Abstract
The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.
Highlights
The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes
In a mutant screen for reduced response to the auxin-like compound indole-3-propionic acid (IPrA), we identified ecr1-1, a mutant carrying a single amino acid change in a RUB activation enzyme
IPrA is structurally similar to the endogenous auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA)
Summary
The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. The ecr mutant is resistant to the auxin-like compound indole-3propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. Ubiquitin and members of the ubiquitin-like protein family modify the stability, localization, activity, or other characteristics of target proteins (for review, see Schwartz and Hochstrasser, 2003) One such ubiquitinlike protein, RELATED TO UBIQUITIN (RUB/NEDD8), is conjugated to CULLIN (CUL) subunits of ubiquitin E3 ligase complexes (Liakopoulos et al, 1998) to regulate E3 activity (for review, see Schwechheimer and Calderon Villalobos, 2004). Auxins and auxin-like compounds are utilized commercially as herbicides and to achieve adventitious rooting of cuttings for clonal plant propagation, with various compounds showing different efficacies depending on the application and species (Hartmann et al, 1990)
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