Phosphoproteome analyses pinpoint the F-box protein SLOW MOTION as a regulator of warm temperature-mediated hypocotyl growth in Arabidopsis.

Hypocotyl elongation is controlled by several signals and is a major characteristic of plants growing in darkness or under warm temperature. While already several molecular mechanisms associated with this process are known, protein degradation and associated E3 ligases have hardly been studied in the context of warm temperature. In a time-course phosphoproteome analysis on Arabidopsis seedlings exposed to control or warm ambient temperature, we observed reduced levels of diverse proteins over time, which could be due to transcription, translation, and/or degradation. In addition, we observed differential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two serine residues. We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also under warm temperature conditions, and protein-protein interaction studies revealed possible interactors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likely SLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth. We propose that warm temperature-mediated regulation of SLOMO activity controls the abundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediated degradation.

Analysis of Proteasome-Associated Ubiquitin Ligase Activity.

The ubiquitin-proteasome system (UPS) is the predominant protein degradation machinery in eukaryotic cells. It is highly conserved among eukaryotes and essential for their survival. Through regulated proteolysis the UPS plays a key role in a myriad of cellular functions, including developmental and stress signaling, cell differentiation, and cell death. Attachment of a ubiquitin chain to a substrate can trigger its recruitment to the proteasome for proteolysis. To efficiently degrade substrates, however, the proteasome employs HECT-type ubiquitin ligases that can further remodel ubiquitin chains of proteasome-captured substrates. It is thought that this remodeling process is necessary to maintain substrate affinity for the proteasome and to completely translocate the substrate into the 20S proteolytic barrel. Here, we describe a protocol for purifying proteasomes and their associated accessory proteins and provide a practical way to detect proteasome-associated E3 ligase activity. This assay is reliable and efficient for assessing the ability of proteasomes to form ubiquitin conjugates and is applicable to a wide range of eukaryotic species.

Proteasome-associated ubiquitin ligase relays target plant hormone-specific transcriptional activators.

The ubiquitin-proteasome system is vital to hormone-mediated developmental and stress responses in plants. Ubiquitin ligases target hormone-specific transcriptional activators (TAs) for degradation, but how TAs are processed by proteasomes remains unknown. We report that in Arabidopsis, the salicylic acid- and ethylene-responsive TAs, NPR1 and EIN3, are relayed from pathway-specific ubiquitin ligases to proteasome-associated HECT-type UPL3/4 ligases. Activity and stability of NPR1 were regulated by sequential action of three ubiquitin ligases, including UPL3/4, while proteasome processing of EIN3 required physical handover between ethylene-responsive SCFEBF2 and UPL3/4 ligases. Consequently, UPL3/4 controlled extensive hormone-induced developmental and stress-responsive transcriptional programs. Thus, our findings identify unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases, which may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates.

Plant proteostasis: a proven and promising target for crop improvement.

The Green Revolution of the 1960s accomplished dramatic increases in crop yields through genetic improvement, chemical fertilisers, irrigation, and mechanisation. However, the current trajectory of population growth, against a backdrop of climate change and geopolitical unrest, predicts that agricultural production will be insufficient to ensure global food security in the next three decades. Improvements to crops that go beyond incremental gains are urgently needed. Plant biology has also undergone a revolution in recent years, through the development and application of powerful technologies including genome sequencing, a pantheon of 'omics techniques, precise genome editing, and step changes in structural biology and microscopy. Proteostasis - the collective processes that control the protein complement of the cell, comprising synthesis, modification, localisation, and degradation - is a field that has benefitted from these advances. This special issue presents a selection of the latest research in this vibrant field, with a particular focus on protein degradation. In the current article, we highlight the diverse and widespread contributions of plant proteostasis to agronomic traits, suggest opportunities and strategies to manipulate different elements of proteostatic mechanisms for crop improvement, and discuss the challenges involved in bringing these ideas into practice.

Harnessing the ubiquitin code to respond to environmental cues.

Ubiquitination is an essential post-translational signal that allows cells to adapt and respond to environmental stimuli. Substrate modifications range from a single ubiquitin molecule to complex polyubiquitin chains, where diverse chain topologies constitute a code that is utilized to modify the functions of proteins in numerous cellular signalling pathways. Diverse ubiquitin chain topologies are generated by linking the C-terminus of ubiquitin to one of seven lysine residues or the N-terminal methionine 1 residue of the preceding ubiquitin. Cooperative action between a large array of E2 conjugating and E3 ligase enzymes supports the formation of not only homotypic ubiquitin chains but also heterotypic mixed or branched chains. This complex array of chain topologies is recognized by proteins containing linkage-specific ubiquitin-binding domains and regulates numerous cellular pathways. Although many functions of the ubiquitin code in plants remain unknown, recent work suggests that specific chain topologies are associated with particular molecular processes. Deciphering the ubiquitin code and how plants utilize it to cope with the changing environment is essential to understand the regulatory mechanisms that underpin myriad stress responses and establishment of environmental tolerance.

SUMO Conjugation to BZR1 Enables Brassinosteroid Signaling to Integrate Environmental Cues to Shape Plant Growth.

Brassinosteroids (BRs) play crucial roles in plant development, but little is known of mechanisms that integrate environmental cues into BR signaling. Conjugation to the small ubiquitin-like modifier (SUMO) is emerging as an important mechanism to transduce environmental cues into cellular signaling. In this study, we show that SUMOylation of BZR1, a key transcription factor of BR signaling, provides a conduit for environmental influence to modulate growth during stress. SUMOylation stabilizes BZR1 in the nucleus by inhibiting its interaction with BIN2 kinase. During salt stress, Arabidopsis plants arrest growth through deSUMOylation of BZR1 in the cytoplasm by promoting the accumulation of the BZR1 targeting SUMO protease, ULP1a. ULP1a mutants are salt tolerant and insensitive to the BR inhibitor, brassinazole. BR treatment stimulates ULP1a degradation, allowing SUMOylated BZR1 to accumulate and promote growth. This study uncovers a mechanism for integrating environmental cues into BR signaling to shape growth.

Root branching toward water involves posttranslational modification of transcription factor ARF7.

Plants adapt to heterogeneous soil conditions by altering their root architecture. For example, roots branch when in contact with water by using the hydropatterning response. We report that hydropatterning is dependent on auxin response factor ARF7. This transcription factor induces asymmetric expression of its target gene LBD16 in lateral root founder cells. This differential expression pattern is regulated by posttranslational modification of ARF7 with the small ubiquitin-like modifier (SUMO) protein. SUMOylation negatively regulates ARF7 DNA binding activity. ARF7 SUMOylation is required to recruit the Aux/IAA (indole-3-acetic acid) repressor protein IAA3. Blocking ARF7 SUMOylation disrupts IAA3 recruitment and hydropatterning. We conclude that SUMO-dependent regulation of auxin response controls root branching pattern in response to water availability.