Auxin and ethylene interactions control mitotic activity of the quiescent centre, root cap size, and pattern of cap cell differentiation in maize

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1 Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment Blackwell Science Ltd 2005? ? Original Article Plant, Cell and Environment (2005) 28, Regulation of root apical meristem development by auxin and ethylene G. Ponce et al. Auxin and ethylene interactions control mitotic activity of the quiescent centre, root cap size, and pattern of cap cell differentiation in maize GEORGINA PONCE 1, PETER W. BARLOW 2, LEWIS J. FELDMAN 3 & GLADYS I. CASSAB 1 1 Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Apdo. Postal 510 3, Cuernavaca, Mor., 62210, México, 2 School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK and 3 Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720, USA ABSTRACT Root caps (RCs) are the terminal tissues of higher plant roots. In the present study the factors controlling RC size, shape and structure were examined. It was found that this control involves interactions between the RC and an adjacent population of slowly dividing cells, the quiescent centre, QC. Using the polar auxin transport inhibitor 1-Nnaphthylphthalamic acid (NPA), the effects of QC activation on RC gene expression and border cell release was characterized. Ethylene was found to regulate RC size and cell differentiation, since its addition, or the inhibition of its synthesis, affected RC development. The stimulation of cell division in the QC following NPA treatment was reversed by ethylene, and quiescence was re-established. Moreover, inhibition of both ethylene synthesis and auxin polar transport triggered a new pattern of cell division in the root epidermis and led to the appearance of supernumerary epidermal cell files with cap-like characteristics. The data suggest that the QC ensures an ordered internal distribution of auxin, and thereby regulates not only the planes of growth and division in both the root apex proper and the RC meristem, but also regulates cell fate in the RC. Ethylene appears to regulate the auxin redistribution system that resides in the RC. Experiments with Arabidopsis roots also reveal that ethylene plays an important role in regulating the auxin sink, and consequently cell fate in the RC. Key-words: auxin; auxin polar transport inhibitors; ethylene; maize root; quiescent centre; root cap. INTRODUCTION Root caps (RC) are the terminal tissue of roots of most plants (Barlow 2003). Some basic functions relating to root biology, such as lubrication of root growth and gravitropism, were ascribed to RCs by Haberlandt (1914, reviewed in Barlow 1975). Within the past 25 years, however, the Correspondence: Gladys I. Cassab. Fax: ; gladys@ibt.unam.mx functions of the RC have been shown to be considerably more diverse and to include regulation of many aspects of root development (Scheres et al. 1996; Tsugeki & Federoff 1999). The RC perceives and processes many environmental stimuli, and mediates the direction of root growth accordingly. Gravity (gravitropism), light (phototropism), obstacles (thigmotropism), gradients of temperature (thermotropism), humidity (hydrotropism), ions and other chemicals (chemotropism) are all examples of environmental stimuli that are perceived and processed by the cap (Hasenstein & Evans 1988; Ishikawa & Evans 1990; Okada & Shimura 1990; Fortin & Poff 1991; Takahashi 1997; Eapen et al. 2003). In roots with a closed type of construction, such as maize (Fig. 1a) and Arabidopsis, a distinct cap meristem with a set of RC initials (RCI) is present. The initial layer consists of the most rapidly dividing and least differentiated cells in the RC. As new RC cells are produced by the RCI, these derivatives are displaced through the RC until they are finally released into the soil as border cells (Hawes & Lin 1990; Hawes et al. 2003). During their passage to the outside of the cap, cells change from statocytes (i.e. gravityperceiving cells) into secretory cells which produce mucilage, and then finally differentiate into border cells which detach from the cap (Barlow 1975; Hawes & Lin 1990). If these cells are allowed to accumulate, and are prevented from being sloughed off (e.g. by suspending the root in air), the RC meristem ceases to produce new cells, suggesting communication between border cells and the RCI (Hawes & Lin 1990; Brighman et al. 1998). There is also evidence that the RCI communicate with adjacent cells located basally in the root proper. If the cap is excised, the adjacent root tissues alter their development and regenerate a new cap (Barlow 1974; Feldman 1976). The new cap regenerates from a population of previously mitotically inactive root cells located at the tip of the root proper and designated as a quiescent centre (QC). In experiments in which both the RC and QC are together excised a new RC reforms, but not until after a new QC re-develops from an even more proximal portion of the root, suggesting that the QC functions as an architectural template (Barlow 1976), because it mainly retards cell differentiation, and not cell division 2005 Blackwell Publishing Ltd 719

2 720 G. Ponce et al. a b c d e f g h Figure 1. Expression pattern of clone C123 in primary root apices treated with 10-5 M of NPA. (a) (d) Untreated 3-day-old maize primary roots grown in a humid chamber 0 h (a), 24 h (b), 48 h (c), and 72 h (d). Note that clone C123 is continually expressed in the lateral RC and root epidermis at all times analysed. (e) (h) Roots treated with 10-5 M NPA for 24 h (t = 0, e) and grown thereafter in a humid chamber without NPA for 24 h ( h, f), 48 h ( h, g), and 72 h ( h, h). After 24 h of NPA treatment (f), the expression of clone C123 in the lateral RC and epidermis was severely diminished. While the closed apical organization of the root apex was apparently lost (f, g), the expression of C123 continues to be absent in the lateral RC and epidermis. As the apex re-organized, clone C123 was now expressed in the lateral RC and epidermis (h) as in untreated roots (a d). (Feldman 1976, 1998). Recent work using laser ablation to destroy one or more QC cells in Arabidopsis roots has extended this view (van den Berg et al. 1997). In Arabidopsis the QC consists of four cells and is surrounded by meristematic initials. When one or more of the QC cells is ablated the contacting RCI, which normally lack starch, differentiate and develop starch grains. Sabatini et al. (1999) have reported that asymmetric auxin distribution, with an apparent high capacity of response to auxin (auxin maximum) in the columella initial/ QC region, establishes an organizer of pattern and polarity in the root meristem. Auxins are considered unique amongst plant hormones in showing a polarity in their movement (Davies 1995; Barlow, Volkmann & Baluska 2004). Indole-3-acetic acid (IAA) is transported into and out of the cell across the plasma membrane through the activities of the auxin influx and efflux carriers, respectively. The identification of auxin transport inhibitors, such as 1- N-naphthylphthalamic acid (NPA), has greatly facilitated our understanding of the physiological importance of auxin transport (Katekar & Geissler 1977). Perturbations in gravitropism, lateral root initiation, vascular differentiation and embryonic patterning are effects reported to follow treatment of plant tissues with NPA (reviewed by Casson & Lindsey 2003). Genetic ablation of RC cells in Arabidopsis has revealed that the roots of transgenic plants (carrying a RC-specific promoter that directs the expression of a diphteria toxin A- chain gene [DT-A tsm ]) have more lateral roots, and these, in turn, are more highly branched than those of wild-type plants (Tsugeki & Federoff 1999). This may indicate that the RC is a complex and dynamic tissue essential for normal root development. Tsugeki & Fedoroff (1999) propose that there is an auxin sink in the RC or that access to the sink is through the RC auxin transport system. Recently, AtPIN4, a novel member of the PIN family of putative auxin efflux carriers, has been linked to the establishment of an auxin sink in the RCI that is essential for auxin distribution and patterning (Friml et al. 2002). Ponce et al. (2000) have explored the role of the QC in the development of the maize RC. They have identified three maize root-specific genes. Two of these are exclusively expressed in the RC, one of them encoding a GDPmannose-4,6-dehydratase (clone C106), and the other, a cysteine-rich protein with unknown function (clone C109). Most likely these two genes are tissue-specific, structural markers of the cap. The third gene, a putative glycine-rich cell wall protein (clone C123), is expressed in the cap and in the root epidermis and conceivably is a positional marker of the cap. It was suggested that the QC and cap initials might regulate the positional and structural expression of these genes in the cap and thereby control RC development (Ponce et al. 2000). Our main goal is to study the effect of stimulating the QC in the intact root and to explore the consequences on the division and differentiation activities of the RC. This can be done by using NPA to block auxin transport since NPA enhances mitotic activity within the QC (Kerk & Feldman 1995). After treating root apices with NPA, the QC was activated at its distal face. As a result, the root body penetrates and ruptures the RC junction and

3 Regulation of root apical meristem development by auxin and ethylene 721 the characteristic closed apical organization changes to open (Jiang, Meng & Feldman 2003). Thus, an interference with auxin movement caused a concomitant change in the pattern of the root apex and in the position of the auxin maximum (Sabatini et al. 1999). In the present experiments, after exposing roots to NPA, in situ hybridization was performed with four cdna clones of RC- specific genes (clones C106, C109, C123, and C103 [a UDP-glucose-4- epimerase, Luján & Cassab, unpublished observation]). Only one RC gene (represented in clone C123) was regulated by the reduction in polar auxin transport towards the root tip. The release of border cells was induced from root apices pre-incubated with NPA, IAA or ethylene. Border cell shedding was increased when NPA perturbed auxin gradients in the RC. However, border cell shedding was significantly decreased in the presence of the ethylene precursor [1-aminocyclopropane-1-carboxylic acid (ACC)]. Concomitantly, the mitotic activity of the QC increased after border cell induction. Our current hypothesis is that auxin and ethylene regulate the fate of cells within the root apex by regulating their apparent movement from one differentiating compartment to another (e.g. QC to RCI, columella to border, and so on). This may also play an essential role in the regulation of the many tropic (gravitropism, hydrotropism, thigmotropism, etc.) responses of the root. For instance, this signal may act as the link between perception of stimuli in the RC and tropic responses in the root. However, ethylene might be important in regulating auxin homeostasis in the RC. In fact, experiments with Arabidopsis roots revealed that ethylene also might regulate the patterning of cells and auxin distribution in the RC. MATERIALS AND METHODS Plant material Caryopses of Zea mays cv. Merit (Asgrow Seed Co., Kalamazoo, MI, USA) were imbibed for 2 h in rapidly running water and then transferred to trays lined with moistened filter paper. Trays were then placed in a light-tight canister in a chamber at 28 C in the dark. Seeds of wild-type Arabidopsis thaliana, ecotype Columbia-0 (Arabidopsis Resource Center, Ohio University, Columbus, OH, USA), were soaked in distilled water for 30 min, and surface sterilized with 10% (v/v) bleach with 0.01% (v/v) Triton X-100 detergent for 10 min. After five washes in sterile distilled water, the seeds were kept in darkness at 4 C for 4 d. The seeds were germinated and grown on sterile control medium as described by Eapen et al. (2003). Seeds were grown in vertically oriented square Petri dishes in 16 h day/ 8 h night cycles at 22 C for 4 d. NPA or auxin treatment For NPA (Uniroyal Chemical Company, Inc., Middlebury, CT, USA) treatments, maize seeds or caryopses were germinated in the dark and then seedlings with roots approximately 3 cm in length were selected. These seedlings were attached to Styrofoam boards covered with moistened filter paper by inserting a pin through the kernel. A 1-cm-wide 1% agar collar (10-4 M NPA or 10-5 M IAA) was then placed over the root at its proximal end according to Kerk & Feldman (1994). Hence, the terminal 2 cm of the root did not have any direct contact with NPA. The roots were returned to a moistened chamber in the dark (28 C) and maintained in a vertical position. At 24 h intervals or times indicated, root tips were excised and fixed as described in Ponce et al. (2000). Border cell production Four-day-old maize root tips were induced to detach border cells by maintaining the seedlings for 10 min in distilled H 2 O, rinsing 70 times with a Pasteur pipette and then allowing them to recover for 0.5 h or the time indicated. Roots were then fixed and processed for in situ hybridization analysis or 5-bromo-2 deoxyuridine (BrdU) immunodetection. In situ hybridization In situ hybridization experiments were performed as described by Ponce et al. (2000). Simultaneous exogenous application of NPA/ ACC, NPA/AVG, IAA/ACC and IAA/AVG to maize roots Application of two compounds to maize roots was made as follows. Three-day-old maize roots were pre-incubated with 10-4 M L-a-(2-aminoethoxyvinil) glycine hydrochloride (AVG) or M ACC, for 1 h, then incubated for 24 h in the presence of: distilled water (control, no treatment), 10-4 M NPA/ M ACC, 10-4 M NPA/10-5 M AVG, 10-5 M IAA/ M ACC, and/or 10-5 M IAA/10-5 M AVG. Pre-incubation with AVG or ACC was not performed in roots treated for 24 h with only one compound. The concentrations of NPA, IAA, ACC and AVG used were 10-4 mm, 10-5 M, M and 10-5 M, respectively. Seedlings in a vertical position were punched onto a piece of Styrofoam and the root tips were inserted in a plastic bag containing a piece of germination paper soaked in the indicated solution. This was done to ensure that root tips were kept moist but not submerged in the solution. Twenty-four hours later, roots were fixed at 4 C, dehydrated, and embedded in paraffin. Paraffin sections of 10 mm were used as indicated. BrdU Incorporation and detection For BrdU immunodetection, roots were incubated for at least 16 h with a solution containing the hormones or inhibitors previously indicated plus BrdU 10-5 M and 5-fluoro-2 deoxyuridine (FdU) 10-6 M. The next day, roots were fixed for 16 h in 2% paraformaldehyde in 0.1 M PO 4 buffer ph 7. BrdU detection was carried out as described by Kerk & Feldman (1995).

4 722 G. Ponce et al. Microscopy For histological analysis, roots were dehydrated in a graded series of EtOH from 10 to 100%, infiltrated in EtOH : Historesin 3 : 1, 1 : 1, 1 : 3 and two changes of pure Historesin and finally embedded in Historesin plus hardener (Historesin; Leica Instruments, Heidelberg, Germany). Sections were cut on a Reichter Jung Ultracut Microtome (Leica, Heidelberg, Germany) to a final thickness of 4 5 mm. Light microscopy on thin sections were collected on glass slides and stained with Safranin-Fast Green (for staining starch granules), or Azur-Methylene Blue, and viewed in a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan). Images were taken with a Nikon D1 digital camera. Epifluorescence microscopy for detecting BrdU incorporation and starch granules on paraffin thin sections was performed in an Axioskop microscope (Zeiss, Jena, Germany) and images were obtained with the MERCATOR software developed by Explora Nova (La Rochelle, France). All images were assembled with Adobe Photoshop Elements 7.0 (Adobe Systems Inc., Mountain View, CA, USA) Exogenous application of auxin, ethylene and inhibitors of polar auxin transport and ethylene biosynthesis to Arabidopsis root tips Four-day-old Arabidopsis seedlings were treated with locally applied compounds (dissolved in molten agar) to root tips in an 8-mm line along and below the tip as described by Rashotte et al. (2000). Controls for these experiments were performed by the addition of an agar line without added compound. After the addition of the agar plus compounds to root tips, seedlings were grown for an additional 7 d under the same conditions as described above. Seedlings were fixed 48 h in formaldehyde : acetic acid : alcohol (FAA) and stained with 0.5% (w/v) I 2 -KI for visualization of starch grains by light microscopy. RESULTS Auxin polar transport regulates gene expression in the root cap Expression of one RC gene (clone C123) in roots whose auxin transport has been halted by NPA treatment for 24 h was completely abolished (Fig. 1e). The expression of this gene continued to be absent for 48 h in roots treated with NPA for 24 h, and then allowed to grow for an additional 24 h or 48 h without NPA ( ; ) (Fig. 1f & g) but reappeared when the morphogenetic pattern of the root meristem was re-established: that is, when cells of the former QC were no longer mitotically active ( ) but had resumed their quiescent state as a new QC (Fig. 1h). Thus, auxin movement apparently regulates the activity of the QC and the RC. Importantly, the absence of auxin supply inhibits the expression of one RC-specific gene. We had previously analysed the expression of clone C123 after removing both the QC and RC by microsurgery (Ponce et al. 2000). The expression of clone C123 was observed in all new nascent cap cells as long as the QC and the RCI had not re-established. This might mean that when auxin is not metabolized as normal, because of the absence of the QC (Kerk, Jiang & Feldman 2000), the communication between the QC and the RC no longer exists, and hence regulation of some genes in the RC is altered. In the present case, high expression of clone C123 was seen in the entire nascent cap. The expression pattern of other RC-specific genes (C106 and C109) was not affected by the NPA treatment (data not shown), nor by the removal of the QC and the RC (Ponce et al. 2000). Thus, neither auxin supply nor the status of the QC regulate the totality of RC activities. Border cell production is correlated with the mitotic activity of QC cells It has been estimated that as much as 20% of photosynthate can be lost to the soil when cells from the edge of the RC are released, and it has been proposed that this release has important developmental consequences for the plant (Hawes et al. 2000). Border cells lost to the soil are now believed to condition the soil to the benefit of the plant (Hawes et al. 2003). Interestingly, when maize roots were induced to shed border cells in vitro, the mitotic activity of QC cells was increased (Fig. 2b). Cell division in the QC cells started 10 min after roots were induced. This probably means that cap differentiation is accompanied by a change in auxin gradients in the cap and QC, and that these gradients can be influenced by specific stimuli. For instance, if roots are mechanically stimulated by abrasion, QC cells are not mitotically activated (Fig. 2c) and remain inactive as in untreated roots (Fig. 2a). We also tested whether QC cells were activated by rotating the root 360 (Fig. 2d) in view of a recent report by Moseyko et al. (2002) that this gentle movement caused the expression of 30% of the genes induced during the early stages of the gravitropic response of Arabidopsis. Nonetheless, this minor movement did not activate cell division in the QC of maize roots. Ethylene and auxin interactions control root cap cell differentiation The effects of ethylene on cap differentiation can be seen on Fig. 3. We first tested the effect of ethylene on the mitotic activity of the QC and RCI cells in maize roots by incubating them with the ethylene precursor, ACC (Fig. 3d). Secondly, we examined the effects of ethylene on the expression of clone C123 and the acquisition of starch granules, a columella marker (Fig. 3e & f). The mitotic activity of QC and RCI is similar in ACC-treated and untreated roots, although fewer initials are apparently stained with BrdU in ACC-treated roots (Fig. 3a & d). Expression of clone C123 in the lateral RC and root epidermis is also not altered after ACC treatment (Fig. 3b & c). However, the number of columella cells that showed starch granules increased in the presence of ethylene

5 Regulation of root apical meristem development by auxin and ethylene 723 a b c d Figure 2. Border cell induction activates mitosis in QC cells. BrdU incorporation in primary root tips of 3-day-old maize seedlings. (a) Control roots, no treatments. (b) Border cell induced RCs. (c) Abraded root tips. (d) Rotated 360 roots. Note that most QC cells increased their rate of division in caps induced to release border cells, compared to untreated and rotated roots. Abrasion resulted in an activation of only the distal region of the QC. QC, quiescent centre; PM, proximal meristem; RC, root cap. (Fig. 3c & f). Nevertheless, the number of border cells shed was diminished (see Table 3). Root elongation of 3-day-old maize seedlings following a 24-h treatment with ACC or NPA was inhibited in contrast to untreated roots (Table 1). A similar effect was observed in roots treated concurrently with ACC and NPA (Table 1). When maize roots were incubated with AVG, the mitotic activity of QC and RCI considerably increased; however, a small QC was still apparent (Fig. 3g). In fact, the number of RCI tiers increased considerably. Concomitantly, the number of columella cells and border cells decreased (Fig. 3i, and see Table 3). Furthermore, expression of clone C123 in the most external cells of the lateral RC is not observed after treatment with AVG (Fig. 3h). Nonetheless, when both ACC and NPA were added to maize roots, the NPA effect on activation of QC cell division was eliminated (Fig. 3j & p) and thus the QC was restored (Jiang et al. 2003). In contrast, roots treated with both NPA and AVG showed BrdU labelling in the QC, but the labelling in the columella portion of RCI was more scattered (Fig. 3m). Additionally, when ethylene levels are modified in maize roots either by treating with ACC or AVG in the presence of NPA, the cell division pattern in the root epidermis was affected and a supernumerary epidermal cell layers were seen at one side of the root, under the lateral RC (Fig. 3s). Here we observed two cell division branch points in the original epidermal cell file, and then each daughter cell lineage itself became secondarily branched. These additional epidermal layers mostly acquired cap-like characteristics, such as starch grains, and perhaps also as a result of positional information showed periclinal divisions, which resembled those of the RCI. Furthermore, most irregularities were restricted to only one side of the meristem (Fig. 3r & s). Experiments were also undertaken to determine the effect of treating maize root tips with IAA in combination with ACC and AVG, particularly in relation to RC morphology and development (Fig. 4). Auxin treatments did Table 1. Effects of an inhibitor of polar auxin transport on root growth of maize seedlings Treatment Root length (mm) C 4.88 ± 0.75 ACC 3.55 ± 1.50* IAA 2.00 ± 0.23*** NPA 3.80 ± 0.40** IAA/ACC 2.33 ± 0.86*** NPA/ACC 3.62 ± 1.31* Three-day-old maize seedlings were treated for 24 h with different compounds. The average and SD of eight seedlings from three separate experiments are reported. The difference was significant with the Student s test, *P = 0.05, **P = and ***P =

6 724 G. Ponce et al. a b c d e f g h i j k l m n o p q r s Figure 3. Ethylene regulation of auxin polar transport influences cap cell differentiation. (a c) Control roots, no treatments. Roots treated with ACC (d f), AVG (g i), NPA (j l). Simultaneous application of NPA/ACC (p s), and NPA/AVG to roots (m o). Roots stained for BrdU (a, d, g, j, m and p) and Safranin-Fast Green for detecting mitotic activity and starch grains, respectively (c, f, i, l, o and r). In situ hybridization analysis with clone C123 (b, e, h, k, n and q). (s) shows a root section stained with Adobe Photoshop Elements 7.0.

7 Regulation of root apical meristem development by auxin and ethylene 725 a b c d e f g h i j k l Figure 4. Auxin and ethylene interactions regulate cell fate in the cap. (a c) Control roots, no treatments. In situ hybridization analysis with clone C123 (b, e, h and k). (d f) Application of IAA to maize roots. Concurrent treatment of IAA/ACC (g i), and IAA/AVG to roots (j l). Roots stained for BrdU (a, d, g and j) and Safranin-Fast Green (c, f, i and l). not activate QC cells and RCI (Fig. 4d) compared with untreated roots (Fig. 4a). In contrast, roots exposed to IAA in the presence of the ethylene synthesis inhibitor AVG exhibited a general increase in the rate of cell division in the QC and initials, to the extent that no QC was apparent (Fig. 4j). Therefore, treating roots with IAA/AVG resulted in the activation of the QC on its proximal and distal faces in contrast with roots treated only with NPA or AVG which showed activation on its distal face (Jiang et al. 2003; Figs 3g & j & 4j). Treatment with either IAA or IAA/ AVG did not alter the expression of the lateral RC marker C123 (Fig. 4b, e & k). Nevertheless, the addition of IAA/ ACC to roots resulted in the absence of expression of C123 on only one side of the lateral RC with the concomitant appearance of a supernumerary epidermal cell layers (Fig. 4h), as seen in roots treated with IAA/AVG (Fig. 5i). Moreover, these adventive columella cells did not express clone C123, a marker of lateral RC cells (Fig. 4h). On the other hand, the presence of starch grains in the central portion of the columella was affected by IAA treatment in combination with ACC or AVG (Fig. 4f, i & l). Treating roots with IAA alone increased the number of columella cells in comparison with untreated roots (Fig. 4c & f, and Table 3). Roots treated with both IAA and ACC showed a similar number of columella cells stained with starch, but the width of the root and RC increased (Fig. 4i) as in roots treated with NPA and ACC (Fig. 3r). An equivalent pattern of starch granules in columella cells was observed after application of IAA/AVG (Fig. 4l). Roots treated with AVG or NPA are shorter than untreated roots, and when both chemicals are used, root length was affected even more (Table 2).

8 726 G. Ponce et al. a b c d e f g h i Figure 5. Auxin and ethylene interactions change the pattern of cell differentiation in the lateral RC and epidermis. Histological analysis in median sections of 3-day-old primary roots of maize stained with Azur-Methylene Blue. Control roots, no treatments (a). Roots treated for 24 h with NPA (b), IAA (c), ACC (d), AVG (e), and with the simultaneous treatment of NPA/ACC (f), NPA/AVG (g), IAA/ACC (h) and IAA/AVG (i). Ethylene modulates the redirection of the distal pattern produced by inhibition of polar auxin transport in the Arabidopsis root In Arabidopsis roots treated for long periods with NPA, it has been shown that the location and shape of the auxin maximum is severely distorted (Sabatini et al. 1999). In these roots, the DR5 peak expression not only included the RCI/QC site, but the peak enlarged and became cupshaped, incorporating flanking proximal cortical cells. In addition, this treatment not only shifted the auxin maximum to a more basal region of cortical cells, but also led to the acquisition of QC identity, RCI and differentiated columella identity in former epidermal, endodermal and cor- Table 2. Effects of an inhibitor of polar auxin transport and ethylene biosynthesis on root growth of maize seedlings Treatment Root length (mm) C 5.20 ± 0.61 AVG 4.43 ± 0.48* IAA 1.55 ± 0.31*** NPA 4.20 ± 0.59** IAA/AVG 1.80 ± 0.34*** NPA/AVG 3.28 ± 0.74*** Three-day-old maize seedlings were treated for 24 h with different compounds. The average and SD of 12 seedlings from three separate experiments are reported. The difference was significant with the Student s test, *P = 0.005, **P = 0.002, and ***P =

9 Regulation of root apical meristem development by auxin and ethylene 727 tical cells, as indicated by the expression of the QC and columella specific markers (Sabatini et al. 1999). This suggests that the ectopic auxin accumulation resulting from inhibition of transport is capable of re-organizing the root cell pattern. Since the pattern of the root apex was not changed in maize roots treated with ethylene and NPA, we therefore analysed the combined effect of NPA with ACC and/or AVG on changes in cell fate in Arabidopsis wildtype roots. Arabidopsis roots were germinated and grown in MS medium for 4 d and then treated by local application to root tips of 100 mm NPA and 0.5 mm ACC or 10 mm AVG for 7 d (Fig. 6). Application of NPA to the root tip abolished the gravity response and reduced root growth (Rashotte et al. 2000). The local addition of NPA alone to Arabidopsis root tips resulted in the apparent loss of the closed meristem and the emergence of supernumerary epidermal cell layers expressing a columella marker (starch grain staining) (Fig. 6g & j). In contrast, Arabidopsis roots treated locally with NPA and ACC, retained the supernumerary cell layers of columella and the typical conical shape of the RC, as well as the closed meristem structure. However, these root tips were wider than those of untreated roots and contained about three columella cell layers that were stained for starch granules (Fig. 6i). Furthermore, the effect of NPA was mitigated when roots were simultaneously exposed to AVG (Fig. 6h), although to a lesser extent than those roots that were treated with NPA/ ACC (Fig. 6i). Root tips grown with local applications of either ACC or AVG showed no apparent pattern misspecification in the RC (Fig. 6b & c). Similar results were obtained by treating root tips locally with only IAA (Fig. 6d), or IAA and AVG (Fig. 6e). Both maize and Arabidopsis root tips simultaneously treated with NPA/ACC or NPA/AVG showed an increase in width (Fig. 3m r and Fig. 6h & i). However, Arabidopsis roots did not develop supernumerary epidermal cell layers on one side of the lateral RC as maize roots did. DISCUSSION The multiple activities of the RC can be achieved by a dynamic interplay of cell division and cell differentiation processes. The fact that cap cells progress from their origin in the cap meristem to their release into the soil in a period of a few days, and continue to perform sensory functions certainly makes the cap one of the most extraordinary parts of the plant. In this paper, we explored the role of auxin and ethylene in the regulation of cell fate within the root apex by regulating their apparent movement from one differentiating compartment to another (e.g. QC to RCI, columella to border cell, and so on). Auxin is widely involved in root cell patterning (Sabatini et al. 1999; Friml et al. 2002; Jiang et al. 2003). However, the dual role of ethylene and auxin on root patterning has been mainly ascribed to the process of root hair initiation and elongation (Cnops et al. 2000). Interestingly, ethylene production is frequently part of the plant s response to stress (Rao, Lee & Davis 2002). As the root advances through the soil, the RC is the first part of the root that confronts various biotic and abiotic challenges/stresses. Thus, we considered the possibility that the RC might have acquired distinctive developmental mechanisms for dealing with different stresses. Recently, Yang et al. (2002) reported the NaCl-stimulated up-regulation of a member of the EREBP family in the rice root tip, which supports the presumed involvement of ethylene in mediating the response of roots to stress. Since the role of auxin in root cell fate determination is generally accepted, we hypothesized that ethylene might act in concert with auxin in the modulation of patterning in response to a variety of challenges. Our results support this hypothesis. Our observations, mainly that auxin and ethylene control cell fate within the root apex, are summarized in Table 3 and Fig. 7. Border cell release is promoted in root tips treated with NPA, but is slightly inhibited in the presence of ACC and AVG. However, ACC apparently counteracted the effect of NPA upon border cell release since QC cells were not mitotically activated. On the other hand, in AVGactivated QC and RCI cells the presence of starch and the detachment of border cells are simultaneously inhibited. In fact, auxin accumulation may induce cap-building cell divisions as it has been observed in Arabidopsis pin4 mutants (Friml et al. 2002). Since Arabidopsis roots do not shed border cells (Hawes et al. 2003), additional tiers of cap cells are seen in the pin4 mutant. In maize roots, no additional Table 3. Summary of maize root tip morphology observed after different treatments % BC RC length (mm) a No. rows c QC b * C123 b Starch bd C yes IAA yes NPA no ACC yes AVG no ACC/IAA no & + + ACC/NPA yes AVG/IAA no & + + AVG/NPA no a Taken from sections shown in Fig. 5 from the cap junction to the tip; b From Figs 3 and 4; c From the cap junction to the tip through the columella; *Indicates BrdU incorporation in RCs (mitotic activity); d Relative abundance of starch grains in columella; & Absence of C123 expression in supernumerary epidermial cell layers.

10 728 G. Ponce et al. a b c d e f g h i j Figure 6. Ethylene and inhibition of polar auxin transport influences cell pattern in Arabidopsis RC. Four-day-old seedling roots were continuously treated with agar with or without compounds for 7 d, and then lugol-stained. Control roots, no compounds (a). Localized application of agar with AVG (b), ACC (c), IAA (d) and NPA (g) at the root tip. Treatment with agar containing IAA/AVG (e), IAA/ACC (f), NPA/AVG (h) and NPA/ACC (i) at the root tip. (g and j) Local application of NPA at root tips resulted in additional layers in the RC. (h, i) Both AVG and ACC reduced the effects of NPA on the morphology of the cap, although ACC was more effective. In both treatments, the width of the root tip and cap increased.

11 Regulation of root apical meristem development by auxin and ethylene 729 Mature cells Acropetal Auxin transport Elongation Zone cells C 2 H 4 C 2 H 4 PIN4 C 2 H 4 NPA Proximal meristem cells IAA IAA QC ACC C 2 H 4 NPA C 2 H 4 ACC IAA RCI IAA ACC C 2 H 4 Statenchyme Mechanical effects Superficial or Border cells Released cells? Figure 7. Model for the proposed interaction of auxin and ethylene in the root tip. Question mark refers to the unknown factors that control border cell release. See text for a detailed discussion. tiers of columella cells were observed, but there was a significant increase in the number of border cells shed (Table 3). In addition, RC length decreased upon NPA treatment indicating a possible loss in the regulation of cap size. Of particular note is the observation that the induction of border cell release and the NPA treatment activated mitosis in QC cells (Table 3, Figs 3 & 7). However, when polar auxin transport was inhibited in the presence of ACC, QC cells were not activated. In contrast, roots treated solely with AVG showed activated QC cells and RCI and fewer columella cells stained for starch grains. However, when roots were treated only with ACC, fewer RCI were activated and consequently there was a considerable increase in the number of columella cells. Hence, changes in the level of ethylene might unbalance the auxin transport system and as a result the rate of cell division in RCI exceeded and/or reduced the rate of columella cell differentiation. Border cell release, on the other hand, is apparently regulated by a mechanical effect and perhaps also by ethylene, as suggested earlier by Russell (1977). Furthermore, border cell differentiation will undoubtedly depend on the source of columella cells that were first differentiated as secretory or peripheral cap cells, before becoming detached cells, a step that seems to be regulated both by ethylene and auxin polar transport. Taken together our results suggest that there is a feedback loop between the QC and the RC controlled by both polar auxin transport and ethylene, which in turn regulates pattern of cell differentiation and size in the cap. Auxin is known to stimulate the production of ethylene (Yang & Hoffman 1984) and this correlates with the fact that the ACC synthase4 gene has been found to be an early auxin-induced gene (Abel et al. 1995). Another ACS gene is expressed in root tips (Rodrigues-Pousada et al. 1999) and corresponds in position to that of the auxin maximum in the root (Sabatini et al. 1999). Furthermore, the elongation of roots is inhibited by both auxin and ACC, which raises the possibility that their action, in this case, is via a common mechanism. One candidate for this common mechanism is the auxin transport machinery (Casson & Lindsey 2003). Root growth of 3-day-old maize seedlings following a 24-h treatment with different compounds

12 730 G. Ponce et al. showed that the combined effect of IAA with ACC or AVG on root elongation was inhibitory and was not significantly different from roots treated with IAA only (Tables 1 & 2). However, maize root growth displayed significantly greater sensitivity to the combined effect of NPA and AVG in comparison with untreated roots (Tables 1 & 2). Ethylene treatment has been shown to reduce auxin polar transport by up to 95% (Morgan & Gausman 1966; Burg & Burg 1967). Furthermore, NPA binding to microsomes was significantly reduced following ethylene treatment due to a reduction in the number of binding sites (Suttle 1988). Since the NPA binding site is believed to be part of the auxin efflux carrier machinery and may regulate its activity (Ruegger et al. 1998), this would suggest that ethylene inhibits auxin efflux by reducing the synthesis or localization of the NPA-binding protein. Recently, ethylene has been involved in the root control of nucleotide sugar flux (Seifert et al. 2004), which in turn requires an intact polar auxin transport. Furthermore, the demonstration that the intracellular level of auxin plays a critical role in regulating the ethylene-mediated growth response in Arabidopsis roots may provide insights into the supposed role of ethylene in the stress response of roots (Rahman et al. 2001). Hence, it is not surprising that ethylene reversed the NPA effect on QC activation. We also observed a decline in border cell release upon ethylene exposure. In contrast, inhibition of polar auxin transport increased the number of detached border cells and activated cell division in the QC (Table 1). When the QC is activated, transport of auxin in the root is perturbed and the auxin maximum is atypically localized (Sabatini et al. 1999; Jiang & Feldman 2003). A protein secreted by border cells into the mucilage seems to control mitosis in the cap meristem (Brighman et al. 1998), but it might also activate mitosis in QC cells after 10 min of border cell induction (Fig. 3b). Therefore, we suggest that the activation of both QC cells and cap initials is a coordinate event that influences cell differentiation in the cap. Most probably, the localization of auxin maximum might be regulated by ethylene, since ethylene could prevent the basipetal auxin transport from the RC to the root elongation and differentiation zones. Thus, the ability of auxin to induce ethylene and ethylene to inhibit polar auxin transport suggests a mechanism by which RC differentiation can be controlled by a feedback loop. Modulation of ethylene levels in roots affected cell division patterns in the maize root epidermis. Maize root tips treated with ACC or AVG in the presence of IAA or NPA presented supernumerary epidermal cell layers that protruded mainly on one side of the proximal meristem (Figs 3s, 4h & i and Table 3). It has been reported that mutations in the TORNADO1 and TORNADO2 genes of Arabidopsis resulted in defective epidermal and lateral RC patterning because lateral RC cells developed in the epidermal position in trn1 and trn2 roots (Cnops et al. 2000). This misspecification seems to indicate that TRN genes repressed lateral RC fate in cells in the epidermal location. Interestingly, tnr1 and trn2 mutants also showed the majority of their irregularities on only one side of the meristem, as was the case in maize roots treated simultaneously with either IAA with ACC (Fig. 4h), or IAA with AVG (Fig. 5i). One clue to the possible defect in trn1 mutants is that this mutant is allelic to the lopped1 mutant (lop1, Carland & McHale 1996). This mutant showed reduced vasculature and was shown to be defective in auxin transport. Given that auxin is proposed to play a significant role in the maintenance of root meristem organization (Sabatini et al. 1999), it is plausible that defective distribution of auxin contributes to the trn mutant defects (Cnops et al. 2000). Hence, ethylene modulation of polar auxin transport in the RC might influence the development and maintenance of the QC and the position of the auxin sink in the root. The additional epidermal layers, and their asymmetrical placement on the root surface beneath the lateral cap resemble the cellular patterns simulated at the root apex of tomato in response to an asymmetry of the growth tensor field (Nakielski 1997). Thus, asymmetry of auxin transport or distribution within the root apex may have an effect on the overall field of growth in which cell division occurs. Similar alterations to cellular layers at the root apex have been reported for roots of gibberellin-deficient tomato (Barlow 1992). Auxin appears to be associated with the development of columella cells and for promoting the cell divisions in the RCI, because the axr1 and axr3-1 mutants of Arabidopsis both show defects in these two characters (Sabatini et al. 1999). In addition, auxin can create a sink towards which more auxin moves (Jiang & Feldman 2003). Nonetheless, the recent finding that AtPIN4 is important in Arabidopsis in generating an auxin sink distal to the QC, suggests that this protein may be part of this mechanism, and that this gene may also be implicated in the acropetal auxin transport (Rashotte et al. 2000; Friml et al. 2002). Interestingly, up to eight additional tiers of columella cells were found in caps of the Atpin4 mutants (Friml et al. 2002), as in wildtype Arabidopsis root tips locally treated with NPA (Fig. 6g & j). Further, both roots showed several outer cap cell layers. This suggests that the impaired basipetal transport of auxin might also impair the usual cell separation process at the cap periphery. In fact, a higher number of border cells were detached in maize roots treated with NPA (Table 3). However, local addition of ethylene or inhibitors of ethylene biosynthesis in the presence of NPA to wild-type Arabidopsis root tips showed that ethylene might modulate or impede the generation of the auxin sink by AtPIN4. This interference or modulation might also influence the acropetal auxin transport in the root. Additionally, when higher concentrations of AVG were used, cells in the position of the columella did not form starch granules, which could not be counteracted by IAA or NPA (data not shown). Although the specificity of action of inhibitors should always be questioned, the ability of AVG to block the various physiological effects of ethylene (Kende 1993) suggests that it might influence the process of differentiation of columella cells by inhibiting ethylene biosynthesis. These results suggest that ethylene is an important player in the pattern of cap cell differentiation. Ethylene might regulate

13 Regulation of root apical meristem development by auxin and ethylene 731 cap size by co-ordinating the release of old cells from the cap and the production of new cells in the cap meristem. This process will consequently be tightly correlated with the establishment of the auxin sink and auxin maximum. No doubt the formation of the auxin sink and maximum are complicated events (Jiang & Feldman 2003). It has been suggested that, in some cases, auxin distribution is related to the placement of vascular bundles (Sabatini et al. 1999). The RC is not in direct contact with vascular bundles and thus, information from this tissue is not likely to organize pattern and polarity in the cap. Because earlier work suggested that auxin is synthesized at low levels in the cap (Feldman 1981) there is the possibility that the distribution of AtPIN4 and AtPIN4-dependent auxin gradient could be mediated by auxin (Jiang & Feldman 2003), ethylene or both. Experiments are underway in order to determine the location of auxin maximum in Arabidopsis wild-type roots upon local treatment of NPA and ethylene. The present results suggest that most probably the position of this maximum will be modified. ACKNOWLEDGMENTS We gratefully acknowledge Aiying Huang for her aid in some experiments, and Dr Patricia Joseph for the epifluorescence microscope facility. This work was supported by grants from the University of California Institute for Mexico and the United States (UC MEXUS), Universidad Nacional Autónoma de México (Dirección General de Asuntos del Personal Académico grant no. IN224103) and Consejo Nacional de Ciencia y Tecnología (CONACyT grant no N). REFERENCES Abel S., Nguyen M.D., Chow W. & Theologis A. (1995) ACS4, a primary indoleacetic acid-responsive gene encoding 1-aminocyclopropane-1-carboxylase synthase in Arabidopsis thaliana characterization, expression in E. coli, and expression characteristics in response to auxin. Journal of Biological Chemistry 270, Barlow P.W. (1974) Regeneration of the cap of primary roots of Zea mays. New Phytology 73, Barlow P.W. (1975) The root cap. In The Development and Function of Roots (eds J.G. Torrey & D.T. Clarkson), pp Academic Press, London, UK. Barlow P.W. (1976) Towards an understanding of the behaviour of root meristems. Journal of Theoretical Biology 57, Barlow P.W. (1992) The meristem and quiescent centre in the gib- 1 mutant of tomato (Lycopersicon esculentum Mill.). Annals of Botany 69, Barlow P.W. (2003) The root cap: cell dynamics, cell differentiation and cap function. Journal of Plant Growth Regulation 21, Barlow P.W., Volkmann D. & Baluska F. (2004) Polarity in roots. In Polarity in Plants (ed. K. Lindsey), pp Blackwells, Oxford, UK. van den Berg C., Willemsen V., Hendriks G., Weisbeek P. & Scheres B. (1997) Short-range control of cell differentiation in the Arabidopsis root meristem. Nature 390, Brighman L.A., Woo H.H., Wen F. & Hawes M.C. (1998) Meristem-specific suppression of mitosis and a global switch in gene expression in the root cap of pea by endogenous signals. Plant Physiology 118, Burg S.P. & Burg E.A. (1967) Molecular requirements for the biological activity of ethylene. Plant Physiology 42, Carland F.M. & McHale N.A. (1996) LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis. Development 122, Casson S.A. & Lindsey K. (2003) Genes and signalling in root development. New Phytologist 158, Cnops G., Wang X., Linstead P., Van Montagu M., Van Lijsebettens M. & Dolam L. (2000) TORNADO1 and TORNADO2 are required for the specification of radial and circumferential pattern in the Arabidopsis root. Development 127, Davies P.J. (1995) Plant Hormones. Kluwer Academic Publishers, Dordrecht, The Netherlands. Eapen D., Barroso M.L., Campos M.E., Ponce G., Corkidi G., Dubrovsky J.G. & Cassab G.I. (2003) A no hydrotropic response root mutant that responds positively to gravitropism in Arabidopsis. Plant Physiology 131, Feldman L.J. (1976) The de novo origin of the quiescent center in regenerating root apices of Zea mays. Planta 128, Feldman L.J. (1981) Effect of auxin on acropetal auxin transport in roots of corn. Plant Physiology 67, Feldman L.J. (1998) Not so quiet quiescent centers. Trends in Plant Sciences 3, Fortin M.-C. & Poff K.L. (1991) Characterization of thermotropism in primary roots of maize: dependence on temperature and temperature gradient, and interaction with gravitropism. Planta 184, Friml J., Benková E., Bilou I., et al. (2002) AtPIN4 mediates sinkdriven auxin gradients and root patterning in Arabidopsis. Cell 108, Hasenstein K.H. & Evans M.L. (1988) Effects of cations on hormone transport in primary roots of Zea mays. Plant Physiology 86, Hawes M.C. & Lin H.J. (1990) Correlation of pectolytic enzyme activity with the programmed release of cells from the root cap of Pisum sativum. Plant Physiology 94, Hawes M.C., Blengough G., Cassab G.I. & Ponce G. (2003) Root caps and rhizosphere. Journal of Plant Growth Regulation 21, Hawes M.C., Gunawardena U., Miyasaka S. & Zhao X. (2000) The role of root border cells in plant defense. Trends in Plant Science 5, Ishikawa H. & Evans M.L. (1990) Electropism of maize roots: role of the root cap and relationship with gravitropism. Plant Physiology 94, Jiang K. & Feldman L.F. (2003) Root meristem establishment and maintenance: the role of auxin. Journal of Plant Growth Regulation 21, Jiang K., Meng Y.L. & Feldman L.J. (2003) Quiescent center formation in maize roots is associated with an auxin-regulated oxidizing environment. Development 130, Katekar G.F. & Geissler A.E. (1977) Auxin transport inhibitors III. Chemical requirements of a class of auxin transport inhibitors. Plant Physiology 60, Kende H. (1993) Ethylene biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 44, Kerk N.M. & Feldman L.J. (1994) The quiescent center in roots of maize: initiation, maintenance and role in organization of the root apical meristem. Protoplasma 183, Kerk N.M. & Feldman L.J. (1995) A biochemical model for the initiation and maintenance of the quiescent center: implications for organization of root meristems. Development 121,