Article Figures & Data
Figures
- Figure 1.
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).
- Figure 2.
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.
- Figure 3.
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.
- Figure 4.
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.
- Figure 5.
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.
- Figure 6.
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.
- Figure 7.
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.
- 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.
Additional Files
Supplemental Data
Files in this Data Supplement:
- Supplemental Figure 1 - Nucleotide sequence of 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 - Phenotype of light-grown keg seedling roots and dark-grown keg seedlings.
- Supplemental Table 1 - Table of primers used in this study.
