Much of plant physiology, growth, and development is controlled by the selective removal of short-lived regulatory proteins. One important proteolytic pathway involves the small protein ubiquitin (Ub) and the 26S proteasome, a 2-MDa protease complex. In this pathway, Ub is attached to proteins destined for degradation; the resulting Ub-protein conjugates are then recognized and catabolized by the 26S proteasome. This review describes our current understanding of the pathway in plants at the biochemical, genomic, and genetic levels, using Arabidopsis thaliana as the model. Collectively, these analyses show that the Ub/26S proteasome pathway is one of the most elaborate regulatory mechanisms in plants. The genome of Arabidopsis encodes more than 1400 (or >5% of the proteome) pathway components that can be connected to almost all aspects of its biology. Most pathway components participate in the Ub-ligation reactions that choose with exquisite specificity which proteins should be ubiquitinated. What remains to be determined is the identity of the targets, which may number in the thousands in plants.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The AB13 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate AB13 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted AB13 protein displays discrete regions of high similarity to the maize viviparous-7 protein.

Small ubiquitin-like modifier (SUMO) is a member of the superfamily of ubiquitin-like polypeptides that become covalently attached to various intracellular target proteins as a way to alter their function, location, and/or half-life. Here we show that the SUMO conjugation system operates in plants through a characterization of the Arabidopsis SUMO pathway. An eight-gene family encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation, and release. A diverse array of conjugates could be detected, some of which appear to be SUMO isoform-specific. The levels of SUMO1 and -2 conjugates but not SUMO3 conjugates increased substantially following exposure of seedlings to stress conditions, including heat shock, H 2 O 2 , ethanol, and the amino acid analog canavanine. The heat-induced accumulation could be detected within 2 min from the start of a temperature upshift, suggesting that SUMO1/2 conjugation is one of the early plant responses to heat stress. Overexpression of SUMO2 enhanced both the steady state levels of SUMO2 conjugates under normal growth conditions and the subsequent heat shock-induced accumulation. This accumulation was dampened in an Arabidopsis line engineered for increased thermotolerance by overexpressing the cytosolic isoform of the HSP70 chaperonin. Taken together, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in plants with initial data indicating that one important function may be in stress protection and/or repair.Post-translational modifications of proteins play a critical role in most cellular processes through their unique ability to alter rapidly and reversibly the functions of preexisting proteins, multiprotein complexes, and intracellular structures. Although originally thought to be restricted to small molecules like phosphate and sugars, emerging data now show that several distinct types of polypeptide tags are important modifiers as well (1-4). These polypeptides become covalently attached to various intracellular targets via mechanistically similar ATPdependent reaction cascades involving activation (E1s) 1 and conjugation (E2s) enzymes. Sometimes an additional enzyme (E3s) also participates in target recognition and ligation. Ultimately, a protein conjugate is formed bearing the tag linked via an isopeptide bond between its C-terminal glycine and free lysyl ⑀-amino groups within the target. Depending on the tag and/or the target protein, the function, location, and/or half-life of the target can be affected. A family of tag-specific proteases also participates in each of the pathways. These proteases help generate the active form of the tag by removing extra residues that cap the C-terminal glycine of the polypeptide and/or are used to disassemble conjugates by cleaving the isopeptide bond between the tag and the target, thus releasing each in an unmodified form.The most pervasive and best understood tag is the 76-amino acid polypeptide ubiquitin (Ub) (5...

Ubiquitination of various intracellular proteins by ubiquitinprotein ligases (or E3s) plays an essential role in eukaryotic cell regulation primarily through its ability to selectively target proteins for degradation by the 26S proteasome. Skp1, Cullin, F-box (SCF) complexes are one influential E3 class that use F-box proteins to deliver targets to a core ligase activity provided by the Skp1, Cullin, and Rbx1 subunits. Almost 700 F-box proteins can be found in Arabidopsis, indicating that SCF E3s likely play a pervasive role in plant physiology and development. Here, we describe the reverse genetic analysis of two F-box proteins, EBF1 and -2, that work coordinately in SCF complexes to repress ethylene action. Mutations in either gene cause hypersensitivity to exogenous ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid. EBF1 and -2 interact directly with ethylene insensitive 3 (EIN3), a transcriptional regulator important for ethylene signaling. Levels of EIN3 are increased in mutants affecting either EBF1 or -2, suggesting that the corresponding SCF complexes work together in EIN3 breakdown. Surprisingly, double ebf1 ebf2 mutants display a substantial arrest of seedling growth and have elevated EIN3 levels, even in the absence of exogenous ethylene. Collectively, our results show that the SCF EBF1/EBF2 -dependent ubiquitination and subsequent removal of EIN3 is critical not only for proper ethylene signaling but also for growth in plants.

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...