CAND1 enhances deneddylation of CUL1 by COP9 signalosome

Cullin-RING ligases (CRLs) regulate diverse cellular functions such as cell cycle progression and cytokine signaling by ubiquitinating key regulatory proteins. The activity of CRLs is controlled by Nedd8 modification of the cullin subunits. Recent reports have suggested that CAND1, which specifically binds to unmodified CUL1 but not to neddylated one, is required for the in vivo function of SCFs, the CUL1-containing CRLs. We show here that CAND1 and COP9 signalosome (CSN), the major deneddylase of cullins, bind to unneddylated CUL1 in a mutually exclusive way. The suppression of CAND1 expression by small inhibitory RNA enhanced the interaction between CUL1 and CSN, suggesting that CAND1 inhibited the binding of CSN to CUL1. We found that the binding of CSN to CUL1 required the four helix bundle in CUL1 C-terminal domain, which was wrapped around by CAND1 in the CAND1-CUL1-Rbx1 complex. CAND1 greatly facilitated CSN-mediated deneddylation of CUL1 in vitro, which was dependent on its binding to CUL1. Our data suggest that enhancement of CSN-mediated deneddylation by CAND1 may contribute to its function as a positive regulator of SCFs in vivo.     

Geminivirus C2 protein might be the key player for geminiviral co-option of SCF-mediated ubiquitination

Viruses are obligate intracellular parasites, and need to create a suitable cell environment for viral propagation to complete their life cycle. In order to achieve this, viruses must usurp or interfere with the cellular machinery. Ubiquitination, a post-translational modification that controls numerous cellular processes, has proven to be a common target for viruses. Recently, geminivirus C2 protein has been shown to interact with the CSN complex and disrupt its activity over CULLIN1, interfering with the function of the CULLIN1-based SCF ubiquitin E3 ligases. Interestingly, over-expression of a given F-box protein may circumvent the general SCF malfunction caused by C2. This result raises the tantalizing idea that geminiviruses might be not only hampering, but also redirecting the activity of SCF complexes, thus co-opting the SCF-mediated ubiquitination pathway. We hypothesize that the mechanism of C2-facilitated co-option of SCF-mediated ubiquitination might not be exclusive for geminiviruses, but rather a common strategy for viruses.Key words: geminivirus, C2 protein, ubiquitination, SCF complex, CSN complex, F-box protein, pathogen co-option     

The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses

The COP9 signalosome (CSN) is an evolutionarily conserved, nucleus-enriched multiprotein complex. CSN plays roles in photomorphogenesis, auxin response, and floral organ formation, possibly via the regulation of ubiquitin-proteasome-mediated protein degradation. COI1 encodes an F-box protein, which is a subunit of SCF(COI1) E3 ubiquitin ligase, and is required for jasmonate (JA) responses. Here, we demonstrate using coimmunoprecipitation and gel-filtration analyses that endogenous as well as epitope-tagged COI1 forms SCF(COI1) and associates directly with CSN in vivo. Like the coi1-1 mutant, CSN reduction-of-function plants exhibited a JA-insensitive root elongation phenotype and an absence of JA-induced-specific gene expression. Genome expression profile analyses indicated that JA-triggered genome expression is critically dependent on COI1 dosage. More importantly, most of the COI1-dependent JA-responsive genes also required CSN function, and CSN abundance was shown to be important for JA responses. Furthermore, we showed that both COI1 and CSN are essential for modulating the expression of genes in most cellular pathways responsive to JA. Thus, CSN and SCF(COI1) work together to control genome expression and promote JA responses.     

A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis

Regulated protein degradation contributes to plant development by mediating signaling events in many hormone, light, and developmental pathways. Ubiquitin ligases recognize and ubiquitinate target proteins for subsequent degradation by the 26S proteasome. The multisubunit SCF is the best-studied class of ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). However, the extent of SCF participation in signaling networks is unclear. SCFs are composed of four subunits: CULLIN 1 (CUL1), ASK, RBX1, and an F-box protein. Null mutations in CUL1 are embryo lethal, limiting insight into the role of CUL1 and SCFs in later stages of development. Here, we describe a viable and fertile weak allele of CUL1, called cul1-6. cul1-6 plants have defects in seedling and adult morphology. In addition to reduced auxin sensitivity, cul1-6 seedlings are hyposensitive to ethylene, red, and blue light conditions. An analysis of protein interactions with the cul1-6 gene product suggests that both RUB (related to ubiquitin) modification and interaction with the SCF regulatory protein CAND1 (cullin associated and neddylation dissociated) are disrupted. These findings suggest that the morphological defects observed in cul1-6 plants are caused by defective SCF complex formation. Characterization of weak cul1 mutants provides insight into the role of SCFs throughout plant growth and development.     

GmCOI1, a soybean F-box protein gene, shows ability to mediate jasmonate-regulated plant defense and fertility in Arabidopsis

The F-box protein gene COI1 from Arabidopsis plays a fundamental role in response to jasmonates, which regulate plant root growth, pollen fertility, wounding and healing, and defense against pathogens and insects. Null mutations in COI1 were previously found to abolish all the jasmonate responses, and the Arabidopsis coil-1 mutant is male sterile and susceptible to pathogen infection. In this study, we isolated an F-box protein gene from soybean, which shares significant homology with the Arabidopsis COI1 and similarly contains an F-box motif and leucine rich repeats (LRR), here designated GmCOI1 (Glycine max L. (Merr.) COI1). To test whether the sequence homology and structural similarity are indicative of functional conservation, we expressed GmCOI1 in the Arabidopsis coil-1 mutant. The transgenic coil-1 plants with expression of the GmCOI1 gene were found to exhibit normal jasmonate responses, including jasmonate-regulated plant defense and fertility. In addition, the chimerical proteins with swapped domain of the F-box motif or LRR between GmCOI1 and COI1 were shown to functionally complement the coil-1 mutation. Furthermore, GmCOI1 was found to assemble into the Skpl-Cullin-F-box (SCF) complexes, similar to the formation of the Arabidopsis SCF(COO1). These data demonstrate the soybean F-box protein gene GmCOI1 is able to mediate jasmonate-regulated plant defense and fertility in Arabidopsis, which implies a generic jasmonate pathway with conserved signal components in different plant species.     

The COP9 signalosome interacts with SCF UFO and participates in Arabidopsis flower development

The COP9 signalosome (CSN) is involved in multiple developmental processes. It interacts with SCF ubiquitin ligases and deconjugates Nedd8/Rub1 from cullins (deneddylation). CSN is highly expressed in Arabidopsis floral tissues. To investigate the role of CSN in flower development, we examined the expression pattern of CSN in developing flowers. We report here that two csn1 partially deficient Arabidopsis strains exhibit aberrant development of floral organs, decline of APETALA3 (AP3) expression, and low fertility in addition to defects in shoot and inflorescence meristems. We show that UNUSUAL FLORAL ORGANS (UFO) forms a SCF(UFO) complex, which is associated with CSN in vivo. Genetic interaction analysis indicates that CSN is necessary for the gain-of-function activity of the F-box protein UFO in AP3 activation and in floral organ transformation. Compared with the previously reported csn5 antisense and csn1 null mutants, partial deficiency of CSN1 causes a reduction in the level of CUL1 in the mutant flowers without an obvious defect in CUL1 deneddylation. We conclude that CSN is an essential regulator of Arabidopsis flower development and suggest that CSN regulates Arabidopsis flower development in part by modulating SCF(UFO)-mediated AP3 activation.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

Role of Jasmonates in the Elicitor- and Wound-Inducible Expression of Defense Genes in Parsley and Transgenic Tobacco

Jasmonates have been proposed to be signaling intermediates in the wound and/or elicitor-activated expression of plant defense genes. We used parsley (Petroselinum crispum) cell cultures and transgenic tobacco (Nicotiana tabacum) plants expressing 4CL1-GUS gene fusions to investigate the potential role played by jasmonates in mediating the wound and/or elicitor activation of phenylpropanoid and other defense-related genes. Jasmonates and [alpha]-linolenic acid strongly induced the expression of 4CL in a dose-dependent manner in parsley cells; methyl jasmonate also activated the coordinate expression of other phenylpropanoid genes and the accumulation of furanocoumarin phytoalexins. However, the response of the cells to optimal methyl jasmonate concentrations was distinct quantitatively and qualitatively from the response of elicitor-treated cells. In transgenic tobacco wound-inducible tobacco 4CL genes and a 4CL1 promoter-GUS transgene were responsive to jasmonates and [alpha]-linolenic acid in a dose-dependent manner. Pre-treatment of parsley cells or tobacco leaves with a lipoxygenase inhibitor reduced their responsiveness to the elicitor and to wounding. These results show that the elicitor response in parsley cells can be partially mimicked by jasmonate treatment, which supports a role for jasmonates in mediating wound-induced expression of 4CL and other phenylpropanoid genes.     

Point mutations in Arabidopsis Cullin1 reveal its essential role in jasmonate response

The SKP1-Cullin/Cdc53-F-box protein ubiquitin ligases (SCF) target many important regulatory proteins for degradation and play vital roles in diverse cellular processes. In Arabidopsis there are 11 Cullin members (AtCUL). AtCUL1 was demonstrated to assemble into SCF complexes containing COI1, an F-box protein required for response to jasmonates (JA) that regulate plant fertility and defense responses. It is not clear whether other Cullins also associate with COI1 to form SCF complexes, thus, it is unknown whether AtCUL1, or another Cullin that assembles into SCF(COI1) (even perhaps two or more functionally redundant Cullins), plays a major role in JA signaling. We present genetic and physiological data to directly demonstrate that AtCUL1 is necessary for normal JA responses. The homozygous AtCUL1 mutants axr6-1 and axr6-2, the heterozygous mutants axr6/AXR6, and transgenic plants expressing mutant AtCUL1 proteins containing a single amino acid substitution from phenylalanine-111 to valine, all exhibit reduced responses to JA. We also demonstrate that ax6 enhances the effect of coi1 on JA responses, implying a genetic interaction between COI1 and AtCUL1 in JA signaling. Furthermore, we show that the point mutations in AtCUL1 affect the assembly of COI1 into SCF, thus attenuating SCF(COI1) formation.     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000     

The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis

Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plant's response to B. cinerea.     

Jasmonate-related mutants of Arabidopsis as tools for studying stress signaling

Jasmonates are naturally occurring signal compounds that regulate plant growth and development, and are involved in plant responses to several environmental stress factors. The mode of action of jasmonates has been investigated traditionally by analysis of the effects of exogenous application of these compounds, including identification of jasmonate-responsive genes and determination of their expression and responsive promoter elements. In addition, jasmonate biosynthesis has been studied by identification of biosynthetic enzymes, use of inhibitors and determination of endogenous jasmonate levels. Recently, several mutants defective in jasmonate biosynthesis and signaling have been isolated and their phenotypes shed new light on the role of jasmonates and jasmonate signaling in plant responses to pathogens, insects and ozone.     

Coordinated activation of metabolic pathways for antioxidants and defence compounds by jasmonates and their roles in stress tolerance in Arabidopsis

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively termed jasmonates, are ubiquitous plant signalling compounds. Several types of stress conditions, such as wounding and pathogen infection, cause endogenous JA accumulation and the expression of jasmonate-responsive genes. Although jasmonates are important signalling components for the stress response in plants, the mechanism by which jasmonate signalling contributes to stress tolerance has not been clearly defined. A comprehensive analysis of jasmonate-regulated metabolic pathways in Arabidopsis was performed using cDNA macroarrays containing 13516 expressed sequence tags (ESTs) covering 8384 loci. The results showed that jasmonates activate the coordinated gene expression of factors involved in nine metabolic pathways belonging to two functionally related groups: (i) ascorbate and glutathione metabolic pathways, which are important in defence responses to oxidative stress, and (ii) biosynthesis of indole glucosinolate, which is a defence compound occurring in the Brassicaceae family. We confirmed that JA induces the accumulation of ascorbate, glutathione and cysteine and increases the activity of dehydroascorbate reductase, an enzyme in the ascorbate recycling pathway. These antioxidant metabolic pathways are known to be activated under oxidative stress conditions. Ozone (O3) exposure, a representative oxidative stress, is known to cause activation of antioxidant metabolism. We showed that O3 exposure caused the induction of several genes involved in antioxidant metabolism in the wild type. However, in jasmonate-deficient Arabidopsis 12-oxophytodienoate reductase 3 (opr3) mutants, the induction of antioxidant genes was abolished. Compared with the wild type, opr3 mutants were more sensitive to O3 exposure. These results suggest that the coordinated activation of the metabolic pathways mediated by jasmonates provides resistance to environmental stresses.     

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.     

Novel chemically synthesized hydroxyl-containing jasmonates as powerful inducing signals for plant secondary metabolism

Novel hydroxyl-containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 microM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl-containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 microM 2,3-dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 +/- 0.5 mg/g (at day 21), whereas the Tc content was 29.2 +/- 0.6 mg/g (on the same day) with addition of 100 microM MJA. Quantitative structure-activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H(2)O(2) production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl-containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures.     

Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function

The ubiquitin-related protein RUB/Nedd8 is conjugated to members of the cullin family of proteins in plants, animals, and fungi. In Arabidopsis, the RUB conjugation pathway consists of a heterodimeric E1 (AXR1-ECR1) and a RUB-E2 called RCE1. The cullin CUL1 is a subunit in SCF-type ubiquitin protein ligases (E3s), including the SCF(TIR1) complex, which is required for response to the plant hormone auxin. Our previous studies showed that conjugation of RUB to CUL1 is required for normal SCF(TIR1) function. The RING-H2 finger protein RBX1 is a subunit of SCF complexes in fungi and animals. The function of RBX1 is to bind the ubiquitin-conjugating enzyme E2 and bring it into close proximity with the E3 substrate. We have identified two Arabidopsis genes encoding RING-H2 proteins related to human RBX1. Studies of one of these proteins indicate that, as in animals and fungi, Arabidopsis RBX1 is an SCF subunit. Reduced RBX1 levels result in severe defects in growth and development. Overexpression of RBX1 increases RUB modification of CUL1. This effect is associated with reduced auxin response and severe growth defects similar to those observed in axr1 mutants. As in the axr1 mutants, RBX1 overexpression stabilizes the SCF(TIR1) substrate AXR2/IAA7. The RBX1 protein is a component of SCF complexes in Arabidopsis. In addition to its direct role in SCF E3 ligase activity, RBX1 promotes the RUB modification of CUL1 and probably functions as an E3 ligase in the RUB pathway. Hypermodification of CUL1 disrupts SCF(TIR1) function, suggesting that cycles of RUB conjugation and removal are important for SCF activity.     

Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases

Selective protein degradation by the ubiquitin-proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The selective components of this pathway are the E3 ubiquitin-ligases which act downstream of the ubiquitin-activating and -conjugating enzymes to identify specific substrates for ubiquitinylation. SCF-type ubiquitin-ligases are the most abundant class of E3 enzymes in Arabidopsis. In a genetic screen for enhancers of the tir1-1 auxin response defect, we identified eta1/axr6-3, a recessive and temperature-sensitive mutation in the CUL1 core component of the SCF(TIR1) complex. The axr6-3 mutation interferes with Skp1 binding, thus preventing SCF complex assembly. axr6-3 displays a pleiotropic phenotype with defects in numerous SCF-regulated pathways including auxin signaling, jasmonate signaling, flower development, and photomorphogenesis. We used axr6-3 as a tool for identifying pathways likely to be regulated by SCF-mediated proteolysis and propose new roles for SCF regulation of the far-red light/phyA and sugar signaling pathways. The recessive inheritance and the temperature-sensitive nature of the pleiotropically acting axr6-3 mutation opens promising possibilities for the identification and investigation of SCF-regulated pathways in Arabidopsis.