Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.Plants, like other eukaryotes, rely on the selective removal of abnormal/nonfunctional polypeptides and key short lived regulatory proteins to maintain homeostasis and control their physiology, growth, and development. Arguably, the main protease in plants is the 26 S proteasome, a 2.5-MDa complex responsible for the bulk of ubiquitin (Ub) 4 -mediated proteolysis (for reviews see Refs. 1, 2). Although the accumulated knowledge of the 26 S proteasome is mainly derived from the analysis of yeast and mammalian complexes, emerging studies indicate that a similar complex exists in plants (3, 4). Its intricate architecture is generated by a 28 subunit core protease (CP) capped at both ends by an 18 subunit or more regulatory particle (RP). The CP is a self-compartmentalized multicatalytic protease created by the assembly of four stacked heptameric rings of ␣ and ␤ subunits in a ␣ 1-7 /␤ 1-7 /␤ 1-7 /␣ 1-7 configuration. A central chamber encloses the active sites for peptidylglutamyl-peptide-hydrolyzing, trypsin-like, and chymotrypsin-like activities provided by the ␤1, ␤2, and ␤5 subunits, respectively. Access to this chamber is guarded by an axial pore created by each ␣ subunit ring, which employs a sophisticated gating mechanism to restrict access such that only unfolded proteins may enter the catalytic chamber (2, 5).The RP binds to one or both ends of the CP and sits directly over the ␣ ring pore. It is composed of two subcomplexes as follows: a base containing six related AAA-ATPases (designated RPT1-6 for regulatory particle triple-A ATPases) and three non-ATPase subunits (designated RPN1, RPN2, and RPN10, for regulatory particle non-triple-A A...

Proteotoxic stress, which is generated by the accumulation of unfolded or aberrant proteins due to environmental or cellular perturbations, can be mitigated by several mechanisms, including activation of the unfolded protein response and coordinated increases in protein chaperones and activities that direct proteolysis, such as the 26S proteasome. Using RNA-seq analyses combined with chemical inhibitors or mutants that induce proteotoxic stress by impairing 26S proteasome capacity, we defined the transcriptional network that responds to this stress in Arabidopsis thaliana. This network includes genes encoding core and assembly factors needed to build the complete 26S particle, alternative proteasome capping factors, enzymes involved in protein ubiquitylation/deubiquitylation and cellular detoxification, protein chaperones, autophagy components, and various transcriptional regulators. Many loci in this proteasome-stress regulon contain a consensus cis-element upstream of the transcription start site, which was previously identified as a binding site for the NAM/ATAF1/CUC2 78 (NAC78) transcription factor. Double mutants disrupting NAC78 and its closest relative NAC53 are compromised in the activation of this regulon and notably are strongly hypersensitive to the proteasome inhibitors MG132 and bortezomib. Given that NAC53 and NAC78 homo-and heterodimerize, we propose that they work as a pair in activating the expression of numerous factors that help plants survive proteotoxic stress and thus play a central regulatory role in maintaining protein homeostasis.

Grain number per panicle (GNP) is a major determinant of grain yield in cereals. However, the mechanisms that regulate GNP remain unclear. To address this issue, we isolate a series of sorghum [Sorghum bicolor (L.) Moench] multiseeded (msd) mutants that can double GNP by increasing panicle size and altering floral development so that all spikelets are fertile and set grain. Through bulk segregant analysis by next-generation sequencing, we identify MSD1 as a TCP (Teosinte branched/Cycloidea/PCF) transcription factor. Whole-genome expression profiling reveals that jasmonic acid (JA) biosynthetic enzymes are transiently activated in pedicellate spikelets. Young msd1 panicles have 50% less JA than wild-type (WT) panicles, and application of exogenous JA can rescue the msd1 phenotype. Our results reveal a new mechanism for increasing GNP, with the potential to boost grain yield, and provide insight into the regulation of plant inflorescence architecture and development.