Overexpression of the ribosomal S30 subunit leads to indole-3-carbinol tolerance in Arabidopsis thaliana.

Indole-3-carbinol (I3C), a hydrolysis product of indole-3-methylglucosinolate, is toxic to herbivorous insects and pathogens. In mammals, I3C is extensively studied for its properties in cancer prevention and treatment. Produced in Brassicaceae, I3C reversibly inhibits root elongation in a concentration-dependent manner. This inhibition is partially explained by the antagonistic action of I3C on auxin signaling through TIR1. To further elucidate the mode of action of I3C in plants, we have identified and characterized a novel Arabidopsis mutant tolerant to I3C, ICT1. This mutant was identified following screening of the Full-length cDNA Over-eXpression library (FOX) seed collection for root growth in the presence of exogenous I3C. ICT1 carries the AT2G19750 gene, which encodes an S30 ribosomal protein. Overexpression, but not knockout, of the S30 gene causes tolerance to I3C. The tolerance is specific to I3C, since ICT1 did not exhibit pronounced tolerance to other indole or benzoxazinoid molecules tested. ICT1 maintains I3C-induced antagonism of auxin signaling, indicating that the tolerance is due to an auxin-independent mechanism. Transcript profiling experiments revealed that ICT1 is transcriptionally primed to respond to I3C treatment.

The COP9 signalosome influences the epigenetic landscape of Arabidopsis thaliana.

MOTIVATION: The COP9 signalosome is a highly conserved multi-protein complex consisting of eight subunits, which influences key developmental pathways through its regulation of protein stability and transcription. In Arabidopsis thaliana, mutations in the COP9 signalosome exhibit a number of diverse pleiotropic phenotypes. Total or partial loss of COP9 signalosome function in Arabidopsis leads to misregulation of a number of genes involved in DNA methylation, suggesting that part of the pleiotropic phenotype is due to global effects on DNA methylation. RESULTS: We determined and analyzed the methylomes and transcriptomes of both partial- and total-loss-of-function Arabidopsis mutants of the COP9 signalosome. Our results support the hypothesis that the COP9 signalosome has a global genome-wide effect on methylation and that this effect is at least partially encoded in the DNA. Our analyses suggest that COP9 signalosome-dependent methylation is related to gene expression regulation in various ways. Differentially methylated regions tend to be closer in the 3D conformation of the genome to differentially expressed genes. These results suggest that the COP9 signalosome has a more comprehensive effect on gene expression than thought before, and this is partially related to regulation of methylation. The high level of COP9 signalosome conservation among eukaryotes may also suggest that COP9 signalosome regulates methylation not only in plants but also in other eukaryotes, including humans. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Protein competition switches the function of COP9 from self-renewal to differentiation.

The balance between stem cell self-renewal and differentiation is controlled by intrinsic factors and niche signals. In the Drosophila melanogaster ovary, some intrinsic factors promote germline stem cell (GSC) self-renewal, whereas others stimulate differentiation. However, it remains poorly understood how the balance between self-renewal and differentiation is controlled. Here we use D. melanogaster ovarian GSCs to demonstrate that the differentiation factor Bam controls the functional switch of the COP9 complex from self-renewal to differentiation via protein competition. The COP9 complex is composed of eight Csn subunits, Csn1-8, and removes Nedd8 modifications from target proteins. Genetic results indicated that the COP9 complex is required intrinsically for GSC self-renewal, whereas other Csn proteins, with the exception of Csn4, were also required for GSC progeny differentiation. Bam-mediated Csn4 sequestration from the COP9 complex via protein competition inactivated the self-renewing function of COP9 and allowed other Csn proteins to promote GSC differentiation. Therefore, this study reveals a protein-competition-based mechanism for controlling the balance between stem cell self-renewal and differentiation. Because numerous self-renewal factors are ubiquitously expressed throughout the stem cell lineage in various systems, protein competition may function as an important mechanism for controlling the self-renewal-to-differentiation switch.

Wounding of Arabidopsis leaves induces indole-3-carbinol-dependent autophagy in roots of Arabidopsis thaliana.

In cruciferous plants insect attack or physical damage induce the synthesis of the glucosinolate breakdown product indole-3-carbinol, which plays a key role in the defense against attackers. Indole-3-carbinol also affects plant growth and development, acting as an auxin antagonist by binding to the TIR1 auxin receptor. Other potential functions of indole-3-carbinol and the underlying mechanisms in plant biology are unknown. Here we show that an indole-3-carbinol-dependent signal induces specific autophagy in root cells. Leaf treatment with exogenous indole-3-carbinol or leaf-wounding induced autophagy and inhibited auxin response in the root. This induction is lost in glucosinolate-defective mutants, indicating that the effect of indole-3-carbinol is transported in the plants. Thus, indole-3-carbinol is not only a defensive metabolite that repels insects, but is also involved in long-distance communication regulating growth and development in plants.

The COP9 signalosome is vital for timely repair of DNA double-strand breaks.

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection-the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability.

The glucosinolate breakdown product indole-3-carbinol acts as an auxin antagonist in roots of Arabidopsis thaliana.

The glucosinolate breakdown product indole-3-carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole-3-carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole-3-carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole-3-carbinol rapidly and reversibly inhibits root elongation in a dose-dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole-3-carbinol and the auxin perception machinery was suggested, as application of indole-3-carbinol rescues auxin-induced root phenotypes. In vitro and yeast-based protein interaction studies showed that indole-3-carbinol perturbs the auxin-dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3-indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole-3-carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole-3-carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.

Multidimensional patterns of metabolic response in abiotic stress-induced growth of Arabidopsis thaliana.

KEY MESSAGE: Contextualization of specific transcriptional responses of Arabidopsis within the stress-tissue-time perspective provides a simplified representation of the cellular transcriptional response pathways to abiotic stress, while reducing the dimensions in gene-oriented response description. Crops resistant to abiotic stresses are a long-term goal of many research programs, thus understanding the progression of stress responses is of great interest. We reanalyzed the AtGenExpress transcription dataset to go beyond gene-level characterization, and to contextualize the discrete information into (1) a process-level signature of stress-specific, time-specific, and tissue-specific responses and (2) identify patterns of response progression across a time axis. To gain a functional perspective, ∼1000 pathways associated with the differentially-expressed genes were characterized across all experiments. We find that the global response of pathways to stress is multi-dimensional and does not obviously cluster according to stress, time or tissue. The early response to abiotic stress typically involves induction of genes involved in transcription, hormone synthesis and signaling modules; a later response typically involves metabolism of amino acids and secondary metabolites. By linking specific primary and secondary response pathways, we outline possible stress-associated routes of response progression. The contextualization of specific processes within stress-tissue-time perspective provides a simplified representation of cellular response while reducing the dimensions in gene-oriented response description. Such simplified representation allows finding stress-specific markers based on process-combinations pointing whether a stress-specific response was invoked as well as provide a reference point for the conductance of comparative inter-plant study of stress response, bypassing the need in detailed orthologous mapping.

Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development.

The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health.

Overexpression of S30 Ribosomal Protein Leads to Transcriptional and Metabolic Changes That Affect Plant Development and Responses to Stress.

ICT1 is an Arabidopsis thaliana line that overexpresses the gene encoding the S30 ribosomal subunit, leading to tolerance to exogenous indole-3-carbinol. Indole-3-carbinol (I3C) is a protective chemical formed as a breakdown of I3M in cruciferous vegetables. The overexpression of S30 in ICT1 results in transcriptional changes that prime the plant for the I3C, or biotic insult. Emerging evidence suggests that ribosomal proteins play important extra-ribosomal roles in various biochemical and developmental processes, such as transcription and stress resistance. In an attempt to elucidate the mechanism leading to I3C and stress resistance in ICT1, and using a multi-pronged approach employing transcriptomics, metabolomics, phenomics, and physiological studies, we show that overexpression of S30 leads to specific transcriptional alterations, which lead to both changes in metabolites connected to biotic and oxidative stress tolerance and, surprisingly, to photomorphogenesis.

Investigation of the Roles of Phosphatidylinositol 4-Phosphate 5-Kinases 7,9 and Wall-Associated Kinases 1-3 in Responses to Indole-3-Carbinol and Biotic Stress in Arabidopsis Thaliana.

Indole-3-carbinol (I3C), a hydrolysis product of indole-3-methylglucosinolate, is toxic to herbivorous insects and pathogens. In mammals, I3C is extensively studied for its properties in cancer prevention and treatment. Produced in Brassicaceae, I3C reversibly inhibits root elongation in a concentration-dependent manner. This inhibition is partially explained by the antagonistic action of I3C on auxin signaling through TIR1. To further elucidate the mode of action of I3C in plants, we have employed a forward-genetic amiRNA screen that circumvents functional redundancy. We identified and characterized two amiRNA lines with impaired I3C response. The first line, ICT2, targets the phosphatidylinositol 4-phosphate 5-kinase family (PIP5K), exhibiting tolerance to I3C, while the second line, ICS1, targets the Wall-Associated Kinases (WAK1-3) family, showing susceptibility to I3C. Both lines maintain I3C-induced antagonism of auxin signaling, indicating that their phenotypes are due to auxin-independent mechanisms. Transcript profiling experiments reveal that both lines are transcriptionally primed to respond to I3C treatment. Physiological, metabolomic, and transcriptomic analysis reveal that these kinases mediate numerous developmental processes and are involved in abiotic and biotic stress responses.

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.

Tryptophol Acetate and Tyrosol Acetate, Small-Molecule Metabolites Identified in a Probiotic Mixture, Inhibit Hyperinflammation.

Probiotic fermented foods are perceived as contributing to human health; however, solid evidence for their presumptive therapeutic systemic benefits is generally lacking. Here we report that tryptophol acetate and tyrosol acetate, small-molecule metabolites secreted by the probiotic milk-fermented yeast Kluyveromyces marxianus, inhibit hyperinflammation (e.g., "cytokine storm"). Comprehensive in vivo and in vitro analyses, employing LPS-induced hyperinflammation models, reveal dramatic effects of the molecules, added in tandem, on mice morbidity, laboratory parameters, and mortality. Specifically, we observed attenuated levels of the proinflammatory cytokines IL-6, IL-1α, IL-1ß, and TNF-α and reduced reactive oxygen species. Importantly, tryptophol acetate and tyrosol acetate did not completely suppress proinflammatory cytokine generation, rather brought their concentrations back to baseline levels, thus maintaining core immune functions, including phagocytosis. The anti-inflammatory effects of tryptophol acetate and tyrosol acetate were mediated through downregulation of TLR4, IL-1R, and TNFR signaling pathways and increased A20 expression, leading to NF-kB inhibition. Overall, this work illuminates phenomenological and molecular details underscoring anti-inflammatory properties of small molecules identified in a probiotic mixture, pointing to potential therapeutic avenues against severe inflammation.

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

Revisiting the COP9 signalosome as a transcriptional regulator.

The COP9 signalosome (CSN) is a highly conserved protein complex that was originally described as a repressor of light-dependent growth and transcription in Arabidopsis. The most studied CSN function is the regulation of protein degradation, which occurs primarily through the removal of the ubiquitin-like modifier Nedd8 from cullin-based E3 ubiquitin ligases. This activity can regulate transcription-factor stability and, therefore, transcriptional activity. Recent data suggest that the CSN also regulates transcription on the chromatin by mechanisms that are not yet clearly understood. Furthermore, the CSN subunits CSN5 and CSN2 seem to act as transcriptional coactivators and corepressors, respectively. Here, I re-evaluate the mechanisms by which the CSN acts as a transcriptional regulator, and suggest that they could extend beyond the regulation of protein stability.

CSN5A Subunit of COP9 Signalosome Is Required for Resetting Transcriptional Stress Memory after Recurrent Heat Stress in Arabidopsis.

In nature, plants are exposed to several environmental stresses that can be continuous or recurring. Continuous stress can be lethal, but stress after priming can increase the tolerance of a plant to better prepare for future stresses. Reports have suggested that transcription factors are involved in stress memory after recurrent stress; however, less is known about the factors that regulate the resetting of stress memory. Here, we uncovered a role for Constitutive Photomorphogenesis 5A (CSN5A) in the regulation of stress memory for resetting transcriptional memory genes (APX2 and HSP22) and H3K4me3 following recurrent heat stress. Furthermore, CSN5A is also required for the deposition of H3K4me3 following recurrent heat stress. Thus, CSN5A plays an important role in the regulation of histone methylation and transcriptional stress memory after recurrent heat stress.

The COP9 signalosome is required for light-dependent timeless degradation and Drosophila clock resetting.

The ubiquitin-proteasome system plays a major role in the rhythmic accumulation and turnover of molecular clock components. In turn, these approximately 24 h molecular rhythms drive circadian rhythms of behavior and physiology. In Drosophila, the ubiquitin-proteasome system also plays a critical role in light-dependent degradation of the clock protein Timeless (TIM), a key step in the entrainment of the molecular clocks to light-dark cycles. Here, we investigated the role of the COP9 signalosome (CSN), a general regulator of protein degradation, in fly circadian rhythms. We found that null mutations in the genes encoding the CSN4 and CSN5 subunits prevent normal TIM degradation by light in the pacemaker lateral neurons (LNs) as does LN-specific expression of a dominant-negative CSN5 transgene. These defects are accompanied by strong reductions in behavioral phase shifts of adult flies lacking normal CSN5 activity in LNs. Defects in TIM degradation and resetting of behavioral phases were rescued by overexpression of Jetlag (JET), the F-box protein required for light-mediated TIM degradation. Flies lacking normal CSN activity in all clock neurons are rhythmic in constant light, a phenotype previously associated with jet mutants. Together, these data indicate that JET and the CSN lie in a common pathway leading to light-dependent TIM degradation. Surprisingly, we found that manipulations that strongly inhibit CSN activity had minimal effects on circadian rhythms in constant darkness, indicating a specific role for the CSN in light-mediated TIM degradation.

Cop9 signalosome subunit 8 (CSN8) is essential for Drosophila development.

The COP9 signalosome (CSN) is a multisubunit regulator highly conserved in evolution. We show here that CSN subunit 8 (CSN8) is essential for Drosophila development. CSN8 is maternally contributed and present throughout development. Null mutants generated in this study are larval lethal, showing phenotypes associated with mutations in either CSN4 (molting defects) or CSN5 (melanotic tumors). Analysis of mitotic and germ-line csn8(null) clones revealed the requirement of CSN8 for multiple developmental processes. The germ-line clones arrested at mid-oogenesis, while the mitotic clones led to deformed adult eyes or wings. CSN8 is present exclusively as part of the CSN holo-complex, and lack of CSN8 in the mutants leads to CSN instability. Consistent with this, Cullin deneddylation is impaired in the csn8(null) mutants.

Role of Cop9 Signalosome Subunits in the Environmental and Hormonal Balance of Plant.

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) is a highly conserved protein complex that influences several signaling and developmental processes. The COP9 signalosome consists of eight subunits, among which two subunits, CSN5 and CSN6, contain an Mpr1/Pad1 N-terminal (MPN) domain and the remaining six subunits contain a proteasome, COP9 signalosome, and initiation factor 3 (PCI) domain. In plants, each MPN subunit is encoded by two genes, which is not the case in other organisms. This review aims to provide in-depth knowledge of each COP9 signalosome subunit, concentrating on genetic analysis of both partial and complete loss-of-function mutants. At the beginning of this review, the role of COP9 signalosome in the hormonal signaling and defense is discussed, whereas later sections deal in detail with the available partial loss-of-function, hypomorphic mutants of each subunit. All available hypomorphic mutants are compared based on their growth response and deneddylation activity.