ASK1 physically interacts with COI1 and is required for male fertility inArabidopsis

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. TheCOI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with theArabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action inplanta. Functional analysis by antisense strategy showed thatASK1 is involved in male fertility.     

ASK1 physically interacts with COI1 and is required for male fertility in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. TheCOI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with theArabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action inplanta. Functional analysis by antisense strategy showed thatASK1 is involved in male fertility.     

F-box蛋白COI1稳定性调控机制

拟南芥F-box蛋白COI1(Coronatine insensitive 1)与ASK1(Arabidopsis serine/Threonine kinase 1)蛋白及CUL1(CULLIN1)蛋白等结合形成SCFCOI1泛素连接酶复合体.COI1感知茉莉素信号、进而调控植物一系列的防御反应和生长发育过程.虽然多种作物的COI1同源蛋白已经被相继鉴定,但是其自身蛋白水平的调控机制仍然未知.本文重点研究了蔬菜作物番茄(Solanum lycopersicum)和经济作物烟草(Nicotiana attenuata)中COI1蛋白稳定性的调控机制.结果证明,这两种作物的COI1蛋白通过与ASK1的相互作用而得到稳定,表明形成SCFCOI1复合体可能有助于COI1蛋白的稳定.同时,26S蛋白酶体抑制剂能够明显抑制COI1的降解,说明泛素-蛋白酶体途径参与了其降解过程.这些结果证明在这两个不同物种中,两条互相拮抗的途径共同发挥作用,平衡并稳定COI1蛋白在细胞内的恰当丰度.该研究为深入研究不同作物的茉莉素信号转导调控机制奠定了良好的基础.     

The intragenic suppressor mutation Leu59Phe compensates for the effect of detrimental mutations in the jasmonate receptor COI1

The phytohormones jasmonates (JAs) control plant development, growth, and defense against insects and pathogens. The Arabidopsis JA receptor Coronatine Insensitive 1 (COI1) interacts with ARABIDOPSIS SKP-LIKE1 (ASK1)/ASK2 to form the SCF^COI1 E3 ligase and mediate JA responses. Here, we performed a genetic suppressor screen using the leaky coi1-2 (COI1^Leu245Phe) mutant for restored sensitivity to JA, and identified the intragenic suppressor mutation Leu59Phe, which was in the region connecting the F-box and leucine-rich repeats domains of COI1. The L59F substitution not only restores the COI1^L245F function, but also the COI1^Gly434Glu (coi1-22^rsp) function in JA responses, through recovering their interactions with ASK1 or ASK2 and their protein levels. The L59F change itself could not enhance the interactions between COI1 and ASK1/2, nor affect JA responses. The present study reveals that the Leu59Phe substitution compensates for the effect of some deleterious mutations in the JA receptor COI1.     

The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF(COI1) complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component of SCF(COI1) complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modification of the AtCUL1 subunit of SCF(COI1) complexes is important for JA signaling.     

The Arabidopsis F-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFCOI1 and Degraded via the 26S Proteasome Pathway

Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCF^COI1) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCF^COI1 complexes, suggesting a function for SCF^COI1 in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCF^COI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses.     

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.     

COS1: an Arabidopsis coronatine insensitive1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence

The Arabidopsis thaliana CORONATINE INSENSITIVE1 (COI1) gene encodes an F-box protein to assemble SCF(COI1) complexes essential for response to jasmonates (JAs), which are a family of plant signaling molecules required for many essential functions, including plant defense and reproduction. To better understand the molecular basis of JA action, we screened for suppressors of coi1 and isolated a coi1 suppressor1 (cos1) mutant. The cos1 mutation restores the coi1-related phenotypes, including defects in JA sensitivity, senescence, and plant defense responses. The COS1 gene was cloned through a map-based approach and found to encode lumazine synthase, a key component in the riboflavin pathway that is essential for diverse yet critical cellular processes. We demonstrated a novel function for the riboflavin pathway that acts downstream of COI1 in the JA signaling pathway and is required for suppression of the COI1-mediated root growth, senescence, and plant defense.     

Proteomics Study of COI1-regulated Proteins in Arabidopsis Flower

Jasmonates (JAs) are a new class of plant hormone that regulate expression of diverse genes to mediate various plant responses. The Arabidopsis F-box protein COM is required for plant defense and male fertility in JA signal pathway. To further investigate the regulatory role of COM in male fertility, we compared the proteomics profiles of Arabidopsis wild type (WT) flowers with coi1-1 mutant male-sterile flowers using two-dimensional difference gel electrophoresis coupled with matrix-assisted laser desoption/ionization-time-of-flight mass spectrometry. Sixteen proteins with potential function in specific biological processes such as metabolism processes and defense/stress responses were differentially expressed in WT and coi1-1 mutant flowers. Verification on a phi class glutathione transferase AtGSTF9, one out of these 16 identified proteins, revealed that the expression of AtGSTF9 was severely downregulated in flowers of coi1-1 mutant compared with that of WT. Further function analyses of these genes would provide new insights into the molecular basis of COI1-regulated male fertility.     

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 COI1 and DFR Genes are Essential for Regulation of Jasmonate-Induced Anthocyanin Accumulation in Arabidopsis

Jasmonates(JAs)are a class of plant hormones that play important roles in the regulation of plant development and plantdefense.It has been shown that Arabidopsis plants produce much higher levels of anthocyanins when treated exogenouslywith methyl jasmonate(MeJA).However,a molecular link between the JA response and anthocyanin production hasnot been determined.The CORONATINE INSENTITIVE1(COI1)gene is a key player in the regulation of many JA-relatedresponses.In the present study,we demonstrate that the COI1 gene is also required for the JA-induced accumulation ofanthocyanins in Arabidopsis.Furthermore,the MeJA-inducible expression of DIHYDROFLAVONOL REDUCTASE(DFR),anessential component in the anthocyanin biosynthesis pathway,was completely eliminated in the coil mutant.Jasmonate-induced anthocyanin accumulation was found to be independent of auxin signaling.The present results indicate that theexpression of both COI1 and DFR genes is required for the regulation of JA-induced anthocyanin accumulation and thatDFR may be a key downstream regulator for this process.     

The COI1 and DFR Genes are Essential for Regulation of Jasmonate-Induced Anthocyanin Accumulation in Arabidopsis

Jasmonates （JAs） are a class of plant hormones that play important roles in the regulation of plant development and plant defense. It has been shown that Arabidopsis plants produce much higher levels of anthocyanins when treated exogenously with methyl jasmonate （MeJA）. However, a molecular link between the JA response and anthocyanin production has not been determined. The CORONATINE INSENTITIVE1 （COI1） gene is a key player in the regulation of many JA-related responses. In the present study, we demonstrate that the COI1 gene is also required for the JA-induced accumulation of anthocyanins in Arabidopsis. Furthermore, the MeJA-inducible expression of DIHYDROFLAVONOL REDUCTASE （DFR）, an essential component in the anthocyanin biosynthesis pathway, was completely eliminated in the coil mutant. Jasmonateinduced anthocyanin accumulation was found to be independent of auxin signaling. The present results indicate that the expression of both COI1 and DFR genes is required for the regulation of JA-induced anthocyanin accumulation and that DFR may be a key downstream regulator for this process.     

Amino acid substitutions of GLY98, LEU245 and GLU543 in COI1 distinctively affect jasmonate-regulated male fertility in Arabidopsis

Jasmonate (JA) regulates various plant defense and developmental processes. The F-box protein CORONATINE INSENSITIVE 1 (COI1) perceives JA signals to mediate diverse plant responses including male fertility, root growth, anthocyanin accumulation, and defense against abiotic and biotic stresses. In this study, we carried out genetic, physiological and biochemical analysis on a series of coi1 mutant alleles, and found that different amino acid mutations in COI1 distinctively affect JA-regulated male fertility in Arabidopsis. All the JA responses are disrupted by the COI1 mutations W467* in coi1-1, Q343* (coi1-6), G369E (coi1-4), G98D (coi1-5), G155E (coi1-7), D452A (coi1-9) and L490A (coi1-10), though the coi1-5 mutant (COI1_G98D) contains adequate COI1 protein (～60% of wild-type). Interestingly, the low basal level of COI1^E543K in the coi1-8 mutant (～10% of wild-type COI1 level) is sufficient for maintaining male fertility (～50% of wild-type fertility); the coi1-2 mutant with low level of COI1^L245F (～10% of wild-type) is male sterile under normal growth condition (22°C) but male fertile (～80% of wild-type fertility) at low temperature (16°C); however, both coi1-2 and coi1-8 are defective in the other JA responses (root growth, anthocyanin accumulation, and plant response to the pathogen Pst DC3000 infection).     

Conservation and divergence of ASK1 and ASK2 gene functions during male meiosis in Arabidopsis thaliana

Selective proteolysis of regulatory proteins mediated by the ubiquitin pathway is an important mechanism for controlling many biological events. The SCF (Skpl-Cullin-F-box protein) class of E3 ubiquitin ligases controls the ubiquitination of a wide variety of substrates, thereby mediating their degradation by the 26S proteasome. The Arabidopsis genome contains 21 genes encoding Skp1-like proteins that are named as ASKs (Arabidopsis Skp1-like). So far, only the ASK1 gene has been characterized genetically, and is known to be required for male meiosis, flower development, and auxin response. The ASK2 gene is most similar to ASK1 in terms of both the amino acid sequence and expression pattern. To compare ASK2 with ASK1 functionally in male meiosis, different transgenic lines over-expressing ASK1 and ASK2 were tested for their ability to complement the male meiosis defect of the ask1-1 mutant. The genomic ASK1 rescued the ask1-1 mutant defects. The 35S::ASK1 transgene restored male fertility to the ask1-1 mutant, although the percentages of normal pollen grains and tetrads were reduced. 35S::ASK2 lines in the ask1-1 background exhibited partial fertility with even fewer normal pollen grains and tetrads than those of the 35S::ASK1 lines. Detailed analysis of chromosome behavior during male meiosis demonstrated that 35S::ASK1 and 35S::ASK2 lines had different fractions of pollen mother cells undergoing normal meiosis. Our results suggest that ASK2 partially substitutes for ASK1 if expressed at higher than normal levels.     

Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana

Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA‐Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F‐box proteins involved in the perception of JA‐Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP₆) as a co‐factor of unknown function, here we explored the possibility that InsP₆ or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two‐hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate‐mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two‐hybrid tests using wild‐type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP₅) and reduced levels of InsP₆ indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1‐1 mutants, also with increased InsP₅ and reduced InsP₆ levels, showed increased defensive capabilities via COI1‐mediated processes, including wound‐induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP₅, contributes to COI1 function.     

The vascular pathogen Verticillium longisporum requires a jasmonic acid-independent COI1 function in roots to elicit disease symptoms in Arabidopsis shoots

Verticillium longisporum is a soil-borne vascular pathogen that causes reduced shoot growth and early senescence in Arabidopsis (Arabidopsis thaliana). Here, we report that these disease symptoms are less pronounced in plants that lack the receptor of the plant defense hormone jasmonic acid (JA), CORONATINE INSENSITIVE1 (COI1). Initial colonization of the roots was comparable in wild-type and coi1 plants, and fungal DNA accumulated to almost similar levels in petioles of wild-type and coi1 plants at 10 d post infection. Completion of the fungal life cycle was impaired in coi1, as indicated by the reduced number of plants with microsclerotia, which are detected on dead plant material at late stages of the disease. Contrary to the expectation that the hormone receptor mutant coi1 should display the same phenotype as the corresponding hormone biosynthesis mutant delayed dehiscence2 (dde2), dde2 plants developed wild-type-like disease symptoms. Marker genes of the JA and the JA/ethylene defense pathway were induced in petioles of wild-type plants but not in petioles of dde2 plants, indicating that fungal compounds that would activate the known COI1-dependent signal transduction chain were absent. Grafting experiments revealed that the susceptibility-enhancing COI1 function acts in the roots. Moreover, we show that the coi1-mediated tolerance is not due to the hyperactivation of the salicylic acid pathway. Together, our results have unraveled a novel COI1 function in the roots that acts independently from JA-isoleucine or any JA-isoleucine mimic. This COI1 activity is required for a yet unknown root-to-shoot signaling process that enables V. longisporum to elicit disease symptoms in Arabidopsis.     

A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway

We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.     

The wound response mutant suppressor of prosystemin-mediated responses6 (spr6) is a weak allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1)

The systemic defense response of tomato plant in response to insect attack and wounding is regulated by the 18 amino acid peptide systemin and the phytohormone jasmonic acid (JA). Recent genetic analyses based mainly on spr (suppressors of prosystemin-mediated responses) mutant screens have led to the hypothesis that systemin acts at, or near, the site of wounding to amplify the production of JA, which in turn functions as a mobile signal to promote the systemic defense response. In order to identify more components involved in the systemin/JA-signaled defense response, we carried out a larger scale screen for new spr mutants in tomato. Here we describe the characterization of spr6, a mutant impaired in wound- and systemin-induced defense gene expression. Using a candidate gene approach based on genetic linkage, we demonstrate that spr6 is allelic to jai1-1, which is a loss-of-function allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. We show several aspects of the spr6 mutant phenotype distinct from that of jai1-1. First, the responsiveness of spr6 plants to exogenous JA shows a dosage dependency, i.e. it is more sensitive to JA than jai1-1 while less sensitive to JA than the wild-type. Secondly, unlike the sterile jai1-1, the spr6 plant displays normal fertility and seed set and thus can be maintained as a pure line and does not require selection. Therefore, spr6 provides a valuable tool, which can complement the limitations of jai1-1, to study JA signaling in tomato. The gene identification process of Spr6 we described herein represents an example showing the convenience of a candidate gene approach, based on genetic linkage, to identify gene functions of genetic loci defined by tomato wound response mutants.