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The Root Cap Determines Ethylene-Dependent Growth and Development in Maize Roots
Molecular Plant
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Volume 1
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Number 2
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Pages 359–367
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March 2008
RESEARCH ARTICLE
The Root Cap Determines Ethylene-Dependent Growth and Development in Maize Roots Achim Hahna,b, Roman Zimmermannc,d, Dierk Wankee, Klaus Hartere and Hans G. Edelmanna,f,1 a Botanisches Institut, Universita¨t zu Ko¨ln, Gyrhofstr. 15, D-50931 Ko¨ln, Germany b Present address: ZMBP Pflanzenphysiologie, Universita¨t Tu¨bingen, Auf der Morgenstelle 1, D-72076 Tu¨bingen, Germany c Institut fu¨r Entwicklungsbiologie, Universita¨t zu Ko¨ln, Gyrhofstr. 17, D-50931 Ko¨ln, Germany d Present address: ZMBP Allgemeine Genetik, Universita¨t Tu¨bingen, Auf der Morgenstelle 28, D-72076 Tu¨bingen, Germany e ZMBP Pflanzenphysiologie, Universita¨t Tu¨bingen, Auf der Morgenstelle 1, D-72076 Tu¨bingen, Germany f Present address: Biologie und ihre Didaktik, Universita¨t Siegen, D-57068 Siegen, Germany
ABSTRACT Besides providing protection against mechanical damage to the root tip, the root cap is involved in the perception and processing of diverse external and internal stimuli resulting in altered growth and development. The transduction of these stimuli includes hormonal signaling pathways such as those of auxin, ethylene and cytokinin. Here, we show that the root cap is essential for the ethylene-induced regulation of elongation growth and root hair formation in maize. Exogenously applied ethylene is no longer able to inhibit elongation growth when the root cap has been surgically removed prior to hormone treatment. Reconstitution of the cap positively correlates with the developing capacity of the roots to respond to ethylene again. In contrast, the removal of the root cap does not per se affect growth inhibition controlled by auxin and cytokinin. Furthermore, our semi-quantitative RT-PCR results support earlier findings that the maize root cap is a site of high gene expression activity with respect to sensing and responding to hormones such as ethylene. From these data, we propose a novel function of the root cap which is the establishment of competence to respond to ethylene in the distal zones of the root.
INTRODUCTION Root caps, surmounting the apical root tip meristems, are a universal feature of angiosperm, gymnosperm and pteridophyte roots (Barlow, 2003). Cap cells are subject to a high turnover and are constantly delivered from the root apical meristem, traverse through the inner cap areas and decay at the outer layer (Barlow, 2003; Iijima et al., 2004). Root caps provide protection against mechanical damage to the root apical meristem and increase penetration strength when grown in compact soils (Barlow, 2003). Besides this protective role, a most intensively studied aspect is the function of the root cap as the gravi-perceiving site of the root, as its removal results in the loss of gravitropic growth (Juniper et al., 1966). According to the Cholodny Went Hypothesis (Cholodny, 1926; Went and Thimann, 1937), changes in root growth direction are brought about by the asymmetric redistribution of auxin within the elongation zone, induced by the signals transduced from the graviperceiving cells within the root cap. The ‘fountain-model’ of auxin-regulated root gravitropism proposed shoot-born IAA to be transported through the roots’ vascular tissue and the root cap into the peripheral parts of the cap. From there, it is radialsymmetrically distributed, being transported upwardly within the root epidermal cells into the elongation zone
where it regulates growth (Hasenstein and Evans, 1988; Ottenschla¨ger et al., 2003; Blilou et al., 2005). In addition, recent studies have demonstrated that auxin is also synthezized within the root tip itself (Ljung et al., 2005; Ru˚zˇicˇka et al., 2007; Swarup et al., 2007). Apart from auxin, other hormones, such as ethylene, were found to be involved in the growth processes coordinated in the root tip (Lee et al., 1990; Aloni et al., 2004, 2006; Buer et al., 2006; Stepanova et al., 2005, 2007; Ru˚zˇicˇka et al., 2007; Swarup et al., 2007). A physiological study in rye has demonstrated that the roots’ capacity for gravitropic bending depends on ethylene (Kramer et al., 2003). Furthermore, blocking ethylene synthesis or signalling disrupts the ability of tomato roots to penetrate compact soil or even loose sand (Clark et al., 1999; Hussain et al., 1999). In Arabidopsis, ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) cause the induction of ectopic root hair formation (Tanimoto et al., 1995;
1 To whom correspondence should be addressed. E-mail: edelmann@biologie. uni-siegen.de, fax 49 271 740 4182, tel. 49 271 740 3118.
ª The Author 2008. Published by Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssm027, Advance Access publication 8 February 2008
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Masucci and Schiefelbein, 1996), an increase in the width of the root (Smalle and Van Der Straeten, 1997) and a rapid but reversible down-regulation of cell elongation (Le et al., 2001). Together with auxin and ethylene, cytokinin regulates root development, vascular differentiation and gravitropism (Ma¨ho¨nen et al., 2000; Aloni et al., 2004, 2006; Ma¨ho¨nen et al., 2006). The functional and molecular interaction of auxin and ethylene became evident in Arabidopsis through the observation that mutations in many auxin transport or signalling components also cause aberrant responses to ethylene (Pickett et al., 1990; Luschnig et al., 1998; Alonso et al., 2003; Stepanova et al., 2005; Ru˚zˇicˇka et al., 2007). Recent studies provide an initial model of how ethylene regulates the growth and development of the Arabidopsis root by controlling auxin biosynthesis, transport and competence in distinct root apical tissues (Ru˚zˇicˇka et al., 2007; Stepanova et al., 2007; Swarup et al., 2007). In the presence of ethylene, auxin accumulation is induced in the root apex by the activation of ASA1 and ASB1 expression (Ljung et al., 2005; Stepanova et al., 2005). The auxin influx carrier AUX1 (Yang et al., 2006) and auxin efflux carrier PIN2 (Wis´niewska et al., 2006) are then essential for mobilizing and transporting auxin from the root apex to the elongation zone. In the elongation zone, components of the auxin response pathway, such as TIR1, AXR2/IAA7, and AXR3/IAA17, are also necessary for ethylene inhibition of root growth, indicating that auxin is required to achieve competence of the cells to respond to ethylene. Despite this progress in understanding the regulatory hormone network controlling root growth and development, little is known about the function of the root cap in these processes. For methodical reasons, however, a surgical removal of the root cap and the analysis of its functional consequences are not possible in Arabidopsis. We therefore studied the function of the root cap in controlling root growth and development using de-capped roots from etiolated maize seedlings. By these experiments, we could show that roots without caps are unable to respond to ethylene with respect to growth inhibition and root hair formation. In addition, RT-PCR studies revealed that the expression of several genes crucial for ethylene biosynthesis and ethylene perception in distant zones of the root is controlled by the cap in an ethylene-dependent way. Our data suggest that (i) the cap is required for the generation and mobilization of a signal, probably auxin, that is transported to the elongation and root hair formation zones, and (ii) that this signal is necessary to establish ethylene competence in the cells of these tissues.
RESULTS AND DISCUSSION Surgical Removal of the Root Cap without Induction of Wounding Stress To study the function of the root cap, the surgical removal of the organ was carried out on the primary roots of 2 d old etiolated maize seedlings. This procedure yielded a cap-less
dome-shaped root tip of intact and physically undamaged surface cells with a residual rim of lateral cap cells (Figure 1A). Using these cap-less roots, we did not observe any gravitropic bending within 20 h after the decapitation, which is in accordance with previous laser ablation studies (Blancaflor et al.,
Figure 1. Surgical Removal of the Cap from the Maize Root is Harmless to the Tip and Does Not Induce Production of Wound Ethylene. (A) Representative binocular images of intact and cap-less root tips showing a dome-shaped root meristem. (B,C) In-situ localization of the transcript of the root cap marker gene ZmNAC5 in cross-sections of tips from intact (B) and de-capped (C) roots. (D,E) Iodide staining of starch granules in cross-sections of tips from intact (D) and de-capped (E) roots 24 h after cap removal. (F) In-situ localization of the transcript of the root cap marker gene ZmNAC5 in a cross-section of a de-capped root 24 h after cap removal. G) Real-time photoacoustic measurement of ethylene emission from intact (black line) and de-capped (red line) maize seedlings over a time course of 24 h after the removal of the caps. The bars represent 600 lm in (A) and 300 lm in (B–F).
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1998). We employed in-situ hybridization using the root cap marker ZmNAC5 (Zimermann and Werr, 2005) and starch granule staining to confirm that the central root cap was completely removed from the root tip. In contrast to the intact roots, no ZmNAC5 signal and starch grains were observed in de-capped roots after surgical removal of the cap (Figure 1B–1E). However, 24 h after the removal, ZmNAC5 expression re-appeared in the outermost four to five cell layers at the tip of the morphologically still cap-less roots, indicating the emerging renewal of the cap (Figure 1F). Although the root tips appeared undamaged from a morphological point of view, the surgical removal of an entire organ may represent a severe injury. Such injuries could induce the production of wound ethylene, as has been found in many other plant systems. Ethylene, however, has been shown to inhibit the elongation growth of roots and aerial parts in a number of plant species (Chadwick and Burg, 1967; Smith and Robertson, 1971; Whalen and Feldman, 1988; Bleecker et al., 1988; Smalle and Van Der Straeten, 1997). However, our removal of the cap neither impaired nor enhanced normal growth rates during the following 24 h (Figure 2A). In addition, the de-capped maize seedlings did not produce more ethylene compared with the intact seedlings, as determined by photo-acoustic ethylene emission measurements (Figure 1G; Kramer et al., 2003). In contrast to earlier reports (Saltveit and Dilley, 1978; Yu and Yang, 1980), our careful removal of the root cap clearly did not induce a major wounding stress response or cause the loss of putative cap-born growthinhibiting or promoting substances. Our findings therefore enabled us to determine the relevance of the root cap for the control of root growth and development of the maize root in response to exogenously applied ethylene and without the interference of wound ethylene.
Root Cap-Dependent Effects of Ethylene on Root Growth and Development According to recent studies in Arabidopsis (Ru˚zˇicˇka et al., 2007; Stepanova et al., 2007; Swarup et al., 2007), the growthinhibiting effect of ethylene is mediated by ethylene-induced auxin synthesis in the root tip region and akropetal translocation of auxin to the elongation zone. If the root cap was not involved in this regulatory network, root growth and development would be expected to continue in response to the application of exogenous ethylene. We, therefore, analyzed the effects of ethylene on root elongation by applying either the ethylene releasing agent ethephon or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to intact and de-capped roots. In the intact organ, the application of ethylene (released from 1 ll ml 1 ethephon) or 1 mM ACC inhibited the elongation growth by more than 60% (Figure 2A and C). In contrast, the elongation growth of de-capped roots was inhibited neither by ethephon nor ACC (Figure 2A and 2F). Similarly, ethylene-induced root hair formation (Tanimoto et al., 1995) was strongly impaired in de-capped roots as compared to intact roots (Figure 3A and 3B). The elimination of any ethylene
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response in intact roots (Figure 2A and 2D) was achieved by treatment with inhibitors of ethylene perception (Ag+; Beyer, 1976; Rodriguez et al., 1999) or biosynthesis (AVG; Yang and Hofman, 1984). Neither of these treatments had any effect on the elongation of de-capped roots (Figure 2A and 2G). In addition, AVG prevented the formation of root hairs in intact roots (Figure 2D). Thus, our results show that the surgical removal of the root cap or the blocking of ethylene biosynthesis or perception, respectively, has very similar effects on maize root development. This is also consistent with our previous findings that the inhibition of ethylene biosynthesis with AVG prevents root gravitropism (Edelmann and Roth, 2006). This observation favors the novel hypothesis that, at least in maize roots, the cap is required for the realization of ethylene-dependent processes such as elongation growth and root hair formation.
Root Cap Regeneration and Reconstitution of Ethylene Sensitivity Earlier studies in etiolated maize seedlings demonstrated that the root cap regenerates within 3–4 d after its removal (Barlow, 1974a, 1974b). We, therefore, were interested in whether the regeneration of the root cap correlates with the reconstitution of ethylene-mediated inhibition of root elongation and hair formation. Under the conditions employed, de-capped roots re-gained their gravitopic competence within 24 h (see Supplemental Figure 1) and their sensitivity to ACC with respect to growth inhibition within 24–48 h after cap removal (Figure 4). Seventy-two hours after the removal of the cap, we could not detect any difference in the growth rates of ethylene-treated intact and de-capped roots (Figure 4), although the root cap was barely regenerated on morphological level in the latter case (Figure 1F). Thus, our findings substantiate our hypothesis that the presence of the root cap is necessary and sufficient for the realization of ethylene-dependent root growth and development in maize.
Effects of Auxin and Cytokinin on the Growth of De-Capped Maize Roots As the root cap appears to be important for the signaling and biosynthesis of other hormones, such as auxin and cytokinin (Miyawaki et al., 2004; Aloni et al., 2005; Ljung et al., 2005), we wondered whether the removal of the root cap also interferes with other hormone response pathways. Therefore, seedlings with intact and de-capped roots were grown for 24 h in the presence of 10 lM auxin (IAA) and 10 lM cytokinin (kinetin), respectively (Figure 5A). In the case of IAA, we found no difference between intact and de-capped roots, despite a strong overall inhibition of elongation growth, illustrating that removal of the root cap per se does not affect auxinmediated growth inhibition and that cells in the elongation zone are still able to respond to the hormone. In contrast, kinetin-treated intact roots were significantly shorter than de-capped roots (Figure 5A). This observation indicates a root cap-dependent processing of cytokinin-induced
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Figure 2. Removal of the Cap Abolishes the Ethylene-Regulated Elongation Growth and Gravitropic Curvature of Maize Roots. (A) Relative growth of intact (white bars) and de-capped (black bars) roots treated with 1 mM ACC, 1 ll ml 1ethephon, 100 lM silver nitrate (Ag+), or 10 lM AVG or mock-treated (control). Root length increase was measured 24 h after the application of the substances. The length increase of intact and mock-treated root was set as 100%. The experiment was repeated three times with at least 20 seedlings per measurement. The data set represents the mean of the three experiments +/–S.E. (n = 3). (B–D) Representative images of horizontally oriented maize seedlings with intact roots. The seedlings were either mock-treated (B) or treated with 1 mM ACC (C) or 100 lM AVG (D). (E–G) Representative images of horizontally oriented maize seedlings with de-capped roots. The seedlings were either mock-treated (E) or treated with 1 mM ACC (F) or 100 lM AVG (G). gYindicates the direction of the gravi vector. The bars represent 10 mm.
signals affecting elongation growth, as suggested in a previous study by Aloni and co-workers (2004). However, in Arabidopsis, several effects of cytokinin have been shown to be mediated by increased ethylene biosynthesis (Cary et al., 1995) due to stabilization of the ACC synthase 5 (ACS5) protein (Vogel et al., 1998; Chae et al., 2003). Therefore, the difference in root elongation growth between cytokinin-treated intact and decapped roots (Figure 5A and 5B) could at least in part be due to
secondary ethylene effects rather than being a direct result of cytokinin action. To test this, we blocked ethylene perception in cytokinin-treated roots by applying the ethylene perception inhibitor Ag+ (Cary et al., 1995). As shown in Figure 5B, we found no difference in the elongation growth of intact and de-capped roots when the seedlings were simultaneously treated with cytokinin in the presence of Ag+. Independently of whether the cap was present or removed, the seedlings
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Figure 3. Removal of the Cap Inhibits the Ethylene-Induced Hair Formation in Maize roots. (A, B) Representative images of 2 d old intact (A) and de-capped (B) roots grown for 24 h on filter paper saturated with a 1-mM ACC solution. The white arrows point to the root hair formation zone. gY indicates the direction of the gravi vector. The white bars represent 5 mm and the yellow bars 2 mm.
Figure 4. The Regeneration of the Cap Reconstitutes the Ethylene Sensitivity to the Maize Root. Two -day-old seedlings were grown on moist filter paper without (–) or with (+) 1 mM ACC. The length increase of the intact (white bars) and de-capped (black bars) roots was measured 24, 48 and 72 h after cap removal. Data are expressed as +/– S.E. (n = 15 roots).
showed a growth rate comparable to that of de-capped, cytokinin-treated roots. These data indicate, firstly, that cytokinin per se is able to inhibit root elongation growth, and, secondly, that this response is root cap independent. Thirdly, the cap-dependent differences in cytokinin-controlled root growth are secondary effects very likely caused by cytokinin-induced ethylene biosynthesis.
Root Cap-Dependent Changes in the Expression of Genes Related to Ethylene Biosynthesis, Perception and Signaling The removal of the root cap could also result in alterations in ethylene biosynthesis and/or efficiency of signaling in distal
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root areas (Young et al., 2004; Gallie and Young, 2004). We, therefore, determined the steady-state transcript levels of several maize genes encoding enzymes and proteins proposed to participate in ethylene biosynthesis or signaling by semiquantitative RT-PCR analysis. To do so, we cut the roots into two segments—the tip and the distal part (Figure 6A, 1 and 2)—and performed a semi-quantitative RT-PCR analysis on RNA extracted from these tissues. With a few exceptions, we found that the majority of the analyzed genes were similarly expressed in the root tip and a segment 5 mm distal from the root tip. Furthermore, this expression pattern was independent of the presence or absence of the root cap (data not shown). However, the transcript of the biosynthetic ZmACO20 isoform (Gallie and Young, 2004) was predominantly detectable in the distal segment while it was absent in the root tip (Figure 6). The treatment of the roots with ACC did not change this expression pattern (Figure 6, ACC). However, after the application of AVG, i.e. inhibition of ethylene synthesis, ZmACO20 expression was also detectable in the root tip (Figure 6, AVG). This suggests a negative transcriptional feedback control from the distal root areas with respect to the root tip. The transcript of the ethylene receptor ZmETR2-40 (Gallie and Young, 2004) was slightly more abundant in the root tip. The early ethyleneresponsive gene ZmERF1 (GenBank: AAT75013), whose homologue is one of the key regulators of ethylene-mediated signal transduction in Arabidopsis (Solano et al., 1998), shows a higher expression level in the root tip compared with that of the distal segment. Intriguingly, the number of ZmERF1 transcripts changed in an ACC-, AVG- and root cap-dependent manner. Although the expression analysis is by no means complete yet, our initial results suggest that ethylene perception may take place in both the apical tip and distal zones of the root, whereas ethylene synthesis might be restricted to distal parts. However, the root cap does not appear to have a major effect on this expression pattern. Intriguingly, a recent microarray study using tissue from maize and a heterologous rice GeneChip has revealed an intensely rich and complex variety of gene expression activities in the root cap (Jiang et al., 2006). The data suggest the operation of many processes in the root cap, including molecular mechanisms for sensing and responding to biotic and abiotic environmental stimuli and for integrating the responses of at least three major growth regulators (auxin, ethylene, jasmonate). In conclusion, our data demonstrate that the root cap is necessary and sufficient for the realization of the ethyleneregulated processes in the maize root, such as elongation growth and root hair formation. Our results strongly suggest that the root cap appears to be functionally responsible for the ethylene-induced accumulation of auxin in the root tip. To a minor extent, the root cap itself is probably the location of auxin biosynthesis. How it triggers its accumulation in other tip cells remains to be answered. In addition to the loss of auxin synthesis, the removal of the cap may also interfere with the transport of auxin from the
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Figure 6. Expression of Genes Involved in Ethylene Biosynthesis, Perception and Signaling in Maize Roots.
Figure 5. The Removal of the Cap Does not Interfere with the Responsiveness of the Root Cells to Auxin and Cytokinin. (A) Relative root growth of intact (white bars) and de-capped (black bars) roots either mock-treated (control) or in the presence of 1 mM ACC, 10 lM Kinetin, or 10 lM IAA. (B) Relative root growth of intact (white bars) and de-capped (black bars) roots either mock-treated or in the presence of 10 lM Kinetin, 10 lM Kinetin + 100 lM Ag+or Ag+alone. Root length increase was measured 24 h after the application of the substances. The experiment was repeated three times with at least 20 seedlings per measurement. The data set represents the mean of the three experiments +/– S.E. (n = 3).
root tip to distal tissues. In Arabidopsis, there is evidence that the lateral cap cells mediate akropetal auxin transport from the root tip to the elongation zone via the auxin influx carrier AUX1, which is required for the gravitropic response of the root and probably for the inhibition of elongation growth and induction of root hair formation (Dharmasiri et al., 2006; Swarup et al., 2007). AUX1-like influx carriers are also expressed in the root cap of maize (Jiang et al., 2006). Because the de-capped maize roots are non-gravitopic (Figure 2E–2G), the AUX1-like carriers were probably removed or inactivated by our surgical procedure. The prevention of the auxin synthesis and akropetal auxin transport abolishes the competence of the distal cells to respond to ethylene. In summary, our study proposes a novel and essential function of the cap in the ethylene-regulated and auxin-mediated developmental processes in the root.
(A) Scheme of the harvested of root segments (1, tip; 2, distal part) for RT-PCR analyses. (B) Steady-state transcript levels of ACO20, ETR2-40, EIN2, ERF1 and tubulin (TUB) in the tip (1) and the distal (2) segment form of intact (+) and de-capped (–) roots (see A) as determined by RT-PCR using gene-specific primers. Before harvest, the seedlings were either mock-treated (H2O) or treated with 1 mM ACC or 100 lM AVG. Tubulin served as RT-PCR control. The analysis was repeated at least three times and one representative data set is shown.
METHODS Plant Materials, Growth Conditions and Cap Removal Vernalized seedlings of maize (Zea mays L., cv. DK 233) were grown in the dark at 25C for 2 d in a vertical position, enrolled in moist filter paper, as described previously (Hahn et al., 2006). Seedlings with an average root length of 15–20 mm were used in all experiments. The root cap of seedlings was removed immediately after harvest under a binocular using a fixed razorblade. Seedlings with intact and de-capped roots were then immobilized between moist filter paper (imbibed in distilled water or appropriate solutions) on blocks of polystyrene in Perspex boxes with water-saturated atmosphere.
Hormone Treatments and Root Cap Regeneration Hormone treatment was conducted by application of solutions of 1 ll l 1 ethephon, 1 mM 1-aminocyclopropane-1-carboxylic acid (ACC), 10 lM kinetin, or 10 lM indoleacetic acid (IAA) to the seedlings. As inhibitors of ethylene biosynthesis and action, 10 lM aminoethoxyvinylglycine (AVG) and 100 lM silver nitrate (Ag+), respectively, were used. Two-day-old seedlings with intact and de-capped roots were placed on moist filter paper (imbibed in distilled water or 1 mM ACC) in 20 3 20 cm Perspex boxes. These boxes were then placed at a 45 position. Over a period of 72 h, the length
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increase of the primary roots was measured every 24 h. The experiment was independently repeated at least three times and one representative data set is shown.
ZmNAC5 In-Situ Localization and Starch Granule Staining RNA in-situ hybridization was performed according to the method of Jackson (1991). Root tips were dissected and fixed at 4C overnight in 4% formaldehyde in phosphate-buffered saline, dehydrated in an ethanol series and embedded in paraffin wax (Paraplast plus, Sigma). Embedded tissue was sectioned by use of a Leica RM 2145 rotary microtome and mounted on coated slides (Super-Frost Plus). Templates for ZmNAC5 in-situ probes were chosen as previously described (Zimmermann and Werr, 2005), cloned in antisense orientation to the T7 promoter and labelled using T7 polymerase and DIG 11-UTP (Roche) as described by Bradley et al. (1993). For starch granule staining, samples were embedded and sectioned as described above. Sections were then dehydrated in an ethanol series and stained with an iodide–potassium iodide solution for 3–5 min, cleared and observed using an Axioskop microscope with nomarsky optics (Zeiss).
Ethylene Measurements Real-time photo-acoustic measurement of ethylene emission of 2-d old maize seedlings were performed using an INVIVO-C2H4-10 laser spectrometer (Invivo GmbH, Sankt Augustin) as described previously (Kramer et al., 2003).
RT-PCR Analysis For RNA extractions, frozen samples were homogenized in RNAwiz (Ambion) according to the manufacturer’s protocol with minor modifications. First strand cDNA synthesis was carried out using the SuperScript III Reverse Transcriptase (Invitrogen) with oligo(dT)20 as the primer following the protocol of the manufacturer. Primer sequences for ACO20, ETR2-40, and EIN2 RT-PCR have been described previously (Gallie and Young, 2004). Primer sequences for ERF1 were ERF1S (5#-TGCGGCGGCGCAATCATCTTCG-3’) and ERF1A (5#-TCATTCAGTAAAGAGCGACAG-3’). Supplemental Figure 1. Intact (+) and De-capped (–) Roots Were Placed Horizontally and the Gravitropic Angle of Each Root Tip Was Measured over 48 h. The angle of each gravi-stimulated root was assigned to one of 12 30 sectors. The length of each bar represents the percentage of roots showing tip growth within that sector. gY indicates the direction of the gravi vector. Data are presented as the mean +/– S.E. of three independent experiments (n > 20 seedlings per experiment).
ACKNOWLEDGMENTS We would like to thank Felicity de Courcy for proofreading the manuscript, Richard Firn for helpful comments on the manuscript, and Jakub Hora´k for help in image processing. We gratefully acknowledge the excellent technical support from Petra Dehring
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and Ralph Ga¨bler. The work was supported by a DFG/AFGN grant to KH (Ha 2146/5–2). No conflict of interest declared.
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RESEARCH ARTICLE
The Root Cap Determines Ethylene-Dependent Growth and Development in Maize Roots Achim Hahna,b, Roman Zimmermannc,d, Dierk Wankee, Klaus Hartere and Hans G. Edelmanna,f,1 a Botanisches Institut, Universita¨t zu Ko¨ln, Gyrhofstr. 15, D-50931 Ko¨ln, Germany b Present address: ZMBP Pflanzenphysiologie, Universita¨t Tu¨bingen, Auf der Morgenstelle 1, D-72076 Tu¨bingen, Germany c Institut fu¨r Entwicklungsbiologie, Universita¨t zu Ko¨ln, Gyrhofstr. 17, D-50931 Ko¨ln, Germany d Present address: ZMBP Allgemeine Genetik, Universita¨t Tu¨bingen, Auf der Morgenstelle 28, D-72076 Tu¨bingen, Germany e ZMBP Pflanzenphysiologie, Universita¨t Tu¨bingen, Auf der Morgenstelle 1, D-72076 Tu¨bingen, Germany f Present address: Biologie und ihre Didaktik, Universita¨t Siegen, D-57068 Siegen, Germany
ABSTRACT Besides providing protection against mechanical damage to the root tip, the root cap is involved in the perception and processing of diverse external and internal stimuli resulting in altered growth and development. The transduction of these stimuli includes hormonal signaling pathways such as those of auxin, ethylene and cytokinin. Here, we show that the root cap is essential for the ethylene-induced regulation of elongation growth and root hair formation in maize. Exogenously applied ethylene is no longer able to inhibit elongation growth when the root cap has been surgically removed prior to hormone treatment. Reconstitution of the cap positively correlates with the developing capacity of the roots to respond to ethylene again. In contrast, the removal of the root cap does not per se affect growth inhibition controlled by auxin and cytokinin. Furthermore, our semi-quantitative RT-PCR results support earlier findings that the maize root cap is a site of high gene expression activity with respect to sensing and responding to hormones such as ethylene. From these data, we propose a novel function of the root cap which is the establishment of competence to respond to ethylene in the distal zones of the root.
INTRODUCTION Root caps, surmounting the apical root tip meristems, are a universal feature of angiosperm, gymnosperm and pteridophyte roots (Barlow, 2003). Cap cells are subject to a high turnover and are constantly delivered from the root apical meristem, traverse through the inner cap areas and decay at the outer layer (Barlow, 2003; Iijima et al., 2004). Root caps provide protection against mechanical damage to the root apical meristem and increase penetration strength when grown in compact soils (Barlow, 2003). Besides this protective role, a most intensively studied aspect is the function of the root cap as the gravi-perceiving site of the root, as its removal results in the loss of gravitropic growth (Juniper et al., 1966). According to the Cholodny Went Hypothesis (Cholodny, 1926; Went and Thimann, 1937), changes in root growth direction are brought about by the asymmetric redistribution of auxin within the elongation zone, induced by the signals transduced from the graviperceiving cells within the root cap. The ‘fountain-model’ of auxin-regulated root gravitropism proposed shoot-born IAA to be transported through the roots’ vascular tissue and the root cap into the peripheral parts of the cap. From there, it is radialsymmetrically distributed, being transported upwardly within the root epidermal cells into the elongation zone
where it regulates growth (Hasenstein and Evans, 1988; Ottenschla¨ger et al., 2003; Blilou et al., 2005). In addition, recent studies have demonstrated that auxin is also synthezized within the root tip itself (Ljung et al., 2005; Ru˚zˇicˇka et al., 2007; Swarup et al., 2007). Apart from auxin, other hormones, such as ethylene, were found to be involved in the growth processes coordinated in the root tip (Lee et al., 1990; Aloni et al., 2004, 2006; Buer et al., 2006; Stepanova et al., 2005, 2007; Ru˚zˇicˇka et al., 2007; Swarup et al., 2007). A physiological study in rye has demonstrated that the roots’ capacity for gravitropic bending depends on ethylene (Kramer et al., 2003). Furthermore, blocking ethylene synthesis or signalling disrupts the ability of tomato roots to penetrate compact soil or even loose sand (Clark et al., 1999; Hussain et al., 1999). In Arabidopsis, ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) cause the induction of ectopic root hair formation (Tanimoto et al., 1995;
1 To whom correspondence should be addressed. E-mail: edelmann@biologie. uni-siegen.de, fax 49 271 740 4182, tel. 49 271 740 3118.
ª The Author 2008. Published by Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssm027, Advance Access publication 8 February 2008
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Masucci and Schiefelbein, 1996), an increase in the width of the root (Smalle and Van Der Straeten, 1997) and a rapid but reversible down-regulation of cell elongation (Le et al., 2001). Together with auxin and ethylene, cytokinin regulates root development, vascular differentiation and gravitropism (Ma¨ho¨nen et al., 2000; Aloni et al., 2004, 2006; Ma¨ho¨nen et al., 2006). The functional and molecular interaction of auxin and ethylene became evident in Arabidopsis through the observation that mutations in many auxin transport or signalling components also cause aberrant responses to ethylene (Pickett et al., 1990; Luschnig et al., 1998; Alonso et al., 2003; Stepanova et al., 2005; Ru˚zˇicˇka et al., 2007). Recent studies provide an initial model of how ethylene regulates the growth and development of the Arabidopsis root by controlling auxin biosynthesis, transport and competence in distinct root apical tissues (Ru˚zˇicˇka et al., 2007; Stepanova et al., 2007; Swarup et al., 2007). In the presence of ethylene, auxin accumulation is induced in the root apex by the activation of ASA1 and ASB1 expression (Ljung et al., 2005; Stepanova et al., 2005). The auxin influx carrier AUX1 (Yang et al., 2006) and auxin efflux carrier PIN2 (Wis´niewska et al., 2006) are then essential for mobilizing and transporting auxin from the root apex to the elongation zone. In the elongation zone, components of the auxin response pathway, such as TIR1, AXR2/IAA7, and AXR3/IAA17, are also necessary for ethylene inhibition of root growth, indicating that auxin is required to achieve competence of the cells to respond to ethylene. Despite this progress in understanding the regulatory hormone network controlling root growth and development, little is known about the function of the root cap in these processes. For methodical reasons, however, a surgical removal of the root cap and the analysis of its functional consequences are not possible in Arabidopsis. We therefore studied the function of the root cap in controlling root growth and development using de-capped roots from etiolated maize seedlings. By these experiments, we could show that roots without caps are unable to respond to ethylene with respect to growth inhibition and root hair formation. In addition, RT-PCR studies revealed that the expression of several genes crucial for ethylene biosynthesis and ethylene perception in distant zones of the root is controlled by the cap in an ethylene-dependent way. Our data suggest that (i) the cap is required for the generation and mobilization of a signal, probably auxin, that is transported to the elongation and root hair formation zones, and (ii) that this signal is necessary to establish ethylene competence in the cells of these tissues.
RESULTS AND DISCUSSION Surgical Removal of the Root Cap without Induction of Wounding Stress To study the function of the root cap, the surgical removal of the organ was carried out on the primary roots of 2 d old etiolated maize seedlings. This procedure yielded a cap-less
dome-shaped root tip of intact and physically undamaged surface cells with a residual rim of lateral cap cells (Figure 1A). Using these cap-less roots, we did not observe any gravitropic bending within 20 h after the decapitation, which is in accordance with previous laser ablation studies (Blancaflor et al.,
Figure 1. Surgical Removal of the Cap from the Maize Root is Harmless to the Tip and Does Not Induce Production of Wound Ethylene. (A) Representative binocular images of intact and cap-less root tips showing a dome-shaped root meristem. (B,C) In-situ localization of the transcript of the root cap marker gene ZmNAC5 in cross-sections of tips from intact (B) and de-capped (C) roots. (D,E) Iodide staining of starch granules in cross-sections of tips from intact (D) and de-capped (E) roots 24 h after cap removal. (F) In-situ localization of the transcript of the root cap marker gene ZmNAC5 in a cross-section of a de-capped root 24 h after cap removal. G) Real-time photoacoustic measurement of ethylene emission from intact (black line) and de-capped (red line) maize seedlings over a time course of 24 h after the removal of the caps. The bars represent 600 lm in (A) and 300 lm in (B–F).
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1998). We employed in-situ hybridization using the root cap marker ZmNAC5 (Zimermann and Werr, 2005) and starch granule staining to confirm that the central root cap was completely removed from the root tip. In contrast to the intact roots, no ZmNAC5 signal and starch grains were observed in de-capped roots after surgical removal of the cap (Figure 1B–1E). However, 24 h after the removal, ZmNAC5 expression re-appeared in the outermost four to five cell layers at the tip of the morphologically still cap-less roots, indicating the emerging renewal of the cap (Figure 1F). Although the root tips appeared undamaged from a morphological point of view, the surgical removal of an entire organ may represent a severe injury. Such injuries could induce the production of wound ethylene, as has been found in many other plant systems. Ethylene, however, has been shown to inhibit the elongation growth of roots and aerial parts in a number of plant species (Chadwick and Burg, 1967; Smith and Robertson, 1971; Whalen and Feldman, 1988; Bleecker et al., 1988; Smalle and Van Der Straeten, 1997). However, our removal of the cap neither impaired nor enhanced normal growth rates during the following 24 h (Figure 2A). In addition, the de-capped maize seedlings did not produce more ethylene compared with the intact seedlings, as determined by photo-acoustic ethylene emission measurements (Figure 1G; Kramer et al., 2003). In contrast to earlier reports (Saltveit and Dilley, 1978; Yu and Yang, 1980), our careful removal of the root cap clearly did not induce a major wounding stress response or cause the loss of putative cap-born growthinhibiting or promoting substances. Our findings therefore enabled us to determine the relevance of the root cap for the control of root growth and development of the maize root in response to exogenously applied ethylene and without the interference of wound ethylene.
Root Cap-Dependent Effects of Ethylene on Root Growth and Development According to recent studies in Arabidopsis (Ru˚zˇicˇka et al., 2007; Stepanova et al., 2007; Swarup et al., 2007), the growthinhibiting effect of ethylene is mediated by ethylene-induced auxin synthesis in the root tip region and akropetal translocation of auxin to the elongation zone. If the root cap was not involved in this regulatory network, root growth and development would be expected to continue in response to the application of exogenous ethylene. We, therefore, analyzed the effects of ethylene on root elongation by applying either the ethylene releasing agent ethephon or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to intact and de-capped roots. In the intact organ, the application of ethylene (released from 1 ll ml 1 ethephon) or 1 mM ACC inhibited the elongation growth by more than 60% (Figure 2A and C). In contrast, the elongation growth of de-capped roots was inhibited neither by ethephon nor ACC (Figure 2A and 2F). Similarly, ethylene-induced root hair formation (Tanimoto et al., 1995) was strongly impaired in de-capped roots as compared to intact roots (Figure 3A and 3B). The elimination of any ethylene
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response in intact roots (Figure 2A and 2D) was achieved by treatment with inhibitors of ethylene perception (Ag+; Beyer, 1976; Rodriguez et al., 1999) or biosynthesis (AVG; Yang and Hofman, 1984). Neither of these treatments had any effect on the elongation of de-capped roots (Figure 2A and 2G). In addition, AVG prevented the formation of root hairs in intact roots (Figure 2D). Thus, our results show that the surgical removal of the root cap or the blocking of ethylene biosynthesis or perception, respectively, has very similar effects on maize root development. This is also consistent with our previous findings that the inhibition of ethylene biosynthesis with AVG prevents root gravitropism (Edelmann and Roth, 2006). This observation favors the novel hypothesis that, at least in maize roots, the cap is required for the realization of ethylene-dependent processes such as elongation growth and root hair formation.
Root Cap Regeneration and Reconstitution of Ethylene Sensitivity Earlier studies in etiolated maize seedlings demonstrated that the root cap regenerates within 3–4 d after its removal (Barlow, 1974a, 1974b). We, therefore, were interested in whether the regeneration of the root cap correlates with the reconstitution of ethylene-mediated inhibition of root elongation and hair formation. Under the conditions employed, de-capped roots re-gained their gravitopic competence within 24 h (see Supplemental Figure 1) and their sensitivity to ACC with respect to growth inhibition within 24–48 h after cap removal (Figure 4). Seventy-two hours after the removal of the cap, we could not detect any difference in the growth rates of ethylene-treated intact and de-capped roots (Figure 4), although the root cap was barely regenerated on morphological level in the latter case (Figure 1F). Thus, our findings substantiate our hypothesis that the presence of the root cap is necessary and sufficient for the realization of ethylene-dependent root growth and development in maize.
Effects of Auxin and Cytokinin on the Growth of De-Capped Maize Roots As the root cap appears to be important for the signaling and biosynthesis of other hormones, such as auxin and cytokinin (Miyawaki et al., 2004; Aloni et al., 2005; Ljung et al., 2005), we wondered whether the removal of the root cap also interferes with other hormone response pathways. Therefore, seedlings with intact and de-capped roots were grown for 24 h in the presence of 10 lM auxin (IAA) and 10 lM cytokinin (kinetin), respectively (Figure 5A). In the case of IAA, we found no difference between intact and de-capped roots, despite a strong overall inhibition of elongation growth, illustrating that removal of the root cap per se does not affect auxinmediated growth inhibition and that cells in the elongation zone are still able to respond to the hormone. In contrast, kinetin-treated intact roots were significantly shorter than de-capped roots (Figure 5A). This observation indicates a root cap-dependent processing of cytokinin-induced
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Figure 2. Removal of the Cap Abolishes the Ethylene-Regulated Elongation Growth and Gravitropic Curvature of Maize Roots. (A) Relative growth of intact (white bars) and de-capped (black bars) roots treated with 1 mM ACC, 1 ll ml 1ethephon, 100 lM silver nitrate (Ag+), or 10 lM AVG or mock-treated (control). Root length increase was measured 24 h after the application of the substances. The length increase of intact and mock-treated root was set as 100%. The experiment was repeated three times with at least 20 seedlings per measurement. The data set represents the mean of the three experiments +/–S.E. (n = 3). (B–D) Representative images of horizontally oriented maize seedlings with intact roots. The seedlings were either mock-treated (B) or treated with 1 mM ACC (C) or 100 lM AVG (D). (E–G) Representative images of horizontally oriented maize seedlings with de-capped roots. The seedlings were either mock-treated (E) or treated with 1 mM ACC (F) or 100 lM AVG (G). gYindicates the direction of the gravi vector. The bars represent 10 mm.
signals affecting elongation growth, as suggested in a previous study by Aloni and co-workers (2004). However, in Arabidopsis, several effects of cytokinin have been shown to be mediated by increased ethylene biosynthesis (Cary et al., 1995) due to stabilization of the ACC synthase 5 (ACS5) protein (Vogel et al., 1998; Chae et al., 2003). Therefore, the difference in root elongation growth between cytokinin-treated intact and decapped roots (Figure 5A and 5B) could at least in part be due to
secondary ethylene effects rather than being a direct result of cytokinin action. To test this, we blocked ethylene perception in cytokinin-treated roots by applying the ethylene perception inhibitor Ag+ (Cary et al., 1995). As shown in Figure 5B, we found no difference in the elongation growth of intact and de-capped roots when the seedlings were simultaneously treated with cytokinin in the presence of Ag+. Independently of whether the cap was present or removed, the seedlings
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Figure 3. Removal of the Cap Inhibits the Ethylene-Induced Hair Formation in Maize roots. (A, B) Representative images of 2 d old intact (A) and de-capped (B) roots grown for 24 h on filter paper saturated with a 1-mM ACC solution. The white arrows point to the root hair formation zone. gY indicates the direction of the gravi vector. The white bars represent 5 mm and the yellow bars 2 mm.
Figure 4. The Regeneration of the Cap Reconstitutes the Ethylene Sensitivity to the Maize Root. Two -day-old seedlings were grown on moist filter paper without (–) or with (+) 1 mM ACC. The length increase of the intact (white bars) and de-capped (black bars) roots was measured 24, 48 and 72 h after cap removal. Data are expressed as +/– S.E. (n = 15 roots).
showed a growth rate comparable to that of de-capped, cytokinin-treated roots. These data indicate, firstly, that cytokinin per se is able to inhibit root elongation growth, and, secondly, that this response is root cap independent. Thirdly, the cap-dependent differences in cytokinin-controlled root growth are secondary effects very likely caused by cytokinin-induced ethylene biosynthesis.
Root Cap-Dependent Changes in the Expression of Genes Related to Ethylene Biosynthesis, Perception and Signaling The removal of the root cap could also result in alterations in ethylene biosynthesis and/or efficiency of signaling in distal
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root areas (Young et al., 2004; Gallie and Young, 2004). We, therefore, determined the steady-state transcript levels of several maize genes encoding enzymes and proteins proposed to participate in ethylene biosynthesis or signaling by semiquantitative RT-PCR analysis. To do so, we cut the roots into two segments—the tip and the distal part (Figure 6A, 1 and 2)—and performed a semi-quantitative RT-PCR analysis on RNA extracted from these tissues. With a few exceptions, we found that the majority of the analyzed genes were similarly expressed in the root tip and a segment 5 mm distal from the root tip. Furthermore, this expression pattern was independent of the presence or absence of the root cap (data not shown). However, the transcript of the biosynthetic ZmACO20 isoform (Gallie and Young, 2004) was predominantly detectable in the distal segment while it was absent in the root tip (Figure 6). The treatment of the roots with ACC did not change this expression pattern (Figure 6, ACC). However, after the application of AVG, i.e. inhibition of ethylene synthesis, ZmACO20 expression was also detectable in the root tip (Figure 6, AVG). This suggests a negative transcriptional feedback control from the distal root areas with respect to the root tip. The transcript of the ethylene receptor ZmETR2-40 (Gallie and Young, 2004) was slightly more abundant in the root tip. The early ethyleneresponsive gene ZmERF1 (GenBank: AAT75013), whose homologue is one of the key regulators of ethylene-mediated signal transduction in Arabidopsis (Solano et al., 1998), shows a higher expression level in the root tip compared with that of the distal segment. Intriguingly, the number of ZmERF1 transcripts changed in an ACC-, AVG- and root cap-dependent manner. Although the expression analysis is by no means complete yet, our initial results suggest that ethylene perception may take place in both the apical tip and distal zones of the root, whereas ethylene synthesis might be restricted to distal parts. However, the root cap does not appear to have a major effect on this expression pattern. Intriguingly, a recent microarray study using tissue from maize and a heterologous rice GeneChip has revealed an intensely rich and complex variety of gene expression activities in the root cap (Jiang et al., 2006). The data suggest the operation of many processes in the root cap, including molecular mechanisms for sensing and responding to biotic and abiotic environmental stimuli and for integrating the responses of at least three major growth regulators (auxin, ethylene, jasmonate). In conclusion, our data demonstrate that the root cap is necessary and sufficient for the realization of the ethyleneregulated processes in the maize root, such as elongation growth and root hair formation. Our results strongly suggest that the root cap appears to be functionally responsible for the ethylene-induced accumulation of auxin in the root tip. To a minor extent, the root cap itself is probably the location of auxin biosynthesis. How it triggers its accumulation in other tip cells remains to be answered. In addition to the loss of auxin synthesis, the removal of the cap may also interfere with the transport of auxin from the
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Figure 6. Expression of Genes Involved in Ethylene Biosynthesis, Perception and Signaling in Maize Roots.
Figure 5. The Removal of the Cap Does not Interfere with the Responsiveness of the Root Cells to Auxin and Cytokinin. (A) Relative root growth of intact (white bars) and de-capped (black bars) roots either mock-treated (control) or in the presence of 1 mM ACC, 10 lM Kinetin, or 10 lM IAA. (B) Relative root growth of intact (white bars) and de-capped (black bars) roots either mock-treated or in the presence of 10 lM Kinetin, 10 lM Kinetin + 100 lM Ag+or Ag+alone. Root length increase was measured 24 h after the application of the substances. The experiment was repeated three times with at least 20 seedlings per measurement. The data set represents the mean of the three experiments +/– S.E. (n = 3).
root tip to distal tissues. In Arabidopsis, there is evidence that the lateral cap cells mediate akropetal auxin transport from the root tip to the elongation zone via the auxin influx carrier AUX1, which is required for the gravitropic response of the root and probably for the inhibition of elongation growth and induction of root hair formation (Dharmasiri et al., 2006; Swarup et al., 2007). AUX1-like influx carriers are also expressed in the root cap of maize (Jiang et al., 2006). Because the de-capped maize roots are non-gravitopic (Figure 2E–2G), the AUX1-like carriers were probably removed or inactivated by our surgical procedure. The prevention of the auxin synthesis and akropetal auxin transport abolishes the competence of the distal cells to respond to ethylene. In summary, our study proposes a novel and essential function of the cap in the ethylene-regulated and auxin-mediated developmental processes in the root.
(A) Scheme of the harvested of root segments (1, tip; 2, distal part) for RT-PCR analyses. (B) Steady-state transcript levels of ACO20, ETR2-40, EIN2, ERF1 and tubulin (TUB) in the tip (1) and the distal (2) segment form of intact (+) and de-capped (–) roots (see A) as determined by RT-PCR using gene-specific primers. Before harvest, the seedlings were either mock-treated (H2O) or treated with 1 mM ACC or 100 lM AVG. Tubulin served as RT-PCR control. The analysis was repeated at least three times and one representative data set is shown.
METHODS Plant Materials, Growth Conditions and Cap Removal Vernalized seedlings of maize (Zea mays L., cv. DK 233) were grown in the dark at 25C for 2 d in a vertical position, enrolled in moist filter paper, as described previously (Hahn et al., 2006). Seedlings with an average root length of 15–20 mm were used in all experiments. The root cap of seedlings was removed immediately after harvest under a binocular using a fixed razorblade. Seedlings with intact and de-capped roots were then immobilized between moist filter paper (imbibed in distilled water or appropriate solutions) on blocks of polystyrene in Perspex boxes with water-saturated atmosphere.
Hormone Treatments and Root Cap Regeneration Hormone treatment was conducted by application of solutions of 1 ll l 1 ethephon, 1 mM 1-aminocyclopropane-1-carboxylic acid (ACC), 10 lM kinetin, or 10 lM indoleacetic acid (IAA) to the seedlings. As inhibitors of ethylene biosynthesis and action, 10 lM aminoethoxyvinylglycine (AVG) and 100 lM silver nitrate (Ag+), respectively, were used. Two-day-old seedlings with intact and de-capped roots were placed on moist filter paper (imbibed in distilled water or 1 mM ACC) in 20 3 20 cm Perspex boxes. These boxes were then placed at a 45 position. Over a period of 72 h, the length
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increase of the primary roots was measured every 24 h. The experiment was independently repeated at least three times and one representative data set is shown.
ZmNAC5 In-Situ Localization and Starch Granule Staining RNA in-situ hybridization was performed according to the method of Jackson (1991). Root tips were dissected and fixed at 4C overnight in 4% formaldehyde in phosphate-buffered saline, dehydrated in an ethanol series and embedded in paraffin wax (Paraplast plus, Sigma). Embedded tissue was sectioned by use of a Leica RM 2145 rotary microtome and mounted on coated slides (Super-Frost Plus). Templates for ZmNAC5 in-situ probes were chosen as previously described (Zimmermann and Werr, 2005), cloned in antisense orientation to the T7 promoter and labelled using T7 polymerase and DIG 11-UTP (Roche) as described by Bradley et al. (1993). For starch granule staining, samples were embedded and sectioned as described above. Sections were then dehydrated in an ethanol series and stained with an iodide–potassium iodide solution for 3–5 min, cleared and observed using an Axioskop microscope with nomarsky optics (Zeiss).
Ethylene Measurements Real-time photo-acoustic measurement of ethylene emission of 2-d old maize seedlings were performed using an INVIVO-C2H4-10 laser spectrometer (Invivo GmbH, Sankt Augustin) as described previously (Kramer et al., 2003).
RT-PCR Analysis For RNA extractions, frozen samples were homogenized in RNAwiz (Ambion) according to the manufacturer’s protocol with minor modifications. First strand cDNA synthesis was carried out using the SuperScript III Reverse Transcriptase (Invitrogen) with oligo(dT)20 as the primer following the protocol of the manufacturer. Primer sequences for ACO20, ETR2-40, and EIN2 RT-PCR have been described previously (Gallie and Young, 2004). Primer sequences for ERF1 were ERF1S (5#-TGCGGCGGCGCAATCATCTTCG-3’) and ERF1A (5#-TCATTCAGTAAAGAGCGACAG-3’). Supplemental Figure 1. Intact (+) and De-capped (–) Roots Were Placed Horizontally and the Gravitropic Angle of Each Root Tip Was Measured over 48 h. The angle of each gravi-stimulated root was assigned to one of 12 30 sectors. The length of each bar represents the percentage of roots showing tip growth within that sector. gY indicates the direction of the gravi vector. Data are presented as the mean +/– S.E. of three independent experiments (n > 20 seedlings per experiment).
ACKNOWLEDGMENTS We would like to thank Felicity de Courcy for proofreading the manuscript, Richard Firn for helpful comments on the manuscript, and Jakub Hora´k for help in image processing. We gratefully acknowledge the excellent technical support from Petra Dehring
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and Ralph Ga¨bler. The work was supported by a DFG/AFGN grant to KH (Ha 2146/5–2). No conflict of interest declared.
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