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DNA methylation plays crucial roles in cellular development and stress responses through gene regulation and genome stability control. Precise regulation of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), thede novoArabidopsis DNA methyltransferase, is crucial to maintain DNA methylation homeostasis to ensure genome integrity. Compared with the extensive studies on DRM2 targeting mechanisms, little information is known regarding the quality control of DRM2 itself.

Here, we conducted yeast two‐hybrid screen assay and identified an E3 ligase, COP9 INTERACTING F‐BOX KELCH 1 (CFK1), as a novel DRM2‐interacting partner and targets DRM2 for degradation via the ubiquitin‐26S proteasome pathway inArabidopsis thaliana. We also performed whole genome bisulfite sequencing (BS‐seq) to determine the biological significance of CFK1‐mediated DRM2 degradation.

Loss‐of‐functionCFK1leads to increased DRM2 protein abundance and overexpression of CFK1 showed reduced DRM2 protein levels. Consistently, CFK1 overexpression induces genome‐wide CHH hypomethylation and transcriptional de‐repression at specific DRM2 target loci.

This study uncovered a distinct mechanism regulatingde novoDNA methyltransferase by CFK1 to control DNA methylation level.

 

Many eukaryotic intracellular processes employ protein ubiquitylation by ubiquitin E3 ligases for functional regulation or protein quality control. In plants, the multi‐subunit Skp1–Cullin1–F‐box (SCF) complexes compose the largest group of E3 ligases whose specificity is determined by a diverse array of F‐box proteins. Although both sequence divergence and polymorphism ofF‐boxgenes well support a broad spectrum of SCF functions, experimental evidence is scarce due to the low number of identified SCF substrates. Taking advantage of the bridge role of Skp1 between F‐box and Cullin1 in the complex, we systematically analyzed the functional influence of a well‐characterizedArabidopsis Skp1‐Like1(ASK1)Dsinsertion allele,ask1, in different Arabidopsis accessions. Through 10 generations of backcrossing with Columbia‐0 (Col‐0), we partially rescued the fertility of this otherwise sterileask1allele in Landsbergerecta, thus providing experimental evidence showing the polymorphic roles of SCF complexes. Thisask1mutant produces twisted rosette leaves, a reduced number of petals, fewer viable pollen grains, and larger embryos and seeds compared to Col‐0. RNA‐Seq‐based transcriptome analysis ofask1uncovered a large spectrum of SCF functions, which is greater than a 10‐fold increase compared with previous studies. We also identified its hyposensitive responses to auxin and abscisic acid treatments and enhanced far‐red light/phyA‐mediated photomorphogenesis. Such diverse roles are consistent with the 20–30% reduction of ubiquitylation events inask1estimated by immunoblotting analysis in this work. Collectively, we conclude that ASK1 is a predominant Skp1 protein in Arabidopsis and that the fertileask1mutant allowed us to uncover a comprehensive set of SCF functions.

 

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 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. 

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. 

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. 

The contemporary capacity of genome sequence analysis significantly lags behind the rapidly evolving sequencing technologies. Retrieving biological meaningful information from an ever-increasing amount of genome data would be significantly beneficial for functional genomic studies. For example, the duplication, organization, evolution, and function of superfamily genes are arguably important in many aspects of life. However, the incompleteness of annotations in many sequenced genomes often results in biased conclusions in comparative genomic studies of superfamilies. Here, we present a Perl software, called Closing Target Trimming (CTT), for automatically identifying most, if not all, members of a gene family in any sequenced genomes on CentOS 7 platform. To benefit a broader application on other operating systems, we also created a Docker application package, CTTdocker. Our test data on the F-box gene superfamily showed 78.2 and 79% gene finding accuracies in two well annotated plant genomes, Arabidopsis thaliana and rice, respectively. To further demonstrate the effectiveness of this program, we ran it through 18 plant genomes and five non-plant genomes to compare the expansion of the F-box and the BTB superfamilies. The program discovered that on average 12.7 and 9.3% of the total F-box and BTB members, respectively, are new loci in plant genomes, while it only found a small number of new members in vertebrate genomes. Therefore, different evolutionary and regulatory mechanisms of cullin-RING ubiquitin ligases may be present in plants and animals. We also annotated and compared the Pkinase family members across a wide range of organisms, including 10 fungi, 10 metazoa, 10 vertebrates, and 10 additional plants, which were randomly selected from the Ensembl database. Our CTT annotation recovered on average 14% more loci, including pseudogenes, of the Pkinase superfamily in these 40 genomes, demonstrating its robust replicability and scalability in annotating superfamiy members in any genomes. 

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.