Functional Analysis of the miR156 Regulatory Network in Arabidopsis Siliques

Siliques are photosynthetically active seed capsules and their development is strongly influenced by embryo development. MicroRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) network is involved in regulating plant growth and development, but the downstream genes of this network are still not fully elucidated. Here, I show that the miR156/SPL2 pathway controls the development of floral organs, regulates pollen production, and thus affects male fertility in Arabidopsis thaliana. I present evidence that SPL2 binds to the 5’UTR of the ASYMMETRIC LEAVES 2 (AS2) gene in vivo, indicating that AS2 acts downstream of SPL2. When compared to wild-type plants, AS2 loss-of-function mutants share certain similarities with SPL2 dominant-negative mutants and miR156 overexpression plants, including defective floral organ growth and partially sterile phenotypes. Furthermore, the transcript levels of AS2 were decreased in both miR156 overexpression plants and SPL2 dominant-negative mutant. My results suggest that the miR156/SPL2 pathway and AS2 affect floral organs and silique development as well as plant fertility, and AS2 is directly activated by SPL2

HECT ubiquitin ligases as accessory proteins of the plant proteasome

The proteasome plays vital roles in eukaryotic cells by orchestrating the regulated degradation of large repertoires of substrates involved in numerous biological processes. Proteasome dysfunction is associated with a wide variety of human pathologies and in plants severely affects growth, development and responses to stress. The activity of E3 ubiquitin ligases marks proteins fated for degradation with chains of the post-translational modifier, ubiquitin. Proteasomal processing of ubiquitinated substrates involves ubiquitin chain recognition, deubiquitination, ATP-mediated unfolding and translocation, and proteolytic digestion. This complex series of steps is made possible not only by the many specialised subunits of the 1.5 MDa proteasome complex but also by a range of accessory proteins that are recruited to the proteasome. A surprising class of accessory proteins are members of the HECT-type family of ubiquitin ligases that utilise a unique mechanism for post-translational attachment of ubiquitin to their substrates. So why do proteasomes that already contain all the necessary machinery to recognise ubiquitinated substrates, harbour HECT ligase activity? It is now clear that some ubiquitin ligases physically relay their substrates to proteasome-associated HECT ligases, which prevent substrate stalling at the proteasome. Moreover, HECT ligases ubiquitinate proteasome subunits, thereby modifying the proteasome’s ability to recognise substrates. They may therefore enable proteasomes to be both non-specific and extraordinarily selective in a complex substrate environment. Understanding the relationship between the proteasome and accessory HECT ligases will reveal how the proteasome controls so many diverse plant developmental and stress responses

Type-enhanced Ensemble Triple Representation via Triple-aware Attention for Cross-lingual Entity Alignment

Entity alignment(EA) is a crucial task for integrating cross-lingual and cross-domain knowledge graphs(KGs), which aims to discover entities referring to the same real-world object from different KGs. Most existing methods generate aligning entity representation by mining the relevance of triple elements via embedding-based methods, paying little attention to triple indivisibility and entity role diversity. In this paper, a novel framework named TTEA -- Type-enhanced Ensemble Triple Representation via Triple-aware Attention for Cross-lingual Entity Alignment is proposed to overcome the above issues considering ensemble triple specificity and entity role features. Specifically, the ensemble triple representation is derived by regarding relation as information carrier between semantic space and type space, and hence the noise influence during spatial transformation and information propagation can be smoothly controlled via specificity-aware triple attention. Moreover, our framework uses triple-ware entity enhancement to model the role diversity of triple elements. Extensive experiments on three real-world cross-lingual datasets demonstrate that our framework outperforms state-of-the-art methods

Proteasome-associated HECT-type ubiquitin ligase activity is required for plant immunity

<div><p>Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in <i>UPL1</i>, <i>UPL3</i> and <i>UPL5</i> significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of <i>upl3</i> mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.</p></div

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 SCF(EBF2) 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

Functional analysis of proteasome-associated ubiquitin ligases in plants

Degradation of intracellular proteins by the ubiquitin-proteasome system (UPS) is a sophisticated mechanism that begins with anchoring ubiquitin molecules to a substrate and ends with proteasome-dependent proteolysis. Initiation of ubiquitination by E3 ligases is a key step in this pathway that selectively labels unstable or damaged proteins. The ubiquitinated substrate is then recognised by proteasome-associated ubiquitin receptors and subsequently degraded by the proteasome. Recent studies have identified several E3 ligases that surprisingly associate with the proteasome as accessory proteins. As substrates are already modified by ubiquitin when they arrive at the proteasome, it is unclear what the role of these proteasome-associated ligases are. In this study, the role of proteasome-associated ubiquitin ligases in proteasomal substrate degradation was characterised and the functional significance of ubiquitin chain remodelling at the proteasome were explored in planta. In Arabidopsis thaliana, HECT-type Ubiquitin Protein Ligases (UPLs) have been identified as proteasome-associated ubiquitin ligases that are required for salicylic acid (SA)-induced plant immunity. Accordingly, the mechanism behind regulation of plant immune response by UPLs is further studied in Chapter 3. Here, it is shown that UPLs control SA-dependent transcriptional reprogramming via regulating homeostasis of the SA-responsive coactivator NPR1. SA-induced accumulation of NPR1 was impaired in upl mutants, which resulted in diminished expression of immune genes. Additionally, proteasome-associated UPLs facilitated polyubiquitination of NPR1, and thereby promoted its proteasomal turnover. This process was indispensable for clearing inactive NPR1 from chromatin. Thus, UPL-mediated remodelling of NPR1-attached ubiquitin chains at the proteasome is required for maximum transcriptional activity of NPR1. In Chapter 4 I show that proteasome-associated UPLs also target other transcription activators, including the developmental and ethylene-responsive EIN3 activator. I demonstrate that by physically interacting with UPL3, the SCFEBF2 ubiquitin ligase complex directly escorted EIN3 to the proteasome. Subsequent ‘eleventh-hour’ ubiquitin chain remodelling by proteasome-associated UPL3/4 was required for processive degradation of EIN3 by the proteasome and was critical for removal of EIN3 from its target gene promoters. Besides targeting substrates destined for the proteasome, I show in Chapter 5 that UPL3 and UPL4 are also involved in polyubiquitination of other E3 ligases. UPL3/4 catalysed ubiquitination of the immune-responsive U-box E3 ligase, PUB22, and controlled its proteasomal turnover. Mutation of PUB22 and its homologues, PUB23 and PUB24, supressed the disease susceptibility phenotype of the upl3 upl4 mutant, indicating that UPL3/4 also regulate immunity via modulating homeostasis of PUB ligases. Overall, my findings indicate that unstable hormone-responsive transcriptional activators are sequentially polyubiquitinated by relays of ubiquitin ligases in which HECT-type ligases prevent the stalling of proteasome-bound substrates. On the other hand, HECT-type ligases also target other E3 ligases for degradation, thereby indirectly influencing substrate levels of these E3 ligases. Thus, my findings demonstrate that proteasomes unexpectedly influence the ubiquitination and stability of both E3 ligases and their substrates to regulate transcriptional programmes in plants

Numerical simulation of detailed flow through forming fabric

The relation between the filament displacement of forming fabric and the wire mark characteristic of final paper product is investigated for the first time. The ultimate goal of this research is to understand the detailed dewatering mechanism of forming section in 3D level as well as provide a guide of forming fabric design and maintenance to obtain paper product with best quality. A 3D computational model of a square weave single-layer forming fabric has been developed by geometry modeling and commercial CFD software to simulate the detailed dewatering process of forming section. The reliability of the geometric model, grid convergence, solver settings, and boundary conditions are checked. Experimental data of wind tunnel screen is used to indirectly validate this model since currently there is no 3D experimental data of forming fabric available. Accuracy analysis is conduct with GCI (grid convergence index) method, which is widely accepted for CFD error estimation. Next, the effect of filament’s displacement on the upstream velocity distribution, which represents the fiber distribution in the formed fiber mat, is revealed. From wire mark theory, this distribution pattern can directly affect the grammage and wire mark level on paper sheet. The 2D/3D velocity plots of geometric structure with filament displacement are compared with those of uniform structure. Pressure drop and mass flow rate under both cases are also discussed. The CFD result shows the displacement of forming fabric filaments has a significant adverse influence on the uniformity of the upstream velocity distribution and mass flow rate through different open areas of the fabric. Good agreement between the simulation outcomes and experimental pressure drop values through wind tunnel screens is achieved. The pressure drop through fabric is independent of the displacement. Therefore, it cannot be used to judge the fabric uniformity. This numerical model can significantly contribute to the understanding of forming fabric with a low cost. However, 3D experimental work should be done in future to validate this model. More complex CFD models such as multi-phase model also should be developed as the next step to obtain more accurate results.Applied Science, Faculty ofMechanical Engineering, Department ofGraduat

AED: a novel visual representation based on AR and empathy computing in manual assembly

Adding personal perceptions to manufacturing engineering can be very challenging, especially when engineering-based methods are used to make artisans understand the designer&#39;s ideas. Unfortunately, the two-dimensional engineering definition can be extremely time-consuming for individuals who lack creativity or imagination, and model-based definition would be incapable of breaking the separation between the virtual space and the real world, which makes the interaction between natural persons exist spatial perception error. The emergence of Augmented Reality (AR), which allows individuals to perceive the intentions and strategies of the designer with visual cues that are attached to actual objects, fills this gap. In this paper, augmented engineering definition (AED) is proposed to enhance the information exchange between natural persons in a succinct, accurate and acceptable form of visual impression. Motivated by visual representation in remote collaboration, the specific empathy scenario bases on the AED design, which leads to the establishment of a mapping relationship between the visual cues and the augmented information. An inquiry had been conducted by involving participants who were paired up for the parts&rsquo; inspection, interacting via 2D visualization data only, interaction with 3D projection data, interaction with 3D visualization data, AED-based communication. The experimental results showed that participants with AED exhibited higher situational appeal and information understanding than using three other interactions. Besides, we discussed the feasibility of using AED in a collaborative manufacturing environment and the impact on AED users