- American Society of Plant Biologists
Abstract
Analysis of the Arabidopsis thaliana RING-ANK (for Really Interesting New Gene-Ankyrin) family, a subgroup of RING-type E3 ligases, identified KEEP ON GOING (KEG) as essential for growth and development. In addition to the RING-HCa and ankyrin repeats, KEG contains a kinase domain and 12 HERC2-like repeats. The RING-HCa and kinase domains were functional in in vitro ubiquitylation and phosphorylation assays, respectively. Seedlings homozygous for T-DNA insertions in KEG undergo growth arrest immediately after germination, suggestive of increased abscisic acid (ABA) signaling, a major phytohormone that plays a key role in plant development and survival under unfavorable conditions. Here, we show that KEG is a negative regulator of ABA signaling. keg roots are extremely sensitive to the inhibitory effects of ABA and exhibit hypersensitivity to exogenous glucose, consistent with the known interaction between glucose and ABA signaling. The observations that KEG accumulates high levels of ABSCISIC ACID-INSENSITIVE5 (ABI5) without exogenous ABA, interacts with ABI5 in vitro, and that loss of ABI5 rescues the growth-arrest phenotype of keg mutant seedlings indicate that KEG is required for ABI5 degradation. In this capacity, KEG is central to ABA signaling by maintaining low levels of ABI5 in the absence of stress.
INTRODUCTION
Protein modification by attachment of one or more ubiquitin molecules has various consequences, including protein degradation, internalization, sorting, and activation (Weissman, 2001; Umebayashi, 2003). Defects within the pathway have been linked to developmental aberrations, abnormal responses to stimuli, and disruption of cell division and growth (Glickman and Ciechanover, 2002). The importance of the ubiquitin pathway to plant development in particular has become apparent in recent years. Protein ubiquitylation and subsequent degradation by the 26S proteasome are involved in almost all aspects of plant growth and development and protection from biotic and abiotic stresses (Smalle and Vierstra, 2004).
Ubiquitin protein ligases (or E3s) along with the ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes catalyze the covalent attachment of ubiquitin to target proteins. A large number of diverse E3s largely control the specificity of ubiquitylation by recruiting appropriate substrates. For example, the Arabidopsis thaliana genome contains >1300 genes predicted to encode E3 ligases (Smalle and Vierstra, 2004), of which >450 belong to the RING (for Really Interesting New Gene) class (Stone et al., 2005). The signature RING domain is a cross-braced scaffold that chelates two zinc ions using an octet of Cys and His residues; this domain forms a docking site for binding the ubiquitin-E2 intermediate during ubiquitin transfer (Barlow et al., 1994; Borden et al., 1995; Zheng et al., 2000). Our initial studies demonstrated that a large number of Arabidopsis RING proteins are capable of mediating protein ubiquitylation (Stone et al., 2005). However, the in vivo roles of all but a few of these proteins remain uncharacterized.
The hormone abscisic acid (ABA) is an important regulator of plant growth and development, especially when exposed to unfavorable environments. It helps direct seed maturation and prolongs seed dormancy to ensure that seeds germinate under conditions favorable for growth. Immediately after germination, ABA can also suspend the growth of young seedlings when stressed, with this postgerminative arrest representing an early developmental checkpoint to slow seedling growth until better conditions arise (Lopez-Molina et al., 2001). As plants mature, the stress-induced accumulation of ABA directs various protective responses that help ameliorate damage induced by salinity, cold, drought, and pathogen attack (Finkelstein et al., 2002).
Although the physiological roles of ABA in seed and seedling development have been well studied, the underlying molecular mechanisms guiding its perception are less well understood. ABA responses involve changes in gene expression, which are regulated by multiple types of transcription factors, including the B3 domain ABI3 (for ABSCISIC ACID-INSENSITIVE3), the APETALA2 domain ABI4, and the bZIP (for basic leucine zipper) ABI5 families (Finkelstein et al., 2002). In Arabidopsis thaliana, ABI5 is a central regulator of ABA signaling during postgerminative growth. ABI5 protein levels remain low in the absence of ABA, but within 1 to 3 d after germination, treatment with ABA dramatically increases ABI5 levels, in large part as a result of reduced proteolysis (Lopez-Molina and Chua, 2000; Lopez-Molina et al., 2001). Previous studies have linked ABI5 degradation to the ubiquitin/26S proteolytic pathway; however, the ligation cascade directing ABI5 ubiquitylation has remained unknown (Lopez-Molina and Chua, 2000; Lopez-Molina et al., 2001, 2003; Smalle et al., 2003).
Here, we demonstrate that a novel RING E3 named KEG (for KEEP ON GOING) is required for ABA signaling and postgerminative growth of Arabidopsis seedlings. KEG contains a RING-HCa domain and a series of ankyrin (ANK) repeats, which are hypothesized to function as a versatile protein–protein interaction module (Sedgwick and Smerdon, 1999). Unlike other members of the RING-ANK family, KEG also contains a Ser/Thr kinase domain and is terminated by 12 HERC2-like repeats. Homozygous seedlings deficient in KEG undergo growth arrest immediately after germination and fail to develop into mature flowering plants. keg seedlings are hypersensitive to the inhibitory growth effects of ABA and sugars. The ability of KEG to interact with ABI5, the extremely high levels of ABI5 protein present in keg seedlings, and the ability of abi5 mutants to rescue the phenotype conferred by keg all support a role for KEG in regulating ABI5 accumulation. In this context, KEG appears to have a central role in ABA signaling through this ability to control ABI5 protein levels.
RESULTS
The RING-ANK Family Contains a Novel Plant-Specific Member
The RING-HCa domain–containing ANK repeat family contains seven members (Stone et al., 2005), one of which (At5g13530) is predicted to also contain a kinase domain (Figure 1A ). Given the novel arrangement of this protein, we analyzed the corresponding genomic locus more closely. Attempts to amplify the open reading frame (ORF) for At5g13530 from cDNA failed using primers based on the available annotation (http://www.Arabidopsis.org). Complete sequencing of the only cDNA for At5g13530 present in the EST database (APZL47g09R) revealed that it also includes sequence from the downstream locus At5g13540, suggesting that the gene is misannotated, with the full ORF likely encompassing both the At5g13530 and At5g13540 loci (Figure 1B). This fusion was confirmed by the isolation and sequencing of products from RT-PCR generated with seedling RNA and a primer from the 5′ end of the predicted At5g13530 ORF paired with one from the 3′ end of the APZL47g09R cDNA.
Reannotation of the KEG Gene.
(A) Scheme and protein similarity tree of the Arabidopsis RING-ANK and kinase-ANK families. The phylogenic tree was generated with ANK repeats. Numbers at branch points represent bootstrap values after 1000 replications. At5g40160 (ANK-only protein) was used as the outgroup. RING-ANK family members tested in in vitro ubiquitylation assays are listed in boldface; those marked with asterisks were confirmed to be active (Stone et al., 2005).
(B) Intron–exon structure of At5g13530 and At5g13540 as determined by The Arabidopsis Information Resource database (top) and reannotation to generate KEG (bottom). Exons are represented by boxes (white, At5g13530; gray, At5g13540) and introns by thick black lines. The cDNA clone APZL47g09R is depicted as a line (below). A scheme of the KEG protein is also shown.
(C) Sequence alignment of the HERC2-like repeats of KEG, an Arabidopsis uncharacterized predicted protein with a HERC region (At4g32250), and similar regions in the HERC2 protein of human (Hs, Homo sapiens) and mouse (Mm, Mus musculus).
The reannotated gene (now referred to as KEG) also has a number of changes in intron–exon organization from the previous prediction (Figure 1B; see Supplemental Figure 1 online). Most notable is the shortening of the last exon of At5g13530 (exon 10), which places the predicted stop codon for At5g13530 within an intron, thus allowing the reading frame to continue into At5g13540 (Figure 1B; see Supplemental Figure 1 online). The final annotation revealed that the KEG locus, from translation start to stop codons, spans 8751 kb with 15 introns and encodes a protein of 1625 amino acids.
Amino acid sequence alignments reveal that KEG is a novel RING protein. In contrast with the six other RING-ANK proteins in Arabidopsis, in which the RING-HCa domain is at the C terminus and is preceded by two to nine ANK repeats (Stone et al., 2005), KEG has the reverse orientation, with an N-terminal RING-HCa domain followed by a kinase domain and nine ANK repeats (Figures 1A and 1B; see Supplemental Figure 2 online). Of the Arabidopsis ANK repeat proteins with either a RING or a kinase domain (Becerra et al., 2004), the kinase-ANK family members formed a single distinct clade that does not include KEG (Figure 1A). The RING-ANK proteins did not fall into a single clade, suggesting more diversity within this subgroup (Figure 1A). The C-terminal region of KEG, which was previously encoded by the At5g13540 locus, was not reported to have any identifiable motifs. However, we detected significant similarity to the mammalian HERC2 (for HECT and RCC1-like) protein, a HECT-type E3 ligase (Garcia-Gonzalo and Rosa, 2005). The similarity between KEG and HERC2 is confined to a 61–amino acid stretch present only once in HERC2 but repeated 12 times in KEG (Figures 1B and 1C). This combination of domains within a single protein is unique to KEG and its orthologs.
Single KEG homologs were found in Oryza sativa (rice; Os KEG), Medicago tuncatula (Mt KEG), and Populus tricocarpa (Pt KEG), but not in fungi or animals, suggesting that KEG is plant-specific (last search, April 31, 2006; see Supplemental Figure 3 online). The predicted rice KEG protein has 60% amino acid identity to Arabidopsis KEG with the same domain architecture, further supporting our reannotation of KEG (see Supplemental Figures 3A and 3B online). The predicted Medicago genome (The Institute for Genomic Research International Medicago Genome Annotation Group) identified three loci that show significant similarity to KEG. The RING-HCa domain, kinase domain, and ANK repeats were encoded by the AC122163_12.1 locus, whereas the HERC2-like repeats were encoded by two downstream loci, AC122163_10.1 and AC122163_11.1. By combining all three genomic regions, a full KEG protein with 69% amino acid sequence identity to Arabidopsis KEG could be assembled, suggesting that the Medicago KEG gene, like its Arabidopsis counterpart, requires reannotation (see Supplemental Figures 3A and 3B online).
KEG Has Both Ubiquitylation and Phosphorylation Activities
The ability of KEG to direct protein ubiquitylation was tested by in vitro ubiquitylation assays (Figure 2A ) (Stone et al., 2005). Given the large size of KEG (∼180 kD), only the region encompassing the RING (R) and kinase (K) domains was assayed as a fusion to glutathione S-transferase (GST-RK). Recombinant GST-RK stimulated E2-dependent ubiquitylation in vitro, as evident by the formation of a heterogeneous collection of high molecular mass proteins detected by immunoblot analysis using anti-ubiquitin antibodies (Figure 2A). Omission of At UBC8 (−E2), GST-RK (−E3), or ubiquitin (−Ub) from the reaction resulted in a loss of protein ubiquitylation (Figure 2A). Substitution of metal ligand positions Cys-3, His-4, and Cys-5 of the RING domain (GST-RKmut) with Ala blocked the ubiquitylation activity of KEG (Figure 2A), indicating that an intact RING-HCa domain is required for ligase activity. In paired reactions with a variety of E2s, GST-RK promoted protein ubiquitylation with the most closely related members of the At UBC8 subgroup—At UBC8, At UBC10, At UBC11, and At UBC28—and very little or no activity was observed with At UBC29 or At UBC30, respectively (Figure 2B), which are more divergent At UBC8 subgroup members (Kraft et al., 2005). No activity was also observed with two other UBCs, UBC34 and UBC35 (Figure 2B).
KEG Has Ubiquitin Ligase and Kinase Activities.
(A) In vitro ubiquitylation assays using GST fusion protein of KEG RING and kinase regions (GST-RK) and RING mutant GST-RKmut, visualized with anti-ubiquitin antibodies. Omission of At UBC8 (−E2), GST-RK (−E3), or ubiquitin (−Ub) from the complete reactions (C) resulted in a loss of ubiquitylation. The top panel confirms the presence of GST-RK proteins in the assay using anti-GST antibody. Arrows in (A) to (C) identify the slower migrating or ubiquitylated form of GST-RK.
(B) Ubiquitylation assays as in (A) using different E2s. The bottom panel indicates the presence of GST-RK proteins in the assay using anti-GST antibody.
(C) Top, ubiquitylation assays using either ATP or 5′-adenylyl imidodiphosphate (AMP-PNP). Bottom, GST pull down of GST-RK after in vitro ubiquitylation assay followed by immuoblotting with anti-ubiquitin or anti-GST antibody.
(D) In vitro phosphorylation assays using [γ-33P]ATP.
(E) Calf intestinal phosphatase (CIP) treatment of phosphorylated GST-RK.
(F) In vitro phosphorylation assays were performed in the presence (+) of MgCl2 and/or MnCl2.
(G) Phosphorylation of the artificial substrates casein (lane 1) and histone IIa (lane 2) but not myelin basic protein (MBP) (lane 3) by GST-RK. The asterisk indicates the position of MBP.
The predicted kinase domain of KEG contains all of the conserved residues that define the catalytic domain of Ser/Thr protein kinases (Hanks et al., 1988), suggesting that KEG should be active in phosphotransfer as well as ubiquitylation (see Supplemental Figure 2C online). This activity was confirmed by in vitro reactions containing recombinant GST-RK and [γ-33P]ATP. GST-RK generated an autophosphorylated form whose abundance was sensitive to calf intestinal phosphatase (Figures 2D and 2E). Autophosphorylation was more robust in the presence of Mn2+ compared with Mg2+, suggesting that the kinase activity of GST-RK prefers Mn2+ (Figure 2F). When added to the nonspecific substrates casein, histone IIa, and mylein basic protein, GST-RK phosphorylated the first two substrates but not the last (Figure 2G).
In ubiquitylation assays with GST-RK, we consistently detected a slower migrating form of GST-RK, which could represent a ubiquitylated and/or a phosphorylated form of KEG (arrows in Figures 2A and 2B). This slower migrating form of GST-RK was still observed in assays containing 5′-adenylyl imidodiphosphate, a β,γ-nonhydrolyzable ATP analog that can be used for ubiquitin activation but that does not support phosphotransfer by protein kinases (Johnston and Cohen, 1991; Leventhal and Bertics, 1991), suggesting that it represents a ubiquitylated form of GST-RK (Figure 2C, top panels). To confirm the presence of ubiquitylated GST-RK, we enriched for all GST-RK forms with pull-down assays using glutathione agarose beads mixed with a completed ubiquitylation reaction and subjected the bead-bound fraction to immunoblot analysis with anti-GST and anti-ubiquitin antibodies. Both antibodies detected the slower migrating form, indicating that KEG is capable of autoubiquitylation at least in vitro (Figure 2C, bottom panels).
KEG Is Essential for Postgerminative Growth and Development
To help define the physiological functions of KEG, we analyzed three Arabidopsis (ecotype Columbia [Col-0]) T-DNA insertion lines, keg-1, keg-2, and keg-3. The keg-1 and keg-2 alleles have a T-DNA insert after nucleotide 1623 (from the ATG) within exon 2 and after nucleotide 2368 within intron 3, respectively. keg-3 has a T-DNA insert at nucleotide 5750 within exon 11, previously annotated as the intergenic region between At5g13530 and At5g13540 (Figure 3A ). To examine whether the insertions affected the accumulation of the KEG transcript, portions of the ∼5-kb ORF were amplified by RT-PCR (Figure 3A).
The KEG Gene Is Essential for Postgerminative Seedling Growth.
(A) Scheme of KEG depicting the positions of T-DNA insertions. Arrows indicate the positions of primers, with letters designating primer pairs used in separate RT-PCRs. RT-PCR for KEG (top three panels; using primer pairs A, B, and C, respectively) and UBQ10 (RNA control; bottom panel) using total RNA isolated from 10-d-old Col-0 and keg-1, keg-2, and keg-3 seedlings. Mock indicates PCR conducted without reverse transcriptase.
(B) Panel a shows the phenotype of a 7-d-old light-grown keg-1 seedling compared with a wild-type sibling. Panels b to d show seedlings of 7-d-old keg-2 (b) and keg-3 (c) and 3-week-old keg-1 and wild-type Col-0 (d). The inset shows a threefold magnification of the keg-1 seedling shown in panel d. All seedlings were grown on GM with 1% sucrose.
(C) Five-day-old dark-grown keg-1, keg-2, and keg-3 and Col-0 seedlings grown on GM with (top) or without (bottom) 1% sucrose. The graph shows hypocotyl and root length after 5 d in the dark for keg-1 and keg-2 and Col-0. Error bars represent sd; n = 12 except for keg-1, with n = 8 and 7 in 0 and 1% sucrose, respectively.
Levels of the KEG transcript were absent or reduced in these insertion lines. From keg-1 seedling mRNA, no RT-PCR products were produced with primer pairs that spanned the insertion site (primer pairs A and C) or with primers that amplified a region downstream of the insertion site (primer pair B). These results suggest that very little or no keg transcript accumulates in this line. For keg-2 mRNA, a low level of product was detected with a primer pair that amplified a region upstream of the insertion site (primer pair A), but no product was detected with primer pairs B and C that span the insertion site. Because the T-DNA is in the intron, this indicates that the T-DNA is not spliced out, but low levels of RNAs with KEG sequence are produced. Similarly, for the keg-3 allele, low levels of PCR products were generated from cDNA with primers that amplify upstream of the insertion site (primer pairs A, B, and C). Given that the phenotypes of all three keg alleles are identical (see below), if a truncated protein is produced from keg-2 or keg-3 transcripts, it is insufficient to supply wild-type function.
To further support the notion that the mutant phenotypes (see below) were caused by the disruption of KEG and not by an unlinked mutation, heterozygous keg-1, keg-2, and keg-3 plants were backcrossed to wild-type Col-0. PCR-based genotyping of F2 seedlings confirmed that the phenotype conferred by keg segregated with each T-DNA insertion (data not shown). In addition, heterozygous keg plants were indistinguishable from wild-type plants, indicating that the phenotype is recessive.
Seedlings homozygous for each of the three keg alleles displayed the same mutant phenotype (Figure 3B, panels a to c). The germination rate of keg seeds, scored as radicle emergence from the seed coat, was normal (data not shown). However, soon after germination, keg mutants displayed a strong postgerminative growth arrest (Figure 3B). Their cotyledons failed to expand and remained white to very pale green compared with wild-type seedlings of the same age (Figure 3B, panels a to c). The morphology of keg roots was also affected by the mutation; however, keg-1 roots were only slightly shorter and keg-2 roots were not shorter than wild-type roots at 7 d after germination (see Supplemental 4A and 4B online). The emergence of true leaves (with trichomes) was greatly delayed in all three keg backgrounds, requiring almost 3 weeks for the first pair of true leaves to emerge. The second pair of true leaves did not emerge, and no further growth was observed (Figure 3B, panel d).
Loss of KEG Affects Dark-Grown Seedling Development
The lack of cotyledon greening and expansion in keg seedlings could be caused by defects in a number of cellular processes, including a block in photomorphogenesis and/or chloroplast biogenesis. To determine whether the keg mutations affect only light-grown seedlings, the phenotype conferred by keg was examined after 3 or 5 d of growth in the dark on growth medium (GM) containing 1% sucrose (Supplemental Figure 4C online and Figure 3C, respectively). Wild-type dark-grown seedlings undergo a skotomorphogenic growth pattern in which chloroplast development is arrested, the hypocotyl elongates rapidly, the apical hook is maintained, and cotyledon expansion is minimal (von Arnim and Deng, 1996). After 3 d of growth in the dark, hypocotyl elongation and cotyledon expansion of all three keg alleles were so minimal that the cotyledons failed to emerge from the seed coat (see Supplemental Figure 4C online). After 5 d of growth on GM, keg-1 and keg-2 hypocotyls were equivalent in length to each other (Figure 3C, top row and graph; P = 0.71 by Student's t test) but were much shorter than Col-0 hypocotyls (P < 0.0001). The lengths of dark-grown roots were also measured. Although the radicle emerged normally, keg roots were substantially shorter than wild-type roots after 3 d of growth (see Supplemental Figure 4C online). However, the growth of keg roots did recover, and by 5 d both keg-1 and keg-2 roots were similar in length to each other and to Col-0 roots (P = 0.88, 0.80, and 0.47, respectively) (Figure 3C, graph). Thus, it appears that KEG controls a general growth process active in both the light and the dark.
keg Seedlings Are Hypersensitive to Sugar
The addition of 6% glucose to GM has been shown to induce postgermination growth arrest in wild-type Arabidopsis seedlings, with a concomitant block in cotyledon expansion and chloroplast development and greening (Zhou et al., 1998; To et al., 2002). Given that keg seedlings display a similar phenotype when grown on 1% sucrose (Figure 3B), we tested whether keg seedlings are sugar-hypersensitive. As shown in Figure 4A , the addition of 4% glucose to the GM strongly exacerbated the phenotype conferred by keg in light-grown seedlings but only minimally affected wild-type seedlings. The phenotype conferred by keg was not enhanced by 1 or 4% mannitol, a nonmetabolizable sugar, demonstrating that the response was not simply the result of osmotic stress (Figure 4A). Consistent with sugar hypersensitivity, the phenotype conferred by keg was attenuated in medium lacking added sugar. The cotyledons expanded and turned green (Figure 4A), but the mutant seedlings still failed to generate new leaves at wild-type rates. Omission of sugar also affected the dark-grown phenotype of keg seedlings (Figure 3C). At 5 d, keg hypocotyls remained significantly shorter than Col-0 hypocotyls (P < 0.0001) despite the fact that keg-1, keg-2, and Col-0 hypocotyls were all longer when grown in the dark without sugar than with 1% sucrose (P < 0.003). However, the morphology and organization of the keg roots were more similar to those of wild-type roots in the absence of sugar. Thus, the sugar hypersensitivity of keg seedlings may account for the defects observed in the organization of keg roots when grown on sugar-containing medium (see Supplemental Figure 4A online).
keg Seedlings Display Glucose Hypersensitivity, and the Severity of the Phenotype Is Modulated by Ethylene.
(A) Seven-day-old keg and Col-0 seedlings grown on GM with 0, 1, or 4% glucose or 1 or 4% mannitol in the light.
(B) keg and Col-0 seedlings grown for 7 d in the light on GM with or without 1% sucrose (top three rows and bottom row, respectively) in the presence of 100 μM silver nitrate (AgNO3) or 50 μM 1-aminocyclopropane-1-carboxylic acid (ACC; second and third rows, respectively). All panels were photographed at the same magnification.
Ethylene Affects the Phenotype Conferred by keg
There is a considerable amount of crosstalk between glucose and ethylene signaling (Leon and Sheen, 2003). For example, ethylene-insensitive mutants such as ethylene-insensitive2 are glucose-hypersensitive (Alonso et al., 1999; Chen et al., 2002), whereas increases in ethylene levels can alleviate the inhibitory effects of high glucose on postgerminative growth (Zhou et al., 1998). Consequently, we tested whether the sugar hypersensitivity of keg seedlings was caused by changes in ethylene perception or synthesis. Blocking ethylene perception by growth in the presence of silver nitrate (Figure 4B) or blocking ethylene synthesis with aminoethoxyvinylglycine (data not shown) strikingly increased the severity of the phenotype conferred by keg in the presence of exogenous sucrose. Treatment of keg seedlings grown on 1% sucrose with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, which is readily converted to ethylene in plants, partially alleviated the inhibitory effects of sugar on keg seedling development (Figure 4B). However, treatment of keg seedlings with 1-aminocyclopropane-1-carboxylic acid in the absence of glucose did not alleviate the mutant phenotype any further (Figure 4B, bottom right). These results suggested that the KEG mutant phenotype was only partially caused by sugar hypersensitivity and that keg seedlings have a functional ethylene signaling pathway.
keg Mutants Are Hypersensitive to ABA
Glucose has been shown to influence the control of postgerminative growth by the hormone ABA via its ability to increase the expression of ABA biosynthetic and signaling genes (Cheng et al., 2002). In fact, many glucose-insensitive mutations are allelic to ABA-deficient or ABA-insensitive mutations (Finkelstein and Gibson, 2002). Furthermore, when wild-type seedlings are exposed to ABA within 24 h after germination, they undergo a developmental arrest similar to that observed for untreated keg seedlings (Figure 5A ; cf. with Figure 3B). Consequently, keg seedlings could be affected in ABA synthesis and/or perception. Treatment of keg seedlings with the ABA biosynthesis inhibitor fluridone, either during or after germination, failed to alleviate the phenotype conferred by keg (data not shown), suggesting that synthesis of the hormone was not increased. By contrast, we found that the keg roots are hypersensitive to exogenous ABA. When 3-d-old keg-1, keg-2, and keg-3 seedlings were exposed to 5 μM ABA, a stronger inhibition of root growth was apparent compared with growth in the wild type (Figure 5B).
keg Seedlings Are Extremely Sensitive to the Inhibitory Effects of ABA.
(A) Col-0 was allowed to germinate for 24 h and then transferred to GM with (right panels) or without (left panel) 5 μM ABA for 5 d. Percentages indicate seedlings with the shown phenotype (total n = 50).
(B) Col-0 and keg seedlings were grown for 3 d on GM and then transferred to GM with (+) or without (−) 5 μM ABA for 5 d. The graph illustrates the percentage difference in root length between ABA-treated (5 μM) and untreated roots.
The ABA-Responsive Transcription Factor ABI5 Accumulates in keg Seedlings
Postgerminative growth arrest induced by ABA treatment coincides with increases in ABI5 mRNA and protein, implying that ABI5 is the causal agent of this arrest (Soderman et al., 2000; Lopez-Molina et al., 2002). Consistent with previous studies (Lopez-Molina et al., 2002; Smalle et al., 2003), we found that ABI5 protein was undetectable in untreated wild-type seedlings and KEG wild-type siblings (Figure 6A ) and became barely detectable upon exposure to 50 μM ABA (Figure 6C). By contrast, seedlings homozygous for each of the three keg alleles contained extremely high levels of ABI5 even without treatment with exogenous hormone (Figures 6A and 6C). RT-PCR analysis showed that ABI5 transcript levels in keg seedlings were indistinguishable from wild-type levels (Figure 6D), indicating that the increase in ABI5 protein was most likely attributable to posttranscriptional effects, such as reduced degradation of the protein in a background of constitutive synthesis.
ABI5 Protein Accumulates in keg Mutants, and KEG Interacts with ABI5.
(A) Levels of ABI5 protein in 6-d-old keg-1, keg-2, and keg-3 seedlings compared with identically grown wild-type siblings (sib), Col-0, ABI5 overexpressor (ABI5-OX), and abi5-1. Anti-PBA1 immunoblot was used as a loading control. PBA1, proteasome β subunit-α
(B) Forms of ABI5 detected in keg-1, keg-2, and keg-3 and ABA0treated rpn10-1 seedlings compared with that found in ABI5-OX seedlings.
(C) Levels of ABI5 protein present in 7-d-old Col, keg, and wild-type siblings in the presence or absence of 50 μM ABA. The left panel shows an equal protein load, and the right panel shows more Col-0 protein extract to visualize ABI5 forms. Anti-PBA1 immunoblot was used as a loading control. The asterisk indicates a form of ABI5 unique to Col-0 treated with ABA.
(D) RT-PCR analysis of ABI5 transcript levels in 6-d-old keg and Col-0 seedlings. UBQ10 transcript was used as a control.
(E) Top, scheme of KEG illustrating regions used in pull-down assays. Plant extracts from 4-d-old abi5-1 and Col-0 seedlings (1 mg of total protein; middle panel) or E. coli lysates containing HA-ABI5 (bottom panel) were used in GST pull-down assays with GST-KEG fusions. Lane − in the bottom panel represents a pull-down assay using beads only. HA-ABI5 represents lysate from E. coli expressing HA-ABI5. IB, immunoblot.
Closer examination of total keg seedling extracts subjected to SDS-PAGE revealed that multiple forms of ABI5 accumulated in the keg-1, keg-2, and keg-3 backgrounds, with apparent molecular masses of ∼50, 51, and 53 kD (Figures 6B and 6C). These forms were present even in extracts prepared under conditions that would minimize posthomogenization proteolysis (hot SDS-PAGE sample buffer), suggesting that they were not artifactually generated during sample preparation. The lower molecular mass protein comigrated with ABI5 that accumulated in an ABI5 overexpression line (ABI5-OX; Figure 6B). ABA treatment of keg seedlings did not generate any new ABI5 species but did slightly increase the accumulation of all preexisting species (Figure 6C). Smalle et al. (2003) previously showed that Arabidopsis seedlings with a mutation in the gene encoding the 26S proteasome subunit RPN10 were also hypersensitive to ABA and had increased levels of ABI5 when treated with ABA. When extracts from ABA-treated rpn10-1 seedlings were examined similarly, these seedlings contained the same three isoforms of ABI5 as seen in keg seedlings without ABA treatment (Figure 6B). Thus, it appears that the two higher mass species represent forms of ABI5 that accumulate when its breakdown is attenuated.
When Col-0 seedlings were treated with ABA, the level of ABI5 increased (Lopez-Molina et al., 2002) (Figure 6C). The migration of ABI5 in Col-0 seedlings treated with ABA was compared with that of untreated keg seedlings (Figure 6C, right). The fastest migrating ABI5 species in Col-0 seedlings comigrated with the fastest form from keg seedlings. In addition, a new slower migrating species became apparent in the ABA-treated Col-0 seedlings that did not correspond to any of the forms seen in untreated or in ABA-treated keg seedlings (Figure 6C, asterisk). Thus, keg affects the nature of the ABI5 proteins present in addition to affecting their abundance.
KEG Interacts Directly with ABI5
The dramatic increase in ABI5 levels in the three keg backgrounds even in the absence of exogenous ABA strongly suggested that KEG is responsible for catalyzing ABI5 ubiquitylation, leading to its subsequent degradation. To determine whether KEG could be directly responsible for ABI5 recognition, we performed GST pull-down assays to determine whether KEG and ABI5 interact physically. We were able to pull down ABI5 from 4-d-old wild-type seedling total protein extracts using a GST fusion protein containing the RING, kinase, and ANK (A) domains of KEG (GST-RKA) (Figure 6E, middle panel). ABI5 was not pulled down using GST-RK, GST-H (HERC repeats), or GST alone (Figure 6E, middle panel). ABI5 was also not detected in pull-down assays using GST-RKA and equivalent protein extract from 4-d-old abi5-1 seedlings (Figure 6E, middle panel), further confirming the identity of the interacting protein as ABI5.
When tested similarly using recombinant hemagglutinin (HA)-tagged ABI5, the same binding preference was observed. HA-ABI5 bound to GST-RKA but not to GST-RK, GST-H, or GST alone (Figure 6E, bottom panel). Because the only protein that interacted contained the ANK repeats, we propose that KEG recognizes ABI5 through one or more of its ANK repeats.
Loss of ABI5 Can Partially Rescue the Phenotype Conferred by keg
The phenotypic similarity of keg mutants in the absence of ABA to that of ABI5-overexpressing plants in the presence of ABA (Lopez-Molina et al., 2003) implied that the phenotype conferred by keg is caused mainly by an overaccumulation of ABI5. To confirm this possibility, we tested whether the loss of ABI5 would rescue some or all of the phenotypes conferred by keg by combining keg-1 with the strong abi5-1 mutant. Consistent with the specific role of ABI5 in regulating ABA signaling, loss-of-function abi5 alleles such as abi5-1 grow well under optimal conditions and are insensitive to concentrations of exogenous ABA that arrest wild-type seedlings (Finkelstein, 1994; Lopez-Molina and Chua, 2000). Homozygous abi5-1 plants were crossed to KEG/keg-1 plants, and F2 progeny homozygous for abi5-1 were identified by their insensitivity to ABA during germination (Finkelstein, 1994). ABA-insensitive plants (abi5-1) were then genotyped by PCR (n = 198) for the keg-1 allele.
F2 abi5-1 plants heterozygous for the KEG mutation (KEG/keg-1) were allowed to self, and F3 progeny were analyzed. Strikingly, this F3 population did not segregate for the phenotype conferred by keg when grown on medium containing 1% sucrose where the phenotype conferred by keg was readily apparent (Figures 3 and 4). By contrast, we detected segregation of a new phenotype that was much milder than that of keg-1. Among seedlings with normally expanded and green cotyledons was a population of seedlings whose hypocotyls were noticeably shorter than wild-type hypocotyls (Figure 7A ). Genotyping showed that the short seedlings (n = 16) were homozygous for keg-1, whereas the wild-type seedlings (n = 104) were either wild type or heterozygous for keg-1 (Figure 7A). Thus, loss of ABI5 was able to substantially, but not completely, rescue the growth-arrest phenotype of the keg-1 seedlings.
Loss of ABI5 Rescues the Growth-Arrest Phenotype of keg Seedlings.
(A) Top, phenotypes of seedlings recovered in the F3 generation from seeds generated by self-pollination of abi5-1 KEG/keg-1 plants. Bottom, genotypes of seedlings as determined by PCR analysis. Lines 1 and 2 refer to progeny from crosses with different KEG/keg parents.
(B) Top, growth of Col-0, abi5-1, and progeny of abi5-1 KEG/keg-1 plants in 3 μM ABA. Bottom, PCR-determined genotypes of ABA-insensitive (Ins) and ABA-sensitive (S) seedlings.
The same F3 seeds were germinated in the presence of 3 μM ABA (Figure 7B). In contrast with homozygous abi5-1 seedlings that are insensitive to this concentration of ABA, a population of ABA-sensitive seedlings was evident in progeny from an abi5-1 KEG/keg-1 parent (Figure 7B). All ABA-sensitive F3 seedlings genotyped (n = 24) were homozygous for keg-1, whereas ABA-resistant seedlings (n = 24) were KEG or KEG/keg-1 (Figure 7B). Thus, ABA sensitivity was restored in keg-1 abi5-1 seedlings. This sensitivity explains our previous inability to identify double mutant F2 seedlings, because we first used ABA insensitivity to select for seedlings homozygous at the abi5-1 locus (see above).
To confirm that the restoration of ABA sensitivity by the loss of KEG was specific for abi5 mutants and not for those affecting other components of ABA signaling, we generated a similar mutant combination with abi4-1. abi4-1 eliminates the expression of ABI4, a transcription factor that also works as a positive regulator of ABA signaling (Finkelstein, 1994; Finkelstein et al., 1998). Unlike ABI3, which forms a complex with ABI5, there is no evidence that ABI4 interacts with ABI5 (Nakamura et al., 2001; Lopez-Molina et al., 2002). F3 progeny of an abi4-1 KEG/keg-1 plant grown on medium containing 1% sucrose segregated for the phenotype conferred by keg, indicating that the loss of ABI4 cannot rescue the phenotype conferred by keg (data not shown).
DISCUSSION
Components of the ubiquitylation pathway, in particular E3 ligases, have been shown to play essential roles in plant growth and development, including multiple phytohormone-signaling pathways, such as auxin, gibberellin, ABA, and ethylene (Moon et al., 2004; Smalle and Vierstra, 2004), through modulation of the stability of proteins involved in the perception of and/or response to hormonal stimuli. The importance of E3 ligases to plant development is further demonstrated by our analysis of the novel RING E3 ligase, KEG, which is shown here to play a significant and essential role in ABA signaling during postgermination development. Identification of conserved domains within KEG suggested biochemical activities that we verified by in vitro assays. KEG is active as a ubiquitin E3 ligase and as a kinase in vitro, implying that KEG posttranslationally modifies one or more proteins, including itself, by ubiquitylation and/or phosphorylation, respectively. Protein phosphorylation has been shown to modulate the self and substrate ubiquitylation activity of the E3 ligase c-Cbl (Yokouchi et al., 2001; Kassenbrock and Anderson, 2004). However, when phosphorylation by KEG was blocked, no effect on in vitro ubiquitylation was seen. Perhaps the kinase domain functions in an in vivo regulatory mechanism not seen in substrate-independent in vitro ubiquitylation assays. The presence of two predicted protein–protein interaction domains also suggests that KEG interacts with and regulates the activity of a number of different proteins, which may account for the various phenotypes conferred by keg.
During postgerminative growth, seedlings sense the quality of their environment and adjust growth accordingly to optimize survival. This response is controlled in large part by ABA, with stress affecting both the synthesis and perception of this hormone. The ABA signaling pathway is not well understood. However, genetic approaches have identified three families of transcription factors that modulate ABA responses, with ABI5 playing a major role in postgerminative growth (Lopez-Molina et al., 2001, 2002). The onset and severity of ABA-induced seedling growth arrest correlate with an increase in ABI5 mRNA and ABI5 protein abundance (Lopez-Molina et al., 2001), suggesting that modulation of ABI5 levels is an important process in regulating postgerminative growth. Previous studies have demonstrated that ABI5 protein abundance is controlled by the ubiquitin/26S proteasome pathway (Lopez-Molina et al., 2001, 2002, 2003). For example, a 26S proteasome mutant affecting the RPN10 subunit results in the overaccumulation of ABI5, especially in the presence of ABA, and generates a hypersensitivity of seedling growth to exogenous ABA (Smalle et al., 2003). Here, we show that KEG is also critical for proper ABA perception. Biochemical analysis of the KEG protein and analyses of loss-of-function KEG alleles support its role in promoting postgerminative growth and modulating ABA responses through the control of ABI5 levels (Figure 8 ).
Model of How KEG Regulates Postgerminative Growth through Modulating ABI5 Levels.
(A) In the presence of KEG, ABI5 levels are low and there is little postgerminative growth arrest unless ABA levels increase and ABI5 degradation is slowed.
(B) In the absence of KEG, ABI5 levels accumulate and seedlings are arrested. Because abi5 keg seedlings arrest growth later than keg mutants, we hypothesize that other ABI5-like transcription factors are regulated by KEG, which controls growth at this stage.
Several lines of evidence identify ABI5 as one potential target for the ubiquitin ligase activity of KEG. In keg mutants, ABI5 levels are significantly higher than in wild-type Col-0 and increase only modestly after the addition of ABA. Loss of both KEG and ABI5 restores postgerminative growth to seedlings, indicating a functional relationship between the two. Finally, recombinant KEG and ABI5 interact in vitro. Together, these data support a model whereby KEG targets ABI5 for ubiquitylation, leading to its subsequent degradation, thus decreasing the ability of ABI5 to suppress postgerminative growth in the absence of ABA. Either the recognition or ubiquitylation of ABI5 by KEG is then inhibited by ABA, allowing ABI5 levels to increase. Our current inability to express full-length recombinant KEG has precluded a direct test of KEG's ability to ubiquitylate ABI5 in vitro. Thus, the possibility remains that KEG modulates ABI5 levels via a different or indirect mechanism.
If ABI5 accumulation soon after germination is responsible for the postgerminative growth arrest, then overexpression of ABI5 should result in a keg-like phenotype. However, this is not the case, as ABI5-OX transgenic seedlings expressing as much or more ABI5 as keg seedlings do not undergo growth arrest in the absence of ABA (Brocard et al., 2002; Lopez-Molina et al., 2003) (Figure 6). Thus, loss of KEG has a more severe effect than simply accumulating excess ABI5. We note that keg and ABI5-OX seedlings differ in the forms of ABI5 that accumulate, and this may contribute to the severity of the phenotype conferred by keg. The molecular basis for the multiple forms of ABI5 in keg seedlings is not yet known. Previous studies have indicated that ABI5 is phosphorylated, particularly in the presence of ABA, and that this form has a slower SDS-PAGE migration (Lopez-Molina et al., 2001, 2002), suggesting that the multiple ABI5 forms we observed in keg may differ in phosphorylation status. We have been unable to modify ABI5 migration by treatment with calf intestinal phosphatase and thus cannot yet confirm that the modified forms we observed are phosphorylated species. If these forms of ABI5 are phosphorylated, their presence in keg seedlings discounts KEG as the responsible kinase, but it remains possible that KEG may phosphorylate ABI5 in a way that does not alter its SDS-PAGE migration. Other possibilities for the kinase activity of KEG is that KEG phosphorylates only itself or other factors associated with either the degradation or activity of ABI5. One intriguing candidate is AFP, a negative regulator of ABA signaling that interacts with and forms a high molecular mass complex with ABI5 (Lopez-Molina et al., 2003). Similar to keg mutants, afp-1 seedlings are ABA-hypersensitive and accumulate ABI5 (Lopez-Molina et al., 2003), suggesting that AFP is required for ABI5 degradation. By targeting AFP for phosphorylation, KEG could promote ABI5 degradation.
If not KEG, what activities could be modifying ABI5? The SnRK kinase family has been reported to phosphorylate AREB1, an ABI5 family member (Furihata et al., 2006). Because the phosphorylation sites in AREB1 are conserved in ABI5, these enzymes could also phosphorylate ABI5 and thus be responsible for the multiple forms of ABI5 observed in keg mutants. However, these kinase activities require ABA, so they would have to be misregulated in keg seedlings. The electrophoretic forms of ABI5 are not identical between Col-0 seedlings treated with ABA and those seen in keg seedlings. This finding further suggests that the nature of ABI5 modifications may be complex, involving multiple kinases.
Although the postgerminative growth of keg seedlings can be improved by the removal of ABI5, the growth of keg-1 abi5-1 seedlings is not identical to that of wild-type seedlings in the absence of exogenous ABA. Hypocotyls of double mutants are shorter, and true leaves are produced more slowly. This altered growth suggests that ABI5 is not the only substrate for KEG, and loss of either the kinase and/or ubiquitin ligase activities affects other targets, leading to growth arrest at a later stage. ABI5 is one of 14 related bZIP transcription factors (Jakoby et al., 2002). Some of these relatives are also ABA-inducible, have similar expression patterns to ABI5, regulate the expression of some of the same ABA-responsive genes as ABI5, and can also generate ABA hypersensitivity when overexpressed (Kim et al., 2002, 2004; Finkelstein et al., 2005; Fujita et al., 2005). Although it is clear that ABI5 is the dominant player with respect to ABA sensitivity in postgerminative growth, it is possible that the accumulation and/or modification of one or more of these homologs is also enhanced in keg seedlings. Their overaccumulation could account for the ABA sensitivity and lack of complete rescue of double mutant keg-1 abi5-1 seedlings and contribute to the severity of the phenotype conferred by keg.
Based on the findings reported here and previous studies, it now appears that ABA signaling is regulated to a large extent by the ubiquitin/26S proteasome system. In addition to the control of ABI5 abundance by KEG (this report), the abundance of the transcription factor ABI3 is controlled by the RING-type E3 ligase AIP2 (Zhang et al., 2005). By maintaining low levels of ABI5 and ABI3 in the absence of hormone, KEG and AIP2 together repress ABA responses under favorable growth conditions. In the presence of the hormone, the degradation of ABI5 and ABI3 is then slowed to allow ABI5 and ABI3 levels to increase and activate their respective signal transduction cascades. An additional process is their ABA-induced phosphorylation. How these posttranslational modifications are affected by ABA will undoubtedly be central steps in ABA signaling.
We note that a remarkably similar situation exists in Arabidopsis and presumably other plants for signaling by another stress hormone, ethylene. Here, the family of EIN3/EIL-positive transcriptional regulators is constitutively degraded by the ubiquitin/26S proteasome system in the absence of ethylene, but it accumulates to high levels in the presence of the hormone as a result of decreased ubiquitylation by a small family of E3s (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). Thus, both ABA and ethylene responses result in deceasing degradation and therefore increasing transcription factor protein levels. The opposite is true for auxin and gibberellic acid signaling. Here, the ubiquitin/26S proteosome system serves to accelerate degradation and decrease levels of the transcriptional regulators, the Aux/IAA and RGA proteins, in response to exposure to auxin and gibberellic acid, respectively (reviewed in Smalle and Vierstra, 2004). Presumably, by placing proteolysis in such central positions in multiple hormonal signaling pathways, especially for ABA and ethylene, which are used to monitor stresses, plants can rapidly and reversibly adjust their growth rates in response to their environment.
METHODS
Sequence Analysis and Domain Definition
The GENSCAN database and manual editing of nucleotide sequences were used to predict ORFs. Domains were identified using the SMART (Simple Modular Architecture Research Tool) and Pfam (Protein families database of alignments and HMMs) databases and by BLAST searches via the National Center for Biotechnology Information. Alignments were generated with the ClustalX program (Thompson et al., 1997); the alignments were revised using the Se-Al sequence editor (Evolutionary Biology Group, University of Oxford). Phylogenetic trees were created by PAUP* (Phylogenetic Analysis Using Parsimony) 4.0 using the neighbor-joining method with 1000 bootstrap replicates.
RT-PCR Cloning and Mutagenesis
Total RNA isolated from Arabidopsis thaliana ecotype Col-0 10-d-old seedlings using the RNeasy plant RNA extraction kit (Qiagen) according to the manufacturer's instructions was used in RT-PCR to amplify the predicted KEG ORF. For RT-PCR for ABI5 (At2g36270), the TRIzol reagent (Invitrogen) extraction method was used to isolate total RNA, according to the manufacturer's instructions. For both KEG and ABI5 cDNAs, 5 μg of total RNA was reverse-transcribed using SuperScript III RNase H reverse transcriptase (Invitrogen), and the resulting cDNAs were used in PCR (see Supplemental Table 1 online for a list of all primers used). The Gateway system (Invitrogen) was used to clone the full-length KEG cDNA (4878 bp) and partial cDNA regions of KEG encoding the RING and kinase (RK; 1383 bp), RING, kinase, and ANK (RKA; 2489 bp), or HERC2-like (H; 2390 bp) domain. KEG cDNAs were introduced into the pDEST15 vector to produce in-frame fusions with the GST tag. To obtain 3× HA-ABI5, the entire ABI5 ORF was amplified from PYAt2g36270 (ABRC) and introduced into pHB1-HA3 (Liu et al., 1998) via the NdeI/BamHI restriction sites. Arabidopsis E2s were described previously (Kraft et al., 2005). For RING mutational analysis, site-directed mutagenesis (Stratagene) was used to make a series of point mutations, Cys to Ala, within the RING region.
Wild-type and mutated KEG cDNAs and the EST cDNA clone APZL47g09R (Asamizu et al., 2000), whose partial sequence was deposited previously (GenBank accession number AV529785), were sequenced in their entirety by automated sequencing.
Protein Expression and Purification
Fusion proteins were expressed in Escherichia coli strain BL21(DE3) pLysS and GST fusion proteins purified using GST-agarose (Sigma-Aldrich) as described previously (Stone et al., 2005). Arabidopsis E2s were expressed and purified as 6× His-tagged fusion proteins as described previously (Kraft et al., 2005).
In vitro Ubiquitylation and Phosphorylation Assays
Ubiquitylation assays were performed as described previously (Hardtke et al., 2002). Briefly, reactions (30 μL) containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.05 mM ZnCl2, 1 mM ATP or 5′-adenylyl imidodiphosphate (Sigma-Aldrich), 0.2 mM DTT, 10 mM phosphocreatine, 0.1 unit of creatine kinase (Sigma-Aldrich), 50 ng of yeast E1, 250 ng of purified 6× His-E2, 500 ng of eluted/bead-bound GST-E3, and 2 μg of ubiquitin (Sigma-Aldrich) were incubated at 30°C for 2 h. Reactions were stopped by adding sample buffer (125 mM Tris-HCl, pH 6.8, 20% [v/v] glycerin, 4% [w/v] SDS, and 10% [v/v] β-mercaptoethanol) and analyzed by SDS-PAGE followed by protein gel blotting using anti-ubiquitin antibodies as described previously (Stone et al., 2005).
In vitro phosphorylation assays used 250 ng of GST-RK or GST incubated at 30°C for 30 min in 30 μL of buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1% Triton X-100, and 10 μCi of [γ-33P]ATP) supplemented with 10 mM MnCl2, 10 mM MgCl2, or 5 mM MnCl2 and 5 mM MgCl2. Reactions were stopped by adding sample buffer and boiled for 5 min. Reactions were analyzed by SDS-PAGE, and gels were stained with Coomassie Brilliant Blue R 250 and dried onto Whatman paper. Phosphorylated proteins were detected by autoradiography or phosphoimagery. For treatment with calf intestinal phosphatase, bead-bound GST-RK was first allowed to autophosphorylate as described above. Beads were then washed in calf intestinal phosphatase buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 1 mM DTT) and incubated with or without 0.01 unit/μL calf intestinal phosphatase for 1 h at 37°C. For substrate phosphorylation, 5 μg of casein, histone IIa, or MBP (Sigma-Aldrich) was added to the in vitro assay.
Plant Material and Growth Conditions
Seeds from Arabidopsis ecotype Col-0 or mutant plant lines surface-sterilized with 30% (v/v) bleach and 0.1% (v/v) Triton X-100 were grown on GM containing 1% (w/v) agar with 1× Murashige and Skoog medium under continuous light. GM contains 1% sucrose unless stated otherwise. Selected seedlings were transferred to soil and grown under photoperiodic cycles of 16 h of light and 8 h of dark at 16°C with 50% RH. For treatments, components were omitted or GM was supplemented as indicated in the text.
Identification of T-DNA Insertional Plants and Microscopy
T-DNA insertional lines were obtained from the ABRC (Alonso et al., 2003). keg1 to keg3 are T-DNA insertion lines (SALK_049542, SALK_018105, and SALK_133445, respectively), all in the Col-0 ecotype background. Plants and seedlings with the T-DNA were identified by PCR analysis of genomic DNA using the Extract N Amp kit (Sigma-Aldrich). Genotyping consisted of PCR using gene-specific primers for the wild-type allele and in combination with T-DNA–specific primers for the mutant allele. PCR products for each of the three lines were sequenced using the T-DNA–specific primers to determine the exact genomic location of the insert. Images of seedlings were acquired using a DC290 digital camera mounted on a Zeiss Stemi SV8 microscope.
ABA Root Assay and Screen
For root assays, wild-type Col-0 and keg seedlings were germinated and grown vertically on solid GM for 3 d. Seedlings were then transferred to vertical GM plates with or without 5 μM ABA (Sigma-Aldrich), and subsequent root growth was measured after 5 d. For ABA screens, seeds were sterilized and grown in liquid GM supplemented with or without 3 μM ABA under continuous light for 7 to 10 d.
Antibody Generation and Immunoblot Analysis
Antibodies against purified ABI5 were produced in a rabbit (Polyclonal Antibody Service, University of Wisconsin-Madison) using recombinant 6× His-ABI5 (a gift of T. Thomas) expressed in E. coli BL21(DE3) pLysS (Novagen). Log-phase cultures were induced with 1 mM isopropylthio-β-galactoside at 37°C for ∼4 h. Insoluble ABI5 protein was extracted using the Bug Buster reagent according to the manufacturer's recommendations (Novagen), solubilized in 8 M urea buffer, and then purified at room temperature via nickel-nitrilotriacetic acid agarose chromatography (Qiagen Sciences). ABI5 eluates were dialyzed, concentrated, and resolved by SDS-PAGE, and gel slices containing the antigen were injected directly into the rabbit. For immunoblot analysis, total plant protein was isolated from 6- to 7-d-old seedlings grown in liquid GM. rpn10-1 plants were treated with 1 μM ABA for 2 h before harvesting. keg-1, keg-2, and keg-3 and wild-type sibling seedlings were treated with 50 μM ABA for 1 h before harvesting. Tissue was frozen and then homogenized in 2:1 (volume:fresh weight) sample buffer, and extracts were analyzed by protein gel blotting with anti-ABI5 antibodies. Anti-PBA1 (proteasome β subunit-α) antibodies were used to confirm equal protein loads (Smalle et al., 2002).
GST Pull-Down Assays
For GST pull-down assays, bead-bound GST or GST-KEG fusions were incubated at 4°C with plant extracts from Col-0 or abi5 seedlings (1 mg of total protein) or bacterial lysate in PBS containing HA-ABI5. Plant extracts were prepared by homogenizing frozen tissue in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 20 mM EDTA, pH 8, 1 mM DTT, 10% glycerol, 1% Nonidet P-40, 50 mM phenylmethylsulfonyl fluoride, and a Complete mini protease inhibitor cocktail tablet (Roche Diagnostics). Extracts were cleared by centrifugation, and protein concentration was determined by Bradford assay. After incubation, beads were washed four times in 1× PBS with 1% Triton X-100. For pull downs from plant extracts, proteins were eluted from beads in SDS sample buffer. After incubation with bacterial lysate, GST-KEG fusion and interacting proteins were eluted from beads using 100 mM glutathione in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.01% Triton X-100. ABI5 was visualized using anti-ABI5 antibodies.
Accession Number
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number DQ315360.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Nucleotide Sequence of the Reannotated At5g13530 and At5g13540 Genomic Region.
Supplemental Figure 2. Illustration of KEG, and Amino Acid Sequence Alignments of Each Domain.
Supplemental Figure 3. Amino Acid Sequence Alignment of Arabidopsis KEG and Homologous Proteins.
Supplemental Figure 4. Phenotypes of Light-Grown keg Seedling Roots and Dark-Grown keg Seedlings.
Supplemental Table 1. Primers Used in This Study.
Acknowledgments
We thank M. Kerber for excellent technical assistance, E. Fuller for help with genotyping, S. Lochhead for controlled pollinations, R. Brown for assisting with sectioning, Joseph Walker for antibody preparation, members of the Callis/Vierstra laboratories for helpful discussions, and the University of California-Davis Controlled Environment Facility and the ABRC for plant growth facilities and Arabidopsis DNAs and seeds, respectively. We thank the Kazusa DNA Research Institute for clone APZL47g09R and T. Thomas for 6× His-ABI5 plasmid. This project was supported in part by National Science Foundation 2010 Grant MCB-00115870 and funds from the Paul K. and Ruth R. Stumpf Endowed Professorship in Plant Biochemistry to J.C. S.L.S. was supported by an International Human Frontier Science Program fellowship.
Footnotes
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The authors 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) are: Richard D. Vierstra (vierstra{at}wisc.edu) and Judy Callis (jcallis{at}ucdavis.edu).
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↵1 Current address: Department of Biology, Life Science Centre, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, Canada B3H 4J1.
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↵[W] Online version contains Web-only data.
- Received August 9, 2006.
- Revised October 30, 2006.
- Accepted November 10, 2006.
- Published December 28, 2006.