Different Phenotypes ofHypomorphic csn2 and csn5 Mutants: Evidence for CSN-Independent Functionsof CSN5

The archetypal CSN present in Arabidopsis is composed of six PCI and two MPN (MPR1, PAD1 N terminal) domain subunits. Among these subunits, CSN5 has some unique features: not only does it harbor the metallopeptidase activity needed for cullin deneddylation16 but it is also the only subunit that is fully stable and detectable as a monomer or in smaller subcomplexes in wild type extracts (reviewed in ref. 17). Also, CSN5 was the only CSN subunit initially identified by sequence comparisons in budding yeast which possesses a more divergent CSN-like complex.18,19 These points suggest that CSN5 might fulfill additional CSN-independent functions inside the cell as supported by studies performed in animal cell culture systems. For example, ectopic expression of HA-Jab1 (CSN5) led to downregulation of the cell cycle regulator p27, although it did not change cullin neddylation and was not detectably incorporated into CSN, but rather present as a monomer and part of a smaller subcomplex.20 In plants, however, no indications for CSN-independent roles of CSN5 have been provided so far and no differences between csn3, csn4 and csn5 mutants could be detected by transcriptional profiling.21 The csn2-5 mutant and the csn5a-1 mutant (which carries a mutation in one of two Arabidopsis CSN5 loci) both accumulate reduced amounts of CSN, but while the CSN5 monomer is unaffected by csn2-5, it is barely detectable in csn5a mutant lines.1,5,17 We compared these two mutants in different biochemical and physiological assays. Very similar results were obtained for defects in cullin deneddylation assessed by western blotting, and physiological assays for presumed CRL-mediated responses to the phytohormones auxin, jasmonate and ethylene. While csn5a-1 mutants displayed a stronger constitutive photomorphogenesis (cop) phenotype and were strongly dwarfed, csn2-5 plants were virtually indistinguishable from wild type (see ref. 1). Thus, physiological defects of csn mutants may not correlate strictly with defects in cullin deneddylation. Also, relatively strong changes in cullin neddylation patterns can be tolerated without major phenotypic consequences. As the presence of the CSN5 monomer seems to be a major difference between the two mutants, CSN-independent functions of this protein, which are retained in csn2-5 mutant but lost in csn5a-1, might account for the above phenotypic differences. Monomeric CSN5 could have important functions in general development but would act independently of CSN's deneddylation activity and CRLs. However, while this explanation for the stronger phenotype of the csn5a-1 mutant is plausible, it should be noted that different results have been reported for Schizosaccharomyces pombe: csn1 and csn2 mutants exhibit more severe phenotypes than csn5 mutant strains.22 These differences highlight the importance of studying multiple model organisms to reach a more fundamental understanding of CSN functions.

Hypomorphic csn Mutants Exhibit Phenotypes Suggesting Defectsin Cytoskeleton Assembly

Auxin is a key player controlling lateral root formation (reviewed in ref. 23). Consistent with CSN's role as an indirect regulator of auxin signaling, defects in lateral root formation were previously described for CSN5 hypomorphic mutants.5,6 When we performed similar analyses with csn2-5 and csn5a-1, we detected differences in lateral root numbers, but these appeared to be mainly due to delayed and reduced growth of mutant primary roots, since no significant differences in lateral root density were found (data not shown). However, under these experimental conditions, we detected a skewed root growth phenotype for the csn5a-1 mutant: the average skewing angle of Col roots was approximately 3° compared to 31° of the csn5a-1 mutant (Fig. 1A; n = 14; p < 0.001). A similar phenotype was not observed for the Ler csn2-5 mutant, possibly due to more pronounced initial root skewing of wild type Ler plants (33.5° ± 7°). The basis for root skewing is currently not well understood, but auxin transport, gravity and root-gel contacts are considered to be of major importance (reviewed in ref. 24). Interestingly, exacerbated root skewing can be induced by cytoskeleton-affecting drugs, and several root skewing mutants are affected in genes regulating cytoskeleton stability. In parallel, we observed a ‘curly hypocotyl’ phenotype reminiscent of defects in cytoskeleton formation when seedlings were grown in darkness (Fig. 1B). Under these conditions, control plants grew straight, but hypocotyls of csn2-5 and csn2-5 sgt1b-3 double mutant seedlings grew in a helix-like shape performing typically 1–2 right-handed turns. In this case, a similar phenotype could not be detected for csn5a-1, but hypocotyls of csn5a-1 seedlings were extremely short because of its dwarfism and strong constitutive photo-morphogenesis (data not shown), which might mask such curly phenotype.

Both the observed root skewing and ‘curly hypocotyls’ could result indirectly from defects in responses to hormones such as auxin and ethylene (reviewed in ref. 24). But although csn mutations confer auxin resistance, we did not detect altered gravitropism response or altered ethylene response (data not shown) in csn5a-1 and csn2-5. Besides, tir1 mutants did not present any agravitropic or modified skewing phenotypes. Therefore, it would be interesting to analyze cytoskeleton assembly and organization in hypomorphic csn mutants further and assess, for example, orientation of cellulose fibrils along the cell wall, which follows the progression of cortical microtubules (reviewed in ref. 25). Such observations would be of particular interest since CRLs and the CSN-dependent Nedd8 deconjugation pathways have been directly implicated in cytoskel eton formation in C. elegans.26,27