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DEVELOPMENT AND HORMONE ACTION

Ethylene Modulates Flavonoid Accumulation and Gravitropic Responses in Roots of Arabidopsis^1,[W]

Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Plant organs change their growth direction in response to reorientation relative to the gravity vector. We explored the role of ethylene in Arabidopsis (Arabidopsis thaliana) root gravitropism. Treatment of wild-type Columbia seedlings with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC) reduced root elongation and gravitropic curvature. The ethylene-insensitive mutants ein2-5 and etr1-3 had wild-type root gravity responses, but lacked the growth and gravity inhibition by ACC found in the wild type. We examined the effect of ACC on tt4(2YY6) seedlings, which have a null mutation in the gene encoding chalcone synthase, the first enzyme in flavonoid synthesis. The tt4(2YY6) mutant makes no flavonoids, has elevated indole-3-acetic acid transport, and exhibits a delayed gravity response. Roots of tt4(2YY6), the backcrossed line tt4-2, and two other tt4 alleles had wild-type sensitivity to growth inhibition by ACC, whereas the root gravitropic curvature of these tt4 alleles was much less inhibited by ACC than wild-type roots, suggesting that ACC may reduce gravitropic curvature by altering flavonoid synthesis. ACC treatment induced flavonoid accumulation in root tips, as judged by a dye that becomes fluorescent upon binding flavonoids in wild type, but not in ein2-5 and etr1-3. ACC also prevented a transient peak in flavonoid synthesis in response to gravity. Together, these experiments suggest that elevated ethylene levels negatively regulate root gravitropism, using EIN2- and ETR1-dependent pathways, and that ACC inhibition of gravity response occurs through altering flavonoid synthesis.

Several mechanisms regulate auxin transport during root gravitropism. One mechanism is the targeting of the auxin efflux facilitator protein PIN3 to unique membranes in response to gravitropic stimulation (Friml et al., 2002). Reversible protein phosphorylation, which may modulate the activity, abundance, or localization of auxin transport proteins, is a second mechanism (Deruère et al., 1999; Christensen et al., 2000; Benjamins et al., 2001; Rashotte et al., 2001; DeLong et al., 2002; Friml et al., 2004). Finally, the localized synthesis of small molecules that regulate auxin transport modulates the rate of gravity response (Murphy et al., 2000; Brown et al., 2001; Buer and Muday, 2004).

Prime candidates for endogenous auxin transport inhibitors are flavonoids. These phenolic compounds competed with synthetic indole-3-acetic acid (IAA) efflux inhibitors, such as naphthylphthalamic acid (Jacobs and Rubery, 1988). The flavonols quercetin and kaempferol had the greatest activity, suggesting that specific members of this chemical family function as auxin transport inhibitors (Jacobs and Rubery, 1988). In vivo studies in Arabidopsis seedlings with mutations in genes encoding flavonoid biosynthetic enzymes demonstrated a role for flavonoid regulation of auxin transport. These tt4 mutants had elevated auxin transport in young seedlings, roots, or inflorescences, consistent with the absence of an endogenous negative auxin transport regulator (Murphy et al., 2000; Brown et al., 2001; Buer and Muday, 2004; Peer et al., 2004). A comparison of the root gravitropic responses of the wild type and the chalcone synthase (CHS) null mutant, tt4(2YY6), identified a lag in gravitropic curvature in tt4(2YY6). Chemical complementation of tt4(2YY6) by naringenin reinstated flavonoid production and restored a wild-type gravity response consistent with a role for flavonoids in controlling the flow of auxin needed for root gravitropism (Buer and Muday, 2004). Mutations that alter flavonoid synthesis affect the abundance of the mRNA-encoding members of the PIN gene family (Peer et al., 2004; Lazar and Goodman, 2006), suggesting that flavonoids may regulate synthesis of auxin transport proteins, not just the activity of existing proteins. Consistent with flavonoid abundance affecting transcription, recent evidence indicated that flavonoid biosynthetic enzymes and flavonoid products accumulate in the nucleus (Saslowsky et al., 2005).

Changing environmental conditions modulate flavonoid synthesis (Winkel-Shirley, 2002), and these changes in flavonoid accumulation may regulate development in several systems (Taylor and Grotewold, 2005). Induction of flavonoid synthesis during gravitropic stimulation could alter the levels of auxin transport and dependent physiological processes. For example, when Arabidopsis seedlings are grown in the dark, flavonoid biosynthesis is off. In the light, there is a dramatic induction in flavonoid accumulation mediated by induced transcription of the genes encoding flavonoid biosynthetic enzymes (Kubasek et al., 1992; Wade et al., 2001; Buer and Muday, 2004). Light levels also regulate auxin transport (Jensen et al., 1998; Rashotte et al., 2003). Flavonoids are quickly and abundantly produced when plants are wounded or during pathogen attacks, conditions that also alter auxin transport (Mathesius et al., 1998; Berleth and Sachs, 2001; Schwalm et al., 2003). Flavonoid abundance also was linked to control of shoot branching through analysis of the max1 mutant, which has increased inflorescence branching and altered expression of genes encoding flavonoid biosynthetic enzymes and IAA transport proteins (Lazar and Goodman, 2006). Finally, reorientation of plants relative to gravity leads to lateral auxin transport (Blancaflor and Masson, 2003) and induction of flavonoid synthesis in the epidermal tissues of Arabidopsis root tips (Buer and Muday, 2004). Therefore, induction of flavonoid synthesis in response to environmental stimuli may alter auxin transport to allow regulation of plant growth and development.

Another potential modulator of root gravitropism is ethylene (Mattoo and Suttle, 1991; Abeles et al., 1992). This plant hormone affects many facets of plant growth, development, and stress responses (for review, see Alonso and Stepanova, 2004; Chen et al., 2005). Exogenous applications of ethylene gas or the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC) were used to test the role of ethylene in gravitropic responses in a range of species, with contradictory results. In some experiments, ethylene treatment clearly reduced gravitropic responses (Wheeler and Salisbury, 1981; Wheeler et al., 1986; Lee et al., 1990; Kiss et al., 1999; Madlung et al., 1999), whereas others showed no effects (Kaufman et al., 1985; Harrison and Pickard, 1986; Woltering, 1991). A recent study indicated that ethylene increased gravitropic curvature in etiolated maize (Zea mays) roots under conditions where ethylene inhibited elongation (Chang et al., 2004). Several studies reported that low concentrations of ethylene affected the rate of gravitropic curvature, but that the plants ultimately reach the same final angle of orientation in the presence of this gas (Wheeler and Salisbury, 1981; Wheeler et al., 1986; Lee et al., 1990). The absence of kinetic data at the early times after gravitropic stimulation in several of the experiments may explain negative results (Kaufman et al., 1985; Harrison and Pickard, 1986; Woltering, 1991). In Arabidopsis roots, the effect of ethylene on root gravitropism has not been directly reported, but ethylene has been shown to alter root waving, a complex growth process mediated by gravity, nutrient conditions, and thigmotropic signals (Buer et al., 2003). These results suggested that ethylene influences the early stages of gravitropic bending, but additional tests are warranted utilizing plants with mutations that alter ethylene synthesis and/or signal transduction.

Few reports in the literature have used the array of Arabidopsis mutants altered in ethylene signaling and/or synthesis to examine a role of ethylene in gravitropic curvature. Extensive studies of the ethylene-signaling pathway using genetic approaches, with a particular focus on isolating Arabidopsis (Alonso and Stepanova, 2004; Chen et al., 2005) and tomato (Lycopersicon esculentum) mutants (Klee, 2004), have identified a number of the key proteins. The etr1 mutant has a defect in a membrane ethylene receptor, which is part of a family of five receptors in Arabidopsis (Chen et al., 2005). The EIN2 protein functions downstream of ETR1 and resembles the NRAMP family of metal transporters, although the biochemical function of EIN2 has not been demonstrated (Alonso and Stepanova, 2004). The gravitropic response of the roots of ein2-1 was reported to be wild type, although the gravitropic angles were measured 3 d after gravitropic reorientation (Roman et al., 1995). Although the eir1 mutant was isolated for an ethylene-insensitive root and has delayed gravitropic responses, the primary defect in this mutant is now linked to auxin transport (Luschnig et al., 1998). The gravitropic response of the tomato mutants Never-Ripe (NR) and epi, which have altered ethylene response and synthesis, respectively, were examined (Madlung et al., 1999). NR encodes an ETR1 homolog that is essential in fruit ripening, whereas epi overproduces ethylene (Klee and Tieman, 2002). Both mutants exhibit delays in shoot gravitropic response (Madlung et al., 1999) consistent with a role for ethylene in the early events of gravitropic response. The NR mutant is insensitive to the effect of exogenous ethylene on hypocotyl gravitropism (Madlung et al., 1999). Additionally, the hypocotyls in the Arabidopsis nph4 mutant have defects in phototropism and gravitropism and are altered in their response to exogenous ethylene (Harper et al., 2000). Together, these results are consistent with ethylene negatively regulating shoot gravitropism.

If ethylene regulates root gravitropism, an important question is whether the action of ethylene is to modulate either auxin movement or synthesis in the root tip to alter auxin gradients needed for root gravitropic responses. In support of this idea, ethylene reduced polar IAA transport in shoot tissues of several species (Morgan and Gausman, 1966; Suttle, 1988). Additionally, ethylene reduced the lateral redistribution of auxin across gravity-stimulated corn roots (Lee et al., 1990). The agravitropic mutant eir1 is insensitive to ethylene inhibition of root growth and has a defect in a gene encoding a protein linked to auxin efflux (Luschnig et al., 1998). Recent isolation of two mutants designated weak ethylene insensitive (wei2 and wei7) suggests that ethylene may positively regulate auxin synthesis in the root tip (Stepanova et al., 2005). The WEI2 and WEI7 genes encode enzymes of Trp synthesis, anthranilate synthase 1 and 1, respectively, whose expression is induced by ethylene. Together, these results suggest that ethylene might act to modulate auxin transport or synthesis during root gravitropism. One mechanism by which ethylene could alter auxin transport is by modulating the synthesis of the endogenous auxin transport regulators, flavonoids.

The goals of our experiments were to determine whether ethylene regulated the response of Arabidopsis roots to gravity and to test whether the action of ethylene was through modulation of flavonoid synthesis in these roots. We examined the effects of elevated endogenous ethylene on root growth and gravitropism in wild-type Arabidopsis, in mutants altered in ethylene responses, and in flavonoid-deficient mutants. Seedlings were treated with the ethylene precursor ACC, and the effect on flavonoid accumulation was examined. Together, these results suggest that ethylene may regulate root gravitropism through modulation of flavonoid biosynthesis.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

ACC Reduces Root Curvature and Elongation in Columbia Wild Type

The compound ACC, a precursor to ethylene synthesis, increases ethylene synthesis in planta (Cameron et al., 1979). We examined the effects of ACC on the gravity response of light-grown wild-type Columbia (Col) and mutant seedlings at various times after ACC treatment and gravitropic stimulation. Seedlings were grown for 5 d on control media, transplanted to media containing ACC, immediately reoriented by 90°, and transferred to the dark. Our first experiment examined the effect of multiple ACC concentrations on Col roots to find the most effective dose. The resulting root elongation and gravitropic curvature after 4-h exposure to ACC are reported in Figure 1 . Increasing ACC concentrations inhibited root growth and gravitropic curvature nearly in parallel, suggesting that these two processes are closely linked. Similar results occurred at 12 h after transfer to ACC (data not shown). Based on these responses, further experiments were performed at 2.5 µM ACC, as this dose closely mimics the levels of ethylene obtained when plants are grown on media in Parafilm-sealed petri dishes (Buer et al., 2003) and in previous experimentation (Larsen and Chang, 2001). The effects of ACC on root-tip curvature were statistically significant relative to the untreated control at all ACC concentrations above 0.1 µM and on root elongation above 0.01 µM (P < 0.0005), but curvature and elongation were not statistically different between 1 and 2.5 µM ACC.

View larger version (9K): [in this window] [in a new window]   Figure 1. ACC inhibits root elongation and tip curvature in Col wild type. Col roots were challenged with a range of ACC concentrations. The data presented are the average and SE of seedlings 4 h after treatment; n = 30 from three independent experiments of 10 seedlings per category. Absence of visible error bars is due to error smaller than the symbol size.

To determine whether the inhibition of gravitropic curvature by ethylene is under the same genetic controls as ethylene-regulated growth, the gravity and growth responses of Arabidopsis roots were compared in several genetic backgrounds. The response of the ethylene mutants, ein2-5 and etr1-3, were compared to Col wild type. Seedlings were germinated on control media and grown for 5 d, then transferred to either control media or media containing 2.5 µM ACC, gravity stimulated by rotating 90°, and placed in the dark. The growth and gravity responses at 2 and 8 h after treatment are shown in Table I . These time points were chosen to reflect events near the initiation and completion of gravitropic responses. These data are presented in a table to facilitate the comparison of several mutants at two time points and in the presence and absence of ACC.

In the presence of 2.5 µM ACC, the elongation and gravity response of wild-type roots was significantly impaired at 2 and 8 h after ACC exposure. In contrast, the gravity response in ein2-5 was significantly different from wild type (P < 0.0005). Indeed, ein2-5 root curvature on ACC is better than on control media at 2 h and equal to the control at 8 h. The growth of ein2-5 on ACC at 2 h was equivalent to the untreated ein2-5 at 2 h, whereas growth is slightly, but significantly, reduced by ACC at 8 h after treatment. The ability of ACC to inhibit root growth in ein2-5 is surprising because previous reports indicate that, in most tissues, ein2 is completely ethylene insensitive (Alonso and Stepanova, 2004; Binder et al., 2004). The ethylene mutant etr1-3 was also unaffected by ethylene at 2 h, but it had depressed root curvature and elongation relative to the untreated control by 8 h. This is consistent with previous reports indicating that etr1-3 responds to ethylene, but with reduced sensitivity in roots (Hall et al., 1999). These results are consistent with elevated ethylene-inhibiting root gravitropism in a pathway mediated by ETR1 and EIN2.

Root Gravitropic Curvature in tt4(2YY6) Is Not Affected by Ethylene at Early Time Points

One of the goals of these experiments was to ask whether the ACC inhibition of gravity response occurred through modulation of flavonoid synthesis. The tt4(2YY6) mutant, which produces no flavonoids, can be used to test this possibility. Previously, the gravity response of this mutant was characterized and found to exhibit a delay in root gravitropism due to a lag in initiation of gravitropic bending, yet the mutant ultimately reached a similar gravitropic angle (Buer and Muday, 2004). The kinetics of root elongation and curvature were quantified in tt4(2YY6) and Col wild type with ethylene levels elevated in two ways. Ethylene was elevated by growth on 2.5 µM ACC, which yielded growth effects similar to 0.5 µL L^–1 of ethylene gas (Larsen and Chang, 2001), or by wrapping plates with Parafilm, which provides an ethylene concentration in the petri plate headspace of approximately 0.3 µL L^–1 (Buer et al., 2003).

In the control treatments, tt4(2YY6) had an initial slower gravity response, but ultimately reached an angle equivalent to wild type (Fig. 2 ). When roots were exposed to elevated ethylene levels through growth on ACC or in wrapped plates, the gravitropic inhibition by ethylene is lost in tt4(2YY6). In fact, ACC treatment restored gravitropic response in tt4(2YY6) to near wild-type levels, suggesting a positive role for ethylene in root gravitropism that is masked in the presence of flavonoids. Curvature of wild-type roots was slightly faster in wrapped plates than on ACC-containing media, suggesting there is more ethylene produced by the ACC plates. Additionally, some acclimation to ethylene conditions could occur in the wrapped-plate experiments. The seedlings were germinated in the sealed plates to allow ethylene accumulation in contrast to the ACC experiments in which seedlings were transferred to ACC immediately before gravity stimulation. These results suggest that transient and long-term elevations in ethylene levels, which reduce the initial gravitropic response, are lost in the tt4(2YY6) mutant.

View larger version (11K): [in this window] [in a new window]   Figure 2. Root curvature in the flavonoid-deficient mutant tt4(2YY6) is resistant to ACC and ethylene. A to D, Curvature kinetics of Col wild type (A and B) and tt4(2YY6) (C and D) on 2.5 µM ACC or in sealed plates to elevate endogenous ethylene. The average and SE are presented with n = 30 for ACC in three independent experiments and n = 20 for wrapped plates in two independent experiments.

View larger version (73K): [in this window] [in a new window]   Figure 3. The ACC-induced root phenotypes of tt4(2YY6) and Col. Shown are differential interference contrast images selected from at least 30 representative roots after 24 h on 2.5 µM ACC or control media in the dark. Bar = 100 µm.

Multiple tt4 Alleles Have Delayed Gravitropic Response and Altered ACC Effects on Gravitropism

After completing our analyses with tt4(2YY6), we learned that a second unlinked mutation, max4-5, was present in this line (Bennett et al., 2006). We therefore repeated our analysis of root gravitropism using a backcrossed tt4(2YY6) allele. In an effort to simplify the nomenclature of the existing tt4 alleles, the mutation in tt4(2YY6) has been renamed tt4-2 in the backcrossed line without the max4-5 mutation (Bennett et al., 2006). We also used the Col tt4 insertion allele, tt4-11, recently characterized from the Salk insertion lines (SALK 020583) and shown to have an insertion within the CHS coding region (M. Ramirez and B. Winkel, personal communication), and the tt4(W85) allele in the Landsberg erecta (Ler) background (Saslowsky et al., 2000), which has been renamed tt4-1 (Murphy et al., 2000). The growth, gravitropic response, and effect of ACC on these responses are summarized in Table III . In the absence of ACC, all the tt4 alleles had a significantly reduced gravitropic response at 2 h after reorientation relative to their wild-type parental lines. By 6 h after reorientation, gravity response is not different from wild type in most lines, consistent with the previous analysis of tt4(2YY6) (Buer and Muday, 2004). In addition, the growth of these alleles is similar to or greater than wild type at 2 and 6 h after treatment.

Two additional experiments verified that the tt4 and not the max4 mutation is linked to the delayed-gravity response and altered ACC response. The gravity response and ethylene sensitivity of a max4 mutant were examined and found to be similar to wild type (data not shown). Additionally, growth of seedlings on naringenin, a flavonoid intermediate in the biosynthetic pathway after the CHS protein, leads to a restoration of wild-type gravitropism in tt4(2YY6) (Buer and Muday, 2004). Therefore, it is clear that the gravitropic defect and the ethylene insensitivity of gravity response are linked to the tt4 mutation.

ACC Induces Flavonoid Accumulation

The altered response of tt4(2YY6) to ACC suggests that ACC may act to induce flavonoid synthesis in wild-type seedlings. The accumulation of flavonoids over time in the presence and absence of ACC was examined by measuring diphenylboric acid 2-amino-ethyl ester (DPBA) fluorescence upon binding flavonoids. The specificity of the stain has been demonstrated because mutants that have a null mutation in the CHS gene do not fluoresce after incubation with DPBA (Peer et al., 2001; Buer and Muday, 2004). DPBA fluorescence is the only technique that allows quantification of flavonoid abundance at a tissue-specific level. Furthermore, it has been shown in several reports that DPBA fluorescence accurately reports flavonoids as detected by microspectrofluorometry, HPLC, or mass spectroscopy (Mathesius et al., 2000; Peer et al., 2001).

Representative images taken from at least 30 DPBA-stained roots examined after ACC treatment are shown in Figure 4 and indicate that DPBA fluorescence increased after ACC treatment in vertical seedlings. Multiple images were also quantified and the results were normalized to the fluorescence in the absence of ACC (Fig. 5A), which indicates that flavonoid accumulation becomes maximally elevated between 4 and 8 h after ACC treatment in vertical seedlings. This sustained flavonoid increase differs from the previously reported transient flavonoid accumulation in response to gravitropic stimulation (Buer and Muday, 2004). To determine whether the ethylene-signaling pathway mediates the DPBA fluorescence changes, the effect of ACC on DPBA fluorescence was examined in the ein2-5 ethylene-insensitive mutant and the DPBA fluorescence is shown in Figure 5A . In contrast to wild type, ein2-5 roots had a constant level of DPBA fluorescence in the presence of ACC. Similarly, etr1-3 roots also showed no increase in DPBA fluorescence upon transfer to ACC (data not shown). These results are consistent with ACC-induced flavonoid accumulation mediated by the ETR1 and EIN2 proteins.

View larger version (82K): [in this window] [in a new window]   Figure 4. ACC delays the gravity-induced flavonoid fluorescence increase in Col. Col roots were challenged with 2.5 µM ACC and/or gravity at different time points as indicated, stained with DPBA, and photographed by gray-scale epifluorescence microscopy. Exposures were normalized to longest exposure that prevented fluorescence saturation. The arrow indicates the direction of the gravity vector relative to the root tips. Bar = 50 µm.

View larger version (7K): [in this window] [in a new window]   Figure 5. ACC affects DPBA fluorescence in Col. A, DPBA fluorescence is not elevated in the ein2-5 mutant when challenged with ACC. Comparison of Col and ein2-5 on 2.5 µM ACC incubated vertically in the dark for the indicated times is shown. Data are the average and percent error of two combined individual experiments. Data from Col are the average and percent error from eight combined independent experiments. B, ACC delays DPBA fluorescence in Col wild type during gravity stimulation in the dark. Plotted is the average and percent error from five independent experiments.

A previous report indicated that reorientation of seedlings relative to the gravity vector induced flavonoid accumulation in the root tip of wild-type Arabidopsis, with a peak about 2 h after reorientation relative to gravity as judged by increased DPBA fluorescence (Buer and Muday, 2004). Therefore, we quantified the effect of ACC on this transient gravity-induced peak of DPBA fluorescence in the tips of wild-type Arabidopsis roots. The previously reported transient increase in DPBA fluorescence with a peak of intensity at 2 h after gravity stimulation was detected in this analysis, but gravity stimulation in the presence of ACC prevented this transient DPBA fluorescence increase (Fig. 4). The fluorescence intensity of multiple roots was quantified and the summarized results are shown in Figure 5B and are consistent with the representative images in Figure 4. The ACC treatment combined with gravitropic stimulation causes a sustained elevation in flavonoid accumulation to occur later than the flavonoid accumulation in response to either individual treatment. These results indicate that ACC prevents the gravity-induced transient flavonoid accumulation consistent with its inhibition of gravitropic curvature.

Confocal Microscopy of DPBA Fluorescence Localization with ACC and Gravity Stimulation

Epifluorescence microscopy is ideal for rapid quantification of overall DPBA fluorescence in multiple roots, but it does not yield precise information on the localization of flavonoid accumulation. We used confocal microscopy to localize flavonoid accumulation using DPBA fluorescence, as shown in Supplemental Figure 1. All micrographs were taken at the identical settings; thus, intensity is directly comparable between images. The increases in DPBA fluorescence at 2 h after gravity stimulation and at 4 h after ACC treatment were evident. The ability of ACC to prevent the spike of DPBA fluorescence at 2 h after gravity stimulation was also evident. These confocal microscopy images indicate that ACC treatment and gravity stimulation cause flavonoid accumulation in similar tissues.

Additionally, we observed a U-shaped pattern of fluorescence in the root tips just above the quiescent center in the endodermal or cortical cell files. This fluorescence is difficult to discern with epifluorescent microscopy due to the thickness of the sample and the background fluorescence from the entire root. Epifluorescent images captured with a color camera indicate that the color of fluorescence in this zone is more gold than in surrounding tissues. A previous report showed that the complex between DPBA and quercetin (Peer et al., 2001) produced gold fluorescence. The biological relevance of this accumulation is not clear because it does not appear to change localization in response to gravity or ACC treatment. The localization occurs in a physiologically relevant area for involvement in auxin transport, however.

ACC Effects on Root Basipetal Auxin Transport

Two previously described procedures were used to examine the effect of ACC treatment on IAA transport (Buer and Muday, 2004). Basipetal IAA transport was examined in living and unwounded roots by application of a cylinder of tritiated IAA at the root tip for wild type and tt4(2YY6) with and without ACC treatment because this polarity of IAA movement is linked to root gravitropism (Rashotte et al., 2000). The tt4(2YY6) mutation does increase both basipetal and acropetal IAA transport relative to wild type (Buer and Muday, 2004), but no consistent differences were found in response to ACC treatment in multiple basipetal transport assays (data not shown). Similarly, using a reporter transport assay in which IAA is applied at the tip of DR5--glucuronidase plants and the distance of the gene expression signal quantified (Buer and Muday, 2004) did not detect IAA transport differences in the presence of ACC (data not shown).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The well-characterized gravity response in Arabidopsis roots combined with the plethora of Arabidopsis mutants with altered responses to ethylene makes this an ideal system to test the hypothesis that ethylene regulates gravitropic curvature. When we elevated ethylene levels in Col roots by exposure to the ethylene precursor ACC or by limiting the diffusion of endogenously synthesized ethylene, root elongation and gravitropic curvature were suppressed in parallel. In the ethylene-insensitive mutants etr1-3 and ein2-5, root growth and gravity responses were less sensitive to the inhibitory effects of ACC. These results indicate that ACC inhibits root growth and gravitropism through similar ethylene-signaling pathways.

These experiments also tested the hypothesis that ethylene inhibition of root gravitropism requires flavonoid synthesis. In three separately isolated tt4 mutant alleles, the inhibitory effect of ACC on initial rates of curvature is lost, whereas the ACC inhibition of root elongation is largely intact. Detailed examination of the kinetics of root curvature using the Multi-ADAPT program further demonstrated that the time for initiation of bending of tt4(2YY6) is unaffected by ACC, whereas curvature initiation in Col is dramatically delayed. Therefore, we conclude that ethylene negatively regulates root gravitropism through a flavonoid-dependent pathway.

These results also reveal the presence of a positive regulatory effect of ethylene on root gravitropism, which is masked in wild-type plants due to the stronger inhibitory ethylene signal. In multiple tt4 alleles and the ein2-5 mutant, we detect a small stimulation of initiation of root gravitropism after ACC treatment. Consistent with our findings, ACC or ethylene treatment of dark-grown maize roots resulted in promotion of gravitropic orientation (Chang et al., 2004). If flavonoid expression is under the same light control in maize seedlings as in Arabidopsis seedlings (Wade et al., 2001; Muller et al., 2005), their dark-grown seedlings may mimic our tt4 mutants with an absence of flavonoid synthesis. This complex role of ethylene, with both stimulatory and inhibitory effects on root gravitropism, may explain the contradictory conclusions in the literature on the question of whether ethylene regulates gravitropism (Wheeler and Salisbury, 1981; Kaufman et al., 1985; Harrison and Pickard, 1986; Wheeler et al., 1986; Lee et al., 1990; Woltering, 1991; Kiss et al., 1999; Madlung et al., 1999). Because both ethylene and flavonoid synthesis are regulated by light, nutrients, and other environmental signals (Winkel-Shirley, 2002; Vandenbussche et al., 2003), it is likely that these contradictory results come from studies in which the synthesis of these molecules is differentially regulated. The ability to dissect these complex actions of ethylene through the use of mutants altered in ethylene and flavonoid synthesis is an important feature of this study.

A second goal of this study was to examine the effects of ACC on flavonoid accumulation to understand the mechanisms by which ethylene, flavonoids, and gravity response are interconnected. ACC had two effects on flavonoid accumulation in the root tip as judged by DPBA fluorescence intensities. DPBA fluorescence increases were detected in vertical Arabidopsis roots with increases beginning at 2 h and extending through 8 h after ACC treatment. These sustained flavonoid increases were completely absent in the etr1-3 and ein2-5 mutants, consistent with ETR1- and EIN2-dependent mechanisms controlling this flavonoid accumulation pattern. Consistent with this result, CHS gene expression is induced in a number of species by environmental factors (Winkel-Shirley, 2002), including ethylene (Ecker and Davis, 1987; Ryder et al., 1987; Schmid et al., 1990; McKhann and Hirsch, 1994). Furthermore, ACC treatment blocks the transient flavonoid accumulation in the root tip that occurs about 2 h after gravitropic stimulation (Buer and Muday, 2004; Figs. 4 and 5). These results suggest that ACC has two distinct effects on flavonoid accumulation in roots. We examined whether the flavonoid accumulation patterns were spatially distinct using confocal microscopy. After analysis of multiple samples, DPBA fluorescence increases were detected in similar locations in epidermal cells both transiently, in response to gravity, or with slower kinetics, in response to ACC treatment (Supplemental Fig. 1).

One mechanism by which ethylene may affect gravity response is through altered root structures. Gravity is sensed in a specific set of cells within the columella in roots (Blancaflor et al., 1998). Within these cells are starch statoliths that sediment and are believed to signal the lower side of the root to initiate root gravitropism (Morita and Tasaka, 2004). Several reports have indicated that morphology of maize and Arabidopsis root tips is changed in response to ethylene treatments for 24 h or longer (Guisinger and Kiss, 1999; Ponce et al., 2005). One study reported that, in Arabidopsis, ethylene results in decreased accumulation of starch (Guisinger and Kiss, 1999), whereas the other focused on developmental changes in the root cap in response to ACC treatment (Ponce et al., 2005). Although it is possible the effect of ACC is indirectly mediated by root structural changes, the previously reported developmental effects occur beyond the times of our examinations. No dramatic changes in the morphology of wild-type and tt4(2YY6) roots were detected upon examination of confocal micrographs of DPBA-stained ACC-treated and -untreated roots. Therefore, our working model does not require developmental changes at the root tip to account for the effects of ethylene on root gravitropism.

An important question remaining concerns the function of flavonoids that are produced in response to gravity and ACC. In the absence of flavonoids, root basipetal auxin transport is elevated (Buer and Muday, 2004). The flavonoid induction in response to gravity may reduce polar IAA transport so that lateral auxin asymmetries establish more readily across the root tip. The absence of flavonoids would then result in a delayed formation of auxin gradients and gravitropic bending in the tt4(2YY6) mutant (Buer and Muday, 2004). We examined the effect of ACC on root basipetal transport in two ways but were unable to obtain reproducible differences in the effect of ACC on auxin transport. Our hypothesis for this result is that, to measure auxin movement, it is necessary to apply exogenous IAA. As IAA induces flavonoid accumulation (Buer and Muday, 2004), it may overwhelm the effect of ACC on flavonoid synthesis and differences between the ACC and untreated controls are no longer significant. Therefore, although it is clear that ACC induces flavonoid synthesis in Arabidopsis roots and that flavonoids negatively regulate auxin transport (Brown et al., 2001), it is not yet clear whether the direct result of this flavonoid induction in response to ACC is to alter auxin transport.

A model summarizing our data is presented in Figure 6 . On the left are the results of previous experiments, which indicate that gravity induces a localized and transient increase in flavonoid accumulation that is linked to the asymmetric distribution of IAA needed for maximal gravity response (Buer and Muday, 2004). The right side of this model is consistent with the data reported here that indicate that ethylene reduces root gravitropism through pathways requiring EIN2 and ETR1 and flavonoid synthesis. Additionally, the sustained induction of flavonoid synthesis in response to ethylene and its ability to block the localized transient synthesis of flavonoids are present in the model. We also included results from a recent paper that indicated that ethylene treatment can induce localized IAA synthesis at the root tip through induction of anthranilate synthase gene expression (Stepanova et al., 2005), which is consistent with a previous report that auxin is required for the growth inhibition by ethylene in Arabidopsis roots (Rahman et al., 2001). The boxed regions of the model represent the areas in which causal and temporal relationships are not yet resolved and will be examined in future experiments. Finally, the bottom of the model indicates the stimulatory effect of ethylene on gravitropic response in plants with tt4 or ein2 mutations. Together, this model provides a testable summary of the complex relationships between flavonoids, auxin, and root gravitropism.

View larger version (9K): [in this window] [in a new window]   Figure 6. A model describing the interactions among flavonoids, ethylene, and root gravitropism. The causal and temporal relationships between the events in the boxes have not been resolved.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Chemicals

Triton X-100 was purchased from Fisher Scientific. Murashige and Skoog formulation salts were purchased from Caisson Labs. All other chemicals were acquired from Sigma.

Plant Material and Growth Conditions

etr1-3 in the Arabidopsis (Arabidopsis thaliana) Col background was obtained from the Arabidopsis Biological Resource Center (ABRC). ein2-5 was generously provided by Paul Larsen (Larsen and Chang, 2001). Col wild type and tt4(2YY6) were used previously (Brown et al., 2001; Buer and Muday, 2004). We also used a newly characterized Salk insertion line (SALK 020583), which is designated tt4-11, shown to have an insertion in the CHS gene (M. Ramirez and B. Winkel, personal communication), and the tt4(W85) allele, renamed tt4-1 in the Ler background (Murphy et al., 2000; Saslowsky et al., 2000). A recent analysis identified a second unlinked max4 mutation in the tt4(2YY6) line, which causes the elevated inflorescence branching previously described for tt4(2YY6) (Sorefan et al., 2003; Bennett et al., 2006). A backcrossed tt4 line without the max4 mutation was isolated and named tt4-2 (Bennett et al., 2006). We have verified that the previously reported root phenotypes of tt4(2YY6), including elevated root branching and delayed initiation of gravitropic curvature (Brown et al., 2001; Buer and Muday, 2004), are equivalent in the tt4(2YY6) and tt4-2 mutant lines and are not affected by the max4 mutation.

Seeds were sterilized by incubation for 1 min in 95% ethanol, then for 5 min in freshly prepared 20% (v/v) bleach plus 0.01% (v/v) Triton X-100, and then washed at least five times with sterile water. The sterilized seeds were sown on control plates: 0.8% (w/v) type M agar (A-4800; Sigma), 1x Murashige and Skoog nutrients (macro and micro salts: MSP0501; Caisson Labs), vitamins (1 µg mL^–1 thiamine, 1 µg mL^–1 pyridoxine HCl, and 0.5 µg mL^–1 nicotinic acid), 1.5% (w/v) Suc, 0.05% (w/v) MES, with pH adjusted to 6.0 with 1 N KOH before autoclaving. This medium has higher Murashige and Skoog and Suc concentrations that are optimal for root growth, but because this medium has been used in previous experiments in our laboratory (Rashotte et al., 2000, 2001, 2003; Brown et al., 2001; Buer and Muday, 2004) we continue to use it for consistency. The unsealed plates were placed vertically in racks and the seedlings were grown under standard light regimes (100 µmol m^–2 s^–1). All assays were performed on 5-d seedlings measured from the time of sowing. Seedlings were maintained at room temperature (23°C). All dark seedling manipulations were under a dim green safelight if required (fluorescent lights covered with green Plexiglas: ACR no. 2092; fluency rate = approximately 1 µmol m^–2 s^–1).

Treatments to Alter Ethylene Levels

Two methods were used to alter ethylene levels. Light-grown seedlings were transferred to control media or media containing a range of ACC concentrations immediately before gravity stimulation. The majority of the experiments were performed with seedlings grown on 2.5 µM ACC. This dose of ACC most closely mimicked the ethylene levels in wrapped plates (0.3 µL L^–1; Buer et al., 2003; see below), and 0.5 µL L^–1 as judged by a previous report that compared growth effects of ACC and ethylene (Larsen and Chang, 2001).

The second treatment to elevate ethylene levels was to limit diffusion of endogenously produced ethylene. For this experiment, seedlings growing on control media plates were wrapped with Parafilm, which caused ethylene to accumulate in the airspace. Seedlings were germinated on these plates to allow sufficient ethylene buildup. This previously used method resulted in accumulation of 0.3 µL L^–1 C₂H₄ (Buer et al., 2003).

Gravity Stimulation

For all experiments, seedlings were grown along the surface of the corresponding media plates in a vertical orientation in the light. Seedlings were transferred to control media or media containing a range of ACC concentrations immediately before gravitropic reorientation. The plates were reoriented 90° relative to the initial growth angle and placed in the dark to prevent phototropism. Experiments with Parafilm-wrapped plates were performed on seedlings that were germinated on the same plates for gravity stimulation to allow ethylene to accumulate in the airspace of the plate. Root elongation and curvature were photographed under low green light with a Sony digital camera, the digital images were imported into Adobe PhotoShop, and measurements were made with PhotoShop measuring tools.

Multi-ADAPT experiments were performed by transplanting seedlings to control media or media containing 2.5 µM ACC, covering the root with molten agar with or without ACC cooled to 35°C, and followed on the Multi-ADAPT computer program (Ishikawa and Evans, 1997). Image analysis began within approximately 5 min following agar set. Initial growth was vertical for approximately 2 h before reorienting the root 90° and the bending root was followed for an additional 7 to 10 h. The seedling was illuminated with an infrared light-emitting diode and the image produced was captured with a CCD camera and analyzed with Multi-ADAPT software every 60 s. We defined the end of the gravity-induced lag as occurring when the root tip remained past 80°, with 90° = horizontal. Root elongation rates were followed in 50-µm segments beginning at the root tip.

Flavonoid Fluorescence Staining and Quantification

Flavonoid accumulation was visualized in vivo by the fluorescence of flavonoid-conjugated DPBA after excitation with blue light (488 nm; Sheahan and Rechnitz, 1992; Murphy et al., 2000; Peer et al., 2001; Buer and Muday, 2004). Assays to determine the effect of ACC on DPBA fluorescence accumulation were performed by transplanting 5-d seedlings to control media or media containing 2.5 µM ACC and incubating for the required times before staining with DPBA and measuring intensities as described below. Comparisons were made to the vertically grown seedling fluorescence on control media or 2.5 µM ACC as percent of control. DPBA staining of whole seedlings was performed according to Murphy et al. (2000), except for the following changes. The staining and transfer of stained seedlings were performed in the dark as much as possible. Staining times were for 5 min using saturated (0.25% [w/v]) DPBA and 0.005% (v/v) Triton X-100. Seedlings were then washed for 5 min with 100 mM sodium phosphate buffer, pH 7.0, plus 0.005% (v/v) Triton X-100. Seedlings were mounted on slides in 50% (v/v) glycerol.

Fluorescence was achieved by excitation with fluorescein isothiocyanate filters (450–490 nm, suppression LP 515 nm) on a Zeiss Axioplan fluorescence microscope equipped with a 0.5 N.A. 20x Zeiss Plan-FLUOR objective. Wide-field epifluorescent gray-scale images of the root tip below the bright fluorescent zone located in the distal elongation zone were collected with a CCD camera (model C4742-95; Hamamatsu) on the epifluorescent microscope and exported into Image-Pro Plus software (version 4.5.1.29; Media Cybernetics). Quantification of fluorescent intensities was performed with Image-Pro Plus software. A freehand line profile was drawn from the root tip through the center of the root and the intensity profile was exported to Microsoft Excel. The intensity in the first 60 µm of the root tip was measured and statistically analyzed by comparative statistics by determining the 2nth maximum to eliminate any skewing by single points of high intensity. Every effort was made to keep exposure times equal. If exposure times were adjusted to prevent saturating the DPBA fluorescence, the resulting intensities were normalized to the longest exposure (2,000 ms; i.e. the intensity at 500 ms would be multiplied by 4 to normalize the intensity to a 2-s exposure). A minimum of 10 roots were analyzed at each time point and compared to vertically oriented, dark-grown controls with and without ACC. Each experiment was independently repeated at least three times. Seedlings incubated with ACC in the light had an increase in DPBA fluorescence at later time points (12–24 h; data not shown).

Confocal Microscopy

Confocal laser-scanning microscopy was performed on a Zeiss 510 confocal laser-scanning microscope. Excitation was with the 488-nm line of an Argon laser with a 505-nm long-pass filter set as previously described (Buer and Muday, 2004). The micrographs were acquired under identical settings to indicate unadjusted fluorescence differences to confirm the epifluorescent imaging, quantification, and image analysis.

	ACKNOWLEDGMENTS

 
We are grateful for the advice and assistance of Anita McCauley with the confocal laser-scanning microscopy and epifluorescence microscopy, and for the communication of unpublished data and sharing of seeds by Tom Bennett, Ottoline Leyser, and Brenda Winkel. Helpful comments on the manuscript from members of the Muday lab and Angus Murphy are appreciated.

Received December 15, 2005; returned for revision January 26, 2006; accepted February 5, 2006.

	FOOTNOTES

 
¹ This work was supported by the National Aeronautics and Space Agency (grant no. NAG2–1507) and with support from Wake Forest University's Science Research and Research and Publication Funds.

² Present address: Australian Research Council Centre of Excellence for Integrative Legume Research, Genomic Interactions Group, Research School of Biological Sciences, Australian National University, Sullivan's Creek Road, Acton, ACT 2601, Australia.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gloria Muday (muday{at}wfu.edu).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.075671.

^* Corresponding author; e-mail muday{at}wfu.edu; fax 336–758–6008.

	LITERATURE CITED

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