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The Arabidopsis EIN3 Binding F-Box Proteins EBF1 and EBF2 Have Distinct but Overlapping Roles in Ethylene Signaling^[W]

^a Department of Botany, University of Wisconsin, Madison, Wisconsin 53706
^b Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706

³ To whom correspondence should be addressed. E-mail vierstra{at}wisc.edu; fax 608-262-2976.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Ethylene signaling in Arabidopsis thaliana converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-Like (EIL) transcription factors to induce various responses. EIN3 BINDING F-BOX1 (EBF1) and EBF2 were recently shown to function in ethylene perception by regulating EIN3/EIL turnover. In the absence of ethylene, EIN3 and possibly other EIL proteins are targeted for ubiquitination and subsequent degradation by Cullin 1–based E3 complexes containing EBF1 and 2. Ethylene appears to block this ubiquitination, allowing EIN3/EIL levels to rise and mediate ethylene signaling. Through analysis of mutant combinations affecting accumulation of EBF1, EBF2, EIN3, and EIL1, we show that EIN3 and EIL1 are the main targets of EBF1/2. Kinetic analyses of hypocotyl growth inhibition in response to ethylene and growth recovery after removal of the hormone revealed that EBF1 and 2 have temporally distinct but overlapping roles in modulating ethylene perception. Whereas EBF1 plays the main role in air and during the initial phase of signaling, EBF2 plays a more prominent role during the latter stages of the response and the resumption of growth following ethylene removal. Through their coordinated control of EIN3/EIL1 levels, EBF1 and EBF2 fine-tune ethylene responses by repressing signaling in the absence of the hormone, dampening signaling at high hormone concentrations, and promoting a more rapid recovery after ethylene levels dissipate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The simple gaseous hydrocarbon, ethylene (C₂H₄), controls a myriad of growth and developmental processes in plants, including seedling germination and growth, leaf and petal abscission, fruit ripening, organ senescence, and stress and pathogen responses (reviewed in Schaller and Kieber, 2002). Ethylene responses are mediated through a signal transduction cascade that has been partially defined in Arabidopsis thaliana using genetic and molecular approaches (Guo and Ecker, 2004; Chen et al., 2005). The hormone is perceived by five ethylene receptors (ETHYLENE RESPONSE1 [ETR1], ETR2, ETHYLENE-INSENSITIVE4 [EIN4], ETHYLENE RESPONSE SENSOR1 [ERS1], and ERS2), which are in a complex with the Raf-like protein kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1). In the absence of ethylene, the receptors stimulate CTR1, which then inhibits downstream events that likely include a mitogen-activated protein kinase (MAPK) cascade. Binding of ethylene to the receptors represses CTR1 action. Once released from inhibition by CTR1, the MAPK cascade eventually activates EIN2 (Alonso et al., 1999), which subsequently induces the accumulation of the transcription factor EIN3 (Chao et al., 1997; Ouaked et al., 2003; Yanagisawa et al., 2003). The pathway between CTR1 and EIN3 is also modulated by the EIN5/XRN4 5'-3' exoribonuclease, but its direct RNA target(s) is currently unknown (Olmedo et al., 2006; Potushak et al., 2006).

EIN3 is a key positive switch in ethylene perception. For example, mutants in EIN3 have reduced responses to ethylene, whereas overexpression of EIN3 results in ethylene hypersensitivity or a constitutive ethylene response (Roman et al., 1995; Chao et al., 1997). It works by promoting the transcription of a variety of ETHYLENE RESPONSE FACTOR genes that ultimately direct the growth and physiological responses under ethylene control (Solano et al., 1998). During activation, EIN3 binds as a dimer to primary ethylene response DNA elements, which are 28-bp imperfect palindromes found in the promoters of various ethylene-responsive genes (Solano et al., 1998).

In addition to EIN3, Arabidopsis encodes five EIN3-Like (EIL) transcription factors, EIL1 to EIL5, that may also contribute to ethylene signaling (Chao et al., 1997; Wang et al., 2002; this work). Mutants in EIL1 have a weak ethylene insensitivity, and overexpression of EIL1 results in a constitutive ethylene response, implying that the EIL1 protein functionally overlaps with EIN3 (Chao et al., 1997). In fact, both growth kinetic and phenotypic analyses of ein3 eil1 double mutants imply that all sustained ethylene responses are generated primarily through the action of EIN3 and EIL1 (Alonso et al., 2003a; Binder et al., 2004a). The cellular roles of the remaining EIL2-5 members of the family remain unclear. The observation that EIL2 overexpression partially complements the ein3 mutant phenotype indicates that EIL2 could play a minor role in ethylene perception (Chao et al., 1997).

It is now apparent that the abundance of EIN3, and possibly the EILs, is a critical regulatory feature governing the strength of ethylene responses. Whereas EIN3 mRNA levels are unaffected by the hormone, abundance of the EIN3 protein rises dramatically upon ethylene exposure due to a strong stabilization of the protein (Guo and Ecker, 2003; Yanagisawa et al., 2003). This stabilization can be reversed by glucose, a well-known antagonist of ethylene, suggesting that multiple signals converge to regulate EIN3 breakdown (Yanagisawa et al., 2003). The observation of Yanagisawa et al. (2003) that MG132, a potent inhibitor of the 26S proteasome, stabilizes EIN3 even in the absence of ethylene first implicated the ubiquitin (Ub)/26S proteasome system in EIN3 turnover. In this system, a polymer of the Ubs is covalently attached to proteins committed to degradation using a large set of Ub-protein ligases (or E3s) for target selection (for review, see Smalle and Vierstra, 2004). More than 1300 different E3s are predicted to exist in Arabidopsis alone, suggesting that each E3 recognizes a limited set of targets through highly specific interactions (Gagne et al., 2002; Smalle and Vierstra, 2004; Gingerich et al., 2005). Once multiple Ubs are bound, the modified target is delivered to the 26S proteasome, which degrades the target but releases the Ub moieties intact for reuse.

How EIN3 is recognized for ubiquitination was recently revealed by us and others through the identification of two E3 components, EIN3 BINDING F-BOX1 (EBF1) and EBF2 proteins, that are essential for proper ethylene perception (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). Like other F-box proteins, they form SCF E3 complexes by each assembling with the core subcomplex containing the SKP1, Cullin 1, and RBX1 subunits. EBF1 and 2 also directly bind their target EIN3, using a conserved C-terminal Leu-rich repeat region, and like EIN3, EBF1/2 appear to be nuclear-localized. Importantly, disruption of either EBF1 or EBF2 increases EIN3 protein levels and induces a hypersensitivity to exogenous ethylene at both the phenotypic and molecular levels. In fact, a dramatic growth arrest phenotype is observed for confirmed ebf1 ebf2 double null mutants, which is more severe than any previously published for other ethylene signaling components (Gagne et al., 2004). The double mutants emerge slowly from their seed coat as severely stunted seedlings and fail to progress further developmentally. Other defects include a substantial increase in root hair number, enhanced anthocyanin accumulation, accelerated senescence of the cotyledons, and a failure to develop leaves, stems, and flowers. The ebf1 ebf2 seedlings have elevated EIN3 levels in the absence of exogenous ethylene, suggesting that ubiquitination of EIN3 (and possibly one or more EILs) by the corresponding SCF^EBF1/2 complexes is essential for plant growth even in the absence of the hormone (Gagne et al., 2004).

Both phenotypic and molecular analyses suggested that EBF1 and EBF2 work coordinately in ethylene signaling (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). For example, the growth of ebf1 mutants is repressed in the absence of exogenous ethylene or at low concentrations but is similar to the wild type at saturating levels, whereas the growth of ebf2 mutants is similar to the wild type at low ethylene concentrations but is markedly more compromised at saturation levels (Guo and Ecker, 2003; Gagne et al., 2004). Here, we demonstrate that EIN3 and EIL1 are the main targets of EBF1 and EBF2. We also delineate the distinct roles of these F-box proteins through the genetic analysis of ebf1 and ebf2 mutants in combination with others affecting ethylene perception and through detailed kinetic analysis of hypocotyl growth inhibition of various mutants in response to exogenous ethylene. Collectively, the data reveal that the SCF^EBF1 and SCF^EBF2 E3 complexes work in concert to fine-tune the abundance of EIN3/EIL1 in response to ambient hormone levels. The synergistic action of EBF1/2 suggests that the breakdown of other Ub/26S proteasome pathway targets also may be precisely regulated by sets of related E3s working together.

	RESULTS

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Growth Arrest by ebf1/2 Mutants Caused by a Defect in Ethylene Signaling
The striking growth arrest of ebf1 ebf2 double null mutants implied that ethylene perception in Arabidopsis is strongly enhanced by a failure to degrade EIN3 and possibly one or more members of the EIL protein family (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). However, given the feed-forward control of ethylene on its own synthesis (Schaller and Kieber, 2002), it remained possible that the growth of the mutant plants was compromised by abnormally high ethylene production in addition to a hypersensitivity to the gas. To test this possibility, we attempted to rescue loss-of-function ebf1-4 ebf2-4 seedlings (in the Wassilewskija background; Gagne et al., 2004) by growth on either L--(2-aminoethoxyvinyl)-glycine (AVG), an ethylene biosynthetic inhibitor, or silver nitrate, a potent inhibitor of perception by the family of ethylene receptors. When used at concentrations that are typically effective (Beyer, 1976; Yang and Hoffman, 1984), neither inhibitor improved the stunted growth of the double homozygous mutant (Figure 1A ). We also exposed the ebf1-4 ebf2-4 seedlings to 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene (Schaller and Kieber, 2002), in an attempt to see if the plants would respond further to the hormone. Under our growth conditions, no additional inhibitory effects of ACC were apparent, suggesting that null ebf1 ebf2 mutants are saturated for ethylene perception (Figure 1A). Finally, the ebf1-4 ebf2-4 seedlings were exposed to high concentrations of glucose (1%), which was previously shown to destabilize EIN3 (Yanagisawa et al., 2003) and thus might partially reverse the effects of the mutations if the sugar activated another degradation pathway. Likewise, glucose was without effect in the presence or absence of ACC, AVG, or silver nitrate, with the only change being a promotion of root hair growth under all conditions that included glucose (Figure 1A).

View larger version (56K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Connection of the ebf1 ebf2 Mutants to Ethylene Synthesis and Perception. (A) Development of homozygous ebf1-4 ebf2-4 seedlings in the Wassilewskija background in response to ACC (0.2 µM), AVG (50 µM), and AgNO3 (100 µM), with or without the inclusion of 2% glucose (Gluc). Seedlings were germinated and grown for 8 d under continuous light. (B) Synergy of the ebf1-3 and ebf2-3 mutations with ctr1-1. Plants were grown for 16 d on growth medium under a long-day photoperiod. ctr1-1 ebf2-3 het indicates a plant that is homozygous for the ctr1-1 allele but heterozygous for the ebf2-3 allele. Bars = 5 mm.

In the absence of ethylene, light-grown ctr1-1 seedlings grow slowly and develop epinastic leaves compared with wild-type Col-0 and homozygous ebf1-3 and ebf2-3 seedlings, which are phenotypically normal under standard growth conditions (Kieber et al., 1993; Figure 1B). We observed a similar ctr1-1–like phenotype for the double mutant of ctr1-1 with ebf1-3, indicating that the corresponding SCF^EBF1 complex does not play a major role during strong ethylene signaling. By contrast, the growth of ctr1-1 ebf2-3 seedlings was substantially arrested beyond that observed for ctr1-1 (Figure 2B ). Like ebf1-3 ebf2-3 mutants, the ctr1-1 ebf2-3 embryos emerged slowly from the seed coat upon germination and then developed into severely stunted seedlings that failed to produce an inflorescence. However, the ctr1-1 ebf2-3 seedlings were not as strongly compromised as ebf1-3 ebf2-3 seedlings; they eventually formed true leaves and a primary root and turned pale green after months of growth. An accentuated ctr1 phenotype was even observed for plants homozygous for ctr1-1 but heterozygous for ebf2-3, implying that one wild-type copy of EBF2 is insufficient to restrict the constitutive ethylene signaling induced by CTR1 inactivation (Figure 1B). This strong additive effect of ebf2-3 with ctr1-1 indicates that the corresponding SCF^EBF2 complex in particular plays a major role in modulating ethylene perception once the signaling pathway is engaged.

View larger version (31K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Description of the Arabidopsis EIN3/EIL Family and Genetic Analysis of ein3-1, eil1-3, and eil1-4 Mutants Alone and in Combination with ebf1-3 and ebf2-3. (A) Schematic representations of the EIN3 and EIL1-5 proteins. The acidic, basic, Pro-, Ser-, Gln-, or Asn-rich domains are indicated by shaded boxes. The predicted nuclear localization sequences are marked with asterisks. The amino acid (aa) sequence length of each polypeptide is indicated on the right. (B) An unrooted phylogenetic tree of the EIN3/EIL family. The bar represents a branch length equivalent to 0.1 amino acid changes per residue. (C) Diagram of the EIL1 gene. Locations of the T-DNA insertion in the eil1-3 and eil1-4 mutants are shown. Arrows indicate the primers used for RT-PCR in (D). (D) RT-PCR analysis of the eil1-3 and eil1-4 mutants. First-strand cDNA was synthesized from total seedling RNA with primer 2 and then amplified by PCR with the primers pairs 1+2. RT-PCR of the PAC1 transcript is included as a control. (E) Hypocotyl elongation of the eil1 mutants in response to ACC. Wild-type Col-0, ein3-1, eil1-3, and eil1-4 were grown for 4 d in the dark with or without 0.1 and 10 µM ACC. Each bar represents the average hypocotyl length (±SD) of at least 12 seedlings. (F) PCR genotyping of the ein3-1 and eil1-3 mutations in combination with the ebf1-3 and ebf2-3 mutations. G, gene-specific primers; T, a left border T-DNA primer used in combination with a gene-specific primer. For EIN3, a pair of gene-specific primers was used for the PCR followed by digestion of the product with HaeIII. The open and closed arrowheads identify the products from the ein3-1 and wild-type Col-0 alleles, respectively. PCR amplification of the PHYA gene is included as a control.

A functional overlap of EIN3 and EIL1 was also revealed by studies showing that EIN3 and EIL1 are the dominant members with respect to ethylene signaling (Chao et al., 1997; Alonso et al., 2003a; Guo and Ecker, 2003; Binder et al., 2004a) and by Potuschak et al. (2003), who found that EIL1, like EIN3, can be recognized by EBF1 and EBF2. Consequently, we tested whether the growth arrest of ebf1 ebf2 double mutants was caused solely by a stabilization of the EIN3 and EIL1 proteins via a phenotypic analysis of triple and quadruple mutant combinations of ebf1-3 and ebf2-3 with strong alleles affecting EIN3 and EIL1. Under the assumption that EIN3 and EIL1 are main targets of SCF^EBF1/2, their removal should synergistically alleviate the growth repression of ebf1 ebf2 double mutants. The ein3-1 mutant used here is a loss-of-function allele previously described by Chao et al. (1997) in the Col-0 ecotype. It contains a premature stop at codon 215, which substantially depresses accumulation of the EIN3 transcript and prevents translation of most of the protein (Figure 3 ; Chao et al., 1997). The eil1-3 and eil1-4 mutants are new eil1 alleles that were identified by us in the SIGNAL Arabidopsis mutant population generated with the Col-0 ecotype. The T-DNAs were inserted in the only exon for EIL1 following nucleotides 719 (from the ATG) and 1145 for eil1-3 and eil1-4, respectively (Figure 2C). RT-PCR analysis of homozygous eil1-3 and eil1-4 seedlings showed that the insertions block accumulation of the full-length EIL1 mRNA and are therefore likely to represent null alleles (Figure 2D). Similar to previously described null eil1 alleles in the Arabidopsis Landsberg erecta (Ler) and Col-0 ecotypes (Alonso et al., 2003a), homozygous eil1-3 and eil1-4 seedlings display a mild insensitivity to exogenous ethylene. At both 0.1 and 10 µM concentrations of ACC, the elongation of etiolated eil1-3 and eil1-4 hypocotyls was less repressed than wild-type Col-0 but still more sensitive than ein3-1 (Figure 2E).

View larger version (95K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. EBF1, EBF2, EIN3, and EIL1 Transcript Levels in Various Mutant and Overexpression Plants Altered in Ethylene Signaling. Total RNA was extracted from 2-week-old light-grown plants and subjected to RNA gel blot analysis with the indicated probes. The ß-Tubulin-4 (TUB4) gene probe was included to verify equal RNA loading.

RNA gel blots of 2-week-old light-grown plants also confirmed the effects of the ein3, eil1, ebf1, and ebf2 mutant combinations on the corresponding transcripts (Figure 3). For example, diminished levels of EIN3 mRNA were observed in all the mutant combinations bearing the ein3-1 allele, whereas the EIL1 transcript was not detected in any line containing either the eil1-3 or eil1-4 allele. As described previously (Gagne et al., 2004), the ebf1-3 mutation induces synthesis of a truncated EBF1 mRNA. This truncation was detected in the single ebf1-3 and triple ein3-1 ebf1-3 ebf2-3 lines as expected but was surprisingly absent in the quadruple mutant (Figure 3). Although it behaves as a null mutation, the ebf2-3 allele directs synthesis of a near full-length EBF2 transcript (Gagne et al., 2004). In addition to having reduced levels in the ebf1-3 lines, the abundance of the EBF2 mRNA was also substantially lower in the ein3-1 and eil1-3 single mutants, the ein3-1 eil1-3 double mutant, and the quadruple mutant (Figure 3). Reduced levels in the ein3/eil1 backgrounds could reflect previous observations that the transcript abundance of EBF2 is positively regulated by ethylene signaling, which is diminished in these mutant backgrounds (Potuschak et al., 2003, 2006; Gagne et al., 2004). Why the EBF2 mRNA was also not reduced in the eil1-4 background was unclear. To avoid this complication, we used the ebf1-3 allele to construct the triple and quadruple mutants.

As described previously (Gagne et al., 2004), Arabidopsis ebf1 ebf2 double null mutants displayed a strong growth arrest following germination even in the absence of ethylene, while the ebf1 and ebf2 single mutants developed normally (Figures 1B and 4A ). Likewise, the ein3-1 and eil1-3 single homozygous seedlings and the previously described ein3-1 eil1-2 double homozygous seedlings (Alonso et al., 2003a) were phenotypically normal in the absence of ethylene in accordance with the nonessential nature of ethylene perception (Figure 4A). Strikingly, when combined with the ebf1-3 ebf2-3 double mutant, either of the eil1-3 and ein3-1 mutations could partially rescue the severely stunted growth of the double mutant (Figure 4). The triple eil1-3 ebf1-3 ebf2-3 seedlings showed only a modest rescue. Unlike ebf1-3 ebf2-3 seedlings, the eil1-3 triple seedlings continued to grow and develop after germination (albeit slowly). Their cotyledons partially expanded and turned pale green following emergence from the seed coat and a stunted root developed that was densely covered with root hairs, all of which were consistent with a partially dampened ethylene response compared with the ebf1-3 ebf2-3 double mutant. Whereas the ebf1-3 ebf2-3 plants failed to develop true leaves, one or more leaf pairs eventually emerged after several weeks from the eil1-3 ebf1-3 ebf2-3 plants (Figure 4A). Unfortunately, the plants never flowered and set seed, thus forcing us to continuously select for eil1-3 triple mutant individuals from segregating populations.

View larger version (50K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Phenotype of Plants Containing Combinations of the ein3, eil1, ebf1, and ebf2 Mutations. (A) Seedlings grown for 16 d under a long-day photoperiod. The inset next to the ein3-1 ebf1-3 ebf2-3 seedling provides a direct size comparison for an ebf1-3 ebf2-3 seedling. Bars = 2 mm. (B) Mature plants grown on soil for 5 weeks under a long-day photoperiod. A ctr1-1 plant is included for comparison.

The most complete phenotypic suppression was observed for the quadruple homozygous ein3-1 eil1-3 ebf1-3 ebf2-3 combination (Figures 4A and 4B). These plants were phenotypically indistinguishable from the wild type at both the juvenile and adult stages when grown under nonstressful conditions in the absence of ethylene. For example, the quadruple mutant plants had flat, fully expanded leaves, tall inflorescences, and morphologically normal flowers that were highly fertile and without a protruding gynoecium. This full development was in stark contrast with plants double homozygous for null ebf1 ebf2 alleles that fail to generate true leaves, ordered inflorescence stems, and flowers (Gagne et al., 2004; Figure 4A) or to double homozygous ebf1 ebf2 plants containing a weak allele for EBF2, which produce supernumerary epinastic leaves, a dwarfed irregular inflorescence, and mis-shaped flowers with protruding gynoecia and reduced fecundity (ebf1-1 ebf2-1; Potuschak et al., 2003). Etiolated ein3-1 eil1-3 ebf1-3 ebf2-3 seedlings also grew normally in the absence of ethylene and exhibited none of the triple response phenotypes (i.e., exaggerated apical hook, swollen hypocotyl, and reduced root growth; Roman et al., 1995) typical of constitutive ethylene signaling (e.g., ctr1-1; Figure 5A ).

View larger version (44K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Hypocotyl Elongation of Various Ethylene-Signaling Mutant Combinations in Response to ACC. (A) Representative plants grown in the dark for 4 d with or without 10 µM ACC. (B) Graphical quantitation of hypocotyl elongation in response to various concentrations of ACC. Each bar represents the average length (±SD) of at least 25 seedlings.

One interesting observation from our ACC sensitivity studies was that seedlings defective in both EIN3 and EIL1 often had substantially unfolded apical hooks even when treated with high concentrations of ACC (Figure 5A). In some cases, the cotyledons were presented upwards without an obvious bend, which was in stark contrast with the exaggerated hooks of ctr1-1 and wild-type seedlings treated similarly. Given the importance of ethylene in forming the apical hook (Lehman et al., 1996; Li et al., 2004; Vriezen et al., 2004), its absence in the ein3-1 eil1-3 seedlings further supports a role for ethylene and suggests that EIN3 and EIL1 are together required for the asymmetric growth near the hook that forms this bend.

EBF1 and 2 Play Temporally Distinct Roles in Ethylene Signaling
The weaker differential effects of the monogenic ebf1 and ebf2 mutants to exogenous ethylene compared with the strong growth arrest of the double mutant suggested previously that the corresponding SCF^EBF1/2 E3s play distinct but overlapping roles in EIN3 and EIL1 turnover (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). The differential sensitivity of the ebf1-3 and ebf2-3 mutants in the ctr1-1 background further supported this possibility (Figure 1B). To more accurately describe their distinct roles, we applied a time-lapsed imaging system developed by Spalding and colleagues (Parks and Spalding, 1999; Folta and Spalding, 2001) and adapted by Binder et al. (2004a, 2004b) to measure the ethylene growth response of Arabidopsis hypocotyls altered in the expression of EBF1, EBF2, EIN3, and EIL1. This highly sensitive system has been exploited previously to more precisely classify ethylene perception mutants within the pathway (Binder et al., 2004a, 2004b; Potushak et al., 2006).

Prior studies of young seedlings revealed two phases of ethylene-induced growth inhibition for wild-type Col-0 hypocotyls (Binder et al., 2004a, 2004b; Figure 6 ). Phase I inhibition begins 10 min after ethylene is applied; within an additional 15 min, a new, lower, steady state growth rate is reached. This steady state is maintained for another 20 min, after which phase II inhibition is initiated, which further depresses growth within another 20 min to an even lower steady state growth rate. This phase II growth depression is maintained for many hours under continuous hormone exposure. However, if ethylene is removed during phase II, hypocotyl elongation rapidly recovers over 90 min back to the initial rate in the absence of hormone. Genetic analyses showed that both phases require EIN2 but that only phase II is dependent on EIN3 and EIL1 (Binder et al., 2004a). In the absence of EIN3 and EIL1, hypocotyls enter the phase I growth arrest but fail to enter phase II. Instead, they resume their normal growth rate, even in the presence of saturating ethylene concentrations (see below). Under the conditions used here for 2-d-old seedlings, wild-type and mutant hypocotyls all elongated in air prior to ethylene treatment at rates ranging from 0.28 to 0.47 mm h^–1 (see Supplemental Table 1 online).

View larger version (54K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Elongation Kinetics of 2-d-Old Etiolated Arabidopsis Hypocotyls in Response to Ethylene for Wild-Type Col-0 Seedlings, Seedlings Homozygous for the ebf1-3 or ebf2-3 Mutations, or EIN3-OX Seedlings. Growth rates were recorded for 1 h in air, followed by a 2-h exposure to 10 µL L–1 ethylene, and then a 5-h recovery in air. Panels on the left show the full time course. Panels on the right show an enlargement of the response immediately after introducing ethylene. The response of wild-type Col-0 seedlings (open squares) is included in each panel. Data represent the average of at least six seedlings ±SD. (A) and (B) ebf1-3. (C) and (D) ebf2-3. (E) and (F) EIN3-OX.

When the ebf1-3 and ebf2-3 single homozygous mutants were examined similarly, we found that the magnitude of the growth inhibition was similar to the wild type, indicating that neither mutant affected the final strength of the ethylene signal by itself. However, significant changes in the kinetics of the ethylene response and its recovery were observed, indicative of distinct temporal roles. ebf1-3 seedlings had a similar onset and strength of the phase I growth inhibition to the wild type, but the onset of phase II growth inhibition was markedly faster (Figure 6A). As shown in an expanded version of the kinetics (Figure 6B), ebf1-3 hypocotyls failed to pause at the intermediate plateau after phase I; instead, they continued to rapidly depress their elongation rate to that eventually reached during a normal phase II response. Following ethylene removal, the elongation rate of ebf1-3 seedlings recovered with a kinetic similar to the wild type, including the dampened oscillations. By contrast, ebf2-3 seedlings had a normal initial response to ethylene with a similar onset kinetic, phase I plateau, and final growth rate depression in phase II (Figures 6C and 6D). Unlike wild-type and ebf1-3 seedlings, their recovery after ethylene removal was substantially delayed, requiring 75 min longer than the wild type to regain the elongation rate present before ethylene exposure.

The delay in growth recovery for the ebf2-3 seedlings suggested that EBF2 is particularly important for removing excess EIN3/EIL1 that is stabilized by the ethylene signal. To test this possibility, we examined the growth kinetics of seedlings overexpressing EIN3 (EIN3-OX) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. These seedlings constitutively express a high level of EIN3 mRNA (Figure 3) and display a heightened ethylene response even in the absence of the exogenous hormone (Chao et al., 1997; Guo and Ecker, 2003). As shown in Figures 6E and 6F, the EIN3-OX seedlings responded remarkably similar to ebf2-3 seedlings. The elongation kinetics for both phase I and phase II were indistinguishable with a substantial delay in growth recovery evident upon ethylene removal. For EIN3-OX seedlings, it took 60 min longer than the wild type to reach the pretreatment growth rate after purging the gas.

To further examine the roles of EBF1 and 2, we analyzed previously described transgenic lines that separately overexpress either of the two proteins under the control of the CaMV 35S promoter (Guo and Ecker, 2003; Potuschak et al., 2006). These lines contain more of the corresponding mRNAs, are less sensitive to ethylene, and have a dampened accumulation of EIN3 after hormone treatment, the latter of which is consistent with a more effective turnover of EIN3 and EIL1. Retesting the hypocotyl elongation response of EBF1-OX and EBF2-OX seedlings in the presence of ACC confirmed their decreased ethylene sensitivity. Whereas the elongation of wild-type hypocotyls was dramatically inhibited by increasing concentrations of ACC, both overexpression lines, like the ethylene-insensitive mutant ein2-1 (Guzman and Ecker, 1990; Alonso et al., 1999), showed little or no response even at saturating concentration (10 µM) (Figure 7A ).

View larger version (31K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Growth of Etiolated Arabidopsis Hypocotyls in Response to ACC and Ethylene for Wild-Type Col-0 Seedlings or Seedlings Overexpressing Either EBF1 or EBF2. (A) Hypocotyl elongation in response to prolonged ACC treatment. Wild-type Col-0, ein2-1, EBF1-OX, and EBF2-OX seedlings were grown for 4 d in the dark with or without various concentrations of ACC. Each bar represents the average hypocotyl length (±SD) of at least 40 seedlings. (B) Growth kinetics of 2-d-old etiolated hypocotyls in response to ethylene. Growth rates for Col-0, EBF1-OX (top panel), and EBF2-OX (bottom panel) were recorded for 1 h in air, followed by a 2-h exposure to 10 µL L–1 ethylene, and then a 5-h recovery in air. Data represent the average of at least six seedlings ±SD. The response of wild-type Col-0 seedlings (open squares) is included in each panel.

The mutational analysis and the kinetic data presented here along with prior interaction data showing that both EBF1 and 2 can bind EIN3 and EIL1 (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004) are consistent with the corresponding SCF^EBF1/2 complexes playing different temporal roles but having similar targets. To further confirm that EIN3 and EIL1 are the main targets, we examined the growth kinetics during prolonged ethylene exposure of the ein3 and eil1 mutants either alone, together, or when combined with ebf1-3 and ebf2-3. (The eil1-3 ebf1-3 ebf2-3 triple mutant was not tested given its already strong growth arrest [see Figure 4A].) Under this prolonged treatment, the elongation rate of wild-type Col-0 seedlings remained strongly inhibited with only a slight recovery after 5 h. In agreement with Binder et al. (2004a), homozygous ein3-1 eil1-2 seedlings exposed continuously to ethylene exhibited a normal phase I response but lacked the phase II component, such that the elongation rate of the mutant slowly returned near to that observed before ethylene exposure (Figure 8A ). When the contributions of the EIN3 and EIL1 proteins in growth were examined separately, we found that EIN3 plays the dominant role in the phase II response (Figure 8B). Whereas the single eil1-3 mutant behaved similar to the wild type during both phase I and II growth responses, the single ein3-1 mutant failed to display a normal phase II response. However, the ein3-1 seedling did retain a modest ethylene sensitivity (as seen by a depression of the elongation rate) that appeared to strengthen after prolonged exposure (>4 h). This sensitivity could reflect the contribution of EIL1 at later times (Figure 8B).

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Growth Kinetics of 2-d-Old Etiolated Arabidopsis Hypocotyls Containing Various Mutant Combinations Affecting EIN3, EIL1, EBF1, and EBF2. Growth rates were recorded for 1 h in air, followed by a 6-h continuous exposure to 10 µL L–1 ethylene. Data represent the average of at least six seedlings ±SD. The response of wild-type Col-0 seedlings is included in (A), (C), and (D). (A) ein3-1 eil1-2 double mutant. (B) ein3-1 and eil1-3 single mutants. (C) ein3-1 eil1-3 ebf1-3 ebf2-3 quadruple mutant. (D) ein3-1 ebf1-3 ebf2-3 triple mutant.

A more complex elongation response was seen for the ein3-1 ebf1-3 ebf2-3 triple mutant (Figure 8D). The triple mutant began responding to ethylene at the same time as the wild type but once engaged did so at a faster rate. The resulting kinetics did not reveal an obvious phase I plateau with the plants appearing to directly enter the phase II response. At longer times, a slight restoration in growth was detected. While the mechanism for this accelerated response is unclear, one possibility is that EIL1 assumes a more prominent role in the absence of both EIN3 and a mechanism to remove EIN3 and EIL1 (i.e., missing the SCF^EBF1/2 E3s). Consistent with this possibility, we found that the abundance of the EIL1 transcript was substantially upregulated in the ein3-1 ebf1-3 ebf2-3 background (Figure 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Previous genetic and biochemical data by us and others revealed that the Ub/26S proteasome pathway is a central regulator of ethylene perception in Arabidopsis through its control of EIN3 (and possibly the family of related EILs) protein abundance, a transcription factor that directs a key limiting step in the ethylene response pathway (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). This regulation is achieved by SCF E3 complexes containing either of the two F-box–containing substrate recognition factors: EBF1 and EBF2. SCF^EBF1/2 presumably promote the ubiquitination and subsequent breakdown of EIN3/EILs in the absence of the hormone, but their activity is blocked in the presence of the hormone, thus allowing EIN3/EILs levels to rise and mediate ethylene responses. Here, we showed that EIN3 and EIL1 are the principal targets of SCF^EBF1/2 and that the two E3s work in a temporally distinct but largely overlapping fashion to control EIN3/EIL1 levels.

Whereas strong ebf1 ebf2 mutants accumulate high concentrations of EIN3 (and likely EIL1) and display a strong growth arrest even in the absence of ethylene (Gagne et al., 2004), sequential genetic inactivation of the EIN3 and EIL1 loci in this background progressively rescues the mutant phenotype with the quadruple ein3 eil1 ebf1 ebf2 mutant now resembling wild-type plants. Consistent with the nonessential nature of ethylene and the epistasis of EBF1 and EBF2 with respect to EIN3 and EIL1, the quadruple mutant grew normally under nonstressed conditions and was almost completely insensitive to prolonged ethylene treatments, at least with respect to etiolated seedling development. Like ein3-1 eil1-3 seedlings, the quadruple mutant still displays a transient ethylene response during hypocotyls elongation. However, this brief response, which was previously shown to require EIN2 (Binder et al., 2004a), is so short that it has little effect on overall hypocotyl length after prolonged hormone treatments.

Our analysis of triple ein3-1 ebf1-2 ebf2-3 and eil1-3 ebf1-3 ebf2-3 seedlings confirmed prior studies that showed that EIN3 plays a greater role in ethylene signaling relative to EIL1. Whereas the ebf1-3 ebf2-3 double mutant was only weakly rescued upon inactivation of EIL1, a much stronger rescue occurred upon further inactivation of EIN3. Although still dwarfed, the ein3-1 ebf1-2 ebf2-3 mutant produced a rosette of true leaves and eventually formed a cluster of inflorescences that flowered and generated viable seeds. The plants phenotypically resembled ctr1 mutants, indicating they were still responding to an apparent ethylene signal in the absence of EBF1 and EBF2. The difference in this response from the wild type likely reflects the action of stabilized EIL1. While there remains speculation that other members of the EIL family (EIL2-5) play important roles in ethylene signaling, both the complete rescue of the ebf1 ebf2 phenotype in the quadruple mutant at both the etiolated and light-grown stages in the absence of ethylene and the failure of the quadruple etiolated seedlings to retain a durable response to exogenous ethylene implies that their role(s) are minor if acting at all. However, we acknowledge that not all ethylene responses in Arabidopsis were evaluated here, leaving open the possibility that EIL2-5 selectively participate in other aspects of ethylene perception (e.g., leaf and floral abscission, and biotic and abiotic stress responses; Schaller and Kieber, 2002). They also could be responsible for the phase I growth response of hypocotyls, which is under EIN2 but not EIN3 or EIL1 control (Figure 8A; Binder et al., 2004b).

The strong phenotypic consequences of simultaneous inactivation of both EBF1 and EBF2 compared with the single mutants clearly indicates that both SCF E3 complexes play essential, overlapping roles in regulating EIN3/EIL1 protein abundance. Distinct functions were first implied from ethylene response studies, which showed only a modest aberrant phenotype for ebf1 mutants in the absence of ethylene or the presence of low hormone concentrations, while the ebf2 mutants displayed an ethylene hypersensitivity only at higher or saturating hormone concentrations (Guo and Ecker, 2003; Gagne et al., 2004). Further differences between EBF1 and EBF2 became apparent from expression studies, which showed that the EBF1 mRNA is abundant even in the absence of ethylene with its level increasing modestly in ethylene, while the EBF2 mRNA levels are lower initially but strongly upregulated upon hormone exposure (Potuschak et al., 2003; Gagne et al., 2004). The differential contributions of EBF1 and EBF2 are further supported here from analysis of double mutants with ctr1. Whereas the strong constitutive ethylene signaling generated by the ctr1-1 mutation was not accentuated upon combination with the ebf1-3 mutation, combining the ctr1-1 and ebf2-3 mutations strongly compromised seedling growth. In fact, the phenotype of these ctr1-1 ebf2-3 seedlings resembled that of ebf1 ebf2 double null mutants (Gagne et al., 2004), the strongest ethylene signaling mutants yet reported. Together, the phenotypes further support the notion that strong ethylene signaling induced by high concentrations of EIN3/EIL1 cannot be overcome by EBF1 but can be by EBF2.

Kinetic analyses of hypocotyl elongation of monogenic ebf1 and ebf2 seedlings indicate that one important distinction in EBF1 and EBF2 function is in the temporal control of EIN3/EIL1 levels. Detailed analysis of ebf1-3 seedlings after ethylene exposure showed that they enter the phase II response, which is dependent on EIN3/EIL1, faster than normal but are indistinguishable from the wild type after ethylene is removed and elongation recovers. Together with the modest upregulation of the EBF1 mRNA upon ethylene exposure and the inability of the ebf1-3 mutation to exacerbate the strong constitutive ethylene signaling generated by ctr1-1, we propose that a main role of the SCF^EBF1 complex is to constitutively target EIN3/EIL1 for degradation, thereby repressing ethylene responses at no or low hormone concentrations. Conversely, detailed kinetic analysis of ebf2-3 seedlings revealed that they are not compromised during the initial stages of ethylene signaling but are substantially delayed in growth recovery following ethylene withdrawal. This pattern was indistinguishable to that of EIN3-overexpressing plants, consistent with the notion that the SCF^EBF2 complex is more important when excess EIN3 (and likely EIL1) is present. Taken together with the data showing that the ebf2-3 mutation strongly accentuates the phenotype of the ctr1-1 mutant and that the EBF2 mRNA is more strongly upregulated by ethylene, we propose that the SCF^EBF2 complex becomes more important once ethylene signaling is engaged and during recovery after hormone withdrawal. Presumably, through the concerted action of SCF^EBF1 and SCF^EBF2, Arabidopsis can repress ethylene signaling in the absence of the hormone, dampen signal at high hormone concentrations, and more rapidly recovery after ethylene levels dissipate.

In addition to playing different temporal roles in ethylene signaling, it is also possible that EBF1 and EBF2 have substrate-specific roles, with one SCF complex recognizing EIN3 and the other recognizing EIL1. The different effects on the elongation kinetics when exposed to ethylene could then reflect the separate temporal functions of the targets and not the degradation machineries. Several observations do not favor such a scenario. First, prior interaction studies showed that EBF1 and EBF2 can both recognize EIN3 and EIL1, indicating that if substrate specificity for the F-box proteins does exist, it is minor (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). Second, plants overexpressing EBF1 or EBF2 have indistinguishable elongation kinetics after ethylene exposure, with both lines failing to enter the phase II inhibition response that requires EIN3/EIL1 protein accumulation. For example, if EIL1 was required early in the response and EIN3 was required later, a differential effect on elongation should be evident for the EBF1-OX and EBF2-OX lines that would reflect the target specificity of each SCF^EBF complex.

Based on the data reported here and elsewhere, we propose a more complete model for ethylene signaling in Arabidopsis (Figure 9 ). In the absence of ethylene, EIN3/EIL1 is constitutively synthesized and degraded. This breakdown is promoted by CTR1 and is achieved by the SCF E3–directed ubiquitination of EIN3/EIL1 using the EBF1 and 2 F-box proteins as the substrate recognition factors. Both genetic and kinetic analyses are consistent with EBF1 playing the main role in this baseline ubiquitination. The ethylene-induced repression of CTR1 blocks the ubiquitination of EIN3/EIL1 by the SCF^EBF1/2 E3s. This inhibition allows the rapid accumulation of EIN3/EIL1, which then direct downstream events in ethylene signaling. Their accumulation also activates a negative feedback loop that modestly and strongly upregulates EBF1 and EBF2 mRNA levels, respectively. Presumably this upregulation in transcript levels then increases the abundance of the corresponding E3s to prevent excess accumulation of EIN3/EIL1, which could inadvertently prolong ethylene signaling. The strong increase in SCF^EBF2 levels in particular would also help remove EIN3/EIL1 once ethylene levels dissipate so the plants can more rapidly resume normal growth. Whereas some of this upregulation likely involves increased transcription of the EBF1 and EBF2 genes, the recent discovery that the EIN5/XRN4 locus encodes a 5'-3' exoribonuclease that depresses the steady state levels of EBF1 and EBF2 mRNAs suggests that the turnover of these transcripts is also directly or indirectly regulated (Olmedo et al., 2006; Potushak et al., 2006).

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Model for the Action of EBF1 and 2 in the Ethylene Signal Transduction Pathway. In the absence of ethylene, the receptor family activates the negative regulator CTR1, which leads to inhibition of EIN2. EIN3 and EIL1 levels are kept low by selective ubiquitination of the proteins by SCFEBF1 and SCFEBF2, which induces their subsequent breakdown by the 26S proteasome. In the presence of ethylene, the receptors are inhibited, thus reducing the output of CTR1 and its subsequent inhibition of EIN2. EIN2 acts in part to directly or indirectly block the interaction of EIN3 and EIL1 with the SCF E3s containing EBF1 and EBF2. The reduction in ubiquitination allows EIN3 and EIL1 levels to rise to mediate ethylene responses. Over a slower time course, EIN2 activation also leads to an increase in EBF1 and EBF2 mRNA and presumably protein levels (shown in inset), which further dampens the accumulation of EIN3 and EIL1. Via an unknown mechanism, the exoribonuclease EIN5/XRN4 dampens the accumulation of the EBF1 and EBF2 transcripts. During ethylene signaling, EBF1 plays a special role at no or low hormone levels to maintain low basal levels of EIN3/EIL1. By contrast, EBF2 accumulates during ethylene signaling to prevent excess accumulation of EIN3/EIL1 and to remove EIN3/EIL1 after ethylene levels dissipate.

The realization that Arabidopsis encodes for almost 700 F-box proteins has placed dramatic emphasis on the importance of selective protein turnover by SCF E3s in plant growth and development (Gagne et al., 2002; Smalle and Vierstra, 2004). This large number certainly reflects in part an equally large number of targets that are subject to ubiquitination. For example, the six F-box proteins in the Arabidopsis TIR1/AFB family, either working individually or together, may be responsible for recognizing many of the 29 auxin/indole-3-acetic acid proteins that negatively regulate auxin signaling (Dharmasiri et al., 2005). Data presented here for the SCF^EBF1/2 complexes also support the notion that some E3s work in concert to degrade individual targets. In fact, the observations that glucose can promote EIN3 turnover suggest that a third E3 is involved in EIN3/EIL1 turnover that is regulated by a sugar-sensing pathway (Yanagisawa et al., 2003). However, the glucose-stimulated action of this E3, if it does exist, is insufficient to overcome the strong stabilization of EIN3 and EIL1 in ebf1 ebf2 seedlings.

Clearly, by using sets of E3s that work in temporally and spatially distinct manners, the abundance of individual targets can be more precisely controlled. While it is clear that EIN3 and EIL1 are key checkpoints in ethylene signaling, a picture is emerging that their action is primarily modulated by the EBF1 and EBF2 F-box proteins and their ability to recognize and presumably ubiquitinate these targets. Coupled with the sophisticated control of EBF1/2 mRNA levels by both the EIN5/XRN4 exoribonuclease and EIN3/EIL1 themselves (Guo and Ecker, 2003; Potuschak et al., 2003, 2006; Gagne et al., 2004), its appears that providing the appropriate levels of SCF^EBF1/2 complexes at the appropriate times are critical determinants in defining the correct degree of signaling before and after ethylene perception.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Alignments and Phylogenetic Analysis
Amino acid sequences for the five members of the EIL family (EIL1-5) were retrieved by BLAST searches against the Arabidopsis thaliana genome database (http://www.arabidopsis.org/blast/). EIN3 and EIL1-5 sequences were aligned, and an unrooted phylogenetic tree with a 1000 bootstrap replicate was generated using ClustalX PC v1.8 (Thompson et al., 1997) and TreeView PC v1.6.0 (Page, 1996). Percentage identities and similarities were calculated using MacBoxshade v2.15 (Institute of Animal Health).

Mutant and Overexpression Lines
Unless indicated, all lines were in the Arabidopsis ecotype Col-0 background. The ebf1-3, ebf1-4, ebf2-3, and ebf2-4 T-DNA insertion mutants were previously described by Gagne et al. (2004). The ebf1-3 and ebf2-3 lines were tracked by PCR using the primers 5'-CTGTGGCATTCTTCAAGC-3' and 5'-TCTTGTCAGGGATCAAGG-3' (ebf1-3) and primers 5'-CCAAAGTATTTGAGATGTCAAGGTCA-3' and 5'-CGCTTTCTTGGGAAAGTTCTGT-3' (ebf2-3) in conjunction with T-DNA left border–specific primer LBa1 (Alonso et al., 2003b). The homozygous ein3-1 eil1-2 double mutant and lines overexpressing EBF1 and 2 under the control of the CaMV 35S promoter (EBF1-OX and EBF2-OX) were as described (Alonso et al., 2003a; Guo and Ecker, 2003; Potuschak et al., 2006). The eil1-2 allele in the ein3-1 eil1-2 double mutant was originally generated by En-1 transposon insertion in the Ler background and contains a TCGA insertion at nucleotide 1145 (relative to the start codon) that is typical of an En-1 transposon footprint (Baumann et al., 1998). This mutation was introgressed in the Col-0 background, but sequencing of the closely linked erecta locus revealed this line still retains the four-point mutation found in the Ler background compared with Col-0 (Torii et al., 1996; data not shown).

The ctr1-1, ein3-1, and ein2-1 mutants were described previously (Guzman and Ecker, 1990; Kieber et al., 1993; Chao et al., 1997; Alonso et al., 2003b). The ctr1-1 diepoxybutane mutation was tracked by genomic PCR using the primers 5'-ACTCCTCAGTTTGTCTTGAAGTTTCAGGT-3' and 5'-ACTATTTAGCTTCCATTGGAAATAGGACC-3'. The products were identified by either DNA sequencing or by digestion with Tsp5091 (New England Biolabs). Whereas the 197-bp CTR1 product from the wild type was not digested with Tsp5091, the T-to-A substitution in the ctr1-1 mutant introduced a Tsp5091 site, which generated two fragments of 161 and 36 bp upon Tsp5091 cleavage. The ein3-1 ethyl methanesulfonate allele was tracked by genomic PCR using the primers 5'-GAGCAAGCTAGGAGGAAGAAATGTCTAG-3' and 5'-TTTAGGCAAACCAAGTTGGATGCCAC-3'. The products were then digested with HaeIII. Whereas the 438-bp EIN3 product from the wild type contains a HaeIII restriction site that upon digestion with HaeIII generates two fragments of 393 and 45 bp, a G-to-A substitution in the ein3-1 mutant removed this site. The glufosinate ammonium-resistant EIN3-OX line was as described (Yanagisawa et al., 2003). PCR products were detected by ethidium bromide staining following electrophoresis.

The eil1-3 and eil1-4 T-DNA insertion alleles (Col-0) were identified in the SIGNAL T-DNA collection (Salk_049679 and Salk_042179) (Alonso et al., 2003b). Both mutants were tracked by genomic PCR using the gene-specific primers 5'-TGAAACGTCTCAAGGAGCAAC-3' and 5'-ATGTTGCATTTTAGCAACCCC-3' and the T-DNA–specific left border primer LBa1. Sequencing the eil1-3 PCR product revealed that the T-DNA is inserted 719 bp downstream of the start codon with the left T-DNA border sequence in 5' orientation relative to the gene. Amplification of the PHYA gene with the primers 5'-GTCAGGCTCTAGGCCG-3' and 5'-ATTGCAATCAACTATCATCC-3' was used as a control in PCR genotyping the various mutant backgrounds. The various mutant combinations were generated by appropriate crosses, and the desired genotypes were identified by PCR testing the progeny.

RNA Isolation and Analysis
RT-PCR of eil1-3 and eil1-4 seedlings was performed on total RNA isolated by Trizol reagent (Invitrogen). First-strand cDNA was synthesized with primer 2 (5'-TACATCTTGCTGCTGCTGC-3') and then amplified by PCR with primers 1 (5'-ATGATGATGTTTAACGAGATG-3') and 2. Amplification of the PAE2 mRNA was used as a control (Downes et al., 2003). RNA gel blot analysis was performed with total RNA isolated by aurintricarboxylic acid/phenol/chloroform extraction followed by sequential lithium chloride and ethanol/potassium acetate precipitations (Hondred et al., 1999). ³²P-labeled RNA probes were synthesized by the riboprobe system using Sp6 or T7 RNA polymerase (Promega) and the EBF1, EBF2, EIN3, and EIL1 cDNAs as templates. The EBF1 and EIL1 probes encompassed the full coding region. The EBF2 and EIN3 probes contained 250 and 302 bp of internal coding sequence, respectively. The ß-TUB4 probe was as described (Smalle et al., 2002).

Plant Growth Conditions
Unless otherwise noted, plants were grown on Gamborg's B5 growth medium (Gibco BRL) at 21°C under a long-day photoperiod (16 h light/8 h dark) following a 4-d stratification of the seeds at 4°C. Effects of ACC on hypocotyl growth were measured on 4-d-old etiolated seedlings grown on agar containing half-strength Murashige and Skoog (MS) medium without sugar (Gagne et al., 2004). For inhibitor studies, ebf1-4 ebf2-4 seeds were germinated on solid half-strength MS medium, pH 5.7, with or without AVG, AgNO₃, ACC, and/or glucose.

Hypocotyl Growth Rate Measurements
Kinetic analyses of hypocotyl elongation were generated using etiolated Arabidopsis seedlings as described previously (Binder et al., 2004a, 2004b). Mutant and wild-type seeds were surface-sterilized by treatment with 70% ethanol for 30 s, placed on sterile filter paper to dry, and plated on half-strength MS medium, pH 5.7, containing 0.8% agar and B5 vitamins (100 mg mL^–1 inositol, 1 mg mL^–1 nicotinic acid, 1 mg mL^–1 pyridoxin HCl, and 10 mg mL^–1 thiamine HCl), and 5 µM AVG (supplied by Tarlochan Dhadialla, Rohm Haas, Philadelphia, PA), with no added sugar.

Seeds were stratified for 2 to 4 d at 4°C and exposed to white light for 2 to 8 h prior to being grown vertically in the dark for 2 d at 22°C. Once seedlings reached a height of 2 to 4 mm, growth rates were measured as described (Binder et al., 2004b). Following a 1-h treatment with air to establish a basal elongation rate, ethylene was introduced at a flow rate of 10 mL min^–1, giving a final concentration of 10 µL L^–1. Gas flow was maintained at 100 mL min^–1 using Side-Trak mass flow meters and controller (Sierra Instruments). Hypocotyl growth was measured from digital images that were captured every 5 min for 7 h with either a EDC-1000N CCD camera (Electrim) or an Infinity 2-1M camera (Luminera) and light provided by an infrared light emitting diode. Growth rates were calculated using custom software generated by Edgar Spalding in LabVIEW 5.0 (National Instruments) as previously described (Parks and Spalding, 1999; Folta and Spalding, 2001). All data presented represent the average of at least four seedlings from a minimum of three separate experiments. Data were normalized to the growth rate in air prior to ethylene treatment.

Accession Numbers
Arabidopsis Genome Initiative accession numbers for genes described in this article are as follows: CTR1, At5g03730; EBF1, At2g25490; EBF2, At5g25350; EIN2, At5g03280; EIN3, At3g20770; EIL1, At2g27050; EIL2, At5g21120; EIL3, At1g73730; EIL4, At5g10120; and EIL5, At5g65100.

Supplemental Data
The following material is available in the online version of this article.

Supplemental Table 1. Average Growth Rate of Etiolated Mutant Seedlings.

	Acknowledgments

 
We thank Joseph Ecker, Eric Schaller, Thomas Potuschak, Pascal Genschik, and Shuichi Yanagisawa for the various mutant lines. Technical support for kinetic measurements was provided by Tobius Zutz and Edgar Spalding. This work was supported by grants from the U.S. National Science Foundation Arabidopsis 2010 Program (MCB-0115870) and the Research Division of the University of Wisconsin College of Agriculture and Life Sciences (Hatch WIS04934) to R.D.V. and by a National Science Foundation grant (MCB-0131564) to A.B.B. This paper is dedicated to the memory of Dr. Tony Bleecker, a great friend and colleague.

	Footnotes

 
¹ These authors contributed equally to this work.

² Current address: Department of Molecular, Cellular, and Developmental Biology, 830 North University Ave., University of Michigan, Ann Arbor, MI 48109.

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.plantcell.org) is: Richard D. Vierstra (vierstra{at}wisc.edu).

^[W] Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.106.048140

Received October 11, 2006; Revision received December 8, 2006. accepted January 25, 2007.

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