ubiquitin-26S Proteasome System Research Articles

Arabidopsis F-box proteins D5BF1 and D5BF2 negatively regulate Agrobacterium-mediated transformation andtumorigenesis.

The pathogen Agrobacterium tumefaciens is known for causing crown gall tumours in plants. However, it has also been harnessed as a valuable tool for plant genetic transformation. Apart from the T-DNA, Agrobacterium also delivers at least five virulence proteins into the host plant cells, which are required for an efficient infection. One of these virulence proteins is VirD5. F-box proteins, encoded in the host plant genome or the Ti plasmid, and the ubiquitin/26S proteasome system (UPS) also play an important role in facilitating Agrobacterium infection. Our study identified two Arabidopsis F-box proteins, D5BF1 and D5BF2, that bind VirD5 and facilitate its degradation via the UPS. Additionally, we found that Agrobacterium partially suppresses the expression of D5BF1 and D5BF2. Lastly, stable transformation and tumorigenesis efficiency assays revealed that D5BF1 and D5BF2 negatively regulate the Agrobacterium infection process, showing that the plant F-box proteins and UPS play a role in defending against Agrobacterium infection.

A pepper RING-finger E3 ligase, CaFIRF1, negatively regulates the high-salt stress response by modulating the stability of CaFAF1.

Controlling protein stability or degradation via the ubiquitin-26S proteasome system is a crucial mechanism in plant cellular responses to stress conditions. Previous studies have revealed that the pepper FANTASTIC FOUR-like gene, CaFAF1, plays a positive role in salt tolerance and that, in this process, CaFAF1 protein degradation is delayed. Here, we sought to isolate the E3 ligases potentially responsible for modulating CaFAF1 protein stability in response to salt stress. The pepper RING-type E3 ligase CaFIRF1 (Capsicum annuum FAF1 Interacting RING Finger protein 1) was found to interact with and ubiquitinate CaFAF1, leading to the degradation of CaFAF1 proteins. In response to high-salt treatments, CaFIRF1-silenced pepper plants exhibited tolerant phenotypes. In contrast, co-silencing of CaFAF1 and CaFIRF1 led to increased sensitivity to high-salt treatments, revealing that CaFIRF1 functions upstream of CaFAF1. A cell-free degradation analysis showed that high-salt treatment suppressed CaFAF1 protein degradation via the 26S proteasome pathway, in which CaFIRF1 is functionally involved. In addition, an in vivo ubiquitination assay revealed that CaFIRF1-mediated ubiquitination of CaFAF1 proteins was reduced by high-salt treatment. Taken together, these findings suggest that the degradation of CaFAF1 mediated by CaFIRF1 has a critical role in pepper plant responses to high salinity.

HECT-type ubiquitin ligase KAKTUS mediates the proteasome-dependent degradation of cyclin-dependent kinase inhibitor KRP2 during trichome morphogenesis in Arabidopsis.

SUMMARYTrichome development is a fascinating model to elaborate the plant cell differentiation and growth processes. A wealth of information has pointed to the contributions of the components associated with cell cycle control and ubiquitin/26S proteasome system (UPS) to trichome morphogenesis, but how these two pathways are connected remains obscure. Here, we report that HECT‐type ubiquitin ligase KAKTUS (KAK) targets the cyclin‐dependent kinase (CDK) inhibitor KRP2 (for kip‐related protein 2) for proteasome‐dependent degradation during trichome branching in Arabidopsis. We show that over‐expression of KRP2 promotes trichome branching and endoreduplication which is similar to kak loss of function mutants. KAK directly interacts with KRP2 and mediates KRP2 degradation. Mutation of KAK results in the accumulation of steady‐state KRP2. Consistently, in kak pKRP2:KRP2‐GFP plants, the trichome branching is further induced compared with the single mutant. Taken together, our studies bridge the cell cycle control and UPS pathways during trichome development and underscore the importance of post‐translational control in epidermal differentiation.

The F-box protein SHORT PRIMARY ROOT modulates primary root meristem activity by targeting SEUSS-LIKE protein for degradation in rice.

Root meristem activity is essential for root morphogenesis and adaptation, but the molecular mechanism regulating root meristem activity is not fully understood. Here, we identify an F-box family E3 ubiquitin ligase named SHORT PRIMARY ROOT (SHPR) that regulates primary root (PR) meristem activity and cell proliferation in rice. SHPR loss-of-function mutations impair PR elongation in rice. SHPR is involved in the formation of an SCF complex with the Oryza sativa SKP1-like protein OSK1/20. We show that SHPR interacts with Oryza sativa SEUSS-LIKE (OsSLK) in the nucleus and is required for OsSLK polyubiquitination and degradation by the ubiquitin 26S-proteasome system (UPS). Transgenic plants overexpressing OsSLK display a shorter PR phenotype, which is similar to the SHPR loss-of-function mutants. Genetic analysis suggests that SHPR promotes PR elongation in an OsSLK-dependent manner. Collectively, our study establishes SHPR as an E3 ubiquitin ligase that targets OsSLK for degradation, and uncovers a protein ubiquitination pathway as a mechanism for modulating root meristem activity in rice.

To Kill or to Be Killed: How Does the Battle between the UPS and Autophagy Maintain the Intracellular Homeostasis in Eukaryotes?

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.

Effects of interaction between ubiquitin/26S proteasome inhibitor MG132 and mycorrhiza on growth and effective components of Salvia miltiorrhiza

Ubiquitin/26 S proteasome system(UPS) is one of the main ways to regulate the degradation of proteins in plants, and plays an important role in physiological processes such as secondary metabolism and plant hormone signal transduction. As indicated recently, UPS is involved in plant-microbe interactions, and presumably regulates arbuscular mycorrhizal symbiosis to affect its effects. This study investigated the effects of interaction between Cbz-leu-leu-leucinal(MG132) and the mycorrhiza on the growth and effective components of Salvia miltiorrhiza by inoculation with Glomus intraradices and spraying MG132 solution. The results showed that the inoculation with G. intraradices could promote the growth of S. miltiorrhiza, increase the accumulation of effective components in the aerial and underground parts, and decrease the relative expression level of JMT. Additionally, MG132 could strengthen the growth-promoting effect of G. intraradices. As compared with the control group, the inoculation with G. intraradices could significantly increase aerial and underground fresh weights by 267% and 95%, respectively, under the treatment with MG132 spraying, while under the MG132 spraying-free condition, the increase was 195% and 32%, respectively. Meanwhile, MG132 spraying could enhance the promotion of mycorrhizal fungi on the accumulation of active components of S. miltiorrhiza. On the other hand, regardless of inoculation with G. intraradices or not, MG132 treatment could promote the root division of S. miltiorrhiza, reduce the content of effective components in the aerial parts, and increase the content in the underground part. The inoculation with G. intraradices could alleviate the inhibitory effect of MG132 on the accumulation of effective components in the aerial part of S. miltiorrhiza. The results show that arbuscular mycorrhizal fungi(AMF) can promote the growth of S. miltiorrhiza and the accumulation of effective components, and MG132 treatment can strengthen such promotion effect, which lays a foundation for the application of MG132 in the mycorrhizal cultivation of S. miltiorrhiza in the future.

A small chromosomal inversion mediated by MITE transposons confers cleistogamy in Brassica napus.

Cleistogamy, self-pollination within closed flowers, can help maintain seed purity, accelerate breeding speed, and aid in the development of ornamental flowers. However, the mechanism underlying petal closing/opening behavior remains elusive. Here, we found that a Brassica napus petal closing/opening behavior was inherited in a Mendelian manner. Fine mapping and positional cloning experiments revealed that the Mendelian factor originated from a short (29.8 kb) inversion mediated by BnDTH9 miniature inverted-repeat transposable elements (MITEs) on chromosome C03. This inversion led to tissue-specific gene promoter exchange between BnaC03.FBA (BnaC03G0156800ZS encoding an F-Box-associated domain-containing protein) and BnaC03.EFO1 (BnaC03G0157400ZS encoding an EARLY FLOWERING BY OVEREXPRESSION 1 protein) positioned near the respective inversion breakpoints. Our genetic transformation work demonstrated that the cleistogamy originated from high tissue-specific expression of the BnaC03.FBA gene caused by promoter changes due to the MITE-mediated inversion. BnaC03.FBA is involved in the formation of an SCF (Skp1-Cullin-F-box) complex, which participates in ubiquitin-mediated protein targeting for degradation through the ubiquitin 26S-proteasome system. Our results shed light on a molecular model of petal-closing behavior.

The ubiquitin-26S proteasome system and autophagy relay proteome homeostasis regulation during silique development.

SUMMARYFunctional studies of the ubiquitin–26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant's life involve UPS‐mediated turnover of abnormal or short‐lived proteins. However, the role of the UPS during development, including in seeds and fruits, remains to be determined in detail, although mutants of several of its core elements are known to be embryonically lethal. Unfortunately, early termination of embryogenesis limits the possibility to characterize the activities of the UPS in reproductive organs. Given both the economic and the societal impact of reproductive production, such studies are indispensable. Here, we systematically compared expression of multiple 26S proteasome subunits along with the dynamics of proteasome activity and total protein ubiquitylation in seedlings, developing siliques, and embryos of Arabidopsis thaliana. Since autophagy plays the second largest role in maintaining proteome stability, we parallelly studied three rate‐limiting enzymes that are involved in autophagy flux. Our experiments unexpectedly discovered that, in contrast to the activities in seedlings, both protein and transcript levels of six selected 26S proteasome subunits gradually decline in immature siliques or embryos toward maturation while the autophagy flux rises despite the nutrient‐rich condition. We also discovered a reciprocal turnover pathway between the proteasome and autophagy. While the autophagy flux is suppressed in seedlings by UPS‐mediated degradation of its three key enzymes, transcriptional reprogramming dampens this process in siliques, which in turn stimulates a bulk autophagic degradation of proteasomes. Collectively, our study of the developmental changes of the UPS and autophagy activities suggests that they relay the proteome homeostasis regulation in early silique and/or seed development, highlighting their interactions during development.

The F-Box/DUF295 Brassiceae specific 2 is involved in ABA-inhibited seed germination and seedling growth in Arabidopsis

To bear harsh environmental threats, plants have developed complex protection mechanisms involving phytohormones, counting abscisic acid (ABA). The function of the F-Box family containing the Domain of Unknown Function 295 (DUF295) has not yet been comprehensively characterized in Arabidopsis (Arabidopsis thaliana). In this study, we evaluated the function of a putative member of the F-Box/DUF295 family in Arabidopsis, F-box/DUF295 Brassiceae specific 2 (FDB2). We found that FDB2 expression was suppressed by ABA and abiotic stresses. FDB2 overexpression (OE) reduced ABA sensitivity during seed germination and seedling growth, but enhanced ABA-sensitivity of seed germination and seedling growth in fdb2 mutants was scored. When treated with ABA, expressions of ABI3, ABI4 and ABI5 showed decreased in OE lines but increased in fdb2 mutants. In addition, ABA-induced FDB2 degradation exhibited sensitive to MG132, suggesting that FDB2 degradation by ABA might be mediated by the ubiquitin-26S proteasome system. Moreover, ABA-induced significant over-accumulation of reactive oxygen species (ROS) at the root tips of fdb2 mutants was observed, this phenomenon was correlated to reduced activities of a set of ROS scavengers in fdb2 mutants relative to Col-0. In summary, our results suggest that Arabidopsis FDB2 is involved in ABA-mediated inhibition of seed germination, seedling growth including modulation of ROS homeostasis in roots.

The ABI3 Transcription Factor Interaction and Antagonism with Ubiquitin E3 Ligase ScPRT1 in Syntrichia caninervis.

The ubiquitination pathway has been found to regulate plant responses to environmental stress. However, the role of E3 ubiquitin ligase in desiccation tolerant moss has not yet been elucidated. Previous research has shown that the abscisic acid (ABA) signaling factor ScABI3 can significantly increase desiccation tolerance and reduce ABA sensitivity in the desert moss Syntrichia caninervis. In this study, we identified a RING-type E3 ubiquitin ligase, ScPRT1, and showed that ScABI3 can directly interact with ScPRT1 in vitro and in vivo. Furthermore, we found that the high expression of ScPRT1 can interfere with the transcription of ScABI3 under ABA treatment. Therefore, we speculate that ScPRT1 may degrade ScABI3 through the ubiquitin-26S proteasome system and participate in ABA-dependent signaling in response to ABA-insensitivity or desiccation tolerance in S. caninervis. The findings from our study may enrich our knowledge of the role of E3 ubiquitin ligase in desiccation tolerance and lay a theoretical foundation for an in-depth study of the relationship between ubiquitination modification and ABA signal transduction under environmental stress.

The ARRE RING-Type E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis thaliana by Controlling ECERIFERUM1 and ECERIFERUM3 Protein Levels.

The outer epidermal cell walls of plant shoots are covered with a cuticle, a continuous lipid structure that provides protection from desiccation, UV light, pathogens, and insects. The cuticle is mostly composed of cutin and cuticular wax. Cuticular wax synthesis is synchronized with surface area expansion during plant development and is associated with plant responses to biotic and abiotic stresses. Cuticular wax deposition is tightly regulated by well-established transcriptional and post-transcriptional regulatory mechanisms, as well as post-translationally via the ubiquitin-26S proteasome system (UPS). The UPS is highly conserved in eukaryotes and involves the covalent attachment of polyubiquitin chains to the target protein by an E3 ligase, followed by the degradation of the modified protein by the 26S proteasome. A large number of E3 ligases are encoded in the Arabidopsis genome, but only a few have been implicated in the regulation of cuticular wax deposition. In this study, we have conducted an E3 ligase reverse genetic screen and identified a novel RING-type E3 ubiquitin ligase, AtARRE, which negatively regulates wax biosynthesis in Arabidopsis. Arabidopsis plants overexpressing AtARRE exhibit glossy stems and siliques, reduced fertility and fusion between aerial organs. Wax load and wax compositional analyses of AtARRE overexpressors showed that the alkane-forming branch of the wax biosynthetic pathway is affected. Co-expression of AtARRE and candidate target proteins involved in alkane formation in both Nicotiana benthamiana and stable Arabidopsis transgenic lines demonstrated that AtARRE controls the levels of wax biosynthetic enzymes ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3). CER1 has also been confirmed to be a ubiquitination substrate of the AtARRE E3 ligase by an in vivo ubiquitination assay using a reconstituted Escherichia coli system. The AtARRE gene is expressed throughout the plant, with the highest expression detected in fully expanded rosette leaves and oldest stem internodes. AtARRE gene expression can also be induced by exposure to pathogens. These findings reveal that wax biosynthesis in mature plant tissues and in response to pathogen infection is controlled post-translationally.

Regulation of Glycosylphosphatidylinositol-Anchored Protein (GPI-AP) Expression by F-Box/LRR-Repeat (FBXL) Protein in Wheat (Triticum aestivum L.).

F-box proteins are substrate recognition components of the Skp1-Cullin-F-box (SCF) complex, which performs many important biological functions including the degradation of numerous proteins via the ubiquitin–26S proteasome system. In this study, we isolated the gene encoding the F-box/LRR-repeat (FBXL) protein from wheat (Triticum aestivum L.) seedlings and validated that the TaFBXL protein is a component of the SCF complex. Yeast two-hybrid assays revealed that TaFBXL interacts with the wheat glycosylphosphatidylinositol-anchored protein (TaGPI-AP). The green fluorescent protein (GFP) fusion protein of TaFBXL was detected in the nucleus and plasma membrane, whereas that of TaGPI-AP was observed in the cytosol and probably also plasma membrane. yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed that TaFBXL specifically interacts with TaGPI-AP in the nucleus and plasma membrane, and TaGPI-AP is targeted by TaFBXL for degradation via the 26S proteasome system. In addition, TaFBXL and TaGPI-AP showed antagonistic expression patterns upon treatment with indole-3-acetic acid (IAA), and the level of TaGPI-AP was higher in tobacco leaves treated with both MG132 (proteasome inhibitor) and IAA than in leaves treated with either MG132 or IAA. Taken together, our data suggest that TaFBXL regulates the TaGPI-AP protein level in response to exogenous auxin application.

Ubiquitylation of ABA Receptors and Protein Phosphatase 2C Coreceptors to Modulate ABA Signaling and Stress Response.

Post-translational modifications play a fundamental role in regulating protein function and stability. In particular, protein ubiquitylation is a multifaceted modification involved in numerous aspects of plant biology. Landmark studies connected the ATP-dependent ubiquitylation of substrates to their degradation by the 26S proteasome; however, nonproteolytic functions of the ubiquitin (Ub) code are also crucial to regulate protein interactions, activity, and localization. Regarding proteolytic functions of Ub, Lys-48-linked branched chains are the most common chain type for proteasomal degradation, whereas promotion of endocytosis and vacuolar degradation is triggered through monoubiquitylation or Lys63-linked chains introduced in integral or peripheral plasma membrane proteins. Hormone signaling relies on regulated protein turnover, and specifically the half-life of ABA signaling components is regulated both through the ubiquitin-26S proteasome system and the endocytic/vacuolar degradation pathway. E3 Ub ligases have been reported that target different ABA signaling core components, i.e., ABA receptors, PP2Cs, SnRK2s, and ABFs/ABI5 transcription factors. In this review, we focused specifically on the ubiquitylation of ABA receptors and PP2C coreceptors, as well as other post-translational modifications of ABA receptors (nitration and phosphorylation) that result in their ubiquitination and degradation.

The Ubiquitin Switch in Plant Stress Response.

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.

Retraction Note: Cloning and characterization of TaPUB4, a U-box gene from common wheat (Triticum aestivum L.) involved in regulation of pollen development by influencing sucrose-starch pathway in anther

Development of functional pollen grains in plants is regulated through complex genetic networks and environmental clues. The ubiquitin-26S proteasome system is important in the post-translational modification of intracellular proteins, and the system includes the ubiquitin activating (E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes. Here, a novel plant U-box-containing cDNA was isolated in anther from common wheat (Triticum aestivum) and designed as TaPUB4. The cDNA encoding comprised 2598 bp, with an open reading frame of 865 amino acids. The deduced amino acid sequence of TaPUB4 showed 41.2–92.3% identity with other plant U-box homologous genes. Self-ubiquitination assay indicated that the bacterially expressed TaPUB4 protein had E3 ligase activity, and subcellular localization results showed that TaPUB4 was located in the nucleus and cytoplasm in onion epidermal cells. Overexpression of TaPUB4 gene in Arabidopsis atpub4 mutant restored partially the fertility of the mutant, increased the number of pollens in anthers, enhanced pollen viability, and displayed normal degradation of tapetum as anther development. The expression levels of genes related to starch deposition also changed greatly in the anthers of transgenic lines. These results support the notion that as a homolog of AtPUB4, TaPUB4 might control male fertility by regulating sucrose-starch metabolism in pollen grains, and this process may be accomplished through ubiquitin-proteasome system. The development and utilization of TaPUB4 as a functional molecular marker may be helpful for improving its fertility in wheat breeding programs in the future.

Identification of Elements Responsible for Maternally-Silenced Imprinted Gene Expression of Upward Curly Leaf1, an F-box Protein Gene that Regulates Curly Leaf in Arabidopsis

Upward Curly Leaf 1 (UCL1) is an Arabidopsis thaliana E3 ligase that targets the Curly Leaf (CLF) SET-domain polycomb-group (PcG) protein for degradation via the ubiquitin-26S proteasome system. UCL1 is a paternally-expressed imprinted gene in the endosperm. To precisely locate the promoter elements required for UCL1 imprinting pattern, various gene constructs were created in which the imprinting control region (ICR), endosperm-specific expression (ENSE) element, and/or the linker sequence were altered. By fusing these constructs with a GUS reporter gene, GUS expression patterns were monitored after reciprocal crosses with wild-type Columbia-0 allowing the determination of parent-of-origin expression. Analysis of publicly-available data on the UCL1 promoter region facilitated the search for allele-specific DNA and H3K27 methylation patterns. Overall, three promoter elements are required for maternal repression of UCL1; the ICR sequence located from − 2.5 to − 2.4 kb upstream of the translation start site, a differentially methylated region 2 (DMR2) that overlaps the short ATLINE1-1 transposable element in the linker region, and a minimal 271 bp ENSE element. In addition, DNA methylation patterns in the DMR2 contribute to the repression of the maternal UCL1 allele. Our findings would help to understand how parent-of-origin epigenetic patterns are created and maintained in the endosperm.

Diverse and dynamic roles of F-box proteins in plant biology.

The SCF complex is a widely studied multi-subunit ring E3 ubiquitin ligase that tags targeted proteins with ubiquitin for protein degradation by the ubiquitin 26S-proteasome system (UPS). The UPS is an important system that generally keeps cellular events tightly regulated by purging misfolded or damaged proteins and selectively degrading important regulatory proteins. The specificity of this post-translational regulation is controlled by F-box proteins (FBPs) via selective recognition of a protein-protein interaction motif at the C-terminal domain. Hence, FBPs are pivotal proteins in determining the plant response in multiple scenarios. It is not surprising that the FBP family is one of the largest protein families in the plant kingdom. In this review, the roles of FBPs, specifically in plants, are compiled to provide insights into their involvement in secondary metabolites, plant stresses, phytohormone signalling, plant developmental processes and miRNA biogenesis.

Aquaporins mediated arsenite transport in plants: Molecular mechanisms and applications in crop improvement

Arsenic (As) is a carcinogen and it is ubiquitous in the environment. Its accumulation in plants can enter the food chain, thereby causing adverse impacts on human health. Based on studies using Xenopus oocytes, engineered microbes, and model plants, aquaporins play key roles in arsenite (AsIII) passage across the cell membrane. Herein, we summarized the roles of aquaporins in AsIII uptake, transport, and accumulation. The coupling action between polar-localized aquaporins and positive transporters probably explains the efficient As transport mediated by bi-directional channels. The expression of aquaporins is regulated at both transcriptional and post-transcriptional levels via microRNAs. However, in response to As, the proteins can be modified through phosphorylation and be degraded via the ubiquitin 26S proteasome system. Further, a proposed model comprising reactive oxygen species, Ca, Ca-dependent kinases, and transcriptional factors is constructed to reveal the signaling pathways and aquaporins’ responses to As in model plants. We also provided strategies to identify and investigate putative aquaporins in plants. Identification and modification of aquaporins’ residues critical for their substrate selectivity, and optimization of their expression pattern by employing appropriate promoters can be employed to reduce As accumulation in crops. Based on the characteristics of their functions and expression patterns, it is promising to employ aquaporins for genetic-engineering of crops to reduce As content and increase food safety.

Diversifying Evolution of the Ubiquitin-26S Proteasome System in Brassicaceae and Poaceae.

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.

Dek40 Encodes a PBAC4 Protein Required for 20S Proteasome Biogenesis and Seed Development.

The 26S proteasome, an essential protease complex of the ubiquitin-26S proteasome system (UPS), controls many cellular events by degrading short-lived regulatory proteins marked with polyubiquitin chains. The 20S proteolytic core protease (CP), the catalytic core of the 26S proteasome, is a central enzyme in the UPS. Its biogenesis proceeds in a multistep and orderly fashion assisted by a series of proteasome assembly chaperones. In this study, we identified a novel maize (Zea mays) kernel mutant named defective kernel40 (dek40), which produces small, collapsed kernels and exhibits delayed embryo and endosperm development. Dek40 was identified by map-based cloning and confirmed by transgenic functional complementation. Dek40 encodes a putative cytosol-localized proteasome biogenesis-associated chaperone4 (PBAC4) protein. DEK40 participates in the biogenesis of the 20S CP by interacting with PBAC3. Loss-of-function of DEK40 substantially affected 20S CP biogenesis, resulting in decreased activity of the 26S proteasome. Ubiquitylome analysis indicated that DEK40 influences the degradation of ubiquitinated proteins and plays an essential role in the maintenance of cellular protein homoeostasis. These results demonstrate that Dek40 encodes a PBAC4 chaperone that affects 20S CP biogenesis and is required for 26S proteasome function and seed development in maize.