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. 2013 Jun 4;36(1):88–96. doi: 10.1007/s10059-013-0097-7

Genetic Identification of a Second Site Modifier of ctr1-1 that Controls Ethylene-Responsive and Gravitropic Root Growth in Arabidopsis thaliana

Kihye Shin 1,3, Rin-A Lee 1,3, Inhye Lee 1, Sumin Lee 1, Soon Ki Park 2, Moon-Soo Soh 1,*
PMCID: PMC3887932  PMID: 23740431

Abstract

Ethylene controls myriad aspects of plant growth throughout developmental stages in higher plants. It has been well established that ethylene-responsive growth entails extensive crosstalk with other plant hormones, particularly auxin. Here, we report a genetic mutation, named 1-aminocyclopropane carboxylic acid (ACC) resistant root1-1 (are1-1) in Arabidopsis thaliana (L.) Heynh. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) encodes a Raf-related protein, functioning as an upstream negative regulator of ethylene signaling in Arabidopsis thaliana. We found that the ctr1-1, a kinase-inactive allele exhibited slightly, but significantly, longer root length, compared to ACC-treated wild-type or ctr1-3, a null allele. Our genetic studies unveiled the existence of are1-1 mutation in the ctr1-1 mutant, as a second-site modifier which confers root-specific ethylene-resistance. Based on well-characterized crosstalk between ethylene and auxin during ethylene-responsive root growth, we performed various physiological analyses. Whereas are1-1 displayed normal sensitivity to synthetic auxins, it showed modest resistance to an auxin transport inhibitor, 1-N-naphthylphthalamic acid. In addition, are1-1 mutant exhibited ectopically altered DR5:GUS activity upon ethylene-treatment. The results implicated the involvement of are1-1 in auxin-distribution, but not in auxin-biosynthesis, -uptake, or -sensitivity. In agreement, are1-1 mutant exhibited reduced gravitropic root growth and defective redistribution of DR5:GUS activity upon gravi-stimulation. Taken together with genetic and molecular analysis, our results suggest that ARE1 defines a novel locus to control ethylene-responsive root growth as well as gravitropic root growth presumably through auxin distribution in Arabidopsis thaliana.

Keywords: Arabidopsis, auxin, ctr1-1, ethylene, gravitropism

INTRODUCTION

Plant hormones regulate myriad aspects of growth and development of higher plants, including communication between cells and integration of environmental signals with the plant’s endogenous developmental programs. Intertwined, extensive crosstalk among plant hormones is currently under scrutiny and has been recognized as a molecular mechanism for fine-tuning development (Depuydt and Hardtke, 2011; Vanstraelen and Benková, 2012).

Ethylene, a gaseous plant hormone, affects various aspects of development, ranging from germination to fruit ripening (Bleecker and Kende, 2000). Most well-characterized ethylene-response is triple response, a set of developmental responses of dark-grown seedling to ethylene; inhibition of hypocotyl growth, exaggeration of the apical hook, and inhibition of root growth (Guzman and Ecker, 1990). For the past two decades, the triple response has been exploited in genetic studies in which several loci involved in ethylene responses have been identified (Ju and Chang, 2012). Upon perception of ethylene through its binding to ethylene receptors, the signal is transduced to inactivate a Raf-like protein kinase, CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) (Hua and Meyerowitz, 1998; Huang et al., 2003). The reduced CTR1 activity induces nuclear translocation of a carboxy-terminal EIN2 fragment from the endoplasmic reticulum (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012). The EIN2-mediated nuclear signaling, as yet unidentified, induces destabilization of the F-box proteins EBF1/EBF2; this destabilization relieves the repression of the transcription factors EIN3/ EIL1 (Alonso et al.,1999; Guo and Ecker 2003; Potuschak 2003; Yanagisawa et al., 2003; Yoo et al., 2008). The activated EIN3/ EIL1 transcription factors regulate transcription by binding to the ethylene binding site (EBS) element in the promoter region of target genes that mediate various developmental processes (An et al., 2010).

The downstream mode of action of ethylene entails extensive crosstalk with other plant hormones, particularly auxin, depending on the developmental context (Muday et al., 2012). Ethylene inhibition of root growth has been shown to be dependent on auxin. Genes encoding auxin biosynthetic enzymes such as WEI2/ASA1, WEI7/ASB1, TAA1, and TAR1 were transcription ally activated, elevating auxin level in the root apex of ethylene-treated plants (Stepanova et al., 2005; 2008). The increased auxin content in the root apex is basipetally distributed via auxin transporters from the root tip to the elongation zone, where cell expansion is inhibited by auxin-perception/signaling (Ruzicka et al., 2007; Stepanova et al., 2007; Swarup et al., 2007). Ethylene-auxin crosstalk is also apparent during apical hook formation. The ethylene-induced HOOKLESS (HLS) regulates auxin distribution via transcriptional regulation of the auxin biosynthetic gene TAR2 as well as the auxin-transport genes AUX1 and PIN3, resulting in asymmetric auxin distribution and hook formation (Lehman et al., 1996; Vandenbussche et al., 2010; Žádníková et al., 2010). Recently, it was also shown that ethylene signaling can inhibit lateral root formation primarily through regulation of the auxin transport system (Lewis et al., 2011).

CTR1, a Raf-related kinase has been thoroughly characterized as an upstream negative regulator of ethylene signaling. The kinase-inactive mutant allele, ctr1-1 has been instrumentally used for epistasis analysis since its identification (Huang et al., 2003; Kieber et al., 1993). Here we presented experimental evidence that the ctr1-1 mutant contained a recessive modifier, designated here as are1-1 (ACC-resistant root1-1). Our physiological analyses implicated that are1-1 is involved in auxin distribution to control ethylene-induced root growth inhibition. In agreement, the are1-1 mutant exhibited additional root growth defect, reduced gravitropic growth. Considering these findings and the results of genetic analyses, we discuss the possible function of ARE1 for auxin distribution during ethylene-mediated inhibition of root growth as well as directional root growth following stimulation by gravity.

MATERIALS AND METHODS

Plant materials and growth conditions

All Arabidopsis mutants used in this study are the Col background. The ctr1-1 (Kieber et al., 1993), ctr1-3 (Kieber et al., 1993), eir1-1 (Luschnig et al., 1998), aux1-7 (Pickett et al., 1990), DR5: GUS (Ulmasov et al., 1997), ASA1:GUS (Stepanova et al., 2005), and PIN2pro:PIN2-GFP (Xu and Scheres, 2005) have been previously described. The are1-1 mutant was isolated from F3 and F4 populations derived from crossing ctr1-1 mutant with the Col wild type. After backcrossing with the wild type at least twice, further physiological analysis was performed. The ctr1-1N lacking the are1-1 mutation was also identified by genetic crossing the ctr1-1 mutant with wild-type. The F2 population derived from the F1 heterozygote was screened to select wild-type-like seedlings on MS media. The wild-type-like seedlings were further grown to set F3 lines. Among the F3 lines, lines heterozygous for the ctr1-like phenotype on MS media (CTR1/ctr1-1) but homozygous for short roots on MS-ACC media (ARE1/ARE1) were chosen to select ctr1-1N lacking the are1-1 mutation. The ctr1-like seedlings from those lines were selected and further grown for F4 homozygous mutant lines, regarded as ctr1-1N. The double mutants including are1-1DR5: GUS, are1-1ASA1: GUS or are1-1PIN2pro:PIN2-GFP were made by crossing are1-1 with DR5: GUS, ASA1:GUS or PIN2pro: PIN2-GFP transgenic lines, respectively. To select double mutant lines, F3 lines homozygous both for the ACC-insensitive root growth phenotype and reporter activity/antibiotic resistance were selected. The presence of PIN2pro:PIN2-GFP in are1-1PIN2pro:PIN2-GFP was further confirmed by PCR-based genotyping using GFP-specific primers (Supplementary Table 1). Unless specified otherwise, stratified seeds were germinated and the seedlings grown on half-strength Murashige and Skoog (MS) medium containing 1% (w/v) sucrose (suc), solidified with 0.8% (w/v) phytoagar. 7-d-old seedlings were transferred to soil and grown to maturity in a growth room under long-day condition (16 h L / 8 h D) at 22–24°C.

Growth response assays

Seeds were sown on MS-suc media containing various concentrations of ACC, 2,4-D, NAA or NPA (all from Sigma). After irradiation with white light for 12 h to promote germination, the plates were incubated for the indicated times under appropriate light conditions. For treatment of gaseous ethylene, ethylene gas was provided by decomposition of ethephon (Sigma) in buffered solution, as described (Zhang and Wen, 2010). The plates were kept in air-tight chamber (2 L) containing beaker with or without ethephon (0.083 μmol) in 100 ml disodium hydrogen phosphate buffer (Na2HPO3, 5 mM), which is estimated to provide 1 μl/L ethylene gas (Zhang and Wen, 2010). Hypocotyl and root length were measured for at least 15 seedlings. ImageJ software (http://rsbweb.nih.gov/ij/) was used for measurement of root or hypocotyl length and curvature. Student’s t-test with paired two-tailed distribution was performed with Microsoft Excel. For the root gravitropism assay, the seeds were sown onto MS-suc media solidified with 1.2% (w/v) phytoagar and vertically grown in darkness or under continuous light for 5 days. For the root-tip re-orientation assay, 5-d-old horizontally light-grown seedlings were transferred and aligned to new plates containing MS media supplemented with 0.8% (w/v) phytagel (Sigma). The plates were rotated 90° and kept in darkness until they were photographed. The angle of bending in response to gravity was measured for at least 70 seedlings.

GUS staining

Seedlings were grown on MS-suc medium with or without ACC (1 μM). After fixation with pre-chilled 90% acetone for 20 min, the seedlings were washed with staining buffer (50 mM phosphate buffer, pH 7.2; 0.5 mM potassium ferro/ferricyanide, and 0.2% Triton X-100) and incubated in fresh staining solution containing 2 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc; Duchefa Biochemie). After incubation at 37°C for the indicated times, seedlings were taken for photograph after clearing with a series of ethanol (25–80%–95%), followed by formalin-acetic-alcohol (FAA) solution (50 ml ethanol, 5 ml glacial acetic acid, 10 ml formaldehyde [37–40%], 35 ml distilled water). To detect DR5:GUS activity after gravi-stimulation, the 4 days old light-grown seedlings, vertically grown on MS-suc media solidified with 0.8% phytagel, were transferred to new plate. After adaptation for 1 h, the plates were rotated at 90° and further incubated for 6 h under light. Then the seedlings were taken for fixation, followed by GUS incubation for 2 h.

Lugol staining and confocal microscopy

Lugol staining was performed with primary root tips using Lugol solution (Sigma), as described (Willemsen et al., 1998). For imaging of propidium iodide-stained roots, 5-d-old seedling roots were stained with 10 μM of propidium iodide for 30 s. After brief washing with distilled water, images were obtained with a Leica TCS SP5 confocal microscopy. The helium/neon laser (543 nm) was used for excitation and emission was detected at 590–620 nm.

Chromosomal mapping and sequencing analysis

The chromosomal location of are1-1 mutation was determined by recombination-based genetic mapping with simple sequence length polymorphism markers, as described (Lukowitz et al., 2000). F2 seedlings derived from crossing are1-1 with Ler wild type were screened ACC-resistant root phenotypes in the presence of 1 μM ACC. The selected mutant-like seedlings were subject to genomic DNA extraction by the cetyltrimethy-lammonium bromide method for genotyping. Molecular markers were designed based on Col/Ler polymorphism (TAIR, www.arabidosis.org). Primers used for mapping are listed in Supplementary Table 1. For sequencing analysis of PIN2 in are1-1 mutant, genomic DNA of PIN2 covering promoter region (−213 bps from the start codon) and 3′-UTR region (+469 bps from the stop codon), was amplified from are1-1 mutant by PCR reactions. Subsequent sequencing and BLAST analysis indicated no sequence alterations in the PIN2 gene. The primers used are listed in Supplementary Table 1.

Expression analysis

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed with total RNA extracted from roots of 7-d-old mutant or wild-type seedlings that had been horizontally grown on MS supplemented with 0.8% (w/v) phytoagar. After treatment of DNase I, 1 μg total RNA was used for cDNA synthesis using Superscript II reverse transcriptase (Invitrogen) with oligo (dT)18 primer. The 10-fold diluted cDNA was used for qRT-PCR analysis with Eco real-time PCR system using the Quantimix SYBR kit (Illumina). The reactions were performed in triplicate for each run with experimental duplicates. The comparative (Δ ΔCT) method was used to evaluate the relative quantities of each amplified product in the samples, normalized against PP2A (At1g13320) as an internal control (Schmittgen and Livak, 2008). Primers used are listed in Supplementary Table 1.

RESULTS

Identification of are1-1 from the ctr1-1 mutant

ctr1-1 is a kinase-inactive allele of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) gene, which encodes a Raf-related kinase, a repressor of ethylene signaling (Huang et al., 2003; Kieber et al., 1993). For the past two decades, ctr1-1 mutant has been used in numerous studies for molecular genetic dissection of ethylene downstream signaling pathway. In the course of our genetic interaction analysis using the ctr1-1 mutant, un-expectedly, we observed that the root length of ctr1-1 mutants was slightly, but significantly, longer than that of wild-type plants grown in the presence of ACC, a biosynthetic precursor of ethylene (Figs. 1A and 1B). Moreover, although the kinase activity of CTR1 was shown to be critical for its function (Huang et al., 2003; Yoo et al., 2008), the ctr1-1 exhibited longer root phenotype, compared to a nonsense allele, ctr1-3 (Figs. 1A and 1B). These findings led us to hypothesize the presence of genetic modifier(s) in the ctr1-1 mutant. To test this hypothesis, we performed genetic analysis. After crossing of ctr1-1 plants with wild-type plants, the resulting F1 seedlings were tested for sensitivity to ACC. All of the F1 plants were normally sensitive to ACC, displaying shortened root length. The F1 plants were further grown and self-pollinated to set F2 seeds. The resulting F2 seedlings examined for the ACC-induced triple response. When grown on MS media supplemented with 1 μM ACC, a small but significant fraction of the F2 plants had longer roots than the wild-type plants, indicating the presence of linked genetic modifier(s) (Fig. 1C). The seedlings with ACC-resistant root growth were chosen for F3 analysis. From the individual F3 lines, we identified homozygous mutant lines for ACC-resistant root growth, designated here as ACC-resistant root1-1 (are1-1). Further genetic analysis was performed with the single are1-1 mutant. The results showed that are1-1 is recessive, monogenic, and displays a Mendelian segregation pattern (Fig. 1C). To verify the existence of the are1-1 mutation in the original ctr1-1 mutant background, we performed a complementation test between the are1-1 single mutant and the original ctr1-1 mutant. The F1 heterozygote seedlings exhibited ACC-insensitive root growth, confirming that original ctr1-1 mutant carried are1-1 as a second-site mutation (Fig. 1C). For phenotypic comparison, we also isolated a ctr1-1 allele that is free from the are1-1 allele, designated as ctr1-1new (ctr1-1N). The ctr1-1N allele exhibited more shortened root than ctr1-1 did. It also was notable that root length of ctr1-1N was comparable to that of ctr1-3, a null allele (Figs. 1A and 1B). Collectively, these genetic analyses led us to conclude that a mutation, designated as ACC-resistant root 1-1 (are1-1), existed in the ctr1-1 mutant as a second-site modifier.

Fig. 1.

Fig. 1.

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Identification of the are1-1 mutant from the ctr1-1 allele. (A) Representative morphology of wild type (WT) and mutant seedlings. The seedlings were grown on half-strength MS-suc media without (MS) or with 10 μM ACC for 5 days under light condition. Scale, 5 mm. (B) Average values of root lengths from seedlings grown as in (A) (n > 15). Error bars indicate standard deviations. WT, wild type. Asterisk indicates significant differences between the ACC-treated wild type (WT) and ctr1-1 plants based on the Student’s t-test (p-value < 0.01). (C) Genetic analysis. Phenotype scoring was performed with 6-d-old seedlings that had been grown on half-strength MS-suc media containing 10 μM ACC. aχ2 was calculated based on the hypothesis of single recessive Mendelian inheritance (3:1), bχ2 was calculated based on the expected segregation ratio of two unlinked recessive epistatic mutations (13:3).

When grown in the presence of exogenous ACC, dark-grown wild-type seedlings exhibited the so-called triple response: shortened hypocotyls, exaggerated apical hooks, and shortened roots (Guzman and Ecker, 1990). As reported (Kieber et al., 1993), the ctr1-1 exhibited the constitutive triple response phenotype in the absence of exogenous ACC. In comparison to the original ctr1-1 allele, ctr1-1N had shorter roots on MS medium, irrespective of the presence of ACC (Figs. 2A and 2B). However, there is no discernible difference between ctr1-1 and ctr1-1N allele in regard to apical hook formation or hypocotyl growth. In accordance, are1-1 mutation reduced ACC-induced root growth inhibition, but not ACC-induced apical hook exaggeration or hypocotyl growth inhibition, compared to wild-type. ACC dose-response analyses revealed that ethylene-insensitivity of are1-1 mutant roots was as severe as that of aux1-7 or eir1-1 mutants, other root-specific ethylene-insensitive mutants with effects to auxin transport, over a range of ACC concentrations (Fig. 2C). To further confirm that are1-1 mutation reduced ethylene-mediated root growth inhibition, the root growth of are1-1 was compared with wild type after treatment with gaseous ethylene. The results showed that are1-1 caused ethylene-insensitivity in regard to root growth inhibition (Fig. 2D). On the contrary to root growth, other ethylene-dependent developmental aspects, including leaf growth inhibition and leaf senescence, appeared to be normal in are1-1 mutant plants (Fig. 2E and unpublished observation). Based on these results, we concluded that are1-1 mutation abrogated ethylene downstream signaling that controls a subset of ethylene-mediated processes, including inhibition of root growth.

Fig. 2.

Fig. 2.

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Ethylene-responsive seedling growth. (A) Representative morphology of wild type (WT), are1-1, ctr1-1, and ctr1-1N mutant seedlings. The seedlings were grown on half-strength MS-suc media without (−) or with 10 μM ACC (+) in darkness for 4 days. Scale, 5 mm. (B) Average values of root (left) and hypocotyl lengths (right) from seedlings grown as in (A) (n > 15). Error bars indicate standard deviations. (C) ACC dose-response analysis. The seedlings were grown on half-strength MS-suc media containing the indicated concentrations of ACC for 5 days under continuous light. Each value represents average of the relative change in root growth over the untreated control, obtained from at least 15 seedlings. Absolute mean values of root elongation on hormone free media were 10.81 mm for wild-type, 11.01 mm for are1-1 mutant, 13.65 mm eir1-1 mutant, 13.71 mm for aux1-7 mutant. Error bars indicate standard deviations. (D) Ethylene-responsive seedling growth. The seedlings were grown on half-strength MS media in air-tight chamber, equipped with beaker containing buffer alone (control) or 0.083 μmol ethephon in buffer (ethephon). Upper, Representative morphology of 5-d-old seedlings. Scale; 5 mm. WT, wild type; Lower, Average values of root length from at least 15 seedlings grown as Upper. Error bars indicate standard deviations. (E) ACC-mediated inhibition of leaf growth. The seeds were sown on half-strength MS-suc media containing various concentrations of ACC, and further grown under continuous light condition for 7 days. Representative seedlings were taken for photograph. Scale, 5 mm; WT, wild type.

Auxin-related phenotypes of the are1-1 mutant

It has been well characterized that ethylene inhibits root growth via activation of auxin biosynthesis at the root apex and subsequent auxin redistribution to elongation zone (Ruzicka et al., 2007; Stepanova et al., 2005; 2008; Swarup et al., 2007). Several auxin biosynthetic genes, including ASA1/WEI2 have been characterized to be ethylene-inducible. We tested whether are1-1 is impaired in ethylene-mediated transcriptional regulation of an auxin biosynthetic gene, ASA1/WEI2 using ASA1: GUS reporter line (Stepanova et al., 2005). As reported, ASA1: GUS activity was increased at the root apex upon ACC treatment. Under the same condition, are1-1 exhibited similar ethylene-inducible ASA1:GUS activity, compared to wild type (Supplementary Fig. 1). The results imply that the ethylene-signaling for transcriptional up-regulation of ASA1 was not affected by are1-1 mutation.

Next, we asked if are1-1 impaired sensitivity to exogenous auxin or auxin-transport inhibitor. To test this possibility, we carried out dose-response analysis of the synthetic auxins 2,4-D and NAA, and the auxin transport inhibitor, NPA. The are1-1 mutant was normally sensitive to the exogenously applied synthetic auxins, but displayed modest resistance to NPA in regard to root growth inhibition compared to wild-type plants (Figs. 3A–3C). The results indicate that auxin-uptake or -sensitivity was not affected by are1-1 mutation.

Fig. 3.

Fig. 3.

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Auxin-responsive phenotypes of are1-1 plants. (A–C) Dose-response analysis of plants to 2,4-D (A), NAA (B), and NPA (C). The seeds of wild-type and are1-1 mutant were sown on half-strength MS-suc media containing various concentrations of the indicated auxin-related chemicals. After growth for 5 days under continuous light condition, average length of roots was obtained from at least 15 seedlings. Each value represents relative value of the root over the untreated control. Absolute mean values of root elongation on hormone free media were 7.92 mm for wild-type and 8.34 mm for are1-1 mutant. Error bars indicate standard deviations. Asterisk indicates significant differences between the wild type (WT) and are1-1 plants based on the student’s t-test (p-value < 0.05). (D) Ethylene-responsive DR5:GUS expression. GUS activity was analyzed with seedlings that were grown on half-strength MS media supplemented with or without ACC (1 μM) for 4 days. Plants were stained for GUS for 2 h and representative seedlings were photographed. Scale, 100 μm.

The altered sensitivity of the are1-1 mutant to NPA, but not to synthetic auxins, raised the possibility that are1-1 is involved in auxin transport/distribution. To assess the possibility, we monitored auxin distribution indirectly with auxin-responsive DR5: GUS reporter activity. After generation of the double-mutant are1-1DR5:GUS transgene by genetic crossing, we examined the reporter activity with or without treatment of ACC. In the absence of ACC, DR5:GUS activity was observed in the columella cells of the wild-type root apex, as reported (Sabatini et al., 1999). In the are1-1 mutant background, it was found to be ectopically expressed in the lateral root cap region (Fig. 3D). ACC treatment caused increased expression of DR5:GUS activity in vascular tissue as well as the lateral root cap region of roots of wild-type plants, as reported (Stepanova et al., 2005; Swarup et al., 2007). In contrast, DR5:GUS activity in the are1-1 mutant did not respond to ACC treatment. These results, together, imply that the are1-1 mutation might alter auxin distribution around the root apex.

Reduced gravitropic root growth of the are1-1 mutant

Auxin redistribution has a prominent role in gravitropic root growth as well as ethylene-induced inhibition of root growth (Grieneisen et al., 2007; Kleine-Vehn et al., 2010). We next investigated whether the are1-1 mutation affected root gravitropism. When seedlings were grown vertically in the dark, hypocotyls of wild-type seedlings grew upward (negative gravitropism) and wild-type roots grew in downward (positive gravitropism). Under the same conditions, the are1-1 mutant roots grew in more random directions whereas the are1-1 mutant hypocotyls showed normal negative gravitropism (Fig. 4A). To better characterize the root gravitropic growth, we performed a gravitropic re-orientation assay. As shown in Fig. 4B, after re-orientation of gravity, the roots of the are1-1 mutant plants did not re-establish positive gravitropism compared to wild-type, and some roots of mutant plants even grew upward after re-orientation. Thus, the are1-1 mutation appears to reduce the positive gravitropic responses of roots.

Fig. 4.

Fig. 4.

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Impaired gravitropic root growth of are1-1 mutant. (A) Gravitropic root growth. For gravitropism assays, seedlings were vertically grown for 7 days under light (upper) or in darkness (lower) on half-strength MS-suc media solidified with 1.6% (w/v) phytoagar. Representative morphology of wild type (WT) and are1-1 mutant seedlings are shown. The asterisks denote the roots that penetrated the medium. (B) Gravitropic re-orientation assay. The 5-d-old seedlings were transferred onto square plates containing half-strength MS-suc media solidified with 0.8% (w/v) phytagel. After reorientation at 90° clockwise, the plates were kept in darkness for 20 h. The black bars represent the percentage of seedlings with the indicated root tip orientation. The curvature of each root was assigned to one of twelve 30° sectors (n > 70). (C) DR5:GUS expression after gravi-stimulation. The vertically grown 4-d-old seedlings were transferred to new plate containing half-strength MS-suc media solidified with 0.8% (w/v) phytagel. After reorientation at 90° clockwise, the plates were kept in light condition for 6 h. After fixation, the seedlings were stained for GUS for 2 h and representative seedlings were photographed. Scale, 100 μm.

It has been well characterized that gravitropic bending of root is accompanied by asymmetric auxin distribution (Kleine-Vehn et al., 2010). Next, we examined whether are1-1 mutation is impaired in auxin redistribution during gravitropic growth by monitoring DR5:GUS activity after gravi-stimulation. As reported (Rasotte et al., 2001), wild type root exhibited increased DR5:GUS activity at the lower side after gravi-stimulation (Fig. 4C). In contrast, are1-1 root did not display asymmetric DR5: GUS activity, as wild type did. The results indicated that are1-1 mutant failed to establish asymmetric auxin distribution after gravi-stimulation. Our observation that are1-1 mutant retained normal cellular context as well as root growth rate ruled out the possibility that the altered gravitropic response of are1-1 is due to abnormality of structural cellular context in the root (Supplementary Fig. 2).

Chromosomal mapping and expression analysis

Several components including auxin transporters, AUX1 and PIN2, have been identified in Arabidopsis to control gravitropic growth as well as ethylene-responsive root growth. Complementation analysis between are1-1 and eir1-1/pin2 or aux1-7 implied that are1-1 is not allelic to eir1-1 or aux1-7, as we observed wild-type segregant among F2 population from each genetic crossing (data not shown). To find the chromosomal location of are1-1 mutation, we performed genetic mapping of ARE1. From the segregating F2 population derived from crossing are1-1 with Ler wild type, seedlings with long roots when grown in 10 μM ACC were selected and subject to genotyping with molecular markers. Recombination frequency was determined for each molecular marker. The results showed that ARE1 locus is present on the lower arm of chromosome 5, between Arabidopsis Genome Initiative (AGI) At5g57050 and At5g57320 (Fig. 5A). Given the possibility that are1-1 is allelic to a known gene, we searched potential candidate genes for the are1-1 mutation (Supplementary Table 2). We found that there are no known genes to affect ACC-induced root growth or root gravitropism within the mapped region of ARE1, except the PIN2 gene (AGI, At5g57090), encoding an auxin efflux carrier. Although the observed phenotypic alterations of are1-1 were quite similar to those of loss-of-function mutation of PIN2, we found no sequence alterations in the coding region of PIN2 gene in are1-1 mutant, consistent with our complementation test. To test if ARE1 affect gene expression of PIN2, we performed expression analysis. The results indicated that the transcript level of PIN2 in are1-1 mutant was not reduced, compared to those in wild type (Fig. 5B). Further, we performed transgenic complementation by introgressing PIN2pro:PIN2-GFP transgene, which was verified to be functional (Xu and Scheres, 2005), into are1-1 mutant. As shown in Fig. 5C, the are1-1PIN2pro:PIN2-GFP double mutant exhibited ACC-resistant root growth phenotype, as are1-1 mutant did, indicating that transgenic PIN2pro:PIN2-GFP could not complement are1-1 mutation. Thus, based on these genetic and molecular analyses, we conclude that ARE1 defines a novel locus for ethylene-responsiveness as well as gravitropism of root in Arabidopsis thaliana.

Fig. 5.

Fig. 5.

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Chromosomal mapping, expression analysis and transgenic complementation. (A) Chromosomal mapping of ARE1. The mapping position of the ARE1 locus is shown along with the actual number of recombinants. (B) Real-time RT-PCR analysis of PIN2 expression. Total RNA was extracted from roots of 7-d-old wild-type (WT) and are1-1 mutant seedlings. One microgram of total RNA was subject to RT-PCR analysis. The relative expression levels of the PIN2 gene of wild-type and are1-1 mutant are presented after normalization against that of the PP2A gene. Data represent the mean of experimental triplicates. Error bars indicate standard deviations. (C) Transgenic complementation analysis. Upper, Representative seedling morphology of wild type(WT), are1-1, PIN2pro: PIN2-GFP, and are1-1 PIN2pro:PIN2 GFP double mutant. The seedlings were grown on MS-suc media containing ACC (1 μM) under continuous light condition for 4 days. Scale, 5 mm. Lower, Genomic PCR results with primers for GFP transgene.

DISCUSSION

Identification of are1-1, as a second-site modifier of the ctr1-1 allele

In this study, we identified the are1-1 mutation from the ctr1-1 mutant, which exists as a second-site modifier in the ctr1-1 mutant allele. Based on our genetic and physiological analyses, we suggest that the presence of are1-1 mutation reduces the effect ctr1-1 on root growth. The ctr1-1N allele, isolated in this study free from are1-1 mutation, exhibited more shortened root growth, compared to ctr1-1 mutant (Figs. 1 and 2). Moreover, the root growth phenotype of ctr1-1N mutant was found to be as severe as that of ctr1-3, a null allele (Kieber et al., 1993). These results are in good agreement with the hypothesis that protein kinase activity of CTR1 protein is critical for its function to repress ethylene signaling (Huang et al., 2003; Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012; Yoo et al., 2008). It was notable that the are1-1 mutation only partially suppressed the short root phenotype of the ctr1-1 mutant. Likewise, the are1-1 mutant displayed shortened root length upon high doses of ACC (Fig. 2C). These subtle effects of are1-1 mutation on the ctr1-induced root growth might make it difficult to identify the are1-1 mutation in the ctr1-1 allele from previous studies. It is noteworthy that the ACC-resistance of are1-1 was apparently specific to root growth. We observed no discernible differences between wild-type and are1-1 mutant plants in other ethylene-responsive developmental aspects, including hypocotyl growth inhibition, apical hook formation, and leaf growth (Figs. 2A–2E). The normal phenotypes of the are1-1 mutant in the aerial part imply that ARE1 does not function in the shoot system. Alternatively, there may exist functionally redundant genes that compensate for the are1-1 mutation in aerial organs.

Role of ARE1 in auxin-mediated processes

It has been extensively characterized that ethylene-induced root growth is dependent on auxin metabolism/transport/ signaling (He et al., 2011; Ruzicka et al., 2007; Stepanova et al., 2005; Swarup et al., 2007). Our physiological analyses implicated that ARE1 is involved in the auxin-ethylene crosstalk, rather than in root-specific ethylene-signaling. Our observation that ASA1:GUS reporter activity was normally activated by ACC in are1-1 mutant suggest that are1-1 may not abrogate ethylene-signaling for transcriptional induction of ASA1/WEI2, encoding an auxin biosynthetic enzyme (Supplementary Fig. 1). Our results that are1-1 mutant retained normal sensitivity to two auxinic compounds, 2,4-D and NAA (Figs. 3A and 3B) precluded the possibility that are1-1 mutation impaired auxin-uptake or -sensitivity. On the contrary, auxin-responsive DR5:GUS reporter line exhibited an ectopic expression pattern in the are1-1 mutant, regardless of ACC treatment, unlike the wild-type DR5:GUS reporter (Fig. 3D). The ectopic DR5:GUS activity in the are1-1 mutant at the lateral root cap region reflects that basipetal auxin redistribution was reduced in are1-1 mutant. The modest resistance of are1-1 mutant to an auxin transport inhibitor, NPA supports the notion that are1-1 might change auxin distribution (Fig. 3C). Moreover, besides ethylene-insensitive root growth phenotype, are1-1 mutant roots had altered directional growth responses to gravity (Figs. 4A and 4B), which requires spatio-temporal dynamic changes of auxin flow and subsequent signaling at root apex (Abas et al., 2006; Lewis et al., 2007; Kleine-Vehn et al., 2010; Rahman et al., 2010; Sedbrook et al., 1998). In consistent, it was found that are1-1 failed to establish asymmetric auxin distribution, monitored by DR5: GUS activity, upon gravi-stimulation (Fig. 4C). Taken together, these results imply that ARE1 might function in auxin distribution to mediate ethylene-induced root growth inhibition as well as directional root growth.

It remains to be determined how ARE1 is involved in auxin distribution. Although a number of loci that control auxin transport/ distribution have been characterized (reviewed in Rosquete et al., 2012), no known loci within the mapped region of ARE1 have been identified to affect auxin distribution, except PIN2 (Fig. 5A). Our genetic and transgenic complementation analyses precluded the possibility that are1-1 is an allele of PIN2 (Fig 5C and data not shown), indicating that ARE1 defines a novel genetic component. Moreover, expression analysis showed that are1-1 mutation did not affect the transcript level of PIN2 (Fig 5B). However, it is noteworthy that the pleiotropic effects of are1-1 are reminiscent of pin2 loss-of-function mutant (Chen et al., 1998; Luschnig et al., 1998; Muller et al., 1998; Stepanova et al., 2007; Swarup et al., 2007; Utsuno et al., 1998), suggestive of close functional relationship between ARE1 and PIN2. It will be of great interest to test whether ARE1 is involved in posttranscriptional regulation of PIN2 that includes cell-type specific polar localization, stability, endocytosis and activity (Ganguly et al., 2012; Kleine-Vehn and Friml, 2008). An alternative possible mode of action of are1-1 is that it might change the cell-type-specific expression of auxin transporters other than PIN2. As exemplified by the finding that ectopic PIN1 overexpression in the root epidermis can phenocopy pin2 mutants (Ruzicka et al., 2007), are1-1 mutation might affect auxin distribution by ectopic expression of other types of auxin transporters (Zažímalová et al., 2010). Obviously, the possibility is also left open that are1-1 mutation affected other processes for auxin distribution, including auxin intracellular compartmentation, auxin conjugation, and transport of metabolic precursor(s) of auxin (Peer et al., 2011; Rosquete et al., 2012). Further studies on the polar auxin transport processes in are1-1 mutant would provide important clues to clarify how the are1-1 mutation altered auxin distribution at the root apex. Undoubtedly, our ongoing map-based cloning of ARE1 would shed light on the molecular mechanism by which ARE1 controls auxin distribution for ethylene-dependent root growth inhibition as well as gravitropic root growth in Arabidopsis thaliana.

Supplementary Material

Acknowledgments

We thank ABRC, NASC, Drs. Malcolm Bennett and Stephen Deslauriers for providing mutant/transgenic seeds used in this study. We also thank Edgar Spalding for critical comments on the present manuscript. This work was partly supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0070016, 2009-0027318), and the Next-Generation Biogreen 21 program (Plant Molecular Breeding Center No. PJ008137) funded by the Rural Development Administration.

Note:

Supplementary information is available on the Molecules and Cells website (www.molcells.org).

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