Phloem-Transported Cytokin

Current Biology 21, 927–932, June 7, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2011.04.049

Reportin Regulates

Polar Auxin Transport and MaintainsVascular Pattern in the Root Meristem

Anthony Bishopp,1,* Satu Lehesranta,1,5 Anne Vaten,1,5

Hanna Help,1 Sedeer El-Showk,1 Ben Scheres,2

Kerttuli Helariutta,3 Ari Pekka Mahonen,1,2

Hitoshi Sakakibara,4 and Yka Helariutta1,*1Institute of Biotechnology and Department of Biosciences,University of Helsinki, FIN-00014 Helsinki, Finland2Section of Molecular Genetics, Department of Biology,Faculty of Science, Utrecht University, Padualaan 8, 3584 CHUtrecht, The Netherlands3Laboratory of Radiochemistry, Department of Chemistry,University of Helsinki, FIN-00014 Helsinki, Finland4RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045,Japan

Summary

Cytokinin phytohormones regulate a variety of develop-mental processes in the root such asmeristem size, vascular

pattern, and root architecture [1–3]. Long-distance transportof cytokinin is supported by the discovery of cytokinins in

xylem and phloem sap [4] and by grafting experimentsbetween wild-type and cytokinin biosynthesis mutants [5].

Acropetal transport of cytokinin (toward the shoot apex)has also been implicated in the control of shoot branching

[6]. However, neither the mode of transport nor a develop-mental role has been shown for basipetal transport of cyto-

kinin (toward the root apex). In this paper, we combine the

use of a new technology that blocks symplastic connectionsin the phloem with a novel approach to visualize radiola-

beled hormones in planta to examine the basipetal transportof cytokinin. We show that this occurs through symplastic

connections in the phloem. The reduction of cytokinin levelsin the phloem leads to a destabilization of the root vascular

pattern in a manner similar to mutants affected in auxintransport or cytokinin signaling [7]. Together, our results

demonstrate a role for long-distance basipetal transport ofcytokinin in controlling polar auxin transport and maintain-

ing the vascular pattern in the root meristem.

Results and Discussion

The phytohormones auxin and cytokinin interact to regulatemultiple processes during root development including forma-tion of the embryonic root, control of rootmeristem size, emer-gence of lateral roots, and control of root vascular pattern [3,7–9]. The polar subcellular localization of the auxin effluxcarriers PIN1, PIN3, and PIN7 directs an intercellular auxinflow rootward through the vascular tissue by a process knownas polar auxin transport [10, 11]. In the columella, this flow isredirected laterally to the root cap and epidermis, where itflows shootward and is recycled back to the vascular tissuein the elongation zone [10]. The concerted action of the PIN

5These authors contributed equally to this work

*Correspondence: anthony.bishopp@helsinki.fi (A.B.), yrjo.helariutta@

helsinki.fi (Y.H.)

proteins generates two auxin response maxima. The firstforms in the quiescent center (QC) [12] and restricts thedomain of the PLETHORA (PLT) proteins, which control rootstem cell specification at this position [13]. As we show else-where in this issue of Current Biology, the PIN proteins alsogenerate an auxin response maximum in the xylem axis thatspecifies protoxylem identity at the marginal positions [7].The level of cytokinin signaling in the root meristem has beenshown to regulate both the cellular efflux of auxin and PINexpression [3, 7, 8, 14, 15]. Because there are indications ofbasipetal transport of cytokinin, it is possible that cytokininproduced in the shoot could regulate PIN proteins and there-fore developmental processes in the root.We first tested whether application of cytokinin to the aerial

parts of the plant could affect cytokinin response in the root.We applied cytokinin to the hypocotyls of 5-day-old seedlingsand assayed the cytokinin response in the root tip using qRT-PCR and pARR5::GUS. Within the vascular cylinder, ARR5 isnormally expressed in intervening procambial cells in the prox-imal meristem (see Figure S1A available online) [7], but there isalso a second peak of ARR5 expression in the transition zoneat the point at which phloem differentiates. After 3 hr, we sawan enhanced GUS signal in the transition zone at the root tip,but longer treatments resulted in the accumulation of elevatedGUS signal in the intervening procambial cells of the proximalmeristem (Figures 1A and 1B; Figure S1A). This indicated thatthe cytokinin signaling pathway is activated in the interveningprocambial cells of the root meristem even if cytokinin is notapplied directly to the roots. In previous studies, labeled cyto-kinins have been applied to leaves of cocoa, pea, and yucca[16–18]. Much of the labeled cytokinin is strongly retained atthe treated site; however, a small proportion is translocatedto other parts such as roots and nodules [16–18]. Thesestudies relied on the fractionation of labeled cytokinin fromwhole roots followed by quantification by scintillation andtherefore offer little insight into the spatial distribution of trans-ported cytokinin within the root. In order to test whether cyto-kinins could translocate to the proximal root meristem, weestablished an assay where we could visualize the migrationof 14C-labeled N6-benzyladenine (BA) (a cytokinin) using animaging plate as a radioactive energy sensor that was pro-cessed with a fluorescent image analyzer (FLA). We appliedlabeled cytokinin to the hypocotyls of 5-day-old plants, andwithin 4 hr we saw a local maximum in the radioactive signalat the root tip, indicating that a proportion of the labeled BAhad been transported to the rootmeristem (Figure 2A). In orderto confirm that the cytokinin transported basipetally corre-spondedwith natural cytokinin, we set up a similar assay usingthe stable isotope-labeled N6-(D2-isopentyl)adenine riboside(iPR). After application of isotope-labeled iPR to the hypocotyl,we measured ARR5 response in the root and observed aresponse similar to the earlier treatments with BA (Figure S1B).We pooled plants, dissected 2 mm sections from the root tipsof 50 individuals, and measured the accumulation of labeledcytokinin in the root tip by mass spectrometry. In addition tothe native cytokinin species, we also observed the accumula-tion of labeled cytokinin species, indicating that the iPR and/oritsmetabolites were transported from the hypocotyl to the root

Figure 1. Cytokinin Signaling Is Increased in the

Intervening Procambial Cells of the Root Meri-

stem in Response to Application of Exogenous

Cytokinin to the Hypocotyls

(A) qRT-PCR shows that 4 hr after application of

1 mMN6-benzyladenine (BA) (+CK) to hypocotyls,

ARR5 expression is increased in the intervening

procambial cells at the root tip. Error bars indi-

cate standard error.

(B) The pARR5::GUS response at the root tip is

increased following a cytokinin application to

the hypocotyls. Leftmost is a mock treatment

that showed the highest ARR5 signal just shoot-

ward of the meristem, close to the longitudinal

domain in which phloem differentiates. Three

hours after treatment, the signal increased inten-

sity close to this position, and by 6 hr, the signal became highly concentrated in the proximal meristem. The signal intensified further with longer treatments

but remained restricted to the intervening procambial cells. In addition to the expression pattern described here, pARR5::GUS signal can also be seen in the

columella root cap. The transition zone is indicated with an arrow.

Current Biology Vol 21 No 11928

(Table 1). Because CYP735A1 and A2, which catalyze trans-zeatin (tZ) synthesis, are predominantly expressed in roots[19], and because N6-(D2-isopentyl)adenine (iP)-type cytoki-nins are the major form in phloem [20, 21], the labeled tZ-typecytokinins are likely to be formed after iP-type translocation tothe root.

Basipetal transport of cytokinin has previously been associ-ated with the phloem, because certain cytokinin species havebeen identified in the phloem sap of Arabidopsis and Sinapis[4, 20, 21]; however, this association has never been tested.We examined this using two transgenic lines, the alteredphloem development (apl) mutant, which lacks phloem [22],

and a line in which we could impair phloem transport byinducing rapid callose biosynthesis in the phloem to blockthe symplastic connections (A.V., Y.H., et al., unpublisheddata) (Figure S2A). Using these lines, we followed the transportof 14C-labeled cytokinin from thehypocotyl to the root proximalmeristem and found it to be severely compromised in both(Figure 2C; Figure S2B), indicating that basipetal transport ofcytokinin occurs through the phloem. Furthermore, the factthat transport of cytokinin was impaired in pAPL::XVE>>cals3mdemonstrates that basipetal cytokinin transport occursthrough symplastic connections in the phloem. We confirmedthese results by quantitativelymeasuring the radioactive signal

Figure 2. Cytokinin and Auxin Are Transported in

a Basipetal Direction through the Phloem

(A and B)When 14C-labeled BA or indole-3-acetic

acid (IAA) is applied to hypocotyls, accumulation

of radiolabeled isotope is seen at the root tip (BA,

n = 15; IAA, n = 13). The leftmost image shows

a heat map showing areas of high radioactivity,

the center image is a light image of the root,

and the rightmost image shows overlay.

(C and D) When plasmodesmatal blockage is

induced using cals3m, transport and unloading

of both BA and IAA are severely affected (BA,

n = 17; IAA, n = 15). It is likely that the small

amount of hormone that is transported rootward

remains trapped in the phloem.

(E and F) Cytokinin transport appears to be unaf-

fected by 1-naphthylphthalamic acid (NPA) treat-

ments, and plants still show a cytokininmaximum

at the root tip in 16 of 17 roots (E). In comparison,

the signal for radiolabeled IAA is barely percep-

tible in NPA-treated roots (n = 17) (F). In this

assay, 10 mM NPA was used; it is likely that the

low auxin signal was observed in NPA-treated

roots because at this concentration, NPA does

not entirely block all polar auxin transport.

Scale bars represent 1 mm. See also Figure S1.

Table 1. Labeled iPR Is Transported to the Root Tip

Control #1 Control #2 3 hr Sample #1 3 hr Sample #2 6 hr Sample #1 6 hr Sample #2

labeled tZ, tZR, and tZRPs ND ND 0.008 (19%) 0.032 (59%) 0.064 (74%) 0.167 (92%)

labeled cZ, cZR, and cZRPs ND ND ND ND ND ND

labeled iP, iPR, and iPRPs ND ND 0.008 (8%) 0.010 (10%) 0.019 (15%) 0.019 (23%)

labeled iP7G and iP9G ND ND 0.034 (32%) 0.115 (68%) 0.145 (67%) 0.137 (73%)

50 pmol of stable isotope-labeled [13C10,15N5]N

6-(D2-isopentyl)adenine riboside (iPR) was applied to hypocotyls, and 2 mm of root tips of 50 plants were

harvested 3 hr and 6 hr after treatment. Labeled and authentic cytokinins were analyzed by mass spectrometry. The applied iPR could be metabolized

to N6-(D2-isopentyl)adenine (iP), iPR 50-monophosphate (iPRMP), trans-zeatin (tZ), tZ riboside (tZR), tZ riboside 50-monophosphate (tZRMP), and further

to other species. The mock samples refer to untreated lines and show no accumulation of heavy cytokinins. However, at 3 hr and 6 hr after treatment of

hypocotyls with labeled cytokinin, accumulation of heavy cytokinin species can be seen at the root tip. Values are in pmol. The amount of stable

isotope-labeled cytokinin is also shown as a percent of total cytokinin (labeled + unlabeled) in the root tip. Numbers 1 and 2 refer to two independent repli-

cates. ND indicates not detectable. See also Figure S2.

Phloem Transport of Cytokinin929

via liquid scintillation counting and observed an approximately4-fold reduction in the root tips of pAPL::XVE>>cals3m (Fig-ure S2C) plants. We also examined the endogenous cytokininresponse following a 24 hr cals3m induction and observedreduced levels of ARR5 signal (Figure 3P), indicating that sym-plastic transport through the phloem provides a significantsource of cytokinin in the root meristem. We then analyzedthe transport of 14C-labeled indole-3-acetic acid (IAA) in wild-type and pAPL::XVE>>cals3m plants and observed that thebulk basipetal flow of labeled auxin transport was also im-paired when phloem transport was blocked (Figures 2B and2D; Figure S2C). We next analyzed the auxin response inpAPL::XVE>>cals3m. Following blockage of the plasmodes-mata in the phloem, we saw only modest changes to the auxinresponse maximum at the QC and columella (a slight expan-sion of auxin response into the epidermis is shown in Fig-ure S3A). This effect is more in keeping with alterations in cyto-kinin signaling, because a similar phenomenon has beenreported in 35S::CKX lines [15]. This suggests that althoughthe phloem is likely to play a role in bulk auxin transport as sug-gested by other researchers [23, 24], additional transportmechanisms or local sites of biosynthesis must exist. Wethen analyzed the transport of 14C-labeled IAA andBA in plantstreated with 1-naphthylphthalamic acid (NPA), which blockspolar auxin transport by compromising PIN activity. Whereascytokinin transport was unaffected in NPA-treated lines, theability of auxin to reach the root tip was severely compromised(Figures 2E and 2F), showing that in addition to the bulk flow ofcytokinin and auxin via phloem, auxin is actively transportedthrough polar transport. Whereas cytokinin regulates auxintransport, cytokinin transport through the phloem appears tofunction independently of polar auxin transport.

Previous studies have postulated directional transport ofspecific cytokinin species where tZ-type cytokinins are usedas an acropetal messenger and iP-type cytokinins as a basip-etal messenger [4, 25]. Recent experiments with graftingbetween cytokinin biosynthetic mutants and wild-type havelent support to this cytokinin species-specificdirectional trans-port [5].However, especially in basipetal transport, thequantityof cytokinin transported is quite low [16–18]. Our transferassays showed that the phloem has the potential to deliverlarge amounts of cytokinins, which can make a substantialproportion of the pool of certain cytokinin species in the rootmeristem (Table 1). To test whether the basipetal transport ofcytokinin plays a role in the development of the root meristem,we examined the root meristems in plants with defects inphloem-mediated cytokinin transport.

In wild-type roots, maximal cytokinin signaling occurs intwo distinct domains flanking the xylem axis and adjacent to

the phloem position [2, 7]. A mutually inhibitory interactionbetween cytokinin and auxin signaling and transport specifiesthe position of these domains and determines vascular patternin the root meristem [7]. High cytokinin signaling promotes theexpression of PIN7 in two domains of intervening procambialcells flanking the xylem axis and regulates the radial distribu-tion of PIN3 and PIN1 in the root meristem; this forces auxininto the xylem axis to create an auxin signaling maximum.High auxin signaling at the marginal protoxylem triggersAHP6 expression and specifies protoxylem identity in a bisym-metric pattern [7] (Figures 3A–3D).We generated a transgenic line to deplete cytokinin within

the phloem (pAPL::XVE>>CKX1:YFP) and used this alongsideapl and pAPL::XVE>>cals3m to investigate cell identity withinthe rootmeristem (Figure S3B). In all three cases, we examinedthe radial patterns of the pPIN7::PIN7:GFP, DR5rev::GFP, andpAHP6::GFP marker genes in the root meristem. In wild-typeplants, all of these markers are invariably expressed at preciselocations that determine the location of distinct domains ofhigh auxin and high cytokinin signaling. However, in thesethree lines we observed fluctuations in these boundaries,and the high auxin signaling domain frequently expanded tobecome two cells wide (Figures 3E–3G, 3I–3K, and 3M–3O).This showed that the basipetal transport of cytokinin throughthe phloem has a role in specifying the spatial regulation ofPIN7 and in the formation of an auxin response maximum inthe correct position. Accordingly, the patterns ofmarker genesthat we observed closely mirrored those previously seen in thepin3 pin7 double mutant [7]. We next observed the vascularpattern in pAPL::XVE>>CKX1:YFP and in apl. In the aplmutant,in addition to the reported switch in cell fate identity at thephloem position, we observed defects in the pattern of proto-xylem differentiation in the root meristem; similar non-cell-autonomous effects on protoxylem identity were seen inpAPL::XVE>>CKX1:YFP (Figures 3H and 3L; Figure S3D).This caused imperfect bisymmetry within the vascular bundle,with plants displaying extra files of protoxylem as well as lossof protoxylem. As a control, we drove expression of theCKX1:YFP gene in immature phloem cells under the controlof the At2g37950 (EPM) promoter [26], and we only veryseldom saw relatively minor effects on protoxylem differentia-tion (Figures S3C and S3D). Together, these results show thatalthough the initial specification of vascular pattern occursduring embryogenesis and preceding the differentiation ofphloem, the basipetal transport of cytokinin through thephloem plays an important role in maintaining this vascularpattern during growth. In order to determine whether thesource for cytokinin in the phloem originated from the shoot,as opposed to the more distal parts of the root, we grafted

Figure 3. Cytokinin Transported through the Phloem Regulates PIN Expression and Reinforces Vascular Pattern

(A) In wild-type plants, PIN7 is expressed in two domains flanking a central xylem axis.

(B) This axis is one cell wide and contains a high auxin response that can be visualized by the DR5rev::GFP reporter.

(C) AHP6 is expressed at the marginal positions of this axis and additionally in the two adjacent pericycle cells.

(D) Later, the cells at the marginal positions differentiate as protoxylem, and this can be visualized by fuchsin staining.

(E–L) Either apl mutants (E–H) or induction of pAPL::XVE>>CKX1:YFP in the phloem (I–L) leads to reduced PIN7 distribution, failure of the auxin response

maximum to form correctly, alterations in the AHP6 domain, and unstable formation of protoxylem files.

(M–P) A 24 hr induction of pAPL::XVE>>cals3m leads to decreased pARR5::GUS signal and a reduced domain of PIN7 coupled with increased domain of

DR5rev and AHP6.

Scale bars represent 10 mm, except in (D), (H), and (L), where they represent 25 mm. Pericycle cells are marked with asterisks. Yellow arrows indicate the

protoxylem position; failure to differentiate protoxylem is indicated with white arrowheads. See also Figure S3.

Current Biology Vol 21 No 11930

35S::CKX2 [15] shoots onto AHP6::GFP rootstocks. Weobserved an expansion in the domain of AHP6 expression ina significant proportion of these roots, but not in control graft-ings (Figure S3E). These data suggest that shoot-to-root trans-port of cytokinin through the phloem is necessary to spatiallyrestrict the domains of hormonal signaling in the proximalroot meristem.

A role for cytokinin has been reported wherein cytokininresponseactivates the repressor of auxin signalingSHY2,whichin turn acts as a negative regulator of PIN activity to controlmeristem size [8]. We examined whether cytokinin transported

via the phloem could play a role in this process by comparingmeristem size between wild-type and pAPL::XVE>>CKX1:YFPplants. We observed no effect on meristem size (wild-type,35.36 0.8 cortex cells compared with pAPL::XVE>>CKX1:YFP,34.8 6 0.8 cells) (Figures S3F and S3G). Transport through thephloem is one source of cytokinin; however, other sourcescertainly exist, and cytokinin biosynthesis and metabolismgenes have been identified in the root meristem [27, 28].One possible way in which the phloem transport of cytokinincould target specific developmental processes is throughdifferent receptor specificity. During vascular development,

Phloem Transport of Cytokinin931

CRE1 is the most significant cytokinin signaling receptor [29],whereas ahk3 exerts the largest effect on meristem size [1].In vitro binding studies suggest that these two receptorshave different ligand specificities; for example, AHK3 displayshigher affinity toward tZ than toward iP, whereas CRE1 hassimilar affinity toward both of these isoforms [30]. It might bepossible that iP transported through the phloem is involved invascular development via CRE1 and that tZ in the root tip isinvolved in maintenance of the root meristem mainly via AHK3.An alternative possibility is that the regulation of root meristemsize may be more robust to small changes in cytokinin thanthe regulation of vascular pattern.

Together, these data demonstrate that the basipetal trans-port of cytokinins through the phloem plays an importantdevelopmental role in regulating PIN activity in the proximalmeristem. Delivery of cytokinin through the phloem enablesthe targeting of specific developmental pathways (e.g., main-taining vascular pattern) while not interfering with other cyto-kinin-regulated pathways (that may be driven by local biosyn-thesis rather than transport). Why has such a regulatorysystem evolved? One possibility is that cytokinin transportedfrom the mature part of the root maintains continuous andsharp domains of hormonal signaling in themeristem to ensurethe formation of continuous vascular pattern.

Experimental Procedures

Hormone Transport Assays

Plants were grown for 5 days on sterile agar plates. NPA and estrogen treat-

ments were performed by moving plants to fresh plates containing 10 mM

NPA or 10 mM 17b-estradiol 24 hr before analysis. Plants were then trans-

ferred to new plates, and sterile glass coverslips were used to isolate the

hypocotyls and aerial parts of the plant from the media. Plates were left

open for a few minutes so that any free water droplets on the gel surface

would evaporate. Labeled or unlabeled hormone was added to the hypo-

cotyls of plants in a 2 ml 1% agar bead and left for 4 hr. For 14C applications,

approximately 50 pmol [8-14C]benzyladenine or [1-14C]indole-3-acetic acid

(American Radiolabeled Chemicals) was added per plant. This corre-

sponded to an estimated 250 pCi of activity. Carboxyfluorescein dye was

added as a control to ensure that the bead did not come in contact with

the agar and that the hormones did not mix with the media. The roots

were removed from the plants, dehydrated briefly in ethanol, mounted on

Whatman filter paper, and exposed to a BAS-TR2025 imaging plate (Fujifilm)

for between 4 and 8 days. The film was then processed using an FLA

scanner. For cytokinin measurements, treatment was performed in the

same manner described earlier but using [13C10,15N5]N

6-(D2-isopentyl)

adenine riboside (Sakakibara laboratory, RIKENPlant Science Center) using

pooled 2 mm sections from 50 root tips as described previously [31]. The

incorporation of the stable isotope increased the molecular mass of the

iPR by 15 Da.

Anatomical Analyses

Anatomical and histological analyses were based on 5-day-old seedlings

grown under long-day conditions. Fuchsin staining, GUS staining, qRT-

PCR, and confocal imaging were performed as described elsewhere [7]. The

inducible system usedwas amodified version of pER8, an estrogen-receptor

based chemical-inducible system [7, 32]. Effector geneswere subcloned into

pDONR(Zeo) (Invitrogen) and recombined into the inducible system. The 2.9

kb of the APL promoter was cloned into the inducible system by amplifying

with the primers 50-CGATCGATTTATCGAATTTAGTGTT-30 and 50-TCTCTCTCTCTCTCTCTCTGACTCTC-30. To drive early phloemexpression, we ampli-

fied 1.6 kb of sequence upstream of the gene At2g37590 [26] with the pri-

mers 50-GTTTATGTGTTGCCTAACTCTTGA-30 and 50-GGTTATTCTCTTTTGA

TTTT-30 and cloned them into the inducible system.CKX1:YFPwas amplified

from the construct pCRE1::XVE/LexAoperator::CKX1:YFP [2] using the pri-

mers 50-GGGACAAGTTTGTACAAAAAAGCAGGCTAAAATGGGATTGACCTC

ATCCTTACG-30 and 50-GGGGACCACTTTGCACAAGAAAGAAAGCTGGGTG

TTACTTGTACAGCTCGTCCATGC-30, and the cals3m entry clone was pre-

pared as will be described elsewhere (A.V., Y.H., et al., unpublished data).

Supplemental Information

Supplemental Information includes three figures and can be found with this

article online at doi:10.1016/j.cub.2011.04.049.

Acknowledgments

We thank Mikko Herpola, Katja Kainulainen, Tia Kaonpaa, and Marja Siitari-

Kauppi for technical assistance and Mikiko Kojima for cytokinin analysis.

Financial support was provided by the European Research Area Network

on Plant Genomics (ERA-PG), the University of Helsinki, the Academy of

Finland, Tekes, the Human Frontier Science Program, and the European

Molecular Biology Organization.

Received: January 19, 2011

Revised: April 7, 2011

Accepted: April 28, 2011

Published online: May 26, 2011

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