The lowdown on breakdown: Open questions in plant proteolysis.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

The Arabidopsis E3 ubiquitin ligase DOA10A promotes localization of abscisic acid (ABA) receptors to the membrane through mono-ubiquitination in ABA signaling.

The endoplasmic reticulum-associated degradation (ERAD) system eliminates misfolded and short-lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain-of-function mutant, doa10a-1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono-ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING-type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono-ubiquitination.

Highlight Induced Transcriptional Priming against a Subsequent Drought Stress in Arabidopsis thaliana.

In plants, priming allows a more rapid and robust response to recurring stresses. However, while the nature of plant response to a single stress can affect the subsequent response to the same stress has been deeply studied, considerably less is known on how the priming effect due to one stress can help plants cope with subsequent different stresses, a situation that can be found in natural ecosystems. Here, we investigate the potential priming effects in Arabidopsis plants subjected to a high light (HL) stress followed by a drought (D) stress. The cross-stress tolerance was assessed at the physiological and molecular levels. Our data demonstrated that HL mediated transcriptional priming on the expression of specific stress response genes. Furthermore, this priming effect involves both ABA-dependent and ABA-independent responses, as also supported by reduced expression of these genes in the aba1-3 mutant compared to the wild type. We have also assessed several physiological parameters with the aim of seeing if gene expression coincides with any physiological changes. Overall, the results from the physiological measurements suggested that these physiological processes did not experience metabolic changes in response to the stresses. In addition, we show that the H3K4me3 epigenetic mark could be a good candidate as an epigenetic mark in priming response. Overall, our results help to elucidate how HL-mediated priming can limit D-stress and enhance plant responses to stress.

Light controls stamen elongation via cryptochromes, phytochromes and COP1 through HY5 and HYH.

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 - oppositely to ARF8 - directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far-red light receptors PHYA/PHYB. In conclusion, different light qualities - sequentially perceived by specific photoreceptors - and the downstream COP1-HY5/HYH module finely tune auxin-induced stamen elongation and thus male fertility.

F-box protein CFK1 interacts with and degrades de novo DNA methyltransferase in Arabidopsis.

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), the de novo Arabidopsis 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 in Arabidopsis thaliana. We also performed whole genome bisulfite sequencing (BS-seq) to determine the biological significance of CFK1-mediated DRM2 degradation. Loss-of-function CFK1 leads 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 regulating de novo DNA methyltransferase by CFK1 to control DNA methylation level.

A Newly Identified Flower-Specific Splice Variant of AUXIN RESPONSE FACTOR8 Regulates Stamen Elongation and Endothecium Lignification in Arabidopsis.

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has been reported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into the nucleus and has tissue-specific localization in Arabidopsis thaliana By inducibly expressing each variant in arf8-7 flowers, we show that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19, encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects on arf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7 caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acid biosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expression of transcription factor gene MYB26 Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 is a tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directly regulating key genes involved in these processes.

ABI4 mediates antagonistic effects of abscisic acid and gibberellins at transcript and protein levels.

Abscisic acid (ABA) and gibberellins (GAs) are plant hormones which antagonistically mediate numerous physiological processes, and their optimal balance is essential for normal plant development. However, the molecular mechanism underlying ABA and GA antagonism still needs to be determined. Here, we report that ABA-INSENSITIVE 4 (ABI4) is a central factor in GA/ABA homeostasis and antagonism in post-germination stages. ABI4 overexpression in Arabidopsis (OE-ABI4) leads to developmental defects including a decrease in plant height and poor seed production. The transcription of a key ABA biosynthetic gene, NCED6, and of a key GA catabolic gene, GA2ox7, is significantly enhanced by ABI4 overexpression. ABI4 activates NCED6 and GA2ox7 transcription by directly binding to the promoters, and genetic analysis revealed that mutation in these two genes partially rescues the dwarf phenotype of ABI4 overexpressing plants. Consistently, ABI4 overexpressing seedlings have a lower GA/ABA ratio than the wild type. We further show that ABA induces GA2ox7 transcription while GA represses NCED6 expression in an ABI4-dependent manner; and that ABA stabilizes the ABI4 protein whereas GA promotes its degradation. Taken together, these results suggest that ABA and GA antagonize each other by oppositely acting on ABI4 transcript and protein levels.

The RING finger E3 ligase STRF1 is involved in membrane trafficking and modulates salt-stress response in Arabidopsis thaliana.

Salt stress is a detrimental factor for plant growth and development. The response to salt stress has been shown to involve components in the intracellular trafficking system, as well as components of the ubiquitin-proteasome system (UPS). In this article, we have identified in Arabidopsis thaliana a little reported ubiquitin ligase involved in salt-stress response, which we named STRF1 (Salt Tolerance RING Finger 1). STRF1 is a member of RING-H2 finger proteins and we demonstrate that it has ubiquitin ligase activity in vitro. We also show that STRF1 localizes mainly at the plasma membrane and at the intracellular endosomes. strf1-1 loss-of-function mutant seedlings exhibit accelerated endocytosis in roots, and have altered expression of several genes involved in the membrane trafficking system. Moreover, protein trafficking inhibitor, brefeldin A (BFA), treatment has increased BFA bodies in strf1-1 mutant. This mutant also showed increased tolerance to salt, ionic and osmotic stresses, reduced accumulation of reactive oxygen species during salt stress, and increased expression of AtRbohD, which encodes a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase involved in H2 O2 production. We conclude that STRF1 is a membrane trafficking-related ubiquitin ligase, which helps the plant to respond to salt stress by monitoring intracellular membrane trafficking and reactive oxygen species (ROS) production.

DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1.

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway.

CRF transcription factors in the trade-off between abiotic stress response and plant developmental processes.

Climate change-induced environmental stress significantly affects crop yield and quality. In response to environmental stressors, plants use defence mechanisms and growth suppression, creating a resource trade-off between the stress response and development. Although stress-responsive genes have been widely engineered to enhance crop stress tolerance, there is still limited understanding of the interplay between stress signalling and plant growth, a research topic that can provide promising targets for crop genetic improvement. This review focuses on Cytokinin Response Factors (CRFs) transcription factor's role in the balance between abiotic stress adaptation and sustained growth. CRFs, known for their involvement in cytokinin signalling and abiotic stress responses, emerge as potential targets for delaying senescence and mitigating yield penalties under abiotic stress conditions. Understanding the molecular mechanisms regulated by CRFs paves the way for decoupling stress responses from growth inhibition, thus allowing the development of crops that can adapt to abiotic stress without compromising development. This review highlights the importance of unravelling CRF-mediated pathways to address the growing need for resilient crops in the face of evolving climatic conditions.

The organization of a CSN5-containing subcomplex of the COP9 signalosome.

The COP9 signalosome (CSN) is an evolutionarily conserved multi-protein complex that interfaces with the ubiquitin-proteasome pathway and plays critical developmental roles in both animals and plants. Although some subunits are present only in an ∼320-kDa complex-dependent form, other subunits are also detected in configurations distinct from the 8-subunit holocomplex. To date, the only known biochemical activity intrinsic to the complex, deneddylation of the Cullin subunits from Cullin-RING ubiquitin ligases, is assigned to CSN5. As an essential step to understanding the structure and assembly of a CSN5-containing subcomplex of the CSN, we reconstituted a CSN4-5-6-7 subcomplex. The core of the subcomplex is based on a stable heterotrimeric association of CSN7, CSN4, and CSN6, requiring coexpression in a bacterial reconstitution system. To this heterotrimer, we could then add CSN5 in vitro to reconstitute a quaternary complex. Using biochemical and biophysical methods, we identified pairwise and combinatorial interactions necessary for the formation of the CSN4-5-6-7 subcomplex. The subcomplex is stabilized by three types of interactions: MPN-MPN between CSN5 and CSN6, PCI-PCI between CSN4 and CSN7, and interactions mediated through the CSN6 C terminus with CSN4 and CSN7. CSN8 was also found to interact with the CSN4-6-7 core. These data provide a strong framework for further investigation of the organization and assembly of this pivotal regulatory complex.

Emerging role of the ubiquitin proteasome system in the control of shoot apical meristem function(f).

The shoot apical meristem (SAM) is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant's life. A plethora of different families of transcription factors (TFs) play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM. Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues, and for development progression. One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system (UPS). However, a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated. Here, we summarize recent evidence supporting a role for the UPS in SAM maintenance and function. We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM, suggesting that they may regulate or act downstream of meristem-specific factors.

The COP9 signalosome: more than a protease.

The COP9 signalosome (CSN) is a conserved protein complex that functions in the ubiquitin-proteasome pathway. After two decades of research, we now know that the CSN is a multi-subunit protease that regulates the activity of cullin-RING ligase (CRL) families of ubiquitin E3 complexes. The CSN is rapidly emerging as a key player in the DNA-damage response, cell-cycle control and gene expression. The independent functions of CSN5 (also known as JAB1) add to the complexity of the CSN machinery. Here, we provide an updated view of the structure, functions and regulation of this protein complex.

Immunoprecipitation of Cullin-Ring Ligases (CRLs) in Arabidopsis thaliana Seedlings.

CRL (Cullin-Ring ubiquitin ligases) are the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-Ring Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: (1) a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.

Cotranslational N-degron masking by acetylation promotes proteome stability in plants.

N-terminal protein acetylation (NTA) is a prevalent protein modification essential for viability in animals and plants. The dominant executor of NTA is the ribosome tethered Nα-acetyltransferase A (NatA) complex. However, the impact of NatA on protein fate is still enigmatic. Here, we demonstrate that depletion of NatA activity leads to a 4-fold increase in global protein turnover via the ubiquitin-proteasome system in Arabidopsis. Surprisingly, a concomitant increase in translation, actioned via enhanced Target-of-Rapamycin activity, is also observed, implying that defective NTA triggers feedback mechanisms to maintain steady-state protein abundance. Quantitative analysis of the proteome, the translatome, and the ubiquitome reveals that NatA substrates account for the bulk of this enhanced turnover. A targeted analysis of NatA substrate stability uncovers that NTA absence triggers protein destabilization via a previously undescribed and widely conserved nonAc/N-degron in plants. Hence, the imprinting of the proteome with acetylation marks is essential for coordinating proteome stability.

The COP9 signalosome: regulating plant development through the control of proteolysis.

The COP9 signalosome (CSN) is a multiprotein complex that was initially identified in plants as a repressor of photomorphogenesis. It is now known to play major roles in several other developmental pathways, from auxin response to flower development. Furthermore, the COP9 signalosome shares homologies with the lid sibcomplex of the proteasome and is evolutionarily conserved from fission yeast to humans. It is important for the proper development of virtually all higher eukaryotes. In recent years, significant progress has been made in unraveling the molecular, cellular, and physiological mode of action of the COP9 signalosome. This review discusses our current understanding of the COP9 signalosome function with particular emphasis on its recently defined role in modulating a wide variety of cellular processes by regulating specific protein degradation events.

CSN1 N-terminal-dependent activity is required for Arabidopsis development but not for Rub1/Nedd8 deconjugation of cullins: a structure-function study of CSN1 subunit of COP9 signalosome.

The COP9 signalosome (CSN) is a multifunctional protein complex essential for arabidopsis development. One of its functions is to promote Rub1/Nedd8 deconjugation from the cullin subunit of the Skp1-cullin-F-box ubiquitin ligase. Little is known about the specific role of its eight subunits in deneddylation or any of the physiological functions of CSN. In the absence of CSN1 (the fus6 mutant), arabidopsis CSN complex cannot assemble, which destabilizes multiple CSN subunits and contributes, together with the loss of CSN1, to the phenotype of fus6. To distinguish CSN1-specific functions, we attempted to rescue the complex formation with deletion or point-mutation forms of CSN1 expressed as transgenes in fus6. We show that the central domain of CSN1 is critical for complex assembly, whereas the C-terminal domain has a supporting role. By expressing the C231 fragment, which contains the structural information but lacks the presumed functional domain located at the N terminus, we have rescued the complex formation and restored the Rub1/Nedd8 deconjugation activity on cullins (fus6/C231). Nonetheless, fus6/C231 exhibits pleiotropic phenotype, including photomorphogenic defects and growth arrest at seedling stage. We conclude that CSN1 N-terminal domain is not required for the Rub1/Nedd8 deconjugation activity of cullins, but contributes to a significant aspect of CSN functions that are essential for plant development.

Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings.

CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.