Abstract
The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCFTIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCFTIR1-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.
Highlights
The phytohormone auxin regulates numerous plant developmental processes, by modulating the expression of auxin responsive genes to control cell division, expansion and differentiation [1]
We have previously reported the identification of several auxin response mutants isolated from a genetic screen for enhancers of the tir1-1 auxin response defect, including eta1/axr6-3 [15], eta2-1/cand1 [52], eta3/sgt1b [53], eta4/ pdr9-1 [54], eta5/iar4 [51] and eta6/csn1-10 [43]
Since our finding that csn3-3 plants exhibited no change in cullin deneddylation was quite surprising, we examined SCFTIR1/AFB activity by monitoring Aux/IAA stability using the previously described HS:AXR3NT-GUS reporter protein [4]. csn1-10 seedlings were again included for comparison. 6-d.o
Summary
The phytohormone auxin (indole-3-acetic acid or IAA) regulates numerous plant developmental processes, by modulating the expression of auxin responsive genes to control cell division, expansion and differentiation [1]. In Arabidopsis, the CSN is required for the proper functions of SCFTIR1/AFB, as well as SCFSLY1, in the degradation of Aux/IAA proteins or the DELLA proteins, respectively [28,31,43] Together, these findings suggest that cullin neddylation/deneddylation is a highly dynamic process essential for maintaining proper CRL function [44,45]. These include mutants lacking one of the two copies of CSN5 and CSN6 encoded in the Arabidopsis genome [23,50], as well as hypomorphic missense alleles of CSN1 and CSN2 [40,43] These viable csn mutants still exhibit diminished deneddylation activity, resulting in the accumulation of neddylated CUL1 and reduced SCF activity [23,28,34,35,40,43]. Our studies suggest that csn is a unique csn mutant that defines a novel functional activity for the CSN3 subunit of the COP9 signalosome in the regulation of auxin signaling
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