The Copper Transporter RAN1 Is Essential for Biogenesis of Ethylene Receptors in Arabidopsis

Plants utilize ethylene as a hormone to regulate multiple developmental processes and to coordinate responses to biotic and abiotic stress. In Arabidopsis thaliana, a small family of five receptor proteins typified by ETR1 mediates ethylene perception. Our previous work suggested that copper ions likely play a role in ethylene binding. An independent study indicated that the ran1 mutants, which display ethylene-like responses to the ethylene antagonist trans-cyclooctene, have mutations in the RAN1 copper-transporting P-type ATPase, once again linking copper ions to the ethylene-response pathway. The results presented herein indicate that genetically engineered Saccharomyces cerevisiae expressing ETR1 but lacking the RAN1 homolog Ccc2p (Δccc2) lacks ethylene-binding activity. Ethylene-binding activity was restored when copper ions were added to the Δccc2 mutants, showing that it is the delivery of copper that is important. Additionally, transformation of the Δccc2 mutant yeast with RAN1 rescued ethylene-binding activity. Analysis of plants carrying loss-of-function mutations in ran1 showed that they lacked ethylene-binding activity, whereas seedlings carrying weak alleles of ran1 had normal ethylene-binding activity but were hypersensitive to copper-chelating agents. Altogether, the results show an essential role for RAN1 in the biogenesis of the ethylene receptors and copper homeostasis in Arabidopsis seedlings. Furthermore, the results indicate cross-talk between the ethylene-response pathway and copper homeostasis in Arabidopsis seedling development.     

RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis.

Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.     

ethylene receptor 1 (etr1) Is Sufficient and Has the Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana

Ethylene influences many processes in Arabidopsis thaliana through the action of five receptor isoforms. All five isoforms use copper as a cofactor for binding ethylene. Previous research showed that silver can substitute for copper as a cofactor for ethylene binding activity in the ETR1 ethylene receptor yet also inhibit ethylene responses in plants. End-point and rapid kinetic analyses of dark-grown seedling growth revealed that the effects of silver are mostly dependent upon ETR1, and ETR1 alone is sufficient for the effects of silver. Ethylene responses in etr1-6 etr2-3 ein4-4 triple mutants were not blocked by silver. Transformation of these triple mutants with cDNA for each receptor isoform under the promoter control of ETR1 revealed that the cETR1 transgene completely rescued responses to silver while the cETR2 transgene failed to rescue these responses. The other three isoforms partially rescued responses to silver. Ethylene binding assays on the binding domains of the five receptor isoforms expressed in yeast showed that silver supports ethylene binding to ETR1 and ERS1 but not the other isoforms. Thus, silver may have an effect on ethylene signaling outside of the ethylene binding pocket of the receptors. Ethylene binding to ETR1 with silver was ～30% of binding with copper. However, alterations in the K(d) for ethylene binding to ETR1 and the half-time of ethylene dissociation from ETR1 do not underlie this lower binding. Thus, it is likely that the lower ethylene binding activity of ETR1 with silver is due to fewer ethylene binding sites generated with silver versus copper.     

Involvement of RTE1 in conformational changes promoting ETR1 ethylene receptor signaling in Arabidopsis

Ethylene is an important regulator of plant growth, development and responses to environmental stresses. Arabidopsis perceives ethylene through five homologous receptors that negatively regulate ethylene responses. RTE1, a novel gene conserved in plants, animals and some protists, was recently identified as a positive regulator of the ETR1 ethylene receptor. Here, we genetically analyze the dependence of ETR1 on RTE1 in order to obtain further insight into RTE1 function. The function of RTE1 was found to be independent and distinct from that of RAN1, which encodes a copper transporter required for ethylene receptor function. We tested the ability of an rte1 loss-of-function mutation to suppress 11 etr1 ethylene-binding domain mis-sense mutations, all of which result in dominant ethylene insensitivity due to constitutive signaling. This suppression test uncovered two classes of etr1 mutations -RTE1-dependent and RTE1-independent. The nature of these mutations suggests that the ethylene-binding domain is a possible target of RTE1 action. Based on these findings, we propose that RTE1 promotes ETR1 signaling through a conformational effect on the ethylene-binding domain.     

Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis

Ethylene signaling in Arabidopsis begins at a family of five ethylene receptors that regulate activity of a downstream mitogen-activated protein kinase kinase kinase, CTR1. Triple and quadruple loss-of-function ethylene receptor mutants display a constitutive ethylene response phenotype, indicating they function as negative regulators in this pathway. No ethylene-related phenotype has been described for single loss-of-function receptor mutants, although it was reported that etr1 loss-of-function mutants display a growth defect limiting plant size. In actuality, this apparent growth defect results from enhanced responsiveness to ethylene; a phenotype manifested in all tissues tested. The phenotype displayed by etr1 loss-of-function mutants was rescued by treatment with an inhibitor of ethylene perception, indicating that it is ethylene dependent. Identification of an ethylene-dependent phenotype for a loss-of-function receptor mutant gave a unique opportunity for genetic and biochemical analysis of upstream events in ethylene signaling, including demonstration that the dominant ethylene-insensitive phenotype of etr2-1 is partially dependent on ETR1. This work demonstrates that mutational loss of the ethylene receptor ETR1 alters responsiveness to ethylene in Arabidopsis and that enhanced ethylene response in Arabidopsis not only results in increased sensitivity but exaggeration of response.     

Ethylene stimulates nutations that are dependent on the ETR1 receptor

Ethylene influences a number of processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptors. In this study, we used high-resolution, time-lapse imaging to examine the long-term effects of ethylene on growing, etiolated Arabidopsis seedlings. These measurements revealed that ethylene stimulates nutations of the hypocotyls with an average delay in onset of over 6 h. The nutation response was constitutive in ctr1-2 mutants maintained in air, whereas ein2-1 mutants failed to nutate when treated with ethylene. Ethylene-stimulated nutations were also eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, further supporting a requirement for ETR1. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. However, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations, but the ETR1 receptor is required and has a contrasting role from the other receptor isoforms in this nutation phenotype. Naphthylphthalamic acid eliminated ethylene-stimulated nutations but had no effect on growth inhibition caused by ethylene, pointing to a role for auxin transport in the nutation phenotype.     

Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Background  

Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.     

The Relationship between Ethylene Binding and Dominant Insensitivity Conferred by Mutant Forms of the ETR1 Ethylene Receptor

Ethylene responses in Arabidopsis are mediated by a small family of receptors, including the ETR1 gene product. Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity. To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast. The etr1-1 and etr1-4 mutations completely eliminated ethylene binding, while the etr1-3 mutation severely reduced binding. Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants. By contrast, the etr1-2 mutation did not disrupt ethylene binding in yeast. These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor. Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors.     

Genetic Analysis of Ethylene Signal Transduction in Arabidopsis Thaliana: Five Novel Mutant Loci Integrated into a Stress Response Pathway

The response of Arabidopsis thaliana etiolated seedlings to the plant hormone ethylene is a conspicuous phenotype known as the triple response. We have identified genes that are required for ethylene perception and response by isolating mutants that fail to display a triple response in the presence of exogenous ethylene. Five new complementation groups have been identified. Four of these loci, designated ein4, ein5, ein6 and ein7, are insensitive to ethylene. The fifth complementation group, eir1, is defined by a novel class of mutants that have agravitropic and ethylene-insensitive roots. Double-mutant phenotypes have allowed the positioning of these loci in a genetic pathway for ethylene signal transduction. The ethylene-response pathway is defined by the following loci: ETR1, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, EIN7, EIR1, AUX1 and HLS1. ctr1-1 is epistatic to etr1-3 and ein4, indicating that CTR1 acts after both ETR1 and EIN4 in the ethylene-response pathway. Mutations at the EIN2, EIN3, EIN5, EIN6 and EIN7 loci are all epistatic to the ctr1 seedling phenotype. The EIR1 and AUX1 loci define a root-specific ethylene response that does not require EIN3 or EIN5 gene activity. HLS1 appears to be required for differential cell growth in the apical hook. The EIR1, AUX1 and HLS1 genes may function in the interactions between ethylene and other plant hormones that occur late in the signaling pathway of this simple gas.     

Reducing jasmonic acid levels causes ein2 mutants to become ethylene responsive

It has previously been shown that jasmonic acid affects the ethylene signaling pathway. EIN2 is a central component of ethylene signaling that is downstream of the receptors. EIN2 has previously been shown to be required for ethylene responses. We found that reducing jasmonic acid levels, either mutationally or chemically, caused ein2 ethylene-insensitive mutants to become ethylene responsive. This effect was not seen with the ethylene-insensitive etr1-1 mutants that affect receptor function. Based upon these results, we propose a model where jasmonic acid is inhibiting ethylene signal transduction down-stream of the ethylene receptors. This may involve an EIN2-independent pathway.     

铜离子与乙烯受体关系研究进展

许多年前 ,人们一直推测金属离子如锌、铜为受体感受乙烯所必需 ,但苦于没有直接证据。最近对拟南芥的生化和遗传学研究证实铜离子参与了乙烯信号的感受和转导。RAN1蛋白与酵母 CCC2蛋白和 Wilson &Menkes疾病蛋白同源 ,这些蛋白为转运铜离子的 P-型ATP酶。RAN1将铜离子整合到乙烯受体中 ,受体才能正常感受乙烯。铜离子投送可能是调节乙烯感受的重要因素     

Tomato BRASSINOSTEROID INSENSITIVE1 is required for systemin-induced root elongation in Solanum pimpinellifolium but is not essential for wound signaling

The tomato Leu-rich repeat receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) has been implicated in both peptide (systemin) and steroid (brassinosteroid [BR]) hormone perception. In an attempt to dissect these signaling pathways, we show that transgenic expression of BRI1 can restore the dwarf phenotype of the tomato curl3 (cu3) mutation. Confirmation that BRI1 is involved in BR signaling is highlighted by the lack of BR binding to microsomal fractions made from cu3 mutants and the restoration of BR responsiveness following transformation with BRI1. In addition, wound and systemin responses in the cu3 mutants are functional, as assayed by proteinase inhibitor gene induction and rapid alkalinization of culture medium. However, we observed BRI1-dependent root elongation in response to systemin in Solanum pimpinellifolium. In addition, ethylene perception is required for normal systemin responses in roots. These data taken together suggest that cu3 is not defective in systemin-induced wound signaling and that systemin perception can occur via a non-BRI1 mechanism.     

Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.     

Lesions in the sterol delta reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis

The brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol Delta7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark-green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13C-labeled precursors in dwf5 verified a deficiency in a sterol Delta7 reductase activity. All six independent alleles contain loss-of-function mutations in the sterol Delta7 reductase gene. These include a putative mRNA instability mutation in dwf5-1, 3' and 5' splice-site mutations in dwf5-2 and dwf5-6, respectively, premature stop codons in dwf5-3 (R400Z) and dwf5-5 (R409Z), and a mis-sense mutation in dwf5-4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild-type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith-Lemli-Opitz syndrome are caused by loss-of-function mutations in a sterol Delta7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.     

The Hyper-Gene Conversion Hpr5-1 Mutation of Saccharomyces Cerevisiae Is an Allele of the Srs2/Radh Gene

The HPR5 gene has been defined by the mutation hpr5-1 that results in an increased rate of gene conversion. This mutation suppresses the UV sensitive phenotype of rad18 mutations in hpr5-1 rad18 double mutants by channeling the aborted repair events into a recombination repair pathway. The HPR5 gene has been cloned and is shown to be allelic to the SRS2/RADH gene, a putative DNA helicase. The HPR5 gene, which is nonessential, is tightly linked to the ARG3 locus chromosome X. The hpr5-1 allele contains missense mutation in the putative ATP binding domain. A comparison of the recombination properties of the hpr5-1 allele and the null allele suggests that recombination events in hpr5 defective strains can be generated by several mechanisms. We propose that the HPR5 gene functions in the RAD6 repair pathway.     

Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression.

Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors. The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors. Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors. Mutations in STE4, 5, 7, and 11 partially reduced the number of binding sites, but this reduction was not sufficient to explain the loss of responsiveness; the products of these genes appear to affect postreceptor steps of the response pathway. As a second method of distinguishing the roles of the various STE genes, we examined the sterile mutants for suppression. Mating of the ste2-3 mutant was apparently limited by its sensitivity to alpha-factor, as its sterility was suppressed by mutation sst2-1, which leads to enhanced alpha-factor sensitivity. Sterility resulting from each of four ste4 mutations was suppressed partially by mutation sst2-1 or by mutation bar1-1 when one of three other mutations (ros1-1, ros2-1, or ros3-1) was also present. Sterility of the ste5-3 mutant was suppressed by mutation ros1-1 but not by sst2-1. The ste7, 11, and 12 mutations were not suppressed by ros1 or sst2. Our working model is that STE genes control the response to alpha-factor at two distinct steps. Defects at one step (requiring the STE2 gene are suppressed (directly or indirectly) by mutation sst2-1, whereas defects at the other step (requiring the STE5 gene) are suppressed by the ros1-1 mutation. The ste4 mutants are defective for both steps. Mutation ros1-1 was found to be allelic to cdc39-1. Map positions for genes STE2, STE12, ROS3, and FUR1 were determined.     

乙烯的生物合成与信号传递

乙烯是气体植物激素, 它在植物的生长发育过程中有很多作用。所以了解乙烯的生物合成及其信号转导是非常重要的。二十年来, 通过筛选有异于正常三重反应的突变体, 人们发现了乙烯信号转导的粗略轮廓。在拟南芥中, 有5个受体蛋白感受乙烯, ETR1、ERS1、ETR2、ERS2、EIN4。它们表现出功能冗余, 是乙烯信号的负调控因子, 在植物体内以二聚体的形式存在。ETR1的N端与乙烯结合时需要 铜离子(Ⅰ)的参与。尽管已经发现ETR1有组氨酸激酶活性, 而其它受体有丝氨酸/苏氨酸激酶活性, 但受体参与乙烯信号转导的机制还不是很清楚。受体与Raf类蛋白激酶CTR1相互作用, CTR1是乙烯反应的负调控因子。CTR1蛋白失活使EIN2蛋白活化。EIN2的N端是跨膜结构域, 与Nramp家族金属离子转运蛋白的跨膜结构域类似。EIN2的C端是一个新的未知结构域, 与乙烯信号途径的下游组分相互作用。EIN3位于EIN2的下游, EIN3和EILs诱导ERF1和其它转录因子的表达, 这些转录因子依次激活乙烯反应目的基因的表达, 表现出乙烯的反应。EIN3受到蛋白酶体介导的蛋白降解途径的调节。由于乙烯是一种多功能的植物激素, 其信号途径与其它信号途径有多重的交叉。