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Plant Cell. Dec 2003; 15(12): 2816–2825.
PMCID: PMC282807

Ethylene Regulates Arabidopsis Development via the Modulation of DELLA Protein Growth Repressor Function

Abstract

Phytohormones regulate plant development via a poorly understood signal response network. Here, we show that the phytohormone ethylene regulates plant development at least in part via alteration of the properties of DELLA protein nuclear growth repressors, a family of proteins first identified as gibberellin (GA) signaling components. This conclusion is based on the following experimental observations. First, ethylene inhibited Arabidopsis root growth in a DELLA-dependent manner. Second, ethylene delayed the GA-induced disappearance of the DELLA protein repressor of ga1-3 from root cell nuclei via a constitutive triple response-dependent signaling pathway. Third, the ethylene-promoted “apical hook” structure of etiolated seedling hypocotyls was dependent on the relief of DELLA-mediated growth restraint. Ethylene, auxin, and GA responses now can be attributed to effects on DELLA function, suggesting that DELLA plays a key integrative role in the phytohormone signal response network.

INTRODUCTION

Germinating seedlings synthesize ethylene when their growth is impeded by the soil. The ethylene promotes the so-called “triple response”: an exaggeration of the transient developmental structure known as the apical hook, together with the inhibition of hypocotyl and root extension growth (Abeles et al., 1992). The triple response is thought to protect the shoot and root apical meristems from damage during growth through the soil.

The triple response is promoted after an ethylene–ethylene receptor interaction. Arabidopsis contains genes that encode five ethylene receptors: ETR1, ERS1, ETR2, ERS2, and EIN4 (Hua and Meyerowitz, 1998). These proteins contain N-terminal, membrane-associated, ethylene sensor domains and intracellular regions homologous with those of His kinases (Hua and Meyerowitz, 1998; Chen et al., 2002). Although His kinase activity has been demonstrated for ETR1 (Gamble et al., 1998), the role of this activity in ethylene signal transduction remains unclear (Wang et al., 2003). However, the His kinase domain of ETR1 has been shown to interact with constitutive triple response (CTR1) (Clark et al., 1998), a downstream element of the ethylene signal transduction pathway (Kieber et al., 1993). CTR1 is a Ser/Thr kinase that is closely related to RAF kinases (Kieber et al., 1993). CTR1 acts as a negative regulator of ethylene responses, and loss-of-function ctr1 mutations result in the constitutive activation of the ethylene response pathway. Therefore, ethylene signaling is thought to act in the following way. In the absence of ethylene, the ethylene receptors activate CTR1, repressing ethylene responses. In the presence of ethylene, the ethylene receptors do not activate CTR1, alleviating the CTR1-mediated repression of ethylene responses. Thus, ethylene derepresses ethylene responses by opposing the activity of the CTR1 signaling pathway (Kieber et al., 1993; Hua and Meyerowitz, 1998).

Gibberellin (GA) also derepresses the hormone response by opposing the effects of the family of proteins now known as the DELLA proteins, a subfamily of the GRAS family of putative transcriptional regulators (Richards et al., 2001). In Arabidopsis, the DELLA family comprises GAI, repressor of ga1-3 (RGA), RGL1, RGL2, and RGL3 (Peng et al., 1997; Silverstone et al., 2001; Lee et al., 2002; Wen and Chang, 2002). DELLA proteins also have been described in wheat, maize, barley, and rice, in which they also regulate GA response (Peng et al., 1999; Fu et al., 2002; Itoh et al., 2002). DELLA proteins are nuclear growth repressors. In many cases, GA opposes the effects of DELLA proteins by promoting DELLA protein destabilization (Dill et al., 2001; Silverstone et al., 2001; Itoh et al., 2002; Fu et al., 2002; Sasaki et al., 2003), although some DELLA proteins are less susceptible to GA-stimulated destabilization than others (Fleck and Harberd, 2002; Wen and Chang, 2002). Recent results indicate that the GA-mediated destabilization of DELLA proteins involves GA-stimulated phosphorylation, polyubiquitination via a specific SCF E3 ubiquitin ligase complex, and subsequent destruction in the 26S proteasome (Fu et al., 2002; McGinnis et al., 2003; Sasaki et al., 2003). Thus, DELLA proteins restrain the growth of plants, whereas GA relieves plants of DELLA-mediated growth restraint by targeting the DELLA proteins for destruction (Harberd, 2003).

Although it has long been clear that both ethylene and GA regulate plant growth, it was not clear how their respective signaling pathways interact with one another in growth regulation. Recent experiments have shown that auxin promotes root growth by modulating DELLA function (Fu and Harberd, 2003). Here, we show that the ethylene signaling pathway also regulates growth by modulating the growth-repressing effects of DELLA proteins. We show that ethylene inhibits the growth of roots via the DELLA proteins GAI and RGA and that ethylene delays the progress of the GA-induced disappearance of RGA via a CTR1-dependent signaling pathway. We also show that the GA-promoted relief of the growth restraint imposed by GAI and RGA is necessary for apical hook maintenance. Our observations indicate that DELLA proteins act as integrators of multiple phytohormone signal inputs, coordinating the elongation and differential growth of plant cells.

RESULTS

Ethylene Inhibits Arabidopsis Root Growth via Its Effects on DELLA Proteins

Ethylene is known to inhibit the growth of vegetative tissues. Seedlings grown in ethylene gas or in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Abeles et al., 1992) have reduced root lengths (Figure 1A). We found that this effect is mediated, at least in part, by GAI and RGA. In the absence of ACC, wild-type, gai-t6 (lacks GAI), rga-24 (lacks RGA), and gai-t6 rga-24 (lacks both GAI and RGA) seedling root lengths were statistically indistinguishable (Figure 1A). However, although wild-type roots were short when grown on medium containing ACC, roots of ACC-grown plants lacking RGA (rga-24) were longer than those of wild-type plants (Figure 1A). Roots of ACC-grown plants lacking both GAI and RGA (gai-t6 rga-24) appeared even longer than those of plants lacking either GAI or RGA alone (Figure 1A), indicating that GAI and RGA together mediate the ethylene-induced inhibition of root growth. When grown on higher concentrations of ACC (>1 μM), the resultant high levels of ethylene caused a dramatic reduction in root length, and the differences between wild-type, gai-t6, rga-24, and gai-t6 rga-24 roots became less apparent (Figure 1B). Thus, at reduced ACC levels (which are likely to generate ethylene levels that are more physiologically representative), the growth of gai-t6 rga-24 roots is more resistant to the effects of ACC than is that of wild-type roots. We also found that the inhibitory effect of ethylene gas on root growth is DELLA dependent. When grown in the presence of ethylene, gai-t6 rga-24 roots were longer than those of wild-type plants (data not shown).

Figure 1.
Ethylene Inhibits Root Elongation via Its Effects on GAI and RGA.

The fact that ACC-grown gai-t6 rga-24 plants did not have roots as long as wild-type plants grown in the absence of ACC (Figure 1A) shows either that additional DELLA proteins (RGL1, RGL2, and RGL3) (Lee et al., 2002) mediate the effect of ethylene on root growth or that, in addition to the DELLA protein pathway of root growth inhibition, ethylene also inhibits root growth via a DELLA protein-independent pathway.

DELLA proteins can be abolished by mutation (see above) or opposed by GA (Silverstone et al., 2001). We next investigated the effect of exogenous GA treatment on ACC-grown wild-type seedlings. We found that GA treatment could substantially overcome the ACC-induced inhibition of seedling root growth (Figure 2), although this treatment had little effect on the growth of seedling roots in the absence of ACC. Thus, GAI and RGA inhibit the growth of roots in response to ethylene, but their effects can be overcome by exogenous GA treatment.

Figure 2.
Ethylene Inhibits Root Growth by Modulating GA Responses.

Ethylene Delays the GA-Mediated Disappearance of Green Fluorescent Protein–RGA from Root Cell Nuclei

We have shown that ethylene inhibits Arabidopsis root growth via its effects on the GAI and RGA proteins. There are two possible explanations for this observation. First, ethylene may alter the properties of GAI and RGA (making them less responsive to GA). Second, ethylene may reduce endogenous GA levels. These explanations are not mutually exclusive, and to further investigate them, we studied the behavior of a green fluorescent protein (GFP)–RGA fusion protein in ethylene-treated roots.

pRGA:GFP-RGA plants express a GFP-RGA fusion protein that is detectable in root cell nuclei and disappears rapidly in response to GA treatments (Dill et al., 2001; Silverstone et al., 2001; Fleck and Harberd, 2002; Fu and Harberd, 2003). Because ethylene inhibits the growth of roots via RGA function, we tried to determine if ethylene altered the response of GFP-RGA to GA. As shown in Figure 3A, nuclear fluorescence resulting from GFP-RGA in both the tip and the elongation zone of the root was reduced greatly after 90 min of GA treatment and had largely disappeared after 3 h of GA treatment. However, when seedlings were grown in air containing ethylene gas, nuclear GFP-RGA was more resistant to the destabilizing effects of GA treatments and was clearly visible at 3 h after the onset of the GA treatment (Figure 3A). Thus, ethylene treatment delays the GA-induced disappearance of GFP-RGA from root cell nuclei.

Figure 3.
Ethylene Delays the GA-Mediated Disappearance of GFP-RGA from Root Cell Nuclei.

One possible explanation for the results shown in Figure 3A would be that ethylene causes an increase in GFP-RGA levels, thus enabling the GFP-RGA signal to persist for a longer period after the onset of GA treatment in the presence than in the absence of ethylene. This increase in GFP-RGA levels could be attributable to an increase in GFP-RGA transcript levels or to post-transcriptional effects. To investigate these possibilities, we first performed a quantitative reverse transcriptase–mediated PCR experiment to compare GFP-RGA and RGA transcript levels in ACC-treated and control seedlings. As shown in Figure 3B, exposure to ACC had no detectable effect on either GFP-RGA (transgene) or RGA (endogenous) transcript levels. Thus, the relative persistence of the GFP-RGA signal in seedlings treated with ethylene and GA cannot be attributed to an ethylene-induced enhancement of GFP-RGA transcript levels. Furthermore, we also tested the effects of ACC treatment on immunologically detectable GFP-RGA (using an anti-GFP antibody). As shown in Figure 3C, levels of immunologically detectable GFP-RGA were not obviously higher in ACC-treated roots before the onset of GA treatment. However, the immunologically detectable GFP-RGA in ACC-treated roots was more resistant to GA treatment than that of non-ACC controls (Figure 3C), in agreement with the confocal microscopy data presented in Figure 3A.

The results presented in Figure 3 are consistent with the idea that ethylene stabilizes GFP-RGA by enhancing resistance to the destabilizing effects of GA.

Ethylene Delays the GA-Mediated Disappearance of GFP-RGA via CTR1-Dependent Signaling

Ethylene is known to derepress many ethylene responses by opposing the activity of a CTR1-dependent signaling pathway. To determine if ethylene also delays the GA-mediated disappearance of GFP-RGA via CTR1-dependent signaling, we inactivated CTR1 via transient systemic RNA interference (RNAi) (Fu and Harberd, 2003). Five-day-old seedlings were cocultivated with Agrobacterium tumefaciens containing a CTR1-RNAi construct for 2 days. As shown in Figure 4A, CTR1 transcript levels were reduced (compared with control transcripts) in roots infected with A. tumefaciens containing a CTR1-RNAi construct but not in roots infected with control A. tumefaciens containing vector only (GV3101). Furthermore, preinfection of pRGA:GFP-RGA roots with A. tumefaciens containing the CTR1-RNAi construct caused GFP-RGA to be relatively resistant to GA treatments (Figure 4B). Although GFP-RGA disappeared rapidly in response to GA treatment in pRGA:GFP-RGA roots infected with control (vector only) A. tumefaciens, GFP-RGA still was clearly detectable in roots infected with A. tumefaciens containing a CTR1-RNAi construct at 4 h after the onset of GA treatment (Figure 4B). These observations indicate that ethylene delays the GA-induced disappearance of GFP-RGA from root cell nuclei by opposing the activity of the CTR1 signaling pathway.

Figure 4.
Ethylene Delays the GA-Induced Disappearance of GFP-RGA via a CTR1-Dependent Pathway.

To confirm this result, we sought to determine if the same effect was observed in plants homozygous for ctr1-1 (the first ctr1 mutant allele) (Kieber et al., 1993) and for the pRGA:GFP-RGA transgene. GFP-RGA fluorescence of young seedling roots was analyzed as described above. As shown in Figure 4C, GFP-RGA was more resistant to GA treatment in the ctr1-1 background than in a wild-type background. GFP-RGA fluorescence was still detectable in root cell nuclei of ctr1-1 pRGA:GFP-RGA at 2 and 3 h after GA treatment, whereas the signal was substantially gone after 2 h of GA treatment in wild-type pRGA:GFP-RGA plants (Figure 4C). Thus, ethylene delays the GA-mediated disappearance of GFP-RGA via a CTR1-dependent signaling pathway.

Apical Hook Maintenance Requires the Opposition of DELLA Proteins by GA

The apical hook is a transient developmental curvature of the hypocotyl that is characteristic of the seedling triple response. The curvature is caused by asymmetric growth of the inner and outer sides of the hypocotyl, which results from differential cell division and elongation (Lehman et al., 1996; Raz and Ecker, 1999; Raz and Koornneef, 2001). In addition, apical hook development can be divided into distinct establishment and maintenance phases (Raz and Ecker, 1999). We found that DELLA proteins repressed the presence of the apical hook in 3-day-old etiolated seedlings (Figure 5A). Although wild-type seedlings exhibited a pronounced apical hook, GA-deficient ga1-3 mutant seedlings (King et al., 2001) did not (Figure 5A). The hypocotyls of 3-day-old etiolated ga1-3 seedlings lacking GAI (gai-t6 ga1-3) or RGA (rga-24 ga1-3) were curved slightly, whereas the combined absence of GAI and RGA (gai-t6 rga-24 ga1-3) suppressed the absence-of-hook phenotype conferred by ga1-3 (Figure 5A). These results indicate that GAI and RGA inhibit the apical hook of 3-day-old ga1-3 seedlings, whereas the GA in wild-type seedlings opposes GAI and RGA activity and promotes the presence of the hook.

Figure 5.
Apical Hook Maintenance Is GA Dependent.

We next found that the GA/DELLA effect on apical hook structure was the result of effects on hook maintenance but not on hook establishment. ga1-3 seedlings initially established an apical hook, but this hook disappeared before the seedlings were 3 days old (Figure 5B). By contrast, wild-type and gai-t6 rga-24 ga1-3 hypocotyls established and maintained an apical hook during this period. It is important to note that the lack of apical hook maintenance exhibited by ga1-3 is not a simple consequence of its reduced growth. When ga1-3 hypocotyls grew to a length equivalent to that of 3-day-old wild-type hypocotyls, no apical hook structure was observed (data not shown).

DELLA Proteins Interact with the Ethylene and Auxin Signaling Pathways in the Maintenance of Apical Hook Structure

The coordination of differential cell growth in the apical hook is orchestrated by several phytohormones, of which auxin and ethylene have been studied extensively (Lehman et al., 1996). Auxin treatments reduce, whereas ethylene enhances, the curvature of the apical hooks of 3-day-old seedlings (Ecker, 1995; Raz and Ecker, 1999). We next investigated the relationships between the GA/DELLA signaling system and the ethylene and auxin signaling pathways in apical hook development, as we had done previously for root growth (Fu and Harberd, 2003). We found that the DELLA proteins act downstream of the effects of ethylene and auxin on apical hook maintenance.

Some of these experiments used the Arabidopsis gai mutant. This mutant shares many of the phenotypic characteristics of GA-deficient mutants, except that its phenotype cannot be restored to normal by treatment with GA (Peng et al., 1997). Like ga1-3, 3-day-old etiolated gai seedlings did not display an apical hook structure (Figure 6). In the presence of GA, ga1-3 seedlings exhibited an apical hook, whereas gai seedlings did not (Figure 6, second row, +GA). In the presence of paclobutrazol (PAC), an inhibitor of GA biosynthesis (Peng et al., 1997), only the gai-t6 rga-24 ga1-3 line (which lacks GAI and RGA), and not ga1-3 or the wild type, exhibited an apical hook, confirming that GA regulates hook maintenance via DELLA signaling (Figure 6, third row, +PAC).

Figure 6.
DELLA Proteins Act Downstream of Ethylene and Auxin Signaling Pathways.

Ethylene promoted the maintenance of the apical hook of 3-day-old wild-type seedlings, whereas ctr1-1 exhibited an exaggerated apical hook in the absence of ethylene (Huang et al., 2003) (Figure 6). Interestingly, PAC reduced the exaggerated hypocotyl curvature conferred by ctr1-1 (Figure 6), whereas ga1-3 hypocotyls were unaffected by ACC (Figure 6, fourth row, +ACC). gai hypocotyls curved slightly in response to ACC treatments (Figure 6), probably because GA responses in gai are reduced but not abolished completely (Koornneef et al., 1985). Combined treatment with both GA and ACC resulted in hypocotyl curvature in ga1-3 (Figure 6, fifth row, +GA, +ACC), similar to that seen in wild-type plants treated with ACC. Thus, for ethylene to promote hook maintenance, the GA-mediated opposition of the DELLA repression of hook maintenance needs to be in operation.

Auxin also coordinates differential cell growth in the apical hook (Lehman et al., 1996). Exogenous auxin inhibits apical hook formation (Schwark and Schierle, 1992). Mutants that accumulate high levels of auxin display a hookless phenotype (Boerjan et al., 1995; Celenza et al., 1995; Lehman et al., 1996), and auxin transport inhibitors (such as 1-naphthylphthalamic acid [NPA] or 2,3,5-tri-iodobenzoic acid) inhibit hook formation (Lehman et al., 1996). We found that etiolated seedlings (even ctr1-1) (Lehman et al., 1996) grown in the presence of NPA were hookless, with the exception of gai-t6 rga-24 ga1-3 (Figure 6, sixth row, +NPA). Thus, as shown previously for root growth (Fu and Harberd, 2003), auxin mediates its effects on apical hook formation via the DELLA proteins GAI and RGA.

DISCUSSION

After organ initiation, much of plant development consists of regulated organ growth. The regulation of organ growth is mediated by the coordination of cell division and expansion. This coordination involves signals that are endogenous to the plant (e.g., phytohormones) and exogenous environmental signals and requires the integration of these multiple signals into a single output.

By investigating phenomena that traditionally have been regarded as ethylene responses, we have identified the previously described DELLA proteins as agents of signal integration in plant growth regulation. Ethylene causes exaggeration of the apical hook, shortening and thickening of the hypocotyls, and inhibition of root growth, a phenomenon known as the triple response (Abeles et al., 1992). Although recent advances identified several components of the signal transduction cascade that specifically modulate plant growth in response to ethylene, the way in which ethylene regulates growth remained unknown. Our results indicate that at least part of the growth regulatory effects of ethylene are mediated via its effects on the DELLA proteins, which act as repressors of growth.

First, we investigated the inhibitory effect of ethylene on root growth and showed that it is mediated, at least in part, by GAI and RGA. We also showed that ethylene delays the GA-mediated disappearance of GFP-RGA from root cell nuclei via a CTR1-dependent signaling pathway. These two types of experiments operate on very different time scales. Although ethylene delays GFP-RGA disappearance (in response to exogenous GA) by only a few hours, the differential effects of the lack of GAI and RGA on ethylene-mediated root growth inhibition are detected over the course of days. It is important to recognize that the exogenous GA treatment experiments merely serve to reveal a difference in the properties of GFP-RGA in ethylene-treated and control roots. The results of the GA treatment experiments suggest that, during the root growth experiments, the DELLA proteins in ethylene-treated roots have altered properties and respond differently (are relatively stable) with respect to endogenous GA than do the DELLA proteins in control roots.

This ethylene-induced increase in DELLA stability, and the consequent effects on cell growth rates integrated over several days, presumably accounts for the observed DELLA-mediated effects of ethylene on growth. The fact that there was no detectable difference in GFP-RGA levels in ACC-treated versus control roots (Figure 3, 0-h points) suggests that in this case differences in GFP-RGA level caused by differential responses to endogenous GA were too small to detect. However, it is likely that differences in DELLA protein level that were not detectable by immunoblot analysis will have clearly detectable effects on the extent of growth when integrated over the course of days. In summary, our experiments suggest that ethylene alters the relationship between GA and the DELLA proteins, making the DELLA proteins more (but not completely) resistant to the opposing effects of GA. The stabilization of DELLA proteins such as RGA would be expected to increase their growth-repressing effects, thus contributing to the ethylene-mediated inhibition of root growth.

Second, we investigated the effects of the DELLA proteins on the development of the ethylene-promoted apical hook structure. The apical hook is a dynamic structure that prevents damage to the shoot apical meristem as plants push through the soil. Apical hook formation is caused by regulated differential cell division and cell elongation (Raz and Ecker, 1999; Raz and Koornneef, 2001), resulting in asymmetric growth of the inner and outer sides of the hypocotyl. We found that GA signaling is crucial to the maintenance of the curvature of the apical hook. The GA-deficient ga1-3 mutant did not exhibit an apical hook, whereas the combined absence of GAI and RGA (in gai-t6 rga-24 ga1-3) restored an apical hook structure to ga1-3 hypocotyls. These observations demonstrate that the maintenance of the apical hook structure requires the GA-mediated opposition of DELLA function. This finding is consistent with the recent report that the Arabidopsis lue1 mutant, which is affected in GA signaling and lacks a functional katanin-like microtubule-severing protein, also has defects in ethylene-promoted apical hook maintenance (Bouquin et al., 2002).

Our further analyses showed that the effects of ethylene (which promotes the apical hook) and auxin transport inhibitors (which inhibit the apical hook) are dependent on the GA/DELLA signaling system. Thus, ethylene-treated (and ctr1 mutant) hypocotyls do not exhibit an apical hook when they are GA deficient (ga1-3 or PAC treated), whereas the auxin transport inhibitor NPA blocks the apical hook in normal hypocotyls but not in hypocotyls lacking GAI and RGA. These observations suggest that the effects of ethylene and auxin on apical hook structure are mediated via the effects of these phytohormones on the DELLA growth repressor proteins.

There have been previous reports indicating the existence of interactions between the ethylene and auxin signaling pathways. For example, ethylene induces the expression of Arabidopsis Hookless (Lehman et al., 1996), a gene that encodes a putative N-acetyltransferase thought to control differential cell growth via the regulation of auxin activity. In addition, Arabidopsis NPH4 encodes an auxin-regulated transcriptional activator (ARF7) that is involved in the differential growth responses of aerial tissues. The characteristic phenotypes of mutants lacking ARF7 are suppressed by ethylene application (Harper et al., 2000). Finally, Arabidopsis EIR1 plays a root-specific role in the transport of auxin. Mutants that lack EIR1 are less sensitive to ethylene, and the lack of EIR1 partially suppresses the phenotype of mutants that lack CTR1, suggesting that EIR1 acts downstream of ethylene signaling (Luschnig et al., 1998). Other studies have indicated an interactive relationship between ethylene and GA in the regulation of plant growth. For example, ethylene and GA are known to interact in promoting the rapid growth of deep-water rice internodes (Sauter et al., 1995). Interaction between auxin, ethylene, and gibberellins also was shown to occur in the promotion of hypocotyl growth and stomatal development in light-grown Arabidopsis seedlings (Saibo et al., 2003).

However, although these previous studies had indicated various connections between ethylene, auxin, and GA, the nature of these connections remained unknown. Here, we suggest that DELLA proteins provide such a connection. Recently, we showed that the properties of the DELLA proteins are modulated by auxin (Fu and Harberd, 2003). Because both ethylene and auxin influence the rate at which the DELLA proteins disappear in response to GA, their respective signaling pathways must affect DELLA function at some point (Figure 7). Our findings indicate that the DELLA proteins are integrators of phytohormonal growth regulatory signals and are molecules that regulate plant growth in response to ethylene, auxin, and GA signals.

Figure 7.
DELLA Proteins Integrate the Effects on Growth of the Ethylene, Auxin, and GA Signaling Pathways.

METHODS

Plant Material

All experiments used the Landsberg erecta laboratory strain of Arabidopsis thaliana as genetic background except in the case of the ctr1-1 mutant (whose genetic background is Columbia). The ctr1-1, ga1-3, gai, gai-t6, and rga-24 mutations and the pRGA:GFP-RGA line were as described previously (Kieber et al., 1993; Fu and Harberd, 2003). The ctr1-1 pRGA:GFP-RGA line was isolated from the F3 progeny of a cross between ctr1-1 and pRGA:GFP-RGA.

Root Growth Experiments

All seeds were surface-sterilized and placed on GM medium plates (King et al., 2001) containing 10−8 to 10−5 M 1-aminocyclopropane-1-carboxylic acid (ACC) at 4°C for 5 days to synchronize germination. Then, plates were placed in a vertical orientation in a growth room (22°C with a 16-h photoperiod). For each growth condition, root lengths of at least 20 7-day-old seedlings were measured from root tip to the base of the hypocotyls.

Analysis of Apical Hook Structure

All seeds were surface-sterilized and pretreated at 4°C with 1 μM gibberellin (GA) for 5 days to synchronize germination. Seeds then were washed thoroughly and placed on GM medium plates containing 1 μM GA, 10 μM ACC, 1 μM paclobutrazol, or 1 μM 1-naphthylphthalamic acid (NPA) and placed vertically in a growth room (22°C in darkness). After 3 days of growth, one representative seedling (from among 50) was chosen and photographed.

Quantitative Reverse Transcriptase–Mediated PCR

Total RNA was extracted from primary roots of 5-day-old seedlings grown in the presence or absence of 1 μM ACC using Trizol reagent (Gibco BRL). cDNA was generated using the Superscript RNase Reverse Transcriptase kit (Gibco BRL). PCR amplification (18 cycles) and blot analyses were performed as described previously (Fu and Harberd, 2003). The primer pairs used for PCR amplification were as follows: for RGA, 5′-TCATGCTCGAGTCCTGATTCTATGG-3′ and 5′-GACAATGATCGATCTGATTCTGC-3′; for GFP, 5′-CGGCACGACTTCTTCAAGAGCGCC-3′ and 5′-GTATTCCAACTTGTGGCCGAGG-3′. The expected DNA fragment sizes for cDNA amplification corresponded to 389 bp (RGA) and 222 bp (GFP). As a control, a fragment from the gene that encodes the eukaryotic protein synthesis initiation factor 4A was amplified using the primers 5′-TTCTCAAACCATAAGCATAAATAC-3′ and 5′-AAACTCAATGAAGTACTTGAGGGA-3′.

Immunoblot Analyses

pRGA:GFP-RGA plants were grown on GM medium in the presence or absence of 1 μM ACC for 5 days. Whole seedlings then were treated with 10 μM GA for 1, 2, and 3 h. The primary roots were harvested and frozen in liquid N2. Total protein was extracted and quantified as described by Silverstone et al. (2001). For each treatment, 20 μg of proteins was separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. Immunodetection was performed using a 2500-fold dilution of anti-GFP monoclonal antibodies from mouse (Chemicon International, Temecula, CA) and a 5000-fold dilution of peroxidase-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL). Signals were detected by chemiluminescence using the enhanced chemiluminescence protein gel blot analysis system (Amersham Biosciences).

Transient Gene Silencing

Transient gene silencing was performed as described previously (Fu and Harberd, 2003). The CTR1 coding fragment sequence (490 bp) was amplified by PCR with the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGACGATGCCGCTTCG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCACGGGATGTCCATATC-3′ and cloned twice in the inverse orientation. Five-day-old pRGA:GFP-RGA seedlings grown on GM medium plates were cocultivated with Agrobacterium tumefaciens strain GV3101 containing RNA interference–inactivated CTR1 or empty vector for 2 days in 50 mL of Murashige and Skoog (1962) liquid medium. The efficiency of the transient silencing was tested using reverse transcriptase–mediated PCR. Total RNA, generation of cDNA, and amplification by PCR (23 cycles) were as described above. The CTR1 primers used for PCR amplification were 5′-GGTGGTTCCCATAGGTAGCCTCTC-3′ and 5′-GACAGTGCCAAAGGAACCTGCTCC-3′. Ubiquitin transcripts (UBQ10) also were amplified (25 cycles) as a constitutive expression control. The UBQ10 primers were 5′-AACTTTCTCTCAATTCTCTCTACC-3′ and 5′-CTTCTTAAGCATAACAGAGACGAG-3′. The expected DNA fragment sizes for cDNA amplification corresponded to 661 bp (CTR1) and 1.4 kb (UBQ10).

Observation of GFP Fluorescence

Confocal microscopy images were obtained with a Leica (Wetzlar, Germany) inverted confocal laser microscope with ×40 objectives. The excitation wavelength for GFP detection was 488 nm. All images were obtained with the same modifications and intensity parameters. For the ethylene treatment, pRGA:GFP-RGA seedlings were grown on GM medium plates in an ethylene chamber (10 ppm ethylene gas, 22°C, all-day photoperiod). Five-day-old seedlings growing in ethylene or in air were treated with 10 μM GA and analyzed for the presence of GFP-RGA. For the transient CTR1 silencing, 5-day-old pRGA:GFP-RGA seedlings were cocultivated as described above and treated for 2 and 4 h with 10 μM GA.

Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact Nicholas P. Harberd, ku.ca.crsbb@drebrah.salohcin.

Acknowledgments

We thank Tai-ping Sun for the pRGA:GFP-RGA line, Grant Calder for technical assistance with confocal microscopy, Paul Linstead for technical assistance with ethylene treatments, Lali Sakvarelidze for computing assistance during the creation of the figures, and Xiangdong Fu, Donald Richards, and Xiuhai Zhang for comments on the manuscript. This work was funded by grants from the European Union Research Training Network (INTEGA: HPRN-CT-RTN1-2000-00090) and the Biotechnology and Biological Science Research Council (a Core Strategic Grant to the John Innes Centre).

Notes

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.015685.

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