Search

Ethylene was previously reported to repress stamen development in both cucumber and

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) in plants enable them to respond to pathogens by activating the production of defence metabolites that orchestrate immune responses

The gaseous nature of ethylene affects not only its role in plant biology but also how you treat plants with the hormone. In many ways, it simplifies the treatment problem. Other hormones have to be made up in solution and applied to some part of the plant hoping the hormone will be taken up into the plant and translocated throughout the plant at the desired concentration. Because all plant cells are connected by an intercellular gas space the ethylene concentration you treat with is relatively quickly reached throughout the plant. In some instances, like mature fruit, treatment with ethylene initiates autocatalytic synthesis of ethylene. However, in most experiments, the exogenous ethylene concentration is saturating, usually1 μL L

With the use of Arabidopsis as a model plant, studies in the past two decades have substantially advanced our understanding towards the ethylene signal transduction. A linear ethylene signal transduction has been proposed based on genetic and biochemical studies. The current model explains how the signaling components are involved in the pathway; yet, there are questions to be answered. For instance, how the different members of the ethylene receptor family may coordinate with the downstream Raf-like protein CONSTITUTIVE TRIPLE-RESPONSE1. It is to be answered for how ETHYLENE INSENSITIVE2 (EIN2) may mediate the ethylene signaling to the transcription factor EIN3. This review focuses on the differential regulation of the ethylene signaling via differential cooperation of different members of the ethylene receptor family, biological significance of the use of multiple ethylene receptor isoforms for the ethylene perception, and possible regulation of the ethylene signaling by a pathway that is independent of the negative regulation of CTR1 on EIN2.

In Arabidopsis, the ethylene-receptor signal output occurs at the endoplasmic reticulum and is mediated by the Raf-like protein CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) but is prevented by overexpression of the CTR1 N terminus. A phylogenic analysis suggested that rice OsCTR2 is closely related to CTR1, and ectopic expression of CTR1p:OsCTR2 complemented Arabidopsis ctr1-1. Arabidopsis ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE SENSOR1 physically interacted with OsCTR2 on yeast two-hybrid assay, and green fluorescence protein-tagged OsCTR2 was localized at the endoplasmic reticulum. The osctr2 loss-of-function mutation and expression of the 35S:OsCTR2 (1-513) transgene that encodes the OsCTR2 N terminus (residues 1-513) revealed several and many aspects, respectively, of ethylene-induced growth alteration in rice. Because the osctr2 allele did not produce all aspects of ethylene-induced growth alteration, the ethylene-receptor signal output might be mediated in part by OsCTR2 and by other components in rice. Yield-related agronomic traits, including flowering time and effective tiller number, were altered in osctr2 and 35S:OsCTR2 (1-513) transgenic lines. Applying prolonged ethylene treatment to evaluate ethylene effects on rice without compromising rice growth is technically challenging. Our understanding of roles of ethylene in various aspects of growth and development in japonica rice varieties could be advanced with the use of the osctr2 and 35S:OsCTR2 (1-513) transgenic lines.

The gaseous nature of ethylene affects not only its role in plant biology but also how you treat plants with the hormone. In many ways, it simplifies the treatment problem. Other hormones have to be made up in solution and applied to some part of the plant hoping the hormone will be taken up into the plant and translocated throughout the plant at the desired concentration. Because all plant cells are connected by an intercellular gas space the ethylene concentration you treat with is relatively quickly reached throughout the plant. In some instances, like mature fruit, treatment with ethylene initiates autocatalytic synthesis of ethylene. However, in most experiments, the exogenous ethylene concentration is saturating, usually >1 μL L-1, and the synthesis of additional ethylene is inconsequential. Also facilitating ethylene research compared with other hormones is that there are inhibitors of ethylene action 1-MCP (1-methylcyclopropene) and 2,5-NBD (2,5-norbornadiene) that are also gases wherein you can achieve nearly 100% inhibition of ethylene action quickly and with few side effects. Inhibitors for other plant hormones are applied as a solution and their transport and concentration at the desired site is not always known and difficult to measure. Here, our focus is on how to treat plants and plant parts with the ethylene gas and the gaseous inhibitors of ethylene action.

Copper ions play an important role in ethylene receptor biogenesis and proper function. The copper transporter RESPONSIVE-TO-ANTAGONIST1 (RAN1) is essential for copper ion transport in Arabidopsis thaliana. However it is still unclear how copper ions are delivered to RAN1 and how copper ions affect ethylene receptors. There is not a specific copper chelator which could be used to explore these questions. Here, by chemical genetics, we identified a novel small molecule, triplin, which could cause a triple response phenotype on dark-grown Arabidopsis seedlings through ethylene signaling pathway. ran1-1 and ran1-2 are hypersensitive to triplin. Adding copper ions in growth medium could partially restore the phenotype on plant caused by triplin. Mass spectrometry analysis showed that triplin could bind copper ion. Compared to the known chelators, triplin acts more specifically to copper ion and it suppresses the toxic effects of excess copper ions on plant root growth. We further showed that mutants of ANTIOXIDANT PROTEIN1 (ATX1) are hypersensitive to tiplin, but with less sensitivity comparing with the ones of ran1-1 and ran1-2. Our study provided genetic evidence for the first time that, copper ions necessary for ethylene receptor biogenesis and signaling are transported from ATX1 to RAN1. Considering that triplin could chelate copper ions in Arabidopsis, and copper ions are essential for plant and animal, we believe that, triplin not only could be useful for studying copper ion transport of plants, but also could be useful for copper metabolism study in animal and human., Author summary Copper ions are cofactors of protein functions, and their disorder is closely related to many human diseases which drive to develop new copper chelator related drugs. In plants, copper ions are essential for ethylene receptors, but many details are unclear. Researchers need novel specific copper chelators to study these questions. Here, by using plant chemical genetics, we identified a novel chemical triplin, which could activate ethylene signaling pathway by chelating copper ions essential for ethylene receptors. Ethylene resistance mutant etr1-1 and ein2 were resistant to triplin, and copper transporter mutants ran1-1 and ran1-2 were hypersensitive to triplin. Using triplin as a molecular genetics tool, we showed copper chaperone ATX1 acts the upstream of RAN1 which transports copper ions to ethylene receptors. We provide a sample that metabolism could be studied by combining chemical genetics and known signaling pathway. Moreover, triplin could chelate copper ions effectively and specifically in Arabidopsis, and could be a useful tool to study copper ion transport in plant, and valuable for designing copper chelator related drug.

The Arabidopsis (Arabidopsis thaliana) ethylene receptor Ethylene Response1 (ETR1) can mediate the receptor signal output via its carboxyl terminus interacting with the amino (N) terminus of Constitutive Triple Response1 (CTR1) or via its N terminus (etr11-349 or the dominant ethylene-insensitive etr1-11-349) by an unknown mechanism. Given that CTR1 is essential to ethylene receptor signaling and that overexpression of Reversion To Ethylene Sensitivity1 (RTE1) promotes ETR1 N-terminal signaling, we evaluated the roles of CTR1 and RTE1 in ETR1 N-terminal signaling. The mutant phenotype of ctr1-1 and ctr1-2 was suppressed in part by the transgenes etr11-349 and etr1-11-349, with etr1-11-349 conferring ethylene insensitivity. Coexpression of 35S:RTE1 and etr11-349 conferred ethylene insensitivity in ctr1-1, whereas suppression of the ctr1-1 phenotype by etr11-349 was prevented by rte1-2. Thus, RTE1 was essential to ETR1 N-terminal signaling independent of the CTR1 pathway. An excess amount of the CTR1 N terminus CTR17-560 prevented ethylene receptor signaling, and the CTR17-560 overexpressor CTR1-Nox showed a constitutive ethylene response phenotype. Expression of the ETR1 N terminus suppressed the CTR1-Nox phenotype. etr11-349 restored the ethylene insensitivity conferred by dominant receptor mutant alleles in the ctr1-1 background. Therefore, ETR1 N-terminal signaling was not mediated by full-length ethylene receptors; rather, full-length ethylene receptors acted cooperatively with the ETR1 N terminus to mediate the receptor signal independent of CTR1. ETR1 N-terminal signaling may involve RTE1, receptor cooperation, and negative regulation by the ETR1 carboxyl terminus.

Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed.

The ethylene response is negatively regulated by a family of five ethylene receptor genes in Arabidopsis (Arabidopsis thaliana). The five members of the ethylene receptor family can physically interact and form complexes, which implies that cooperativity for signaling may exist among the receptors. The ethylene receptor gene mutations etr1-1 ( C65Y )(for ethylene response1-1), ers1-1 ( I62P ) (for ethylene response sensor1-1), and ers1C65Y are dominant, and each confers ethylene insensitivity. In this study, the repression of the ethylene response by these dominant mutant receptor genes was examined in receptor-defective mutants to investigate the functional significance of receptor cooperativity in ethylene signaling. We showed that etr1-1 ( C65Y ), but not ers1-1 ( I62P ), substantially repressed various ethylene responses independent of other receptor genes. In contrast, wild-type receptor genes differentially supported the repression of ethylene responses by ers1-1 ( I62P ); ETR1 and ETHYLENE INSENSITIVE4 (EIN4) supported ers1-1 ( I62P ) functions to a greater extent than did ERS2, ETR2, and ERS1. The lack of both ETR1 and EIN4 almost abolished the repression of ethylene responses by ers1C65Y, which implied that ETR1 and EIN4 have synergistic effects on ers1C65Y functions. Our data indicated that a dominant ethylene-insensitive receptor differentially repressed ethylene responses when coupled with a wild-type ethylene receptor, which supported the hypothesis that the formation of a variety of receptor complexes may facilitate differential receptor signal output, by which ethylene responses can be repressed to different extents. We hypothesize that plants can respond to a broad ethylene concentration range and exhibit tissue-specific ethylene responsiveness with differential cooperation of the multiple ethylene receptors.

Ethylene influences many processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptor isoforms. We used high-resolution, time-lapse imaging of dark-grown Arabidopsis seedlings to better understand the roles of each isoform in the regulation of growth in air, ethylene-stimulated nutations, and growth recovery after ethylene removal. We found that ETHYLENE RECEPTOR1 (ETR1) is both necessary and sufficient for nutations. Transgene constructs in which the ETR1 promoter was used to drive expression of cDNAs for each of the five receptor isoforms were transferred into etr1-6;etr2-3;ein4-4 triple loss-of-function mutants that have constitutive growth inhibition in air, fail to nutate in ethylene, and take longer to recover a normal growth rate when ethylene is removed. The patterns of rescue show that ETR1, ETR2, and ETHYLENE INSENSITIVE4 (EIN4) have the prominent roles in rapid growth recovery after removal of ethylene whereas ETR1 was the sole isoform that rescued nutations. ETR1 histidine kinase activity and phosphotransfer through the receiver domain are not required to rescue nutations. However, REVERSION TO SENSITIVITY1 modulates ethylene-stimulated nutations but does not modulate the rate of growth recovery after ethylene removal. Several chimeric receptor transgene constructs where domains of EIN4 were swapped into ETR1 were also introduced into the triple mutant. The pattern of phenotype rescue by the chimeric receptors used in this study supports a model where a receptor with a receiver domain is required for normal growth recovery and that nutations specifically require the full-length ETR1 receptor.

Pathogens induce pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) in plants. PAMPs are microbial molecules recognized by host plants as nonself signals, whereas pathogen effectors are evolved to aid in parasitism but are sometimes recognized by specific intracellular resistance proteins. In the absence of detectable ETI determining classical incompatible interactions, basal resistance exists during compatible and nonhost interactions. What triggers the basal resistance has remained elusive. Here, we provide evidence that ETI contributes to basal resistance during both compatible and nonhost Arabidopsis–Pseudomonas syringae interactions. Mutations in RAR1 and NDR1, two genes required for ETI, compromise basal resistance in both compatible and nonhost interactions. Complete nonhost resistance to P. syringae pv. tabaci required a functional type III secretion system. PTI appears to play a greater role in nonhost resistance than basal resistance during compatible interactions, because abrogation of PTI compromises basal resistance during nonhost but not compatible interactions. Strikingly, simultaneous abrogation of ETI and flagellin-induced PTI rendered plants completely susceptible to the nonadapted bacterium P. syringae pv. tabaci, indicating that ETI and PTI act synergistically during nonhost resistance. Thus, both nonhost resistance and basal resistance to virulent bacteria can be unified under PTI and ETI.

Ethylene is a gaseous plant hormone used in many physiological studies examining its role in plant growth and development. However, ethylene gas may not be conveniently available to many laboratories for occasional use, and therefore several chemicals can be used as replacements. Here we report that the kinetics of the ethylene response induced by ethylene and two widely-used ethylene replacements are different. ACC failed to efficiently replace prolonged ethylene treatments, while the decomposition products of ethephon may cause non-specific responses and the efficiency of ethephon conversion to ethylene was relatively low. A cost-effective method to prepare ethylene gas was developed. Analyzed by gas chromatography, the chemically produced ethylene exhibited an identical chromatogram to that from the commercial source. Our synthetic ethylene gave the same dose-response curve in Arabidopsis as gaseous ethylene. Our study shows that the use of the ethylene gas is essential to experiments that are sensitive to treatment duration and dosage. When ACC and ethephon are used as replacements, caution should be taken in the experimental design. For laboratories that do not have an ethylene tank, ethylene gas can be easily prepared by a chemical approach without further purification.

Unlike other plants, bamboo (Bambusoideae) flowering is an elusive physiological phenomena, because it is unpredictable, long-periodic, gregarious, and uncontrollable; also, bamboo plants usually die after flowering. The flowering mechanism in Arabidopsis thaliana, a eudicot model species, is well established, but it remains unknown in bamboo species. We found 4470 and 3878 expressed sequence tags in the flower bud and vegetative shoot cDNA libraries, respectively, of the bamboo species, Bambusa oldhamii. Different genes were found expressed in bamboo flower buds compared to vegetative shoots, based on the Munich Information Center for Protein Sequences functional categorization; flowering-related genes were also identified in this species. We also identified Arabidopsis flowering-specific homologs that are involved in its photoperiod in this bamboo species, along with autonomous, vernalization and gibberellin-dependent pathways, indicating that bamboos may have a similar mechanism to control floral transition. Some bamboo expressed sequence tags shared high similarity with those of rice, but others did not match any known sequences. Our data lead us to conclude that bamboo may have its own unique flowering genes. This information can help us understand bamboo flowering and provides useful experimental methods to study the mechanisms involved.

Ethylene is a gaseous plant hormone. The finding that unveils regulation of ethylene signalling at the translational level adds complexity to the ethylene signalling ‘regulatome’ and generates insightful questions that may advance our understanding of the pathway.

The gaseous plant hormone ethylene, involved in various biological processes throughout development, is perceived by the ethylene receptor family members. Signaling of the gas is mediated by ETHYLENE INSENSITIVE2 (EIN2) to nuclear EIN3/EIN3-LIKE1 (EIL1) transcription factors, with EIN2-activated ethylene signaling directly inhibited by the serine/threonine kinase CONSTITUTIVE ETHYLENE RESPONSE1 (CTR1). The receptors, CTR1, and EIN2 are all localized at the endoplasmic reticulum (ER). How the ER-associated proteins mediate ethylene signaling to the nucleus was not resolved until it was found that the EIN2 C terminus (CEND) can mediate ethylene signaling from the ER to the nucleus (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012).

Arabidopsis thaliana has five ethylene hormone receptors, which bind ethylene and elicit responses critical for plant growth and development. Here we describe a negative regulator of ethylene responses, REVERSION-TO-ETHYLENE SENSITIVITY1 ( RTE1 ), which regulates the function of at least one of the receptors, ETR1 , in Arabidopsis . RTE1 was identified based on the ability of rte1 mutations to suppress ethylene insensitivity of the dominant gain-of-function allele etr1-2 . rte1 loss-of-function mutants have an enhanced ethylene response that closely resembles the etr1 null phenotype. The etr1 rte1 double null mutant is identical to the etr1 and rte1 single null mutants, suggesting that the two genes act in the same pathway. rte1 is unable to suppress the etr1-1 gain-of-function allele, placing RTE1 at or upstream of ETR1 . rte1 also fails to suppress gain-of-function mutations in each of the four other ethylene receptor genes. RTE1 encodes a previously undescribed predicted membrane protein, which is highly conserved in plants and protists but absent in fungi and prokaryotes. Ethylene treatment induces RTE1 expression, and overexpression of RTE1 confers reduced ethylene sensitivity that partially depends on ETR1 . These findings demonstrate that RTE1 is a negative regulator of ethylene signaling and suggest that RTE1 plays an important role in ETR1 function.

The mitogen-activated protein kinase kinase kinase (MAPKKK) Constitutive Triple-Response1 (CTR1) plays a key role in mediating ethylene receptor signaling via its N-terminal interaction with the ethylene receptor C-terminal histidine kinase (HK) domain. Loss-of-function mutations of CTR1 prevent ethylene receptor signaling, and corresponding ctr1 mutants show a constitutive ethylene response phenotype. We recently reported in Plant Physiology that expression of the truncated ethylene receptor Ethylene Response1 (ETR1) isoforms etr1 ( 1-349) and dominant ethylene-insensitive etr1-1 ( 1-349) , lacking the C-terminal HK and receiver domains, both suppressed the ctr1 mutant phenotype. Therefore, the ETR1 N terminus is capable of receptor signaling independent of CTR1. The constitutive ethylene response phenotype is stronger for ctr1-1 than ctr1-1 lines expressing the etr1 ( 1-349) transgene, so N-terminal signaling by the full-length but not truncated ETR1 is inhibited by ctr1-1. We address possible modulations of ETR1 N-terminal signaling with docking of CTR1 on the ETR1 HK domain.

Library of Congress Control Number: 2014951662Springer Dordrecht Heidelberg New York London© Springer Science+Business Media Dordrecht 2015This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part ofthe material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission orinformation storage and retrieval, electronic adaptation, computer software, or by similar or dissimilarmethodology now known or hereafter developed. Exempted from this legal reservation are briefexcerpts in connection with reviews or scholarly analysis or material supplied specifically for thepurpose of being entered and executed on a computer system, for exclusive use by the purchaser of thework. Duplication of this publication or parts thereof is permitted only under the provisions ofthe Copyright Law of the Publisher’s location, in its current version, and permissionfor use must alwaysbe obtained from Springer. Permissions for use may be obtained through RightsLink at the CopyrightClearance Center. Violations are liable to prosecution under the respective Copyright Law.The use of general descriptive names, registered names, trademarks, service marks, etc. in thispublication does not imply, even in the absence of a specific statement, that such names are exemptfrom the relevant protective laws and regulations and therefore free for general use.While the advice and information in this book are believed to be true and accurate at the date ofpublication, neither the authors nor the editors nor the publisher can accept any legal responsibility forany errors or omissions that may be made. The publisher makes no warranty, express or implied, withrespect to the material contained herein.Printed on acid-free paperSpringer is part of Springer Science+Business Media (www.springer.com)

In Arabidopsis, the DELLA subfamily of GRAS regulatory genes consists of GAI, RGA, RGA-LIKE1 (RGL1), RGL2, and RGL3. GAI and RGA are known to be negative regulators of gibberellin (GA) responses. We found that RGL1 is a similar repressor of GA responses, as revealed by RGL1 gain-of-function and loss-of-function phenotypes. Repression of GA responses in Arabidopsis was conferred by a dominant 35S-rgl1 transgene carrying a DELLA domain deletion analogous to the GA-insensitive gai-1 mutation. As in GA-deficient Arabidopsis, the transgenic plants were dark green dwarfs with underdeveloped trichomes and flowers. Expression levels of GA4, a feedback-regulated GA biosynthetic gene, were increased correspondingly. Conversely, a loss-of-function rgl1 line had reduced GA4 expression and exhibited GA-independent activation of seed germination, leaf expansion, flowering, stem elongation, and floral development, as detected by resistance to the GA biosynthesis inhibitor paclobutrazol. RGL1 plays a greater role in seed germination than do GAI and RGA. The expression profile of RGL1 differed from those of the four other DELLA homologs. RGL1 message levels were predominant in flowers, with transcripts detected in developing ovules and anthers. As with RGA, green fluorescent protein (GFP)–tagged RGL1 protein was localized to the nucleus, but unlike GFP-RGA, there was no degradation after GA treatment. These findings indicate that RGL1 is a partially redundant, but distinct, negative regulator of GA responses and suggest that all DELLA subfamily members might possess separate as well as overlapping roles in GA signaling.

Ethylene gas is an important plant hormone that can be chemically prepared or biologically synthesized by microbes and plants. The gas can be commercially obtained in a pressurized gas cylinder or chemically prepared with necessary purifications. Laboratories that wish to perform experiments involving ethylene treatment need a convenient setup for ethylene preparation and delivery. When the use of a pressurized ethylene gas cylinder is not feasible, an ethylene response can be initiated in the plant or plant organs by treating the plant or organ with an aqueous solution of the natural plant precursor to ethylene, 1-aminocyclopropane-1-carboxylic (ACC), or 2,4-dichlorophenoxyacetic acid (ethephon), which decomposes slowly to make ethylene at a pH above 4.0. However, the release of ethylene for these applications is dynamic and not experimentally controllable, and, moreover, the replacement may produce unwanted side effects that can affect data interpretation. Therefore, a direct ethylene treatment is often favorable over the replacement. An alternative to a pressurized tank of ethylene is the chemical synthesis of ethylene by ethanol dehydration or ethephon decomposition, with a specific setup to collect the produced ethylene. This chapter discusses the advantages and disadvantages of direct ethylene treatment using ethylene gas or a replacement. Also discussed are the underlying chemical and biochemical reactions for ethylene production, and the setup for ethylene treatment in a closed system or a flow-through system. The goal is to provide readers with the necessary tools for ethylene treatment with easily accessed laboratory devices.

Ethylene, the simple but vital gaseous hormone, affects an extensive array of developmental processes and responses to external and internal cues in plants. Extensive molecular genetic investigations during the past two decades have established a linear ethylene signaling pathway starting from endoplasmic reticulum (ER) membrane-spanning receptors to nuclear-localized transcription factors in the model plant Arabidopsis thaliana. The pathway involves negative regulation of ethylene signaling by ethylene receptor family members and Raf-like CONSTITUTIVE TRIPLE-RESPONSE1 (CTR1) and positive regulation by ER-associated ETHYLENE INSENSITIVE2 (EIN2) and nuclear-localized EIN3 and EIN3-LIKE1 (EIL1). Although ethylene is the signaling molecule that switches off the negative regulation by the receptors, several components fine-tune the signaling. In this chapter, we briefly summarize studies of ethylene signal transduction to give an overall picture of the ethylene signaling cascade. We also discuss regulatory components modifying the signaling components in the ethylene signaling pathway. Finally, we pose intriguing questions related to ethylene actions.

Antheridiogen (A CE ) is a pheromone that is required for the development of male gametophytes in the homosporous fern Ceratopteris richardii . Subtractive hybridization of cDNAs isolated from A CE -treated and non-A CE -treated gametophytes was used to isolate genes that are induced by the pheromone. The expression of one gene, ANI1 (for antheridiogen induced), was induced within 3 hr of A CE treatment, but its expression was transient. Patterns of ANI1 expression in wild-type and mutant gametophytes show that ANI1 expression inversely correlates with the predicted activity of one of the sex-determining genes, TRANSFORMER5 ( TRA5 ). These data suggest that ANI1 transcription or transcript accumulation is directly or indirectly prevented by TRA5 in the absence of A CE and that A CE inactivates the TRA5 gene or its product, leading to the upregulation of ANI1 . Cycloheximide (no A CE ) induced the expression of ANI1 , also indicating that ANI1 expression is subject to negative regulation in the absence of A CE . The sequence and inferred protein structure of ANI1 suggest that it is a novel, extracellular protein. The secreted portion of the ANI1 protein potentially forms a β barrel with superficial similarities to lipocalins, which bind small hydrophobic molecules such as pheromones, steroids, and odorants. ANI1 may be an extracellular carrier of A CE that is required to initiate the male program of development as the sexual fate of the young gametophyte is determined.

The MADS genes encode a family of transcription factors, some of which control the identities of floral organs in flowering plants. To understand the role of MADS genes in the evolution of floral organs, five MADS genes (CMADS1, 2, 3, 4, and 6) were cloned from the fern Ceratopteris richardii , a nonflowering plant. A gene tree of partial amino acid sequences of seed plant and fern MADS genes showed that the fern genes form three subfamilies. All members of one of the fern MADS subfamilies have additional amino-terminal amino acids, which is a synapomorphic character of the AGAMOUS subfamily of the flowering plant MADS genes. Their structural similarity indicates a sister relationship between the two subfamilies. The temporal and spatial patterns of expression of the five fern MADS genes were assessed by Northern blot analyses and in situ hybridizations. CMADS1, 2, 3, and 4 are expressed similarly in the meristematic regions and primordia of sporophyte shoots and roots, as well as in reproductive structures, including sporophylls and sporangial initials, although the amount of expression in each tissue is different in each gene. CMADS6 is expressed in gametophytic tissues but not in sporophytic tissues. The lack of organ-specific expression of MADS genes in the reproductive structures of the fern sporophyte may indicate that the restriction of MADS gene expression to specific reproductive organs and the specialization of MADS gene functions as homeotic selector genes in the flowering plant lineage were important in floral organ evolution.

Multilevel interactions of the plant hormones ethylene and auxin coordinately and synergistically regulate many aspects of plant growth and development. This study isolated the AUXIN RESISTANT1 (AUX1) allele aux1(rcr1) (RCR1 for REVERSING CTR1-10 ROOT1) that suppressed the root growth inhibition conferred by the constitutive ethylene-response constitutive triple response1-10 (ctr1-10) allele. The aux1(rcr1) mutation resulted from an L126F substitution at loop 2 of the plasma membrane-associated auxin influx carrier protein AUX1. aux1(rcr1) and the T-DNA insertion mutant aux1-T were both defective in auxin transport and many aspects of the auxin response. Unexpectedly, expression of the auxin-response reporter DR5:GUS in the root apex was substantially prevented by the aux1(rcr1) but not the aux1-T mutation, even in the presence of the wild-type AUX1 allele. Following treatment with the synthetic auxin 1-naphthaleneacetic acid (NAA), DR5:GUS expression in aux1(rcr1) and aux1-T occurred mainly in the root apex and mature zone. NAA-induced DR5:GUS expression in the root apex was markedly prevented by ethylene in genotypes with aux1(rcr1) but not in aux1-T genotypes and the wild type. The effect of aux1(rcr1) on DR5:GUS expression seemed to be associated with AUX1-expressing domains. Green fluorescence protein-fused aux1(rcr1) was localized in the cytoplasm and probably not to the plasma membrane, indicating important roles of the Lys(126) residue at loop 2 in AUX1 targeting. The possible effects of aux1(rcr1) on DR5:GUS expression are discussed.

The homosporous fern Ceratopteris richardii is a model system for studying the genetic and epigenetic factors that are involved in sex-determining processes in plants. The sex of the Ceratopteris gametophyte is determined by antheridiogen, a gibberellin-like pheromone that is secreted by the hermaphroditic gametophyte and promotes male development of sexually immature gametophytes. Abscisic acid blocks the antheridiogen response in Ceratopteris. Several mutations that affect sex expression have been isolated and characterized; these mutations define at least some of the major regulatory genes that are involved in determining the sex of the Ceratopteris gametophyte. By studying the epistatic interactions among these mutants, it is possible to determine how these genes interact with one another to form a regulatory genetic hierarchy that controls sex determination and expression in Ceratopteris. While a genetic approach is useful in identifying genes that are involved in sex determination, other molecular a...

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.

The gaseous hormone ethylene is perceived in Arabidopsis by a five member receptor family that consists of the subfamily 1 receptors ETR1 and ERS1 and the subfamily 2 receptors ETR2, ERS2, and EIN4. Previous work has demonstrated that the basic functional unit for the ethylene receptor, ETR1, is a disulfide-linked homodimer. We demonstrate here that ethylene receptors isolated from Arabidopsis also interact with each other through noncovalent interactions. Evidence that ETR1 associates with other ethylene receptors was obtained by co-purification of ETR1 with tagged versions of ERS1, ETR2, ERS2, and EIN4 from Arabidopsis membrane extracts. ETR1 preferentially associated with the subfamily 2 receptors compared with the subfamily 1 receptor ERS1, but ethylene treatment affected the interactions and relative composition of the receptor complexes. When transgenically expressed in yeast, ETR1 and ERS2 can form disulfide-linked heterodimers. In plant extracts, however, the association of ETR1 and ERS2 can be largely disrupted by treatment with SDS, supporting a higher order noncovalent interaction between the receptors. Yeast two-hybrid analysis demonstrated that the receptor GAF domains are capable of mediating heteromeric receptor interactions. Kinetic analysis of ethylene-insensitive mutants of ETR1 is consistent with their dominance being due in part to an ability to associate with other ethylene receptors. These data suggest that the ethylene receptors exist in plants as clusters in a manner potentially analogous to that found with the histidine kinase-linked chemoreceptors of bacteria and that interactions among receptors contribute to ethylene signal output.

Arabidopsis (Arabidopsis thaliana) RTE1 encodes a membrane protein and negatively regulates ethylene responses. Genetic and transformation studies suggest that the function of the wild-type RTE1 is primarily dependent on ETR1 and can be independent on the other receptors. Ethylene insensitivity caused by the overexpression of RTE1 is largely masked by the etr1-7 mutation, but not by any other receptor mutations. The wild-type ETR1 N terminus is sufficient to the activation of the RTE1 function and the ectopic expression of etr1(1–349) restored ethylene insensitivity conferred by 35S∷gRTE1 in etr1-7. The RTE1 N terminus is not essential to the etr1-2 function and the expression of rte1(NΔ49), which has an N-terminal deletion of 49 amino acid residues, restored ethylene insensitivity in etr1-2 rte1-2. The ectopic expression of GREEN FLUORESCENT PROTEIN (GFP)-RTE1 conferred ethylene insensitivity in wild type and the GFP fusion displayed fast movement within the cytoplasm. The GFP-RTE1 and EYFP-NAG proteins colocalized and the Brefeldin A treatment caused aggregation of GFP-RTE1, suggesting RTE1 is a Golgi-associated protein. Our results suggest specificity of the RTE1 function to ETR1 and that endomembranes may play a role in the ethylene signal transduction.

It has been more than a centaury since the identification of ethylene as a gaseous plant hormone, and the effects of ethylene on plants have enabled broad applications in agriculture. Efforts had been made to understand ethylene signal transduction in plants, but it had not been successful until the identification and cloning of the first plant hormone receptor gene, ETR1, in Arabidopsis a decade ago. Comprehensive genetic studies allow the dissecting of the signaling pathway to reveal the components involved, which makes the studies of ethylene signal transduction become one the most-understood signal transduction pathways in plants. The identification of the signaling components and the genetic model of ethylene signal transduction pathway are described in this review.