilinal orientation during the initial phases of lateral root primordium (LRP) organogenesis to the perilinal ell membranes, thus direting the auxin transport stream toward the primordia tips. However, the polarizing ues and underlying mehanisms that diret the auxin flow during primordia formation are still unknown. Developmental regulation of the root system by ytokinin is partially mediated by interferene with the endoyti traffiking. The ytokinin-stimulated PIN lyti degradation from stage-i LRP ell membranes orrelates with rression of primordia organogenesis []. To get a broader view on the developmental role of the ytokinin-ontrolled PIN degradation, we haraterized the ytokinin effets on the LRPs at the more advaned developmental stage III, when distint PIN polarities at either the antilinal or perilinal membranes (transversal and longitudinal to the primary root growth axis, respetively) are established (igure A). Cytokinin at low ( nm) or high (. mm) onentrations rapidly dleted the PIN-P fusion signal from antilinal membranes when ompared to untreated primordia (igure A D and ), but had no or a weaker impat at perilinal membranes (igures A C, E, and ). The differential ytokinin sensitivity of PIN at antilinal versus perilinal LRP membranes implies that ytokinin might modulate the PIN polarity index (the PIN ratio at perilinal versus antilinal membranes) in favor of the perilinal loation and, onsequently, enhane the auxin flow toward the primordia tips to promote their development. Evaluation of the PIN polarity index revealed a lear shift toward the perilinal membranes of LRPs treated with ytokinin (igures SA and SB available online). Importantly, appliation of nm ytokinin on stage-v LRPs onward signifiantly stimulated the primordia outgrowth when ompared to ontrol primordia (igures SC, SD, and S). In ontrast, high ytokinin onentrations, despite the modified PIN polarity index, retarded primordia outgrowth, probably due to high overall derease in PIN abundane (igures SC, SE, and S; igures and ). These results indiate that ytokinin differentially targets PIN at antilinal or perilinal LRP membranes. Consequently, beause the PIN polarization index is altered in favor of perilinal PIN, the auxin flow toward the primordia tips might be enhaned and promote primordia development. Cytokinin signaling mediated through the ARABIDOPSIS HISTIDINE KINASE (AHK)/CYTOKININ RESISTANT (CRE) ytokinin retor was found to ontribute to this regulatory pathway []. Expression of ytokinin retors AHK, AHK, and AHK and the TCS::P ytokinin rorter was observed in the inner layers of LRPs (igures S and SK). However, whereas expression of ytokinin retors ould be deteted from developmental stage I on (igure S) [], the ytokinin response was signifiantly ativated from stage III on, orrelating with the PIN polarization time and

2 Current Biology Vol No μM_.8 6. C.6.9 nm_ B PIN::PIN-P A PIN::PIN-P an linal membranes 8 6 nm E Rela ve fluoresene (%) Rela ve fluoresene (%) D 8 6 nm. PIN::PIN-P an linal membranes 8 6.μM Rela ve fluoresene (%) Rela ve fluoresene (%) perilinal membranes PIN::PIN-P. PIN::PIN-P perilinal membranes 8 6.μM.. igure. PIN Dletion from Antilinal LRP Membranes Stimulated by Cytokinin Real-time monitoring of PIN-P at membranes in stage-iii LRPs untreated (A) and treated with ytokinin (B and C). Cytokinin sensitivity of PIN-P was higher at the antilinal (D and ) than at the perilinal (E and ) membranes. PIN-P primordia were treated for. hr, hr, and hr with nm (B, D, and E) and. mm (C,, and ), respetively. Sale bars, mm. Error bars indiate the SEM ompared to primordia at time hr (p <., p <., p <.; n = LRPs). White and yellow asterisks mark antilinal and perilinal membranes, respetively. The insets show lose-up views, with doubleheaded arrows indiating membranes at whih PIN-P signal was quantified., ontrol medium;, ytokinin derivative N6-benzyladenine. See also igure S.

3 Cytokinin Controls Polarity of PIN need for the auxin flow rediretion along the new primordia axis (igure SH SK). To examine the impat of the ytokinin pertion on the PIN polarity, we analyzed ytokinin retor mutant LRPs. Whereas the PIN polarity index did not hange in the single re-/ahk (igures A, B, and D), it signifiantly shifted in favor of antilinal PIN in multiple re-/ahk,ahk and re-/ahk,ahk mutants (igures A, C, and E H). PIN at antilinal membranes of re-/ ahk,ahk LRPs was insensitive to ytokinin, supporting a role of ytokinin signaling in ytokinin-stimulated elimination of PIN from antilinal membranes (igures SL and SM). This PIN polarity index shift in ytokinin retor mutants orrelated with a defetive LRP patterning, refleted by a smaller number of idermal ells in stage-iv LRPs than in the ontrol (igures I and J). These results indiate that an intat ytokinin pertion pathway ontributes to proper PIN polarity establishment and rediretion of auxin fluxes along new growth axes during primordia organogenesis. To examine the PIN sensitivity to ytokinin at different polar domains and ell types, we used root idermal and ortial ells as a model system. Previously, hemagglutinin (HA)- tagged PIN or a PIN-P fusion, when produed etopially in root idermal ells (driven by PIN or S promoters), had been shown to exhibit an apolar and/or basal (rootward) membrane loalization [, ]. Thus, the loalization of PIN in idermal ells differs from that of the innate idermal PIN, PIN-P, or PIN-P fusions, whih are loated at apial (shootward) membranes [, ]. In ontrast, in ortex ells lose to the meristem, both PIN and PIN predominantly loalize to basal membranes []. This olletion of transgeni lines with well-defined PIN loalizations provided a onvenient model system to test the ytokinin sensitivities at different polar domains. Short-term exposure to ytokinin signifiantly redued the PIN-P membrane signal in idermal ells (igures A and D). In ontrast, apially loated PIN-P and PIN-P in root idermal ells were fully insensitive to ytokinin (igures B, C, E, and ). In ortex ells, in whih all PIN variants are loated at basal membranes, ytokinin signifiantly dereased the PIN abundane, regardless of the PIN variants (igures A ). Immunoloalization of HA-tagged PIN enabled us to sore the number of idermal ells with nonpolar, basal, or apial PIN due to the partial ell saration in fixed root samples. Cytokinin preferentially dereased PIN at the basal, but not the apial, membranes, and the proportion of ells with only apially loalized PIN- HA was higher in ytokinin-treated roots (6.% 6 8%; n = 9) than in untreated roots, with most ells exhibiting nonpolar (% 6 %, n = ) or basal (% 6 %; n = ) loalizations (igures and H). These data support our observations that the PIN sensitivity to ytokinin dends primarily on the PIN polar loalization rather than on ell type or developmental ontext. In root idermal ells, the apially loalized PIN mediates the shootward auxin stream, whih is indispensable for gravity response [,,, 6]. The etopially expressed PIN-HA and PIN-P fail to resue the agravitropi root phenotype of pin due to their predominantly nonpolar or basal loalizations [, 6]. We hypothesized that ytokinin, through elimination of PIN at the basal, but not apial, membranes of the idermal ells, might restore the shootward auxin flux and, onsequently, the root gravitropism. To verify this assumption, we examined the auxin redistribution in idermal ells with the DRrev::P auxin rorter [8]. As expeted, hr of gravistimulation signifiantly enhaned the auxin response at the bottom (stimulated) side of ontrol roots (igures SA and SB). In gravistimulated PIN::PIN-HA/eir-, the auxin response on both sides of the roots was not statistially different (igures SA and SB), but when PIN::PIN-HA/ eir- seedlings were grown in the presene of ytokinin, gravistimulation resulted in an asymmetri DR response, indiating that ytokinin restored the shootward auxin transport (igures SA and SB). Next, we tested whether ytokininmediated rair of the auxin redistribution in PIN::PIN-HA/ eir- orrelates with an improved root gravitropism. Cytokinin slightly delayed the gravitropism of wild-type roots and did not restore the agravitropi phenotype of eir-/pin (igures SC and SD). In ontrast, agravitropi PIN::PIN-HA as well as S::PIN roots were able to reat to gravitropi stimulation when treated with ytokinin (igures SC and SD). Measurement of auxin transport in roots revealed that the rootward transport in ontrol, pin, and PIN::PIN-HA lines was as strongly redued by ytokinin as in the untreated pin, onsistent with observations that the PIN-mediated rootward transport is primary ytokinin target (igure SE) []. In ontrast, shootward transport was defetive in pin and PIN::PIN-HA, but not in ontrol and pin roots (igure S) []. When ontrol, pin, and pin roots were treated with ytokinin, the shootward transport was slightly redued, orroborating that the auxin flow mediated by PIN is modestly affeted by ytokinin at the transriptional [], but not at the posttranslational, level []. Importantly, ytokinin resued the shootward auxin transport in PIN::PIN-HA, but not in pin, onfirming that the ytokinin effet on PIN has a diret impat on the diretional auxin transport (igure S). In summary, these results demonstrate that ytokinin promotes the PIN degradation seletively from the basal membranes. Hene, this membrane-speifi derease in PIN at the basal side of idermal ells in the presene of ytokinin restores the shootward auxin transport and, onsequently, the gravitropi response. The protein phosphatase A (PPA) and AC kinases at antagonistially on the phosphorylation of PIN proteins and affet their apial-basal loalization [8 ]. A PIN polarity shift toward the apial membrane is failitated in ppa mutants, by overexpression of the AC kinases PINOID (PID), WA, and WA or by phosphomimi mutations of PIN. Conversely, the ag or PIN loss-of-phosphorylation mutations indue an apial-to-basal PIN polarity swith [8 ]. We argued that hanged PIN loalization in mutants defetive in PPA or AC funtions might result in PIN- modulated ytokinin sensitivity. Cytokinin treatment redued the PIN membrane signal in the ppaa single mutant similarly as in the wild-type. However, in the ppaa,ppaa and ppaa,ppaa double mutants laking two of the three regulatory A subunits of the PPA omplex, the ytokinin sensitivity was signifiantly redued (igures A and B). In roots overexpressing PID, the PIN sensitivity to ytokinin was muh lower than that in ontrol roots (igures C and D). Thus, in lines with more phosphorylated and, hene, more apially loalized PIN proteins, the PIN degradation is less sensitive to ytokinin. To examine the ytokinin sensitivity of a phosphorylated PIN subpopulation, we used antibodies raised against the PIN ptide arrying phosphorylated S-P (PIN-a- S-P), previously shown to be targeted by PID []. Typially, ytokinin signifiantly redued the PIN membrane signal in root meristem ells when immunodeteted with standard anti-pin antibodies reognizing the hydrophili loop of PIN, but no derease ould be deteted with

4 Current Biology Vol No 9 A PIN::PIN-P B C re/pin::pin-p re ahk/pin::pin-p D.. perilinal/an linal Rela ve expression.8.6. PIN-P_ re-_ E... perilinal/an linal Rela ve expression.8.6. PIN-P_ re- ahk-_. PIN::PIN-P re ahk/pin::pin-p H Rela ve expression perilinal/an linal PIN-P_ re- ahk-_.. rel. intensity I PIN::PIN-P re ahk/pin::pin-p re ahk/pin::pin-p J Avegrage number of ells re/ahk re/ahk igure. PIN Polarity Index in Cytokinin Retor Mutants (A H) Shift of the polarity index of the PIN-P membrane abundane in favor of the antilinal LRP loalization of re,ahk (C; quantified in E) and re,ahk (; quantified in H), but not signifiant in re (B; quantified in D) ytokinin retor mutants. LRPs were monitored for hr ompared to wildtype PIN-P (p <., p <.; n = LRPs). (I and J) Redued numbers of idermal ells at stage-iv LRPs when ompared to ontrol in both re,ahk and re,ahk (n= LRPs) (I; quantified in J). White and yellow asterisks mark antilinal and perilinal membranes, respetively. A semi-quantitative olor-oded heat-map of the P fluoresene intensity is provided. Sale bars, mm. Error bars indiate the standard error of the mean., ontrol medium. See also igure S. PIN-a-S-P antibodies (igures E and ), indiating that a phosphorylated PIN subpopulation is reruited for degradation in response to ytokinin with redued effiieny. The loss-of-phosphorylation mutations PIN-P(Ala) and PIN-PS,A and the phosphomimi mutations PIN- P(Asp) and PIN-PS,E were orrelated with basal

5 Cytokinin Controls Polarity of PIN A B C PIN::PIN-P PIN::PIN-P PIN::PIN-P D E PIN::PIN-P PIN::PIN-P Rela ve fluoresene (%) 8 6 Rela ve fluoresene (%) 8 6 Rela ve fluoresene (%) 8 6 PIN::PINP or H or or PIN::PIN-HA Polar ell loaliza on (%) 8 6 PIN::PIN- HA apial non-polar basal igure. Basally Loalized PIN Proteins from the Plasma Membranes Are Dleted by Cytokinin (A ) Cytokinin-dleted basally loalized PIN-P in ortex and idermal ells (A; quantified in D), but not apially loalized PIN-P (B; quantified in E) and PIN-P (C; quantified in ) in root idermal ells. The PIN-P membrane signal was measured in root ortial (or) and idermal () ells. Roots were treated for. hr with. mm. Error bars indiate the SEM ompared to untreated samples (p <.; n = roots, with ells per root). ( and H) Cytokinin-dleted PIN-HA from basal membranes of root idermal ells and inreased proportion of ells with only apially loalized PIN. The PIN membrane signal was deteted by immunoloalization. Seedlings were grown 6 days on.6 mm (; quantified in H). Sale bars, mm. ; ontrol medium;, ytokinin derivative N 6 -benzyladenine. and apial membrane loalizations, respetively [, ]. We found that whereas the ytokinin sensitivity in PIN-P(Ala) and PIN-PS,A was unhanged, the phosphomimi versions PIN-P(Asp) and PIN-PS,E were onsiderably less sensitive to ytokinin (igures and H; igures SA and SB). The phosphorylation status of PIN affeted its ytokinin sensitivity also in developmentally more advaned LRPs. Unlike in ontrol roots, in whih ytokinin eliminated PIN from antilinal, but less from perilinal, membranes (igures D and ), it dereased signifiantly PIN-PS,A from both antilinal and perilinal membranes (igures SC and SD). In ontrast, PIN-PS,E was insensitive to ytokinin treatment on both antilinal and perilinal membranes (igures SE and S). Thus, phosphorylated PIN proteins are muh less sensitive to ytokinin than dhosphorylated ones. Next, we tested the extent to whih the phosphorylation status of PIN affets the ytokinin-ontrolled root development. Whereas under ontrol onditions, 86% of the wildtype LRPs developed from stage I or II to stage III or IV (igure S), after ytokinin treatment 8% of the LRPs were fully arrested (igures S and SA). LRP development was mildly affeted in PIN-PS,(A) and PIN-PS,E lines, with % and % arrested primordia, respetively, probably due to a defiient PIN polarity establishment. In the PIN- PS,A mutants, ytokinin inreased the proportion of arrested LRPs to %, whereas only % of the primordia expressing the PIN-PS,E were arrested (igures S and SB), suggesting that modulations mimiking PIN phosphorylation redue the ytokinin sensitivity of the PIN protein and, as a onsequene LRP sensitivity to ytokinin might be attenuated.

6 Current Biology Vol No 9 6 A ppaa ppaa ppaa ppaa ppaa B expression (%) C D 8 6 E PIN ppaa ppaa ppaa ppaa ppaa PIN-a-S-P S::PID Rela ve expression (%) 8 6 expression (%) 8 6 S::PID PIN PIN-a-S-P PIN-P PIN-P PIN-P PIN-P PIN-P I (Ala) a) (Asp) (Asp) H Rela ve fluoresene (%) 8 6 PIN-P PIN-P(Ala) PIN-P(Asp) igure. Redued Cytokinin Sensitivity by Enhaned PIN Phosphorylation (A D) PIN ytokinin insensitivity in the endodermal (en) ells of ppaa ppaa and ppaa ppaa (A and B) and S::PID (C and D) roots ompared to untreated samples (p <., p <.; n = roots, with ells per root). The PIN signal was deteted by immunoloalization and quantified in endodermal ells of -day-old root meristem. hr after. mm treatment (B and D). Sale bars, mm (A) and mm (C). (E and ) Cytokinin-redued PIN membrane signal in root meristem ells by 8% 6 % (n = ), when immunodeteted with antibodies reognizing the PIN hydrophili loop, but not with PIN-a-S-P antibodies. ( and H) Cytokinin insensitivity of phosphomimi PIN-P(Asp), but not of loss-of-phosphorylation PIN-P(Ala) allele, ompared to untreated samples (p <., p <.; n = LRPs). White arrowheads mark vauoles with P aumulation. The PIN-P membrane signal was measured in stage-i LRPs. hr after treatment with. mm. Sale bars, mm. Error bars indiate the SEM., ontrol medium;, ytokinin derivative N 6 -benzyladenine. See also igures S and S. The auxin flow diretion is largely defined by the membrane polarity of PIN transporters [, ]. The PIN polarity is highly dynami, and its hange during organogenesis or tropi responses [,, 8,, ] is ruial for proper developmental output. However, the polarizing ues and mehanisms that underlie the rapid auxin flow rediretion during the formation of new organs are still sarely understood. Here, we demonstrate that ytokinin not only regulates the overall auxin flow

7 Cytokinin Controls Polarity of PIN apaity, but might also at as a polarizing ue and ontrol the auxin stream diretionality. In LRPs, beause of the more effiient PIN elimination at antilinal membranes, ytokinin alters the overall polarization in favor of perilinal PIN, thereby direting the auxin stream toward the tips of newly formed primordia and, onsequently, promoting LRP development. Similarly, by elimination of PIN from the basal membranes in the root idermal ells, ytokinin an restore the gravitropi growth aused by the inorret PIN polarity in roots that etopially express PIN. This seletive ytokinin sensitivity of PIN dends on phosphorylation and it is redued by an inreased phosphorylation degree. In onlusion, this ytokinin-driven differential degradation rresents a ontually novel mehanism to fine-tune the PIN polarity that operates in a developmental program involving a rapid auxin transport rediretion. Supplemental Information Supplemental Information inludes four figures and Supplemental Experimental Proedures and an be found with this artile online at doi.org/.6/j.ub... Aknowledgments We thank Candela Cuesta for tehnial assistane, Doron Solnik for sharing expression and imaging results prior to publiation, Jürgen Kleine-Vehn for disussions and ritial reading of the manusript, and Annik Bleys and Martine De Cok for help in praring it. This work was supported by a European Researh Counil Starting Indendent Researh rant (ERC--Stg-6-HCPO to E.B.), the Division of Energy Biosienes, US Dartment of Energy (grant DE--6ER8 to A.S.M.), and an EMBO for a postdotoral fellowship (LT 9- to E..). Reeived: January, Revised: Marh, Ated: April, Published: April, Referenes and Notes. Müller, A., uan, C., älweiler, L., Tänzler, P., Huijser, P., Marhant, A., Parry,., Bennett, M., Wisman, E., and Palme, K. (998). AtPIN defines a lous of Arabidopsis for root gravitropism ontrol. 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