Report. Phloem-Transported Cytokinin Regulates Polar Auxin Transport and Maintains Vascular Pattern in the Root Meristem

Transcription

1 Current Biology 21, , June 7, 2011 ª2011 Elsevier Ltd All rights reserved DOI /j.cub Phloem-Transported Cytokinin Regulates Polar Auxin Transport and Maintains Vascular Pattern in the Root Meristem Report Anthony Bishopp, 1, * Satu Lehesranta, 1,5 Anne Vatén, 1,5 Hanna Help, 1 Sedeer El-Showk, 1 Ben Scheres, 2 Kerttuli Helariutta, 3 Ari Pekka Mähönen, 1,2 Hitoshi Sakakibara, 4 and Ykä Helariutta 1, * 1 Institute of Biotechnology and Department of Biosciences, University of Helsinki, FIN Helsinki, Finland 2 Section of Molecular Genetics, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands 3 Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki, FIN Helsinki, Finland 4 RIKEN Plant Science Center, Tsurumi, Yokohama , Japan Summary Cytokinin phytohormones regulate a variety of developmental processes in the root such as meristem size, vascular pattern, and root architecture [1 3]. Long-distance transport of cytokinin is supported by the discovery of cytokinins in xylem and phloem sap [4] and by grafting experiments between 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 developmental role has been shown for basipetal transport of cytokinin (toward the root apex). In this paper, we combine the use of a new technology that blocks symplastic connections in the phloem with a novel approach to visualize radiolabeled hormones in planta to examine the basipetal transport of cytokinin. We show that this occurs through symplastic connections in the phloem. The reduction of cytokinin levels in the phloem leads to a destabilization of the root vascular pattern in a manner similar to mutants affected in auxin transport or cytokinin signaling [7]. Together, our results demonstrate a role for long-distance basipetal transport of cytokinin in controlling polar auxin transport and maintaining the vascular pattern in the root meristem. Results and Discussion The phytohormones auxin and cytokinin interact to regulate multiple processes during root development including formation of the embryonic root, control of root meristem size, emergence of lateral roots, and control of root vascular pattern [3, 7 9]. The polar subcellular localization of the auxin efflux carriers PIN1, PIN3, and PIN7 directs an intercellular auxin flow rootward through the vascular tissue by a process known as polar auxin transport [10, 11]. In the columella, this flow is redirected laterally to the root cap and epidermis, where it flows shootward and is recycled back to the vascular tissue in the elongation zone [10]. The concerted action of the PIN 5 These 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 first forms in the quiescent center (QC) [12] and restricts the domain of the PLETHORA (PLT) proteins, which control root stem cell specification at this position [13]. As we show elsewhere in this issue of Current Biology, the PIN proteins also generate an auxin response maximum in the xylem axis that specifies protoxylem identity at the marginal positions [7]. The level of cytokinin signaling in the root meristem has been shown to regulate both the cellular efflux of auxin and PIN expression [3, 7, 8, 14, 15]. Because there are indications of basipetal transport of cytokinin, it is possible that cytokinin produced in the shoot could regulate PIN proteins and therefore 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 seedlings and assayed the cytokinin response in the root tip using qrt- PCR and parr5::gus. Within the vascular cylinder, ARR5 is normally expressed in intervening procambial cells in the proximal meristem (see Figure S1A available online) [7], but there is also a second peak of ARR5 expression in the transition zone at the point at which phloem differentiates. After 3 hr, we saw an enhanced GUS signal in the transition zone at the root tip, but longer treatments resulted in the accumulation of elevated GUS signal in the intervening procambial cells of the proximal meristem (Figures 1A and 1B; Figure S1A). This indicated that the cytokinin signaling pathway is activated in the intervening procambial cells of the root meristem even if cytokinin is not applied directly to the roots. In previous studies, labeled cytokinins have been applied to leaves of cocoa, pea, and yucca [16 18]. Much of the labeled cytokinin is strongly retained at the treated site; however, a small proportion is translocated to other parts such as roots and nodules [16 18]. These studies relied on the fractionation of labeled cytokinin from whole roots followed by quantification by scintillation and therefore offer little insight into the spatial distribution of transported cytokinin within the root. In order to test whether cytokinins could translocate to the proximal root meristem, we established an assay where we could visualize the migration of 14 C-labeled N 6 -benzyladenine (BA) (a cytokinin) using an imaging plate as a radioactive energy sensor that was processed with a fluorescent image analyzer (FLA). We applied labeled cytokinin to the hypocotyls of 5-day-old plants, and within 4 hr we saw a local maximum in the radioactive signal at the root tip, indicating that a proportion of the labeled BA had been transported to the root meristem (Figure 2A). In order to confirm that the cytokinin transported basipetally corresponded with natural cytokinin, we set up a similar assay using the stable isotope-labeled N 6 -(D 2 -isopentyl)adenine riboside (ipr). After application of isotope-labeled ipr to the hypocotyl, we measured ARR5 response in the root and observed a response similar to the earlier treatments with BA (Figure S1B). We pooled plants, dissected 2 mm sections from the root tips of 50 individuals, and measured the accumulation of labeled cytokinin in the root tip by mass spectrometry. In addition to the native cytokinin species, we also observed the accumulation of labeled cytokinin species, indicating that the ipr and/or its metabolites were transported from the hypocotyl to the root

2 Current Biology Vol 21 No Figure 1. Cytokinin Signaling Is Increased in the Intervening Procambial Cells of the Root Meristem in Response to Application of Exogenous Cytokinin to the Hypocotyls (A) qrt-pcr shows that 4 hr after application of 1 mmn 6 -benzyladenine (BA) (+CK) to hypocotyls, ARR5 expression is increased in the intervening procambial cells at the root tip. Error bars indicate 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 shootward of the meristem, close to the longitudinal domain in which phloem differentiates. Three hours after treatment, the signal increased intensity 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. (Table 1). Because CYP735A1 and A2, which catalyze transzeatin (tz) synthesis, are predominantly expressed in roots [19], and because N 6 -(D 2 -isopentyl)adenine (ip)-type cytokinins are the major form in phloem [20, 21], the labeled tz-type cytokinins are likely to be formed after ip-type translocation to the root. Basipetal transport of cytokinin has previously been associated with the phloem, because certain cytokinin species have been 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 altered phloem development (apl) mutant, which lacks phloem [22], and a line in which we could impair phloem transport by inducing rapid callose biosynthesis in the phloem to block the symplastic connections (A.V., Y.H., et al., unpublished data) (Figure S2A). Using these lines, we followed the transport of 14 C-labeled cytokinin from the hypocotyl to the root proximal meristem and found it to be severely compromised in both (Figure 2C; Figure S2B), indicating that basipetal transport of cytokinin occurs through the phloem. Furthermore, the fact that transport of cytokinin was impaired in papl::xve>> cals3m demonstrates that basipetal cytokinin transport occurs through symplastic connections in the phloem. We confirmed these results by quantitatively measuring the radioactive signal Figure 2. Cytokinin and Auxin Are Transported in a Basipetal Direction through the Phloem (A and B) When 14 C-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 unaffected by 1-naphthylphthalamic acid (NPA) treatments, and plants still show a cytokinin maximum at the root tip in 16 of 17 roots (E). In comparison, the signal for radiolabeled IAA is barely perceptible 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.

3 Phloem Transport of Cytokinin 929 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 (19%) (59%) (74%) (92%) labeled cz, czr, and czrps ND ND ND ND ND ND labeled ip, ipr, and iprps ND ND (8%) (10%) (15%) (23%) labeled ip7g and ip9g ND ND (32%) (68%) (67%) (73%) 50 pmol of stable isotope-labeled [ 13 C 10, 15 N 5 ]N 6 -(D 2 -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 N 6 -(D 2 -isopentyl)adenine (ip), ipr 5 0 -monophosphate (iprmp), trans-zeatin (tz), tz riboside (tzr), tz riboside 5 0 -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 replicates. ND indicates not detectable. See also Figure S2. via liquid scintillation counting and observed an approximately 4-fold reduction in the root tips of papl::xve>>cals3m (Figure S2C) plants. We also examined the endogenous cytokinin response following a 24 hr cals3m induction and observed reduced levels of ARR5 signal (Figure 3P), indicating that symplastic transport through the phloem provides a significant source of cytokinin in the root meristem. We then analyzed the transport of 14 C-labeled indole-3-acetic acid (IAA) in wildtype and papl::xve>>cals3m plants and observed that the bulk basipetal flow of labeled auxin transport was also impaired when phloem transport was blocked (Figures 2B and 2D; Figure S2C). We next analyzed the auxin response in papl::xve>>cals3m. Following blockage of the plasmodesmata in the phloem, we saw only modest changes to the auxin response maximum at the QC and columella (a slight expansion of auxin response into the epidermis is shown in Figure S3A). This effect is more in keeping with alterations in cytokinin signaling, because a similar phenomenon has been reported in 35S::CKX lines [15]. This suggests that although the phloem is likely to play a role in bulk auxin transport as suggested by other researchers [23, 24], additional transport mechanisms or local sites of biosynthesis must exist. We then analyzed the transport of 14 C-labeled IAA and BA in plants treated with 1-naphthylphthalamic acid (NPA), which blocks polar auxin transport by compromising PIN activity. Whereas cytokinin transport was unaffected in NPA-treated lines, the ability of auxin to reach the root tip was severely compromised (Figures 2E and 2F), showing that in addition to the bulk flow of cytokinin and auxin via phloem, auxin is actively transported through polar transport. Whereas cytokinin regulates auxin transport, cytokinin transport through the phloem appears to function independently of polar auxin transport. Previous studies have postulated directional transport of specific cytokinin species where tz-type cytokinins are used as an acropetal messenger and ip-type cytokinins as a basipetal messenger [4, 25]. Recent experiments with grafting between cytokinin biosynthetic mutants and wild-type have lent support to this cytokinin species-specific directional transport [5]. However, especially in basipetal transport, the quantity of cytokinin transported is quite low [16 18]. Our transfer assays showed that the phloem has the potential to deliver large amounts of cytokinins, which can make a substantial proportion of the pool of certain cytokinin species in the root meristem (Table 1). To test whether the basipetal transport of cytokinin plays a role in the development of the root meristem, we examined the root meristems in plants with defects in phloem-mediated cytokinin transport. In wild-type roots, maximal cytokinin signaling occurs in two distinct domains flanking the xylem axis and adjacent to the phloem position [2, 7]. A mutually inhibitory interaction between cytokinin and auxin signaling and transport specifies the position of these domains and determines vascular pattern in the root meristem [7]. High cytokinin signaling promotes the expression of PIN7 in two domains of intervening procambial cells flanking the xylem axis and regulates the radial distribution of PIN3 and PIN1 in the root meristem; this forces auxin into the xylem axis to create an auxin signaling maximum. High auxin signaling at the marginal protoxylem triggers AHP6 expression and specifies protoxylem identity in a bisymmetric pattern [7] (Figures 3A 3D). We generated a transgenic line to deplete cytokinin within the phloem (papl::xve>>ckx1:yfp) and used this alongside apl and papl::xve>>cals3m to investigate cell identity within the root meristem (Figure S3B). In all three cases, we examined the radial patterns of the ppin7::pin7:gfp, DR5rev::GFP, and pahp6::gfp marker genes in the root meristem. In wild-type plants, all of these markers are invariably expressed at precise locations that determine the location of distinct domains of high auxin and high cytokinin signaling. However, in these three lines we observed fluctuations in these boundaries, and the high auxin signaling domain frequently expanded to become two cells wide (Figures 3E 3G, 3I 3K, and 3M 3O). This showed that the basipetal transport of cytokinin through the phloem has a role in specifying the spatial regulation of PIN7 and in the formation of an auxin response maximum in the correct position. Accordingly, the patterns of marker genes that we observed closely mirrored those previously seen in the pin3 pin7 double mutant [7]. We next observed the vascular pattern in papl::xve>>ckx1:yfp and in apl. In the apl mutant, in addition to the reported switch in cell fate identity at the phloem position, we observed defects in the pattern of protoxylem differentiation in the root meristem; similar non-cellautonomous effects on protoxylem identity were seen in papl::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 loss of protoxylem. As a control, we drove expression of the CKX1:YFP gene in immature phloem cells under the control of the At2g37950 (EPM) promoter [26], and we only very seldom saw relatively minor effects on protoxylem differentiation (Figures S3C and S3D). Together, these results show that although the initial specification of vascular pattern occurs during embryogenesis and preceding the differentiation of phloem, the basipetal transport of cytokinin through the phloem plays an important role in maintaining this vascular pattern during growth. In order to determine whether the source for cytokinin in the phloem originated from the shoot, as opposed to the more distal parts of the root, we grafted

4 Current Biology Vol 21 No 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. 35S::CKX2 [15] shoots onto AHP6::GFP rootstocks. We observed an expansion in the domain of AHP6 expression in a significant proportion of these roots, but not in control graftings (Figure S3E). These data suggest that shoot-to-root transport of cytokinin through the phloem is necessary to spatially restrict the domains of hormonal signaling in the proximal root meristem. A role for cytokinin has been reported wherein cytokinin response activates the repressor of auxin signaling SHY2, which in turn acts as a negative regulator of PIN activity to control meristem size [8]. We examined whether cytokinin transported via the phloem could play a role in this process by comparing meristem size between wild-type and papl::xve>>ckx1:yfp plants. We observed no effect on meristem size (wild-type, cortex cells compared with papl::xve>>ckx1:yfp, cells) (Figures S3F and S3G). Transport through the phloem is one source of cytokinin; however, other sources certainly exist, and cytokinin biosynthesis and metabolism genes have been identified in the root meristem [27, 28]. One possible way in which the phloem transport of cytokinin could target specific developmental processes is through different receptor specificity. During vascular development,

5 Phloem Transport of Cytokinin 931 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 receptors have different ligand specificities; for example, AHK3 displays higher affinity toward tz than toward ip, whereas CRE1 has similar affinity toward both of these isoforms [30]. It might be possible that ip transported through the phloem is involved in vascular development via CRE1 and that tz in the root tip is involved in maintenance of the root meristem mainly via AHK3. An alternative possibility is that the regulation of root meristem size may be more robust to small changes in cytokinin than the regulation of vascular pattern. Together, these data demonstrate that the basipetal transport of cytokinins through the phloem plays an important developmental role in regulating PIN activity in the proximal meristem. Delivery of cytokinin through the phloem enables the targeting of specific developmental pathways (e.g., maintaining vascular pattern) while not interfering with other cytokinin-regulated pathways (that may be driven by local biosynthesis rather than transport). Why has such a regulatory system evolved? One possibility is that cytokinin transported from the mature part of the root maintains continuous and sharp domains of hormonal signaling in the meristem to ensure the formation of continuous vascular pattern. Experimental Procedures Hormone Transport Assays Plants were grown for 5 days on sterile agar plates. NPA and estrogen treatments were performed by moving plants to fresh plates containing 10 mm NPA or 10 mm 17b-estradiol 24 hr before analysis. Plants were then transferred 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 hypocotyls of plants in a 2 ml 1% agar bead and left for 4 hr. For 14 C applications, approximately 50 pmol [8-14 C]benzyladenine or [1-14 C]indole-3-acetic acid (American Radiolabeled Chemicals) was added per plant. This corresponded 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 [ 13 C 10, 15 N 5 ]N 6 -(D 2 -isopentyl) adenine riboside (Sakakibara laboratory, RIKEN Plant 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 used was a modified version of per8, an estrogen-receptor based chemical-inducible system [7, 32]. Effector genes were 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 5 0 -CGATCGATTTATCGAATTTAGTGTT-3 0 and 5 0 -TCTCTC TCTCTCTCTCTCTGACTCTC-3 0. To drive early phloem expression, we amplified 1.6 kb of sequence upstream of the gene At2g37590 [26] with the primers 5 0 -GTTTATGTGTTGCCTAACTCTTGA-3 0 and 5 0 -GGTTATTCTCTTTTGA TTTT-3 0 and cloned them into the inducible system. CKX1:YFP was amplified from the construct pcre1::xve/lexaoperator::ckx1:yfp [2] using the primers 5 0 -GGGACAAGTTTGTACAAAAAAGCAGGCTAAAATGGGATTGACCTC ATCCTTACG-3 0 and 5 0 -GGGGACCACTTTGCACAAGAAAGAAAGCTGGGTG TTACTTGTACAGCTCGTCCATGC-3 0, and the cals3m entry clone was prepared 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: /j.cub Acknowledgments We thank Mikko Herpola, Katja Kainulainen, Tia Kaonpää, 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 References 1. Dello Ioio, R., Linhares, F.S., Scacchi, E., Casamitjana-Martinez, E., Heidstra, R., Costantino, P., and Sabatini, S. (2007). Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Curr. 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