The RUB/Nedd8 conjugation pathway is required for early development in Arabidopsis

Abstract

The related-to-ubiquitin (RUB) protein is post-translationally conjugated to the cullin subunit of the SCF (SKP1, Cullin, F-box) class of ubiquitin protein ligases. Although the precise biochemical function of RUB modification is unclear, studies indicate that the modification is important for SCF function. In Arabidopsis, RUB modification of CUL1 is required for normal function of SCF(TIR1), an E3 required for response to the plant hormone auxin. In this report we show that an Arabidopsis protein called RCE1 functions as a RUB-conjugating enzyme in vivo. A mutation in the RCE1 gene results in a phenotype like that of the axr1 mutant. Most strikingly, plants deficient in both RCE1 and AXR1 have an embryonic phenotype similar to mp and bdl mutants, previously shown to be deficient in auxin signaling. Based on these results, we suggest that the RUB-conjugation pathway is required for auxin-dependent pattern formation in the developing embryo. In addition, we show that RCE1 interacts directly with the RING protein RBX1 and is present in a stable complex with SCF. We propose that RBX1 functions as an E3 for RUB modification of CUL1.

Fig. 1. Pattern of RCE1 gene expression analyzed by RNA blot and GUS staining. (A) RNA blot showing expression of RCE1 in all the tissues examined. The lower panel shows an ethidium bromide stained gel to demonstrate equal loading. (B–G) GUS staining in RCE1::GUS plants. These plants carry a translational fusion between RCE1 and GUS under control of the RCE1 promoter. (B) GUS staining in 4-day-old light grown seedling. (C) Shoot apical region of a 7-day-old light grown seedling. (D) Root apex of a 10-day-old seedling. (E) Root segment from a 10-day-old seedling showing GUS staining in lateral root primordium. (F) Primary leaf from 10-day-old seedling. (G) A magnified view of a trichome from (F) showing intense staining at the basal region.

Fig. 2. A mutation in RCE1 confers morphological defects similar to auxin-resistant mutants. (A) RNA blot showing expression of RCE1 in 6-day-old wild-type and rce1-1 seedlings. (B) Response of 6-day-old seedling roots to gravity. (C) Twenty-one-day-old rosettes. (D) Thirty-six-day-old plants. For both (C) and (D), Ler is on the left and rce1-1 is on the right.

Fig. 3. RCE1 is required for RUB modification of CUL1. Ten micrograms of total proteins were loaded in each lane. Proteins were blotted and treated with α-CUL1 antiserum. (A) Protein blot of Ler (lane 1) and rce1-1 (lane 2). (B) Protein blot of Col-0 (lane 1), 35S::RBX1-2B RCE1 (lane 2), rce1-1 (lane 3) and 35S::RBX1-2B rce1-1 (lane 4).

Fig. 4. RCE1 is required for auxin and jasmonate response. (A) Inhibition of seedling roots on media containing 2,4-D. Col (diamonds), Ler (triangles), rce1-1 (squares) and axr1-12 (circles). (B) Growth of rce1-1, rce1-1, 35S::Myc-RCE1 and Col-0 roots on medium containing 85 nM 2,4-D. Black line represents the position of the root tip at time of transfer to auxin medium. (C) Lateral root formation in seedling roots in response to 85 nM 2,4-D. (D) RNA blot showing the induction of IAA2 in 6-day-old seedlings treated with or without 20 µM 2,4-D for 60 min. The ethidium bromide stained gel is shown in the bottom panel. (E) GUS staining of 7-day-old seedlings. The seedlings were treated with or without 20 µM 2,4-D for 2 h. (F) Pulse–chase analysis of IAA7/AXR2 in Col-0 and rce1-1 seedlings. The half-lives represent the mean of three separate experiments ± SD. (G) Inhibition of seedling roots by methyl jasmonate. Col-0 (diamonds), Ler (triangles), rce1-1 (squares) and axr1-12 (circles).

Fig. 5. The RUB conjugation pathway is required during embryogenesis. (A) Phenotypes of wild-type and mutant seedlings. The rce1-1 axr1-12 double mutant seedlings were 6 days old when photographed. These seedlings died within 10–12 days of germination. All other seedlings were 12–14 days old when photographed. (B) Protein blot showing the levels of unmodified and modified CUL1 for each genotype. Total proteins were extracted from 6-day-old seedlings and immunoblotted with α-CUL1 antiserum. Ten micrograms of protein was loaded in each lane. (C) Ratio of RUB–CUL1 to CUL1 from (B) determined using NIH image. (D) Vascular patterning in cotyledons of 6-day-old seedlings.

Fig. 6. RCE1 interacts with the SCF and the RUB activating enzyme. (A) [³²P]RBX1 (20 µl) was added to glutathione beads loaded with 3 µg of either GST or GST–RCE1. After washing, proteins retained on the beads were analyzed by SDS–PAGE. (B) Protein extracts were prepared from 35S::Myc-RCE1 seedlings. Proteins were recovered after pull-down with GST–RBX1 or immunoprecipitation with α-CUL1 antiserum and analyzed by immunoblot using α-Myc antiserum. (C) GST–RCE1 pulldowns were performed using protein extracts prepared from GVG::TIR1-Myc or 35S::LRF1-Myc seedlings. Recovered proteins were analyzed by immunoblot using α-Myc antiserum. (D) Protein extracts from 35S::Myc-RCE1 seedlings were immunoprecipitated with α-AXR1 antiserum and immunoblotted with α-Myc antibody. Lanes 1 and 3, Columbia extract; lanes 2, 4 and 5, 35S::Myc-RCE1 extract.