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In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different poly-Ub (UBQ) gene overexpression plants of Arabidopsis thaliana. One is transformed with hexa-6His-UBQ (designated 6HU), driven by the cauliflower mosaic virus 35S promoter, while the other expresses hexa-6His-TEV-UBQ (designated 6HTU), driven by the endogenous promoter of UBQ10. We discovered that 6HU and 6HTU had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both 6HU and 6HTU. Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of UBQ expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply. 

Abstract Protein ubiquitylation is a post-translational modification (PTM) process that covalently modifies a protein substrate with either mono-ubiquitin moieties or poly-ubiquitin chains often at the lysine residues. In Arabidopsis, bioinformatic predictions have suggested that over 5% of its proteome constitutes the protein ubiquitylation system. Despite advancements in functional genomic studies in plants, only a small fraction of this bioinformatically predicted system has been functionally characterized. To expand our understanding about the regulatory function of protein ubiquitylation to that rivalling several other major systems, such as transcription regulation and epigenetics, I describe the status, issues, and new approaches of protein ubiquitylation studies in plant biology. I summarize the methods utilized in defining the ubiquitylation machinery by bioinformatics, identifying ubiquitylation substrates by proteomics, and characterizing the ubiquitin E3 ligase-substrate pathways by functional genomics. Based on the functional and evolutionary analyses of the F-box gene superfamily, I propose a deleterious duplication model for the large expansion of this family in plant genomes. Given this model, I present new perspectives of future functional genomic studies on the plant ubiquitylation system to focus on core and active groups of ubiquitin E3 ligase genes. 

In the crowded and confined space of a cell, numerous proteins work collaboratively in various subsystems, such as metabolic pathways, organelle compartments, and complexes, to regulate cell growth and development [...] 

The ubiquitin-26S proteasome system and autophagy are two major protein degradation machineries encoded in all eukaryotic organisms. While the UPS is responsible for the turnover of short-lived and/or soluble misfolded proteins under normal growth conditions, the autophagy-lysosomal/vacuolar protein degradation machinery is activated under stress conditions to remove long-lived proteins in the forms of aggregates, either soluble or insoluble, in the cytoplasm and damaged organelles. Recent discoveries suggested an integrative function of these two seemly independent systems for maintaining the proteome homeostasis. One such integration is represented by their reciprocal degradation, in which the small 76-amino acid peptide, ubiquitin, plays an important role as the central signaling hub. In this review, we summarized the current knowledge about the activity control of proteasome and autophagosome at their structural organization, biophysical states, and turnover levels from yeast and mammals to plants. Through comprehensive literature studies, we presented puzzling questions that are awaiting to be solved and proposed exciting new research directions that may shed light on the molecular mechanisms underlying the biological function of protein degradation. 

Retrograde signaling modulates the expression of nuclear genome-encoded organelle proteins to adjust organelle function in response to environmental cues. MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2) was initially recognized as a plastidial RNA-editing factor but recently shown to interact with GUN1. Given the central role of GUN1 in chloroplast retrograde signaling and the unviable phenotype of morf2 mutants that is inconsistent with many viable mutants involved in RNA editing, we hypothesized that MORF2 has functions either dosage dependent or beyond RNA editing. Using an inducible Clustered Interspaced Short Palindromic Repeat interference (iCRISPRi) approach, we were able to reduce the MORF2 transcripts in a controlled manner. In addition to MORF2-dosage dependent RNA-editing errors, we discovered that reducing MORF2 by iCRISPRi stimulated the expression of stress responsive genes, triggered plastidial retrograde signaling, repressed ethylene signaling and skotomorphogenesis, and increased accumulation of hydrogen peroxide. These findings along with previous discoveries suggest that MORF2 is an effective regulator involved in plastidial metabolic pathways whose reduction can readily activate multiple retrograde signaling molecules possibly involving reactive oxygen species to adjust plant growth. In addition, our newly developed iCRISPRi approach provided a novel genetic tool for quantitative reverse genetics studies on hub genes in plants. 

The F-box proteins function as substrate receptors to determine the specificity of Skp1-Cul1-F-box ubiquitin ligases. Genomic studies revealed large and diverse sizes of the F-box gene superfamily across plant species. Our previous studies suggested that the plant F-box gene superfamily is under genomic drift evolution promoted by epigenomic programming. However, how the size of the superfamily drifts across plant genomes is currently unknown. Through a large-scale genomic and phylogenetic comparison of the F-box gene superfamily covering 110 green plants and one red algal species, I discovered four distinct groups of plant F-box genes with diverse evolutionary processes. While the members in Clusters 1 and 2 are species/lineage-specific, those in Clusters 3 and 4 are present in over 46 plant genomes. Statistical modeling suggests that F-box genes from the former two groups are skewed toward fewer species and more paralogs compared to those of the latter two groups whose presence frequency and sizes in plant genomes follow a random statistical model. The enrichment of known Arabidopsis F-box genes in Clusters 3 and 4, along with comprehensive biochemical evidence showing that Arabidopsis members in Cluster 4 interact with the Arabidopsis Skp1-like 1 (ASK1), demonstrates over-representation of active F-box genes in these two groups. Collectively, I propose purifying and dosage balancing selection models to explain the lineage/species-specific duplications and expansions of F-box genes in plant genomes. The purifying selection model suggests that most, if not all, lineage/species-specific F-box genes are detrimental and are thus kept at low frequencies in plant genomes. 

Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating, and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number expansion of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana . Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neural network machine learning approach to predict whether an unknown FBX member is likely functionally active in Arabidopsis, thereby facilitating its future functional characterization. 

Ubiquitin is a 76 amino acid polypeptide common to all eukaryotic organisms. It functions as a post-translationally modifying mark covalently linked to a large cohort of yet poorly defined protein substrates. The resulting ubiquitylated proteins can rapidly change their activities, cellular localization, or turnover through the 26S proteasome if they are no longer needed or are abnormal. Such a selective modification is essential to many signal transduction pathways particularly in those related to stress responses by rapidly enhancing or quenching output. Hence, this modification system, the so-called ubiquitin-26S proteasome system (UPS), has caught the attention in the plant research community over the last two decades for its roles in plant abiotic and biotic stress responses. Through direct or indirect mediation of plant hormones, the UPS selectively degrades key components in stress signaling to either negatively or positively regulate plant response to a given stimulus. As a result, a tightly regulated signaling network has become of much interest over the years. The ever-increasing changes of the global climate require both the development of new crops to cope with rapid changing environment and new knowledge to survey the dynamics of ecosystem. This review examines how the ubiquitin can switch and tune plant stress response and poses potential avenues to further explore this system. 

Genome amplification and sequence divergence provides raw materials to allow organismal adaptation. This is exemplified by the large expansion of the ubiquitin-26S proteasome system (UPS) in land plants, which primarily rely on intracellular signaling and biochemical metabolism to combat biotic and abiotic stresses. While a handful of functional genomic studies have demonstrated the adaptive role of the UPS in plant growth and development, many UPS members remain unknown. In this work, we applied a comparative genomic study to address the functional divergence of the UPS at a systematic level. We first used a closing-target-trimming annotation approach to identify most, if not all, UPS members in six species from each of two evolutionarily distant plant families, Brassicaceae and Poaceae. To reduce age-related errors, the two groups of species were selected based on their similar chronological order of speciation. Through size comparison, chronological expansion inference, evolutionary selection analyses, duplication mechanism prediction, and functional domain enrichment assays, we discovered significant diversities within the UPS, particularly between members from its three largest ubiquitin ligase gene families, the F-box (FBX), the Really Interesting New Gene (RING), and the Bric-a-Brac/Tramtrack/Broad Complex (BTB) families, between Brassicaceae and Poaceae. Uncovering independent Arabidopsis and Oryza genus–specific subclades of the 26S proteasome subunits from a comprehensive phylogenetic analysis further supported a diversifying evolutionary model of the UPS in these two genera, confirming its role in plant adaptation.