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Report

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

Peter Marhavy´ , 1 , 2 , 3 Je´ roˆ me Duclercq, 1 , 2 , 7 Benjamin Weller, 4 Elena Feraru, 1 , 2 , 8 Agnieszka Bielach, 1 , 2 Remko Offringa, 5 Ji rı´ 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

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: eva.benkova@ist.ac.at

1190 Vienna, Austria *Correspondence: eva.benkova@ist.ac.at growth axis [ 6, 7 ] and of newly initiated organs

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 1 A). Cytokinin at low (5 nM) or high (0.1 m M) 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 S1 A 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 S1 C, 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 S1 C, 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 S1 G and S1K). However, whereas expression of cytokinin receptors could be detected from developmental stage I on ( Figure S1 G) [12 ], the cytokinin response was significantly activated from stage III on, correlating with the PIN1 polarization time and

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A PIN1::PIN1-GFP Ø 69.534 52.998 0 43.463 45.749 54.612 65.232 1.5 42.268 65.731 73.98 77.897
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5nM_BA 70.852 60.703 0 58.802 68.39 89.93 93.4 1.5 64.124 62.29 109.665 123.915 42.743 3
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Real-time monitoring 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 nM BA (B, D, and E) and 0.1 mM BA (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 < 0.0001; n = 10 LRPs). White and yellow asterisks mark anticlinal and periclinal membranes, respectively. The insets show close-up views, with double- headed arrows indicating membranes at which PIN1-GFP signal was quantified. Ø, control medium; BA, cytokinin derivative N 6 -benzyladenine. See also Figure S1.

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need for the auxin flow redirection along the new primordia axis ( Figure S H–S1K). To examine the impact of the cytokinin perception on the PIN1 polarity, we analyzed cytokinin recep- tor 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,ahk3 and cre1-12/ahk4,ahk2 mutants ( Figures 2 A, 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 S1 L and S1M). This PIN1 polarity index shift in cytokinin receptor mutants corre- lated 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 cyto- kinin 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 PIN1-GFP2 fusion, when produced ectopi- cally in root epidermal cells (driven by PIN2 or 35S promoters), had been shown to exhibit an apolar and/or basal (rootward) membrane localization [13, 14 ]. Thus, the localization of PIN1 in epidermal cells differs from that of the innate epidermal PIN2, PIN2-GFP, or PIN1-GFP3 fusions, which are located at apical (shootward) membranes [ 13, 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 PIN1-GFP2 membrane signal in epidermal cells ( Figures 3 A and 3D). In contrast, apically located PIN2-GFP and PIN1-GFP3 in root epidermal cells were fully insensitive to cytokinin ( Figures 3 B, 3C, 3E, and 3F). In cortex cells, in which all PIN variants are located at basal membranes, cyto- kinin significantly decreased the PIN abundance, regardless of the PIN variants ( Figures 3A–3F). 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 = 209) than in untreated roots, with most cells exhibiting nonpolar (52% 6 11%, n = 113) or basal (24% 6 7%; n = 25) localizations ( Figures 3 G and 3H). These data support our observations that the PIN sensitivity to cytokinin depends pri- marily 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, 13, 16 ]. The ectopically expressed PIN1-HA and PIN1-GFP2 fail to rescue the agravitropic root phenotype of pin2 due to their predominantly nonpolar or basal localizations [ 13, 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 grav- istimulation significantly enhanced the auxin response at the

bottom (stimulated) side of control roots ( Figures S2 A and S2B). In gravistimulated PIN2::PIN1-HA / eir1-1 , the auxin response on both sides of the roots was not statistically different ( Figures S2 A and S2B), but when PIN2::PIN1-HA / eir1-1 seedlings were grown in the presence of cytokinin, grav- istimulation resulted in an asymmetric DR5 response, indi- cating that cytokinin restored the shootward auxin transport ( Figures S2 A and S2B). Next, we tested whether cytokinin- mediated 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 S2 C and S2D). In contrast, agravitropic PIN2::PIN1-HA as well as 35S::PIN1 roots were able to react to gravitropic stimulation when treated with cytokinin ( Figures S2 C 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 S2 E) [12 ]. In contrast, shootward transport was defective in pin2 and PIN2::PIN1-HA , but not in control and pin1 roots ( Fig- ure S2 F) [ 13 ]. 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 S2 F). In summary, these results demonstrate that cytokinin pro- motes the PIN1 degradation selectively from the basal mem- branes. 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 AGC3 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 AGC3 kinases PINOID (PID), WAG1, and WAG2 or by phosphomimic mutations of PIN1. Conversely, the agc3 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 AGC3 functions might result in PIN1- modulated cytokinin sensitivity. Cytokinin treatment reduced the PIN1 membrane signal in the pp2aa1 single mutant simi- larly as in the wild-type. However, in the pp2aa1,pp2aa2 and pp2aa1,pp2aa3 double mutants lacking two of the three regu- latory A subunits of the PP2A complex, the cytokinin sensitivity was significantly reduced ( Figures 4 A and 4B). In roots overex- pressing 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 S231-P (PIN1-a- S231-P), previously shown to be targeted by PID [ 20 ]. Typi- cally, 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

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Figure 2. PIN Polarity Index in Cytokinin Receptor Mutants

(A–H) Shift of the polarity index of the PIN1-GFP membrane abundance in favor of the anticlinal LRP localization of cre1,ahk2 (C; quantified in E) and cre1,ahk3 (G; quantified in H), but not significant in cre1 (B; quantified in D) cytokinin receptor mutants. LRPs were monitored for 7 hr compared to wild- type PIN1-GFP (*p < 0.05, **p < 0.001; n = 10 LRPs). (I and J) Reduced numbers of epidermal cells at stage-IV LRPs when compared to control in both cre1,ahk3 and cre1,ahk2 (n= 30 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, 20 mm. Error bars indicate the standard error of the mean. Ø, control medium. See also Figure S2.

PIN1-a-S231-P antibodies ( Figures 4E and 4F), indicating that a phosphorylated PIN1 subpopulation is recruited for degra- dation in response to cytokinin with reduced efficiency.

The loss-of-phosphorylation mutations PIN1-GFP(Ala) and PIN1-GFPS1,3A and the phosphomimic mutations PIN1- GFP(Asp) and PIN1-GFPS1,3E were correlated with basal

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Figure 3. Basally Localized PIN Proteins from the Plasma Membranes Are Depleted by Cytokinin

(A–F) Cytokinin-depleted basally localized PIN1-GFP in cortex and epidermal cells (A; quantified in D), but not apically localized PIN2-GFP (B; quantified in E) and PIN1-GFP3 (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 1.5 hr with 0.1 mM BA. Error bars indicate the SEM compared to untreated samples (*p < 0.05; n = 10 roots, with 10 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 0.6 m M BA (G; quantified in H). Scale bars, 15 mm. Ø; control medium; BA, cytokinin derivative N 6 -benzyladenine.

and apical membrane localizations, respectively [ 20, 22 ]. We found that whereas the cytokinin sensitivity in PIN1-GFP(Ala) and PIN1-GFPS1,3A was unchanged, the phosphomimic ver- sions PIN1-GFP(Asp) and PIN1-GFPS1,3E were considerably less sensitive to cytokinin ( Figures 4G and 4H; Figures S3 A and S3B). 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 ( Fig- ures 1D and 1G), it decreased significantly PIN1-GFPS1,3A from both anticlinal and periclinal membranes ( Figures S3 C and S3D). In contrast, PIN1-GFPS1,3E was insensitive to cytokinin treatment on both anticlinal and periclinal mem- branes ( Figures S3 E and S3F). Thus, phosphorylated PIN pro- teins are much less sensitive to cytokinin than dephosphory- lated ones.

Next, we tested the extent to which the phosphorylation status of PIN1 affects the cytokinin-controlled root develop- ment. Whereas under control conditions, 86% of the wild- type LRPs developed from stage I or II to stage III or IV ( Fig- ure S3 G), after cytokinin treatment 85% of the LRPs were fully arrested (Figures S3 G and S4 A). LRP development was mildly affected in PIN1-GFPS1,3(A) and PIN1-GFPS1,3E lines, with 30% and 33% arrested primordia, respectively, probably due to a deficient PIN1 polarity establishment. In the PIN1- GFPS1,3A mutants, cytokinin increased the proportion of arrested LRPs to 77%, whereas only 30% of the primordia ex- pressing the PIN1-GFPS1,3E were arrested (Figures S3 G and S4 B), suggesting that modulations mimicking PIN1 phosphor- ylation reduce the cytokinin sensitivity of the PIN1 protein and, as a consequence LRP sensitivity to cytokinin might be attenuated.

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Figure 4. Reduced Cytokinin Sensitivity by Enhanced PIN1 Phosphorylation

(A–D) PIN1 cytokinin insensitivity in the endodermal (en) cells of pp2aa1 pp2aa3 and pp2aa1 pp2aa2 (A and B) and 35S::PID (C and D) roots compared to untreated samples (*p < 0.05, **p < 0.001; n = 10 roots, with 10 cells per root). The PIN1 signal was detected by immunolocalization and quantified in endo- dermal cells of 5-day-old root meristem 1.5 hr after 0.1 m M BA treatment (B and D). Scale bars, 30 mm (A) and 40 m m (C).

(E and F) Cytokinin-reduced PIN1 membrane signal in root meristem cells by 38% 6 11% (n = 10), when immunodetected with antibodies recognizing the

PIN1 hydrophilic loop, but not with PIN1-a-S231-P antibodies.

(G and H) Cytokinin insensitivity of phosphomimic PIN1-GFP(Asp), but not of loss-of-phosphorylation PIN1-GFP(Ala) allele, compared to untreated samples

(*p < 0.05, **p < 0.001; n = 10 LRPs). White arrowheads mark vacuoles with GFP accumulation. The PIN1-GFP membrane signal was measured in stage-I LRPs 1.5 hr after treatment with 0.1 mM BA.

Scale bars, 20 mm. Error bars indicate the SEM. Ø, control medium; BA, cytokinin derivative N 6 -benzyladenine. See also Figures S3 and S4 .

The auxin flow direction is largely defined by the membrane polarity of PIN transporters [13, 23 ]. The PIN polarity is highly dynamic, and its change during organogenesis or tropic re- sponses [3, 4, 8, 24, 25 ] is crucial for proper developmental

output. However, the polarizing cues and mechanisms that un- derlie the rapid auxin flow redirection during the formation of new organs are still scarcely understood. Here, we demon- strate that cytokinin not only regulates the overall auxin flow

Cytokinin Controls Polarity of PIN1

1037

capacity, but might also act as a polarizing cue and control the auxin stream directionality. In LRPs, because of the more effi- cient 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 gravi- tropic growth caused by the incorrect PIN polarity in roots that ectopically express PIN1. This selective cytokinin sensi- tivity 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 Experi- mental Procedures and can be found with this article online at http://dx. doi.org/10.1016/j.cub.2014.04.002 .

Acknowledgments

We thank Candela Cuesta for technical assistance, Doron Scolnik for sharing expression and imaging results prior to publication, Ju¨ rgen 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-2007-Stg-207362-HCPO to E.B.), the Division of Energy Biosci- ences, US Department of Energy (grant DE-FG02-06ER15804 to A.S.M.), and an EMBO for a postdoctoral fellowship (LTF 795-2012 to E.F.).

Received: January 21, 2014 Revised: March 25, 2014 Accepted: April 1, 2014 Published: April 24, 2014

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Seifertova´ , D., Wi sniewska,

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

Rela ve Increase of transversal length of LRP (%)

A

periclinal/an clina 2 **** **** 1.8 1.6 * 1.4 1.2 PIN1-GFP_Ø 1 PIN1-GFP_5nM BA 0.8
periclinal/an clina
2
****
****
1.8
1.6
*
1.4
1.2
PIN1-GFP_Ø
1
PIN1-GFP_5nM BA
0.8
0.6
0.4
0.2
0
0
1.5
3
7h
Rela
ve expression

C

PIN1::PIN1-GFP 0 18h Ø
PIN1::PIN1-GFP
0
18h
Ø

B

1.6 periclinal/an clinal * 1.4 1.2 1 PIN1-GFP_Ø 0.8 PIN1-GFP_0.1µM BA 0.6 0.4 0.2 0
1.6
periclinal/an clinal
*
1.4
1.2
1
PIN1-GFP_Ø
0.8
PIN1-GFP_0.1µM BA
0.6
0.4
0.2
0
0
1.5
3
7h
Rela
ve express ion

D

18h

PIN1::PIN1-GFP 0 18h 5nM_BA
PIN1::PIN1-GFP
0
18h
5nM_BA

E

F

PIN1::PIN1-GFP PIN1::PIN1-GFP PIN1::PIN1-GFP 0 0 0 18h 18h 18h 0.1µM_BA high_BA high_BA
PIN1::PIN1-GFP
PIN1::PIN1-GFP
PIN1::PIN1-GFP
0
0 0
18h
18h
18h
0.1µM_BA
high_BA high_BA

G

0 0 0 18h 18h 18h 0.1µM_BA high_BA high_BA G 120 100 80 60 40 20

120

100

80

60

40

20

0

*** PIN1-GFP_Ø 0 PIN1-GFP_5nM_BA PIN1-GFP_0.1µM_BA0 ****
***
PIN1-GFP_Ø
0
PIN1-GFP_5nM_BA
PIN1-GFP_0.1µM_BA0
****
CRE/AHK4::GUS III_stage
CRE/AHK4::GUS
III_stage
AHK2::GUS III_stage
AHK2::GUS
III_stage
III_stage AHK3::GUS
III_stage
AHK3::GUS

H

PIN1::PIN1-GFP II_stage
PIN1::PIN1-GFP
II_stage

I

J

K

TCS:GFP
TCS:GFP

II_stage PIN1::PIN1-GFP

K TCS:GFP II_stage P I N 1 : : P I N 1 - G F

III_stage TCS:GFP

III_stage
III_stage
L ** *** 45 ** 40 ** ** 35 30 25 20 15 10 5
L
**
***
45
**
40
**
**
35
30
25
20
15
10
5
0
0
1.5
3
7h
Rela ve fluorescence
an clinal membranes
an clinal
membranes

PIN1-GFP_ Ø0 0 1.5 3 7h Rela ve fluorescence an clinal membranes PIN1-GFP_ BA cre ahk2 _

PIN1-GFP_ BA7h Rela ve fluorescence an clinal membranes PIN1-GFP_ Ø cre ahk2 _ Ø cre ahk2 _

cre ahk2_ Ø _ Ø

cre ahk2_ BA _ BA

*** ** M * 45 * 40 35 30 25 20 15 10 5 0
***
**
M
*
45
*
40
35
30
25
20
15
10
5
0
0
1.5
3
7h
Rela ve fluorescence

periclinal

membranes

PIN1-GFP_ Ø15 10 5 0 0 1.5 3 7h Rela ve fluorescence periclinal membranes PIN1-GFP_ BA cre

PIN1-GFP_ BA15 10 5 0 0 1.5 3 7h Rela ve fluorescence periclinal membranes PIN1-GFP_ Ø cre

cre ahk2_ Ø _ Ø

cre ahk2_ BA _ BA

Figure S1

A

DR5::GFP
DR5::GFP
Ø
Ø
DR5::GFP
DR5::GFP
BA
BA

B

14 ** 12 10 8 ** 6 4 * 2 0 DR5-GFP Ø DR5-GFP BA
14
**
12
10
8
**
6
4
*
2
0
DR5-GFP Ø
DR5-GFP BA
PIN2::PIN1-HA Ø
PIN2::PIN1-HA BA
Ra
o of DR5
-GFP fluorescence

D

90°

Col Ø
Col
Ø

PIN2::PIN1-HA Ø PIN2::PIN1-HA Ø

BA B A

BA -GFP fluorescence D 90° Col Ø PIN2::PIN1-HA Ø B A Shootward IAA transport eir1-1 Ø 35S::PIN1

Shootward IAA transport

eir1-1 Ø
eir1-1
Ø
35S::PIN1 Ø
35S::PIN1
Ø

t/0hØ B A BA Shootward IAA transport eir1-1 Ø 35S::PIN1 Ø t/4h C PIN2::PIN1-HA Ø Col_Ø

t/4hA BA Shootward IAA transport eir1-1 Ø 35S::PIN1 Ø t/0h C PIN2::PIN1-HA Ø Col_Ø PIN2::PIN1-HA BA

C

PIN2::PIN1-HA Ø
PIN2::PIN1-HA
Ø

Col_Øeir1-1 Ø 35S::PIN1 Ø t/0h t/4h C PIN2::PIN1-HA Ø PIN2::PIN1-HA BA BA 90° eir1-1 _ Ø

PIN2::PIN1-HA BA
PIN2::PIN1-HA
BA

BA

90° eir1-1 _ Ø
90°
eir1-1 _ Ø
Col_BA
Col_BA
BA
BA

BA Ø Col_Ø PIN2::PIN1-HA BA BA 90° eir1-1 _ Ø Col_BA BA eir1-1 _ BA 0° 30°

eir1-1_ BA _ BA

0° 30° 60° BA 90° 90° PIN2::PIN1 PIN2::PIN1 35S::PIN1_Ø 35S::PIN1_BA -HA _ Ø -HA _
30°
60°
BA
90°
90°
PIN2::PIN1
PIN2::PIN1
35S::PIN1_Ø
35S::PIN1_BA
-HA _ Ø
-HA _ BA
E
F
Rootward IAA transport
1400
1400
control
kin
a
1200
a
1200
a
1000
1000
800
b
b
800
b
b
b
600
600
400
400
200
200
0
0
Col-0
pin1
pin2
PIN2::PIN1-HA
DPM . 10 seedlings -1
DPM . 10 seedlings -1
a control kine n a a a a b b c Col-0 pin1 pin2 PIN2::PIN1-HA
a
control
kine
n
a
a
a
a
b
b
c
Col-0
pin1
pin2
PIN2::PIN1-HA

Figure S2

A

C

B PIN1-GFP PIN1-GFP PIN1-GFP Ø BA S1,3A Ø BA S1,3E Ø BA D Rela ve
B
PIN1-GFP
PIN1-GFP
PIN1-GFP
Ø
BA
S1,3A
Ø
BA
S1,3E
Ø
BA
D
Rela
ve fluorescence
60 50 40 ** Ø 30 ** BA 20 10 0 PIN1-GFP PIN1-GFP S1,3A PIN1-GFP
60
50
40
**
Ø
30
**
BA
20
10
0
PIN1-GFP
PIN1-GFP S1,3A
PIN1-GFP S1,3E
120 PIN1::PIN1-GFP S1,3A 100 80 * * * * 60 Ø_ 0.1µM BA_ 40 20
120
PIN1::PIN1-GFP S1,3A
100
80
*
*
*
*
60
Ø_
0.1µM BA_
40
20
0
0
h
1.5 h
3 h
7 h
E
F
120
PIN1::PIN1-GFP S1,3E
100
80
60
Ø_
0.1µM BA_
40
20
0
0
h
1.5 h
3 h
7 h
ve fluorescence (%)Rela
Rela ve fluorescence (%)
120 PIN1::PIN1-GFP S1,3A 100 * 80 * 60 Ø_periclinal 0.1µM BA_periclinal 40 20 0 0
120
PIN1::PIN1-GFP S1,3A
100
*
80
*
60
Ø_periclinal
0.1µM BA_periclinal
40
20
0
0
h
1.5 h
3 h
7 h
140
PIN1::PIN1-GFP S1,3E
120
100
80
Ø_periclinal
60
0.1µM BA_periclinal
40
20
0
0
h
1.5 h
3 h
7 h
Rela ve fluorescence (%)
Rela ve fluorescence (%)

G

Stages I and II

PIN1-GFP

PIN1-GFPS1,3A

PIN1-GFPS1,3E

Control

2 µM BA

Control

2 µM BA

Control

2 µM BA

Total LRP

43

47

37

31

48

43

Developing LRP

37

7

26

7

20

30

Arrested LRP

6*

40*

11*

24*

16*

13

Arrested/Total

6*/43

40*/47

11*/37

24*/31

10*/30

8*/20

Inhibition (%)

13.95

85.1

29.72

77.41

33.33

30.00

*, Fully arrested; BA, cytokinin derivative N 6 -benzyladenine.

Figure S3

A

0 2 PIN1-GFP Ø
0
2
PIN1-GFP
Ø
0 2 PIN1-GFP BA
0
2
PIN1-GFP
BA

B

0 2 PIN1-GFP (Asp) Ø
0
2
PIN1-GFP
(Asp)
Ø
4 6
4
6
8
8
4 6 8
4
6
8
4 6
4
6
8
8
0 2 4 6 PIN1-GFP (Asp) BA
0
2
4
6
PIN1-GFP
(Asp)
BA

Figure S4

8
8
10
10
10
10
10
10
10
10
12 14 16h
12
14
16h
12 14 16h
12
14
16h
12 14 16h
12
14
16h
12 14 16h
12
14
16h

Supplemental Figure Legends

Figure S1- Related to Figure1.Cytokinin modulates PIN polarsity index and affect LRP growth

(A, B) Real-time monitoring of membrane PIN1-GFP in stage-III LRP. Periclinal/anticlinal

polarity index of the PIN1-GFP membrane abundance is shifted in favor of the periclinal

localization after cytokinin treatment when compared to the nontreated control (*P<0.05;

***P<0.001; ****P<0.0001; n=10 LRP). Roots were treated for 7 h with low (5 nM) and high

(0.1 µM) cytokinin concentrations. (C to F) Shift of the periclinal/anticlinal polarity index of the

PIN1-GFP membrane abundance after treatment with 5 nM BA, correlated with enhanced

primordia growth (D and F). Growth of LRP treated with 0.1 µM BA is repressed, despite the

changed polarity index, probably due to an overall PIN1 depletion from membranes (E and F)

compared to primordia at time 0 h (*P<0.05; **P<0.001; ***P<0.0001; n=20 LRP). LRP

outgrowth was monitored for 18 h and growth increase is marked by a white arrow. Scale bars,

45 µm. (G) AHK2, AHK3 and AHK4/CRE1 expression in stage-III LRP. Scale bars, 30 µm. (H

and J) PIN1-GFP 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, 30 µm. (L and M) Real-time monitoring of

membrane PIN1-GFP in stage-III LRP in wild type (Col-0) and cre,ahk2. Cytokinin sensitivity

of PIN1-GFP 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, 30 µm. Error bars mark the standard error of the mean. Ø, control

medium; BA, cytokinin derivative N 6 -benzyladenine.

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 0 h (*P<0.05;

**P<0.001; error bars indicate standard error of the mean, n=20 roots). Arrowheads mark

gravistimulated root sides. (C and D) Gravitropic response of PIN2::PIN1-HA,pin2/eir1 and

35S::PIN1-GFP roots rescued by cytokinin, but not of pin2/eir1. White and red arrows indicate

change in gravity-stimulation. (E and F) 3 H-indole-3-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< 0.05; error bars indicate standard deviation, n= 3

pools of 10 seedlings for assays). Seven-day-old roots grown on medium with 0.1 µM BA (A

and B) and 0.6 µM BA (C and D) or without cytokinin were exposed to a 24-h gravistimulus.

Scale

bars,

30 µm

(A),

benzyladenine.

0.5 cm

(D).

Ø,

control

medium;

BA,

cytokinin

derivative

N 6 -

Figure S3- Related to Figure4. Reduced cytokinin sensitivity by enhanced PIN1 phosphorylation.

(A and B)

cytokinin

insensitivity

of

phosphomimic PIN1-GFPS1,3E,

but

not

of

loss-of-

phosphorylation PIN1-GFPS1,3A alleles (*P<0.05; **P<0.001; n=10 LRP). White arrowheads

indicate vacuoles with GFP accumulation. The PIN1-GFP membrane signal was measured in

stage-I LRP 1.5 h after treatment with 0.1 µM BA. (C to F) Real-time monitoring of PIN1-GFP

at anticlinal and periclinal membranes in control and cytokinin-treated stage-III LRP. Loss-of-

phosphorylation PIN1-GFPS1,3A at both anticlinal and periclinal membranes is cytokinin

sensitive (C and D), whereas the phosphomimic PIN1 -GFPS1,3E variant is insensitive (E and F)

compared to primordia at time 0 h (* P<0.05; ** P<0.001; n=5 LRP) . Error bars indicate the

standard error of the mean. (G) Phosphorylation status of PIN1 modulates the cytokinin-

controlled lateral root development. LRP development monitored for 12 hours. Ø, control

medium; BA, 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 PIN1-GFP degradation and inhibiting LRP

development

(A)

and

cytokinin-insensitive

PIN1-GFP(Asp)

correlating

with

cytokinin-

insensitive LRP development (B). Representative real-time analysis of LRP development on

control medium (Ø) and in the presence of 0.1 μM N 6 -benzyladenine (BA) (n =10 primordia per

line and treatment). Scale bars, 20 µm.

Supplemental Experimental Procedures

Plant material

The

transgenic

Arabidopsis

thaliana

(L.)

Heynh.

lines

have

been

described

elsewhere:

PIN1::PIN1-GFP and DR5rev::GFP [S1]; PIN2::PIN2-GFP [S2]; PIN2::PIN1-HA, eir1-1/pin2,

PIN2::PIN1-GFP,

and

PIN2::PIN1-GFP3

[S2];

PIN2::PIN1-HA/eir1/DR5::GFP

[S3];

35S::PIN1-GFP [S4]; 35::PID [S5]; pp2aa1, pp2aa1, pp2aa2, and pp2aa1,pp2aa3 [S6];

PIN1::PIN1-GFP(Asp)

and

PIN1::PIN1-GFP(Ala)[S7];

PIN1::PIN1-GFPS1.3A

and

PIN1::PIN1-GFPS1.3E [S8], eir1-1 [S9] ; pin1, and pin2 [S10] cre1-12,PIN1::PIN1-GFP;

cre1-12,ahk2,PIN1::PIN1-GFP;

cre1-12,ahk3,PIN1::PIN1-GFP

[S11];

TCS:GFP

CRE/AHK4::GUS, AHK2::GUS, AHK3::GUS [S11].

Growth conditions

[S12];

Seeds of Arabidopsis (accession Columbia-0) were plated on half-strength (0.5) Murashige and

Skoog (MS) medium (Duchefa) with 1% sucrose, 0.5 MS medium (Duchefa) and 0.8% agar (pH

5.7). For the root gravity assays, 3.5 % 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 (BA) and incubated for 1.5 h to 7 h in the dark at 22°C. The

concentrations

used

were

5 nM,

0.1 μM,

0.6 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 FV10 ASW confocal microscope

(Olympus) and LSM700 (Zeiss) with a 20 or 60 (water immersion) objective. For all the

experiments, the scans were done with identical microscopes and laser settings. At minimum, 10

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

[S13,

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: 1:1000

for rabbit anti-PIN1 [S16], 1:500 for mouse anti-HA (AbCam/HA.C5), and 1:600 for Cy3 anti-

rabbit (Sigma). Signal intensities were evaluated with the quantification function of Image J 1.41

software (http://rsbweb.nih.gov/ij/) and Cellset software. The rabbit PIN1-a-S231-P antibody

was generated with the phosphorylated synthetic peptide LSATPRP-S(PO 3 H 2 )-NLTNA followed

by

affinity

purifications

against

the

nonphosphorylated

and

phosphorylated

peptide

at

Eurogentec (Liège, Belgium). Col-0 seedlings were immunostained with rabbit PIN1-a-S231-P

(dilution 1:300).

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 20 stage-V LRP were processed.

For root gravitropism response analyses, seedlings were grown on 3.5% agar (pH 5.7) and 0.5

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 90 degree for 24 h and the

gravitropic responses were measured with the ImageJ software. The obtained values were

processed in Excel 11.3.3 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 0.25 MS (pH 5.5) ± kinetin (0.5 μM) for a 2-h acclimation preceding the

application of 3 H-indole-3-acetic acid (30 Ci mmol -1 ).

Supplemental References

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Friml, J. (2003). Local, efflux-dependent auxin gradients as a common module for plant

organ formation. Cell 115, 591–602.

S2.

Wiśniewska, J., Xu, J., Seifertová, D., Brewer, P. B., Růžička, K., Blilou, I., Rouquié, D.,

Benková, E., Scheres, B., and Friml, J. (2006). Polar PIN localization directs auxin flow in

plants. Science 312, 883.

S3. Feraru, E., Feraru, M. I., Kleine-Vehn, J., Martinière, A., Mouille, G., Vanneste, S.,

Vernhettes, S., Runions, J., and Friml, J. (2011). PIN polarity maintenance by the cell wall

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S4.

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S8.

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S9.

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S10.

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Petrášek, J., Friml, J., Kleine-Vehn, J., et al. (2011). Cytokinin modulates endocytic

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804.

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W., Tarkowski, P., and Benková, E. (2012). Spatiotemporal regulation of lateral root

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S13.

Žádníková,

P.,

Petrášek,

J.,

Marhavý,

P.,

Raz,

V.,

Vandenbussche,

F.,

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Z.,

Schwarzerová, K., Morita, M. T., Tasaka, M., Hejátko, J., et al. (2010). Role of PIN-

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S14. Pound, M. P., French, A. P., Wells, D. M., Bennett, M. J., and Pridmore, T. P. (2012).

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