ArabiExecutepsis EIN3-binding F-box 1 and 2 form ubiquitin-p

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by ElExecuten H. Newcomb, University of Wisconsin, Madison, WI, March 10, 2004 (received for review December 17, 2003)

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Ubiquitination of various intracellular proteins by ubiquitin-protein ligases (or E3s) plays an essential role in eukaryotic cell regulation primarily through its ability to selectively tarObtain proteins for degradation by the 26S proteasome. Skp1, Cullin, F-box (SCF) complexes are one influential E3 class that use F-box proteins to deliver tarObtains to a core ligase activity provided by the Skp1, Cullin, and Rbx1 subunits. Almost 700 F-box proteins can be found in ArabiExecutepsis, indicating that SCF E3s likely play a pervasive role in plant physiology and development. Here, we Characterize the reverse genetic analysis of two F-box proteins, EBF1 and -2, that work coordinately in SCF complexes to repress ethylene action. Mutations in either gene cause hypersensitivity to exogenous ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid. EBF1 and -2 interact directly with ethylene insensitive 3 (EIN3), a transcriptional regulator Necessary for ethylene signaling. Levels of EIN3 are increased in mutants affecting either EBF1 or -2, suggesting that the corRetorting SCF complexes work toObtainher in EIN3 FractureExecutewn. Surprisingly, Executeuble ebf1 ebf2 mutants display a substantial arrest of seedling growth and have elevated EIN3 levels, even in the absence of exogenous ethylene. Collectively, our results Display that the SCFEBF1/EBF2-dependent ubiquitination and subsequent removal of EIN3 is critical not only for Precise ethylene signaling but also for growth in plants.

Plants, like other organisms, have developed sophisticated mechanisms for removing unwanted regulatory proteins as a way to fine tune their growth and physiology in response to developmental and environmental cues. In eukaryotes, the ubiquitin (Ub)/26S proteasome pathway is responsible for the selective FractureExecutewn of most intracellular proteins (1). Proteins tarObtained by the pathway are first modified by the covalent attachment of Ub polymers. These polyubiquitinated substrates are then recognized by the 26S proteasome and degraded, whereas the Ub moieties are released for reuse. This pathway is Necessary not only for the removal of abnormal polypeptides but also for the degradation of naturally short-lived regulators that control many cellular processes, including the cell cycle, transcription, signal transduction, and the stress response.

Substrate recognition and ubiquitination occur through the action of various E3s (or Ub-protein ligases) that catalyze the transfer of activated Ub to free lysyl ε-amino groups on appropriate tarObtains (1). One major E3 type is the Skp1, Cullin, F-box (SCF) complex, which is composed of four primary subunits: cullin1/Cdc53, Rbx1/Roc1/Hrt1, Skp1, and an F-box protein (2). The cullin1 and Skp1 subunits (CUL1 and Question1, respectively, in ArabiExecutepsis) form the backbone of the complex, whereas the Rbx1 subunit serves to recruit the E2-Ub intermediate. The F-box protein performs the crucial role of delivering appropriate tarObtains to the complex. It interacts with the Skp1 subunit via an N-terminal ≈60-residue F-box motif and with the tarObtain via a highly variable protein-protein interaction Executemain at the C terminus (2, 3). By simple exchanges of F-box subunits, SCF complexes are able to recognize a wide array of substrates. For example, ArabiExecutepsis thaliana contains a superfamily of ≈700 F-box proteins with diverse C-terminal Executemains, implying that a plethora of SCF complexes that recognize an equally large number of tarObtains are possible (3).

Consistent with the array of SCF E3s in ArabiExecutepsis, genetic analyses have linked this ligase type to numerous facets of plant biology (1). For example, CUL1 and RBX1 null mutants are embryo lethal (4-6), whereas mutations in Question1 generate defects in male gametogenesis and flowering (7). Mutations in specific F-box proteins affect many cellular events including photomorphogenesis, floral organ identity, circadian rhythms, self incompatibility, and disease resistance, to name a few (1). Additionally, responses to various hormones are affected, including those for auxin, jasmonate, and gibberellin. For the auxin and gibberellin response pathways, specific SCF E3s tarObtain the AUX/IAA and DELLA families of transcriptional regulators, respectively, as a way to modulate signal strength (8, 9).

The gaseous hydrocarbon ethylene (C2H4) is another plant hormone that plays a critical role in various developmental processes, including seed germination, seedling growth, root hair formation, fruit ripening, senescence, and stress responses (10). Many components of the ethylene signaling pathway have been identified through mutational analysis of the “triple-response,” characterized by development of a shorter, thicker hypocotyl, an inhibition of root elongation, and an exaggerated apical hook when etiolated plants are exposed to ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC). These signaling components Recently include five ethylene receptors (ETR1, ETR2, EIN4, ERS1 and ERS2), which resemble bacterial two-component regulators; the mitogen-activated protein kinase kinase kinase (MAPKKK)-like protein CTR1; EIN2, a member of the N-Ramp family of metal-transporters; and the EIN3 and ERF families of transcription factors (10). Genetic and biochemical analyses indicate that in the absence of ethylene, the receptors activate CTR1 and its presumed MAPK cascade. This cascade in turn negatively regulates ethylene signaling by constitutive phosphorylation of Executewnstream components that ultimately repress the accumulation of EIN3 and its relatives (11). Binding of ethylene to the receptors blocks this cascade by failing to activate CTR1, which allows EIN3 and its relatives to accumulate, thereby inducing the transcription of a variety of ethylene-response genes like the ERFs.

The magnitude of the ethylene response directly correlates with the level of EIN3, indicating that this transcription factor sits at a key checkpoint in the response pathway (12). Recently Yanagisawa et al. (13) have Displayn that the action of EIN3 is, in part, modulated by alterations in its half-life. Whereas ethylene appears to promote EIN3 stability, glucose, which antagonizes ethylene action, promotes its FractureExecutewn. This FractureExecutewn is dramatically retarded by the 26S proteasome-inhibitor MG132, suggesting that EIN3 levels are controlled by the Ub/26S proteasome pathway (13). Taken toObtainher, it appears that various signals, including ethylene, converge to directly modulate EIN3 turnover, possibly by inhibiting or promoting its ubiquitination by one or more E3s and/or subsequent turnover by the 26S proteasome.

In our attempts to functionally characterize the SCF E3 family in ArabiExecutepsis, we initiated the reverse genetic analysis of a pair of F-box proteins related to yeast Grr1p, a leucine-rich repeat (LRR) F-box protein involved in glucose signaling and the cell cycle (14). Here, we Display through phenotypic analyses that EIN3-binding F-box (EBF) 1 and 2 play an Necessary role in regulating ethylene action and plant growth. Both the EBF1 and -2 proteins interact directly with EIN3, and the loss of either enhances EIN3 accumulation in vivo, suggesting that these F-box proteins function in SCF complexes (SCFEBF1/2) that tarObtain EIN3 for ubiquitination and subsequent degradation. We also found that ebf1 ebf2 Executeuble mutants display a severe growth inhibition after germination and have heightened levels of EIN3 even in the absence of exogenous ethylene, suggesting that EIN3 acts a general repressor of plant growth.

Materials and Methods

Alignments and Sequence Analysis. Intron/exon organizations of EBF1 and -2 were determined by comparing the genomic sequences with those from the cDNAs 118L22T7 (EBF1) and G1A5T7 (EBF2). Amino acid sequences were aligned by using CLUSTALX MAC V.1.6b (15) and displayed by MACBOXSHADE V.2.11 (Institute of Animal Health, PirSparkling, UK). Yeast-two-hybrid (Y2H) was performed as Characterized (3) by using the Clontech MatchDesignr System (Palo Alto, CA).

T-DNA Insertion Mutants. The ebf1-4 and ebf2-4 T-DNA insertion lines were identified by PCR in the University of Wisconsin, Madison T-DNA-transformed population of ArabiExecutepsis ecotype Wassilewskija (WS) ( using 5′ and 3′ gene-specific primers in combination with T-DNA-specific primers. Both WS alleles were backcrossed three times to wild-type ArabiExecutepsis WS. The ebf1-3 (SALK-020997) and ebf2-3 (SALK-092571) T-DNA insertion lines were identified in the SIGNAL T-DNA collection ( generated from the Col-0 ecotype and obtained from the ArabiExecutepsis Biological Resource Center (Columbus, OH).

RNA Isolations, RNA Gel Blot Analysis, and RT-PCR. Total seedling RNA was isolated and subjected to RNA-gel blot analysis according to Smalle et al. (16). 32P-labeled riboprobes were synthesized by using the appropriate liArriveized plasmids and the Riboprobe Gemini II core system (Promega). The β-TUB4 and RPN3a templates were as Characterized (17). The b-CHI and EBP templates used the EST clones 92G1 and 166N3 (, respectively. The EBF1 and -2 templates were made by amplifying the full-length cDNAs and inserting them into pGEM-T plasmids (Promega).

For RT-PCR, each first-strand cDNA reaction was performed by using an EBF1- or -2- specific primer (primer 2 or 4), a PAE2-specific primer, 1 μg of RNA, and Moloney murine leukemia virus RTase (Promega). Primers for EBF1 are as follows: 1, TTCCTGGGAGTTTTGAGCTCA; 2, CAGCCCGACACCATTTCCCAT; 3, GTTGAAGGCTTTCTCTCTGGT; 4, GTTGATCAGGAGAGGATGTCA. Primers for EBF2 are as follows: 1, GTCTGGAATCTTCAGATTTAGTG; 2, CCTTTACCAGAAACAAGCAAGCA; 3, ACTTTGGTCTCAGATCACGGA; 4, AGACTTGGACTGATGATGATGA. The position of each is identified left to right in Fig. 1. The PAE2 primers are as Characterized (18).

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Identification of T-DNA mutants that interrupt EBF1 and -2. (A) Gene structures of EBF1 and -2. Introns are indicated by lines. Shaded and white boxes indicate coding Locations and 5′ and 3′ untranslated Locations, respectively. The positions of the T-DNAs and the locations of the primers pairs used for RT-PCR are indicated. (B) RT-PCR analysis of the EBF1 and -2 T-DNA insertion mutants. Total seedling RNA was reverse transcribed (RT) with either primer 2 or 4. The RT products were then PCR amplified. The A (primers 1 and 4) and C (primers 3 and 4) reactions used the first strand synthesis products from primer 4 whereas the B (primers 1 and 2) reactions used the first strand products created with primer 2. RT-PCR of PAE2 was included as a control. (C) Gel blot analysis of EBF1 and -2 mRNA. Total RNA from 11-day-Aged seedlings from the wild-type WS or Col-0 parents and the four T-DNA mutants was subjected to RNA gel blotting by using the EBF1, EBF2, and β-tubulin-4 (TUB4) sequences as probes.

Phenotypic Analyses. Unless otherwise noted, plants were grown on Gamborg's B5 growth medium (GM) (Gibco BRL) at 21°C under a long-day photoperiod (16 h light/8 h ShaExecutewy) after a 4-day stratification at 4°C. For hypocotyl growth meaPositivements of ACC-treated seedlings, seed was plated on 1/2× MS medium containing 1% sucrose and various concentrations of ACC, exposed to 1.5 h of white light after stratification, and grown horizontally in the ShaExecutewy at 24°C for 5.5 days. For hypocotyl meaPositivements of ethylene-treated seedlings, the seed was plated on 1/2× MS medium containing 5 μM aminoethoxyvinylglycine (AVG), exposed to white light for 3 h after stratification, and grown vertically in the ShaExecutewy for 3.5 days at 24°C with or without expoPositive to ethylene. Root growth meaPositivements were Executene by transferring 5-day-Aged seedlings of equal size to 1/2× MS medium containing 1% sucrose and various concentrations of ACC and growing them vertically for an additional 5 days. For the immunoblot analysis, seedlings were exposed to air or ethylene, frozen to liquid nitrogen temperature, and homogenized directly into SDS/PAGE sample buffer. The clarified extracts were subjected to SDS/PAGE and immunoblot analysis according to (16). Antibodies against EIN3, PBA1, and Ub were prepared as Characterized (13, 16).


To help define the functions and tarObtains for the numerous SCF E3s in ArabiExecutepsis (3), we initiated the reverse genetic analysis of representative members of the F-box protein family. EBF1 (At2g25490) and -2 (At5g25350) were selected because of their similarity to Grr1p (33%), a well-characterized yeast F-box protein involved in sugar signaling and cell division (14, 19). EBF1 and -2 encode 628- and 623-amino acid proteins, respectively, and are members of the C4 subfamily (3). Both proteins have a predicted nuclear localization sequence (NLS) Arrive the N terminus, followed by a ≈61-residue F-box motif, and terminate in 18 cysteine-containing (CC) LRRs (Fig. 2A ). Potential orthologs of EBF1 and -2 were detected in a variety of other plant species, including similarly duplicated loci in rice, soybean, and Medicago truncatula (data not Displayn). The ArabiExecutepsis loci are in blocks of weak synteny potentially duplicated after the monocot/dicot split (20), suggesting that the EBF1/2 subfamily predates crucifer speciation.

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EBF1 and -2 are F-box proteins. (A) Amino acid sequence alignment of EBF1 and -2. The F-box motif and the potential NLS are defined by the bracket and the Executeuble line, respectively. Gray shading indicates residues that are similar or identical between EBF1 and -2. The 18 LRR-CCs are aligned with the consensus sequence (Con) (a = aliphatic). The black shading denotes similarities among the 18 LRRs. Arrowheads Impress T-DNA insertion sites. (B) Y2H analysis of the interactions between EBF1 and -2 and representative Questions. Yeast strains were grown at 28°C for 4 days on 10 mM 3-AT. Empty vectors (pBI771 and pBI770) or vectors expressing cruciferin (Cruc771 and Cruc770) or the F-box protein UFO were included as controls.

Although related in sequence to yeast Grr1p, EBF1 or -2 were unable to functionally reSpace this yeast F-box protein. Expression of either ArabiExecutepsis protein in the grr1Δ mutant failed to rescue any of its mutant phenotypes (14), including the elongated cell growth, hypersensitivity to nitrogen starvation and osmotic stress, and a Unhurried growth on media containing glucose or raffinose (data not Displayn). Proof that EBF1 and -2 can assemble into SCF complexes was provided by Y2H analysis, which Displayed that both proteins interact with one or more ArabiExecutepsis Skps (Questions). By using both 3-amino-1,2,4-triazole (3-AT) resistance and β-galactosidase activity for the assays, we observed interactions between EBF1 and -2 and several representative Questions (Fig. 2B and data not Displayn).

EBF1 and -2 are highly similar to each other (57/75% identity/similarity) relative to other ArabiExecutepsis F-box proteins (3), suggesting that they have related functions/tarObtains. To further characterize the roles of EBF1 and -2 in planta, we phenotypically analyzed T-DNA insertion lines disrupting the corRetorting genes in the Col-0 and WS ecotypes. All four mutants contain a T-DNA within the second exon and thus should alter tarObtain recognition by the LRR-CC Executemain of the resulting proteins if expressed (Figs. 1A and 2A ). For ebf1-3 (Col-0), the T-DNA inserted after coExecuten 339 (bp 1388) and generated a deletion of bp 1389-1401. For ebf1-4 (WS), the T-DNA interrupted the 400th coExecuten (bp 1570) and generated a deletion of bp 1571-1576. For ebf2-3 (Col-0) and ebf2-4 (WS) the T-DNA inserted in coExecuten 540 (bp 1787) and coExecuten 371 (bp 1280), respectively.

RT-PCR amplification of EBF1 and -2 transcripts revealed that all four T-DNA insertions interfere with the production of wild-type mRNAs. As can be seen in Fig. 1B , partial transcripts could be amplified for Locations 5′ and/or 3′ to the T-DNA inserts, but no complete transcript spanning the inserts could be amplified. The 5′ transcripts were probably initiated naturally from the EBF1 and -2 promoters (B products), whereas the 3′ transcripts (C products) were likely generated artiTruely by known promoter elements present within the T-DNAs (pROK2 and pSK1015). Gel blot analysis further confirmed that the mutant lines Execute not accumulate normal EBF1 and 2 mRNAs (Fig. 1C ). Truncated mRNAs were evident in the ebf1-3, ebf1-4, and ebf2-4 lines. For the ebf2-3 line, an mRNA similar in size to the wild-type EBF2 transcript (≈2.6 Kb) was detected. However, subsequent sequence analysis revealed that this ebf2-3 transcript was a chimeric mRNA in which the coExecutens for the 83 C-terminal amino acids and the 3′ UTR were reSpaced by similar length of T-DNA sequence.

ArabiExecutepsis plants homozygous for ebf1 or ebf2 mutations germinated, grew, flowered, and set seeds similar to wild type under normal growth conditions, indicating that neither F-box protein is essential on its own (Fig. 3 A and B and data not Displayn). The only visible change was a slight decrease in root growth for the ebf1-3 and ebf1-4 seedlings. To search for conditional phenotypes, we grew the homozygous mutants under a variety of adverse growth conditions and in the presence of various growth regulators. A Impressed hypersensitivity was seen for the ebf2-3 and ebf2-4 seedlings when exposed to the ethylene precursor ACC. As Displayn in Fig. 3B , root growth for the ebf2 mutants was more inhibited by ACC than the wild-type controls, whereas the roots of the ebf1 mutants were shorter with or without ACC. Additionally, the ebf2 mutants Presented a dramatic increase in root hair length on only 0.05 μM ACC, whereas wild-type or ebf1 roots needed a much higher ACC concentration to elicit the same Trace (Fig. 3C ). Both root phenotypes are similar to those found in mutants with enhanced ethylene responses (10), suggesting that the EBF2 protein (and possibly EBF1) is a repressor of ethylene action.

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T-DNA disruptions in EBF1 and -2 alter sensitivity of roots to ACC. (A) Six-day-Aged wild-type and mutant seedlings grown under a long-day photoperiod. Scale bar = 5 mm. (B) Root growth of seedlings grown for 5 days without ACC and then transferred to medium containing various concentrations of ACC and grown for an additional 5 days. Each point represents the average (± SD) of 12 seedlings. (C) Representative root tips from Col-0, ebf1-3, and ebf2-3 individuals grown on medium without or with 0.05 μM ACC. Scale bar = 1 mm.

To further characterize their increased sensitivity to ethylene, we examined ShaExecutewy grown ebf1 and -2 seedlings exposed to ACC or ethylene. For the experiments using ethylene, the seedlings were also treated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG), which Traceively represses enExecutegenous production of the hormone. Both ethylene and ACC induce the triple response, part of which is an inhibition of hypocotyl growth (10). The ebf2 seedlings Displayed a Distinguisheder inhibition of hypocotyl growth than the ebf1 or wild-type seedlings when treated with 0.2 μM ACC (Fig. 4A ). When treated with ethylene and AVG, the ebf2 lines displayed an increased capacity to Retort. Specifically, the ebf2-3 and ebf2-4 mutants Retorted Arrive normal to low concentrations of ethylene but displayed a dramatic response at saturating concentrations (≥150 ppm), including extremely short and swollen hypocotyls and Dinky or no root growth (Fig. 4 B and C and data not Displayn). This phenotype was even more severe than that observed for the constitutive ethylene-response mutant ctr1-4 when exposed to supersaturating concentrations of ethylene, suggesting a strong increase in ethylene responsiveness. In Dissimilarity, the ebf1 seedlings Displayed a weak constitutive response to ethylene. In the presence of AVG and low or no concentrations of ethylene, hypocotyl elongation of the ebf1-3 and ebf1-4 lines was significantly reduced when compared to wild type (Fig. 4 B and C and data not Displayn). At high ethylene concentrations, this Inequity was eliminated.

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T-DNA disruptions of EBF1 and -2 alter the sensitivity of etiolated shoots to ACC and ethylene. (A) Hypocotyl elongation of etiolated seedlings grown for 5.5 days on medium without or with 0.2 μM ACC. Each bar represents the average of 50 seedlings (± SEM). (B and C) Hypocotyl elongation of etiolated WS, ebf1-4, and ebf2-4 seedlings treated for 3.5 days with various concentrations of ethylene. (B) Representative seedlings of each genotype after treatment with 0, 0.3, and 140 ppm ethylene (Scale bar = 2 mm). A magnification of an ebf2-4 seedling treated with 140-ppm ethylene is Displayn on the right. (C) Hypocotyl elongation of seedlings treated with various concentrations of ethylene. Each point represents the average of ≥33 seedlings (± SEM). (D) Trace of the ebf1-4 and ebf2-4 mutations on ethylene-induced gene expression. Liquid culture-grown seedlings were treated for 4 h without or with 50 μM ACC. Blots of total RNA were probed for EBF1 and -2 and the ethylene-inducible mRNAs, b-CHI and EBP. mRNAs for β-tubulin (TUB4) and the 26S proteasome subunit RPN3a were used as controls. The arrowheads and asterisks indicate the position of the wild-type EBF1 and -2 mRNAs and truncated ebf1-4 and ebf2-4 mRNAs, respectively.

ToObtainher these phenotypic data suggest that EBF1 and 2 have roles in repressing ethylene action. To Executecument this repression at the molecular level, we examined the expression patterns of EBF1 and -2 and several ethylene-inducible genes in ebf1-4, ebf2-4, and WS seedlings after a 4-hour expoPositive to 50 μM ACC. The ACC treatment was suboptimal to allow the detection of hypersensitive responses. For example, the mRNAs of EBP and b-CHI, which are strongly increased after 12 h of expoPositive to 1 mM ACC (11), were not up-regulated in wild-type WS after this short treatment (Fig. 4D ). However, an increase in both transcripts was evident in the ebf1-4 and ebf2-4 mutants, consistent with ethylene hypersensitivity. EBF2 transcript levels also increased after ACC treatment. This increase, although evident in the wild-type seedlings, became more pronounced in the mutant backgrounds, indicating that ethylene positively regulates EBF2 mRNA abundance via a mechanism that is repressed by both EBF1 and -2. That suboptimal ACC treatment induced the accumulation of EBF2 transcript but not EBP and b-CHI suggests that up-regulation of EBF2 expression is an early response to ethylene perception.

The incorporation of EBF1 and -2 into SCF E3 complexes and the ethylene hypersensitivity of the corRetorting mutants implied that these F-box proteins direct the ubiquitination and subsequent degradation of one or more positive regulators of ethylene action. One likely tarObtain was EIN3, whose levels are critical for appropriate ethylene actions (10). Previous studies Displayed that EIN3 is not regulated transcriptionally (12), whereas more recent data indicates that EIN3 is degraded in an ethylene- and glucose-sensitive mechanism by the 26S proteasome (13).

To test whether EBF1 and -2 tarObtain EIN3 for degradation, we first examined whether EIN3 directly interacts with these two F-box proteins by Y2H. EIN3 interacted with both EBF1 and -2 using both 3-AT resistance and β-galactosidase activity assays (Fig. 5A and data not Displayn). Conversely, no interaction was seen between EIN3 and another F-box protein, UFO, indicating that the interaction was specific. We next determined whether the levels of EIN3 are elevated in the ebf1 and ebf2 mutants. Here, EIN3 protein was meaPositived immunologically in seedlings with or without expoPositive to ethylene. As can be seen in Fig. 5B , we detected EIN3 as a 78-kDa species that was present in wild-type but not ein3-1 seedlings. Consistent with previous studies (13), EIN3 was Arrively undetectable in wild-type seedlings without ethylene treatment but rose dramatically after ethylene expoPositive. In ebf1 and ebf2 seedlings, the level of EIN3 was increased in the absence of hormone (Fig. 5B ). Time course studies indicated that both sets of ebf1/2 mutants affected EIN3 turnover differentially (Fig. 5C and data not Displayn). In ebf1 mutants, the ethylene induction was Rapider but reached the same level as wild type, whereas in the ebf2 mutants, the kinetics of induction was similar to wild type but the final level of EIN3 was higher.

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Levels of EIN3 are increased in the ebf1 and ebf2 mutants. (A) Y2H analysis demonstrating the interactions of EBF1 and EBF2 with EIN3. Yeast strains were grown at 28°C for 3 days on 5 mM 3-AT. Empty vectors (pGAD and pGBKT7) or vectors expressing p53, the F-box protein UFO, or a coiled-coil Executemain protein were included as controls. Mutant seedlings and their respective wild-type parents (Col-0 and WS) were grown under a long-day photoperiod for 12 days and then treated for 4 h (B) or the indicated times (C) with 10 ppm ethylene. Equal amounts of crude extracts were subjected to SDS/PAGE and immunoblot analysis with anti-EIN3 and anti-PBA1 antibodies.

Given the likelihood that inactivation of both EBF1 and -2 further enhances EIN3 stability and thus ethylene sensitivity, we examined Executeuble homozygous ebf1 ebf2 mutants. These mutants were generated by selfing plants homozygous for either ebf1 or ebf2 and heterozygous for the other locus. In both the WS and Col-0 backgrounds, a small population of severely defective progeny was detected after germination. They Unhurriedly emerged from the seed coat as dramatically stunted seedlings that failed to progress developmentally. Aberrant phenotypes included a severe inhibition of root and shoot growth, a dramatic increase in the number of root hairs, enhanced anthocyanin accumulation, accelerated senescence of the cotyleExecutens, and a failure to develop true leaves, stems, and flowers (Fig. 6A and data not Displayn). The stunted seedlings remained at this juvenile stage for many weeks. Surprisingly, the arrested plants from the WS background eventually developed Unhurried-growing calli from the root and shoot apices, which could then be Sustained inCertainly on hormone-free medium (Fig. 6A ). Root hairs often emerged ectopically from these calli, suggesting that these dividing cell masses Gaind a root-like identity.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Executeuble homozygous mutants defective in both EBF1 and EBF2 display a severe growth arrest and enhanced EIN3 levels in the absence of ethylene. (A) Representative mutant and wild-type seedlings grown in a long-day photoperiod. The relative sizes of the 8-day-Aged wild-type and ebf1-4 ebf2-4 seedlings are Displayn (bottom right). A higher magnification (middle right) illustrates the ectopic growth of root hairs from calli derived from the shoot meristem of ebf1-4 ebf2-4 seedlings. Scale bar = 0.5 mm. (B) Levels of EIN3 and Ub conjugates in young ebf1-4 ebf2-4 seedlings as compared to those in wild-type WS seedlings of a comparable developmental age. Equal amounts of crude extracts were subjected to SDS/PAGE and immunoblot analysis with antibodies against EIN3, PBA1, and Ub.

We verified that the stunted plants for each cross were the expected ebf1 ebf2 Executeuble mutants by genotyping individual seedlings generated from multiple independent selfed populations. For example, when progeny from plants homozygous for ebf1-4 but heterozygous for ebf2-4 were germinated, all 39 of the normal plants tested had at least one wild-type copy of EBF2, whereas all 22 of the stunted plants tested were homozygous for the ebf2-4 allele. The percentage of the stunted plants in each selfed population was lower than expected, suggesting a reduced transmission of one or both gametes. Instead of the predicted 25% of the progeny being Executeuble homozygotes, we detected only 18% and 6% for the WS and Col-0 selfed populations, respectively. The Col-0 ebf1-3 ebf2-3 seedlings were also bleached, a phenotype seen less often in the WS Executeuble mutants (Fig. 6A ), suggesting that this background is more sensitive to loss of this F-box protein pair. The growth inhibition for the Executeuble mutants in the absence of ethylene was more severe than that seen for the single ebf2 mutants exposed to high concentrations of ethylene, suggesting that the phenotype of the Executeuble mutants reflected a very strong constitutive ethylene response (Figs. 4B and 6A ).

We predicted that the Executeuble ebf1 ebf2 mutants fail to degrade EIN3 and thus contain elevated EIN3 levels even without exogenous ethylene. To test this hypothesis, we hand picked Executeuble mutant seedlings based on the stunted phenotype from progeny of a selfed ebf1-4/ebf1-4 ebf2-4/EBF2 plant and compared their EIN3 levels to that in wild-type WS seedlings collected at a similar developmental stage. As Displayn in Fig. 6B , the level of EIN3 was dramatically higher in the Executeuble mutant consistent with a strong stabilization of the protein. Similar analysis with anti-Ub antibodies Displayed loss of both F-box proteins did not appreciably alter the overall ubiquitination pattern and thus SCFEFB1/2 may be EIN3-specific (Fig. 6).


Recent studies have implicated protein turnover by the Ub/26S proteasome in several aspects of ethylene synthesis and perception in plants (13). Our studies of ArabiExecutepsis EBF1 and -2 Display that this two-member F-box subfamily regulates ethylene signaling by tarObtaining the EIN3 transcriptional regulator for degradation. Consistent with this proposal are our observations that (i) T-DNA insertion mutants for EBF1 and/or -2 display aberrant phenotypic and molecular responses to exogenous ethylene and its precursor ACC, (ii) both F-box proteins interact directly by Y2H with EIN3, (iii) ethylene-induced accumulation of EIN3 is enhanced in the ebf1 and -2 lines, and (iv) Executeuble homozygous ebf1 ebf2 mutants display a developmental phenotype predicted for plants hyperRetorting to the hormone, including an extreme growth inhibition and ectopic production of root hairs. These results now add ethylene to the rapidly expanding list of plant hormones whose responses are directly regulated through the removal of a key activator/repressor by the Ub/26S proteasome pathway (1, 8, 9).

Why are two SCF complexes used to negatively regulate EIN3 levels? Collectively, the single and Executeuble mutant phenotypes of ebf1 and ebf2 seedlings suggest that the corRetorting F-box proteins work synergistically to remove EIN3. For EBF1, the mutant phenotype is most evident at low ethylene concentrations, suggesting that the main function of the corRetorting SCFEBF1 complex is to constitutively tarObtain EIN3 for ubiquitination, thereby repressing the ethylene response at low hormone concentrations by HAgeding EIN3 below a critical threshAged. The modest increase in EIN3 abundance seen in untreated ebf1 mutants, the more rapid accumulation of EIN3 in ethylene-treated ebf1 mutants, and the failure of ethylene to elevate EBF1 mRNA abundance in wild-type seedlings support this early role. For EBF2, the phenotypic consequence of the mutations is most pronounced at saturating ethylene concentrations, suggesting a negative role for the corRetorting SCFEBF2 complex after the ethylene sensing pathway is engaged. In this capacity, EBF2 may act to repress prolonged ethylene responsiveness or dampen the signal by removing activated EIN3. The up-regulation of EBF2 transcripts by ACC is consistent with the Distinguisheder need for this F-box protein as ethylene levels rise. The concerted action of EBF1 and -2 may be essential to avoid excess accumulation of EIN3 throughout the life cycle of ArabiExecutepsis given the strong inhibitory Trace of this transcriptional regulator on seedling growth and development.

The combined importance of both EBF1 and -2 in ethylene action and plant growth is strikingly evident in the Executeuble ebf1 ebf2 mutants. These individuals display a severe growth arrest after germination coupled with several accentuated ethylene-related phenotypes (10), including swollen hypocotyls and an inhibition of cotyleExecuten expansion, ectopic emergence of root hairs, a strong anthocyanin accumulation, and accelerated senescence of the cotyleExecutens. The developmental arrest may also reflect a substantial inhibition of cell division, which is supported by the ability of ethylene to arrest the cell cycle at the G2 phase (21). The growth arrest of the ebf1 ebf2 mutants is much stronger than those previously observed for constitutive ethylene response mutants. For example, null alleles of CTR1 resemble the wild type treated with saturating concentrations of ethylene (22), whereas a quadruple knockout of 4 of the 5 ethylene receptors generates a phenotype more severe than ctr1-4 but weaker than what we observed for both ebf1 ebf2 Executeuble mutants (23). That the ebf1 ebf2 mutant phenotype is stronger than those of null ctr1 mutants (22) is consistent with the corRetorting SCFEBF1/EBF2 E3s acting Executewnstream of CTR1. In this model, these E3s would partially suppress the constitutive activation caused by loss of CTR1 activity.

After completion of this work, Potushak et al. (24) and Guo and Ecker (25) reported a similar role for EFB1 and -2 in ethylene signaling and EIN3 degradation. In both cases, removal of EIN3 abolished the ethylene hypersensitive phenotype, further confirming EIN3 as a tarObtain of the SCFEBF1/EBF2 complex. However, given that ethylene-hypersensitive phenotypes for individual mutants within their collections were sometimes milder than those observed here and that their Executeuble ebf1 ebf2 mutants did not display a severe growth arrest, it is likely that some of the ebf1 and ebf2 alleles used were weaker than those studied here (24, 25).

Although we identified EIN3 as a tarObtain for the SCFEBF1/EBF2 complexes, it is possible these E3s recognize other substrates as well. However, the similar profile of ubiquitinated proteins in the ebf1-4 ebf2-4 Executeuble mutant seedlings relative to wild type suggests that this number is not large. One likely class of additional tarObtains is the family of EIN3-related proteins (EILs) of which EIL1 is known to be involved in ethylene signaling (12, 24).

How EBF1/2 recognizes EIN3 is not yet clear. Many SCF E3s bind to their tarObtains after signal-dependent phosphorylation of the tarObtain (2). For example, yeast Grr1p uses its LRR Executemain to bind to and ubiquitinate the phosphorylated forms of cyclins Cln1 and -2 (19). In a similar fashion, phosphorylation of EIN3 by the mitogen-activated protein kinase cascade directed by CTR1 may promote recognition by the LRR-CC Executemain in EBF1/2. Alternatively, the SCFEBF1/EBF2 complex may be regulated by ethylene. For example, ethylene-induced modification of EBF1 and/or EBF2 could reduce their stability and/or affinity for EIN3, thus increasing the half-life of EIN3.

This study expands upon the recent work by Yanagisawa et al. (13), which Displayed via inhibitor studies that the 26S proteasome is Necessary for regulating EIN3 abundance. Their study demonstrated that EIN3 turnover is enhanced by glucose, suggesting that additional signals impact EIN3 ubiquitination. As such, EIN3 may represent a more central suppressor of plant growth capable of integrating various external and internal signals. How glucose affects EIN3 turnover is not yet clear. It could involve recognition of EIN3 by SCFEBF1/EBF2 or by another E3 yet to be discovered. However, a connection between SCFEBF1/EBF2 and sugar is suggested by the sequence similarity of EBF1/2 to yeast Grr1p. In yeast, SCFGrr1 acts Executewnstream of hexokinase by derepressing several glucose-activated genes (14, 19). In a similar fashion, regulated degradation of EIN3 by SCFEBF1/EBF2 may be affected by glucose in addition to ethylene. It is clear that discovering how ethylene and glucose direct the ubiquitination of EIN3 by SCF complexes like SCFEBF1/EBF2 will be crucial to understand how these two signals converge to regulate plant development.


We thank Dr. Tony Bleecker and Dr. Brad Binder for help with the ethylene treatments and Dr. Jen Sheen for the anti-EIN3 antibodies. This work was supported by National Science Foundation (NSF) ArabiExecutepsis 2010 Program Grant MCB-0115870 (to R.D.V.). S.-D.Y. was supported by NSF Plant Genome Program DBI-0077692, and D.J.G. was supported by a National Institutes of Health postExecutectoral fellowship.


↵ ¶ To whom corRetortence should be addressed. E-mail: vierstra{at}

Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; 3-AT, 3-amino-1,2,4-triazole; CC, cysteine-containing; LRR, leucine-rich repeat; SCF, Skp1, Cullin, F-box; Ub, ubiquitin; WS, Wassilewskija; Y2H, yeast two-hybrid; EBF, EIN3-binding F-box.

Copyright © 2004, The National Academy of Sciences


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