Cytokinin Growth Responses in Arabidopsis Involve the 26S Proteasome Subunit RPN12

Abstract

The 26S proteasome is an ATP-dependent eukaryotic protease responsible for degrading many important cell regulators, especially those conjugated with multiple ubiquitins. Bound on both ends of the 20S core protease is a multisubunit regulatory particle that plays a crucial role in substrate selection by an as yet unknown mechanism(s). Here, we show that the RPN12 subunit of the Arabidopsis regulatory particle is involved in cytokinin responses. A T-DNA insertion mutant that affects RPN12a has a decreased rate of leaf formation, reduced root elongation, delayed skotomorphogenesis, and altered growth responses to exogenous cytokinins, suggesting that the mutant has decreased sensitivity to the hormone. The cytokinin-inducible genes CYCD3 and NIA1 are upregulated constitutively in rpn12a-1 , indicating that feedback-inhibitory mechanisms also may be altered. rpn12a-1 seedlings also showed changes in auxin-induced growth responses, further illustrating the close interaction between auxin and cytokinin regulation. In yeast, RPN12 is necessary for the G1/S and G2/M transitions of the cell cycle, phases that have been shown to be under cytokinin control in plants. We propose that RPN12a is part of the Arabidopsis 26S proteasome that controls the stability of one or more of the factors involved in cytokinin regulation. INTRODUCTIONRegulated protein turnover provides a mechanism to adjust rapidly to changing ligand concentrations and/or environmental conditions and is essential for many signal response pathways. In eukaryotes, the ubiquitin/26S proteasome pathway is particularly important, being responsible for removing most short-lived intracellular proteins (Hershko and Ciechanover, 1998;Callis and Vierstra, 2000). In this proteolytic pathway, proteins committed for degradation first are modified by the covalent attachment of multiple ubiquitins. This conjugation is directed by an ATP-dependent reaction cascade involving the sequential action of E1s, E2s, and E3s, which ultimately attach one or more ubiquitins to appropriate targets. In most cases, the ubiquitinated proteins then are recognized and degraded by the 26S proteasome, a multisubunit ATP-dependent protease with broad substrate specificity.The 26S proteasome is a 2-MD complex assembled from two particles: the 20S core particle (CP) and the 19S regulatory particle (RP) (Voges et al., 1999). The proteolytic activities reside within the central chamber of the 28-subunit CP, whereas the functions that direct substrate recognition, unfolding, and subsequent entry into the 20S particle reside within the Ͼ 18-subunit RP. The RP can be divided further in two subcomplexes, the lid and base (Glickman et al., 1998). The base contains six ATPase subunits, RPT1 to RPT6, which presumably use ATP hydrolysis to unfold target proteins, and three non-ATPase subunits, RPN1, RPN2, and RPN10. The lid contains nine additional RPN subunits (RPN3, RPN5 to RPN9, and RPN11 to RPN13). Many of the lid RPN subunits share sequence motifs with components of the COP9/signalosome and EIF3 co...

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

“…Whereas the decline in ATG1a and ATG13a during 24 or 72 h of Suc/N limitation proceeded normally in the 26S proteasome mutant rpn12a-1, which compromises proteasome integrity (Smalle et al, 2002), their levels were dramatically stabilized in the strong autophagy mutant atg5-1 Phillips et al, 2008) (Figure 9B). The transcript levels of both ATG1a and ATG13a were unaffected by the atg5-1 mutation, indicating that the stabilization reflected a posttranscriptional event and not atg5-1-mediated induction of the two genes (see Supplemental Figure 7D online).…”

“…Each mutant was backcrossed three times to wild-type Col-0 before phenotypic analysis. The atg5-1, atg7-2, and rpn12a-1 mutants in the Col-0 background were as previously reported (Smalle et al, 2002;Chung et al, 2010).…”

“…For RNA gel blot analysis, total RNA was isolated with the TRIzol regent (Invitrogen) according to Smalle et al (2002). 32 P-labeled riboprobes were synthesized with T7, SP6, or T3 RNA polymerase using the RiboprobeR system and the linearized pGEMT (Promega) or pBluescript (Stratagene) cDNA constructs for ATG1a, ATG1b, ATG13a, ATG13b, and TUB1.…”

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

“…For example, the non-ATPase RPN12 subunit has been demonstrated to be involved in cytokinin responses. A T-DNA insertion mutant of RPN12 exhibits reduced leaf formation and root elongation, delayed skotomorphogenesis, and decreased growth responses to exogenous cytokinin (Smalle et al, 2002). On the other hand, a mutation in the RPN10 gene results in a pleiotropic phenotype in Arabidopsis.…”

Arabidopsis thaliana26S Proteasome Subunits RPT2a and RPT5a Are Crucial for Zinc Deficiency-Tolerance

“…RPN12a and RPN10 are involved in the responses to cytokinin and abscisic acid respectively. 4,5) RPT2a is required for the maintenance of root apical meristems. 6) In A. thaliana, most of the RP subunits were encoded by two genes, suggesting that various combinations of 26SP subunits are present in A. thaliana, this might contribute to functional diversity.…”