Stone Lab

at Dalhousie University, Nova Scotia, Canada

Research

Ubiquitin is a small, highly conserved, ubiquitously expressed eukaryotic protein with immensely important and diverse regulatory functions. The covalent attachment of one or more ubiquitin molecules to intracelluar proteins (referred to as ubiquitination) can influence activity, abundance, interaction, trafficking or localization. Ubiquitination is a multi-step reaction involving the sequential action of three enzymes: E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme) and E3 (ubiquitin ligase) (Fig. 1). The conjugation process involves the transfer of ubiquitin attached to the E1 to the E2 enzyme forming a thioester linked E2-ubiquitin (E2-ub) intermediate. The substrate recruiting E3 facilitates transfer of the ubiquitin from the E2 onto a lysine residue of the substrate. The most notable function of ubiquitin is its role in selective proteolysis, where the attachment of a chain of ubiquitin molecules (referred to as polyubiquitination) promotes recognition and destruction of the modified substrate by the 26S proteasome (Fig. 1). Plants utilize the ubiquitin proteasome system (UPS) to facilitate changes in cellular protein content required for continuous growth, development and survival of their ever-changing environment. In response to an environmental stimulus or developmental cue, ubiquitination of proteins, such as transcription regulators, can be either promoted or inhibited leading to increased degradation or stabilization, which results in the required cellular response.


Figure 1. The Ubiquitin Proteasome System. Ubiquitination pathway showing the function of the ubiquitin enzymes, E1, E2 and E3 (single and complex E3s).

 

The Arabidopsis thaliana (Arabidopsis; model species) genome is predicted to encode for over 1,400 proteins associated with the UPS, of which ~500 are classified as single subunit RING-type E3s (Fig. 1) (1). Ubiquitin ligases are involved in many aspects of plant growth and development as well as defense against pathogen attack (2). E3s have emerged as major modulators of plant response to abiotic stresses including drought, salinity, radiation and nutrient deprivation. A single E3 can regulate the abundance of multiple proteins, allowing an enzyme to regulate different types of processes. For example, a single E3 may regulate responses multiple abiotic stresses such as drought and high salinity, or have diverse roles such as regulating pathogen defense and flower development. The impact of the ubiquitin ligases is usually associated with regulating the biosynthesis, perception, signal transduction and/or output of phytohormones. This exemplified by the finding that over 20 E3s regulate the action of the stress hormone abscisic acid (ABA) (Fig. 2) (3).


Figure 2. Ubiquitin ligases that regulate ABA signaling. E3s are categorized by involvement in facilitating inhibition (1), activation (2), and attenuation or termination (3) of hormone signallingthrough proteasome-dependent degradation. Not all E3 ligases are shown, mainly those with identified substrates. Ref. International Review of Cell and Molecular Biology 2019, 343:65-110.


Figure 3. ANKYRIN repeat-containing RING-type E3 family of Arabidopsis contains six members named XBAT (XB3 ortholog in Arabidopsis) 31 to 35 and KEG (Keep on Going).

 

Our research focuses mainly on the function of the RING-type E3s, specifically the Anyrin-repeat containing E3 ligase, which includes KEG, XBAT31 to XBAT35 (Fig. 3). KEG is a negative regulator of ABA signalling, required to maintain low levels of the ABA responsive transcription factor Abscisic Acid Insensitive 5 (ABI5) (4,6) (Fig. 2 and 4). Increase in ABA levels in response to abiotic stress leads to ABI5 accumulation and suspension of seedling growth. Our studies show that ABA promotes KEG self-ubiquitination and proteasome-dependent degradation, which would allow for the accumulation of ABI5 and post-germinative growth arrest (5,6). The identification of Calcineurin B-like (CBL) Interacting Protein Kinase (CIPK) 26, which phosphorylates ABI5, as a target expands the role of the KEG as a negative regulator of ABA signalling (7,8) (Fig. 2). Other KEG interactors include the stress-responsive enzyme Formate Dehydrogenase (FDH). In accordance with KEG promoting FDH degradation, overexpression of the E3 increases seedling formate sensitivity (9). XBAT32 is involved lateral root development and targets ethylene biosynthesis enzymes, aminocyclopropane-1-carboxylic acid synthase 4 (ACS4) and ACS7, to the 26S proteasome for degradation (Fig. 5)(10,11). XBAT35 undergoes alternative splicing to produce two isoforms; nuclear-localized XBAT35.1 and golgi-localized XBAT35.2 (12). XBAT35.2 was found to regulate cell death and promote defense against pathogen attack (Fig. 6) (12). We are also investigating a potential role for both isoforms in abiotic stress tolerance. XBAT31 is also alternatively spliced producing two isoforms; nuclear-localized XBAT31.1 and membrane-bound XBAT31.2. Preliminary studies suggest a role for XBAT31 in iron deficiency stress response.


Figure 4. KEG mutants accumulate phosphorylated ABI5 and undergo growth arrest in the absence of ABA. Ref. Plant Cell 2010,18(12):3415-28.


Figure 5. Response of xbat32 mutant and wild type seedlings to inhibition of ethylene signaling. Three-day-old seedlings were treated with silver nitrate (AgNO3) for five days. Ref. Plant Physiology 2010, 153(4):1587-96.


Figure 6. XBAT35.2 overexpression leads to cell death in tobacco leaves. Tobacco leaf epidermal cells were transiently transformed with Agrobacteria carrying the constructs for expression of XBAT35.2-YFP-HA or non-functional XBAT35.2AA-YFP-HA. Circles indicate site of infiltration. Ref. Plant Physiology 2017, 175(3):1469-1483

See the publications list for a more information on our research.