Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model

The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein.     

Members of the tomato LeEIL (EIN3-like) gene family are functionally redundant and regulate ethylene responses throughout plant development

The plant hormone ethylene regulates many aspects of growth, development and responses to the environment. The Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) protein is a nuclear-localized component of the ethylene signal-transduction pathway with DNA-binding activity. Loss-of-function mutations in this protein result in ethylene insensitivity in Arabidopsis. To gain a better understanding of the ethylene signal-transduction pathway in tomato, we have identified three homologs of the Arabidopsis EIN3 gene (LeEILs). Each of these genes complemented the ein3-1 mutation in transgenic Arabidopsis, indicating that all are involved in ethylene signal transduction. Transgenic tomato plants with reduced expression of a single LeEIL gene did not exhibit significant changes in ethylene response; reduced expression of multiple tomato LeEIL genes was necessary to reduce ethylene sensitivity significantly. Reduced LeEIL expression affected all ethylene responses examined, including leaf epinasty, flower abscission, flower senescence and fruit ripening. Our results indicate that the LeEILs are functionally redundant and positive regulators of multiple ethylene responses throughout plant development.     

Myo-Inositol-Dependent Sodium Uptake in Ice Plant

The phytohormone ethylene regulates many aspects of plant growth, development, and environmental responses. Much of the developmental regulation of ethylene responses in tomato (Lycopersicon esculentum) occurs at the level of hormone sensitivity. In an effort to understand the regulation of ethylene responses, we isolated and characterized tomato genes with sequence similarity to the Arabidopsis ETR1 (ethylene response 1) ethylene receptor. Previously, we isolated three genes that exhibit high similarity to ETR1 and to each other. Here we report the isolation of two additional genes, LeETR4 and LeETR5, that are only 42% and 40% identical to ETR1, respectively. Although the amino acids known to be involved in ethylene binding are conserved, LeETR5 lacks the histidine within the kinase domain that is predicted to be phosphorylated. This suggests that histidine kinase activity is not necessary for an ethylene response, because mutated forms of both LeETR4 and LeETR5 confer dominant ethylene insensitivity in transgenic Arabidopsis plants. Expression analysis indicates that LeETR4 accounts for most of the putative ethylene-receptor mRNA present in reproductive tissues, but, like LeETR5, it is less abundant in vegetative tissues. Taken together, ethylene perception in tomato is potentially quite complex, with at least five structurally divergent, putative receptor family members exhibiting significant variation in expression levels throughout development.     

The tomato ethylene receptor gene family: Form and function

Phytohormones are essential for integrating many aspects of plant development and responses to the environment. Regulation of hormonally controlled events occurs at multiple levels: synthesis, catabolism and perception (Trewavas 1983, Bradford and Trewavas 1994). At the level of perception, sensitivity to hormones can be regulated both spatially and temporally during the life cycle. An example of spatial regulation is the differential response to a hormone that occurs during organ abscission. Temporally, sensitivity of an organ to a hormone may change during maturation, as occurs during fruit ripening. In this review, we will focus on the initial event in recognition of one hormone, ethylene. The ethylene receptor was the first plant hormone receptor to be unambiguously identified. Over the last few years, great progress has been made in elucidating the genes involved in ethylene action. Nonetheless, the mechanisms of signal transduction remain to be established. Here, we will address the status of the tomato receptor gene family and the evidence that regulation of receptor gene expression can influence the response of the plant to the hormone.     

LeCTR2, a CTR1-like protein kinase from tomato, plays a role in ethylene signalling, development and defence

Arabidopsis AtCTR1 is a Raf-like protein kinase that interacts with ETR1 and ERS and negatively regulates ethylene responses. In tomato, several CTR1-like proteins could perform this role. We have characterized LeCTR2, which has similarity to AtCTR1 and also to EDR1, a CTR1-like Arabidopsis protein involved in defence and stress responses. Protein–protein interactions between LeCTR2 and six tomato ethylene receptors indicated that LeCTR2 interacts preferentially with the subfamily I ETR1-type ethylene receptors LeETR1 and LeETR2, but not the NR receptor or the subfamily II receptors LeETR4, LeETR5 and LeETR6. The C-terminus of LeCTR2 possesses serine/threonine kinase activity and is capable of auto-phosphorylation and phosphorylation of myelin basic protein in vitro . Overexpression of the LeCTR2 N-terminus in tomato resulted in altered growth habit, including reduced stature, loss of apical dominance, highly branched inflorescences and fruit trusses, indeterminate shoots in place of determinate flowers, and prolific adventitious shoot development from the rachis or rachillae of the leaves. Expression of the ethylene-responsive genes E4 and chitinase B was upregulated in transgenic plants, but ethylene production and the level of mRNA for the ethylene biosynthetic gene ACO1 was unaffected. The leaves and fruit of transgenic plants also displayed enhanced susceptibility to infection by the fungal pathogen Botrytis cinerea , which was associated with much stronger induction of pathogenesis-related genes such as PR1b1 and chitinase B compared with the wild-type. The results suggest that LeCTR2 plays a role in ethylene signalling, development and defence, probably through its interactions with the ETR1-type ethylene receptors of subfamily I.     

Ligand-induced alterations in the phosphorylation state of ethylene receptors in tomato fruit

Perception of the plant hormone ethylene is essential to initiate and advance ripening of climacteric fruits. Since ethylene receptors negatively regulate signaling, the suppression is canceled upon ethylene binding, permitting responses including fruit ripening. Although receptors have autophosphorylation activity, the mechanism whereby signal transduction occurs has not been fully determined. Here we demonstrate that LeETR4, a critical receptor for tomato (Solanum lycopersicum) fruit ripening, is multiply phosphorylated in vivo and the phosphorylation level is dependent on ripening stage and ethylene action. Treatment of preclimacteric fruits with ethylene resulted in accumulation of LeETR4 with reduced phosphorylation whereas treatments of ripening fruits with ethylene antagonists, 1-methylcyclopropene and 2,5-norbornadiene, induced accumulation of the phosphorylated isotypes. A similar phosphorylation pattern was also observed for Never ripe, another ripening-related receptor. Alteration in the phosphorylation state of receptors is likely to be an initial response upon ethylene binding since treatments with ethylene and 1-methylcyclopropene rapidly influenced the LeETR4 phosphorylation state rather than protein abundance. The LeETR4 phosphorylation state closely paralleled ripening progress, suggesting that the phosphorylation state of receptors is implicated in ethylene signal output in tomato fruits. We provide insights into the nature of receptor on and off states.     

Tomato transcriptional changes in response to Clavibacter michiganensis subsp. michiganensis reveal a role for ethylene in disease development

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive actinomycete, causing bacterial wilt and canker disease in tomato (Solanum lycopersicum). Host responses to gram-positive bacteria and molecular mechanisms associated with the development of disease symptoms caused by Cmm in tomato are largely unexplored. To investigate plant responses activated during this compatible interaction, we used microarray analysis to monitor changes in host gene expression during disease development. This analysis was performed at 4 d postinoculation, when bacteria were actively multiplying and no wilt symptoms were yet visible; and at 8 d postinoculation, when bacterial growth approached saturation and typical wilt symptoms were observed. Of the 9,254 tomato genes represented on the array, 122 were differentially expressed in Cmm-infected plants, compared with mock-inoculated plants. Functional classification of Cmm-responsive genes revealed that Cmm activated typical basal defense responses in the host, including induction of defense-related genes, production and scavenging of free oxygen radicals, enhanced protein turnover, and hormone synthesis. Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response. After inoculation with Cmm, Never ripe (Nr) mutant plants, impaired in ethylene perception, and transgenic plants with reduced ethylene synthesis showed significant delay in the appearance of wilt symptoms, compared with wild-type plants. The retarded wilting in Nr plants was a specific effect of ethylene insensitivity, and was not due to altered expression of defense-related genes, reduced bacterial populations, or decreased ethylene synthesis. Taken together, our results indicate that host-derived ethylene plays an important role in regulation of the tomato susceptible response to Cmm.     

Effect of salt and osmotic stress upon expression of the ethylene receptor ETR1 in Arabidopsis thaliana

In hormone perception, varying the concentrations of hormone, receptor, or downstream signaling elements can modulate signal transduction. Previous research has demonstrated that ethylene biosynthesis in plants is regulated by abiotic factors. Here we report that exposure of Arabidopsis plants to NaCl reduced expression of the ethylene receptor ETR1. The change in gene expression was reflected at the protein level based on immunoblot analysis. Further analysis supports a general effect of osmotic stress upon the expression level of ETR1. The reduction in ETR1 levels should cause increased sensitivity of the plant to ethylene. These results suggest that plant responses to abiotic stress are modulated by changes in the expression level of ethylene receptors.     

Cooperative ethylene receptor signaling

The gaseous plant hormone ethylene is perceived by a family of five ethylene receptor members in the dicotyledonous model plant Arabidopsis. Genetic and biochemical studies suggest that the ethylene response is suppressed by ethylene receptor complexes, but the biochemical nature of the receptor signal is unknown. Without appropriate biochemical measures to trace the ethylene receptor signal and quantify the signal strength, the biological significance of the modulation of ethylene responses by multiple ethylene receptors has yet to be fully addressed. Nevertheless, the ethylene receptor signal strength can be reflected by degrees in alteration of various ethylene response phenotypes and in expression levels of ethylene-inducible genes. This mini-review highlights studies that have advanced our understanding of cooperative ethylene receptor signaling.     

Regulation of Ethylene-mediated Responses at the Level of the Receptor

The ethylene receptor is currently the best characterized ofplant hormone receptors. Mutant and biochemical analyses haverevealed much about the mode(s) of action and biological regulationof ethylene responses. The receptors are related to bacterialtwo-component regulators, which are histidine protein kinases.In both arabidopsis and tomato, a family of genes encodes astructurally divergent set of receptors. While one arabidopsisreceptor, ETR1, has been shown to be a functional histidinekinase, the significant structural divergence among the familymembers makes the actual mode of action of the receptors unclear.Molecular and genetic evidence indicates that quantitative alterationsin receptor levels can modify the sensitivity of a plant ortissue to ethylene. However, our current knowledge of receptorgene regulation does not address how a plant regulates differentialresponsiveness of a tissue to ethylene. Copyright 2001 Annalsof Botany Company Ethylene sensitivity, ERS, ETR, histidine kinase, LeETR, Lycopersicon esculentum     

The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches

Ethylene has long been regarded as the main regulator of ripening in climacteric fruits. The characterization of a few tomato mutants, unable to produce climacteric ethylene and to ripen their fruits even following treatments with exogenous ethylene, has shown that other factors also play an important role in the control of climacteric fruit ripening. In climacteric peach and tomato fruits it has been shown that, concomitant with ethylene production, increases in the amount of auxin can also be measured. In this work a genomic approach has been used in order to understand if such an auxin increase is functional to an independent role played by the hormone during ripening of the climacteric peach fruits. Besides the already known indirect activity on ripening due to its up-regulation of climacteric ethylene synthesis, it has been possible to show that auxin plays a role of its own during ripening of peaches. In fact, the hormone has shown the ability to regulate the expression of a number of different genes. Moreover, many genes involved in biosynthesis and transport and, in particular, the signalling (receptors, Auxin Response Factors and Aux/IAA) of auxin had increased expression in the mesocarp during ripening, thus strengthening the idea that this hormone is actively involved in the ripening of peaches. This study has also demonstrated the existence of an important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene and genes in the ethylene domain regulated by auxin.     

Evidence that CTR1-Mediated Ethylene Signal Transduction in Tomato is Encoded by a Multigene Family Whose Members Display Distinct Regulatory Features

Ethylene governs a range of developmental and response processes in plants. In Arabidopsis thaliana, the Raf-like kinase CTR1 acts as a key negative regulator of ethylene responses. While only one gene with CTR1 function apparently exists in Arabidopsis, we have isolated a family of CTR1- like genes in tomato ( Lycopersicon esculentum ). Based on amino acid alignments and phylogenetic analysis, these tomato CTR1- like genes are more similar to Arabidopsis CTR1 than any other sequences in the Arabidopsis genome. Structural analysis reveals considerable conservation in the size and position of the exons between Arabidopsis and tomato CTR1 genomic sequences. Complementation of the Arabidopsis ctr1-8 mutant with each of the tomato CTR genes indicates that they are all capable of functioning as negative regulators of the ethylene pathway. We previously reported that LeCTR1 expression is up-regulated in response to ethylene. Here, quantitative real-time PCR was carried out to detail expression for LeCTR1 and the additional CTR1 -like genes of tomato. Our results indicate that the tomato CTR1 gene family is differentially regulated at the mRNA level by ethylene and during stages of development marked by increased ethylene biosynthesis, including fruit ripening. The possibility of a multi-gene family of CTR1 -like genes in other species besides tomato was examined through mining of EST and genomic sequence databases.     

Characterization of an ethylene receptor homolog gene from rice

Ethylene is a gaseous hormone and plays important roles in plant growth and development, including seed germination, root hair development, flowering, pollination, abscission, and fruit ripening[1]. It is also involved in plant responses to biotic stress such as pathogen attack, and abiotic stresses such as wounding, drought and freezing[1]. Mutational and genetic analysis of Arabidopsis has led to the identification of five ethylene receptor genes, i.e. ETR1, ERS1, ETR2, EIN4 and ERS2. …     

Genetic mapping of ripening and ethylene-related loci in tomato

 The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response. Received: 26 October 1998 / Accepted: 13 November 1998     

Characterization of an ethylene receptor homolog gene from rice

Ethylene plays important roles in plant growth, development and stress responses. Its receptor genes have been studied in dicots such asArabidopsis, tobacco and tomato. However, no research has been reported for the ethylene receptors from monocots currently. In the present study, we cloned an ethylene receptor geneOSPK2 from rice and found that its encoded protein was divergent from the ethylene receptors from dicots. OSPK2 had a long extension in its N-terminal, followed by three transmembrane segments, a GAF domain, a putative kinase domain and a putative receiver domain. Although most of the domains were conserved, the expected phosphorylation site His and the phosphate receiver Asp have been replaced by Gln and Asn, respectively. This fact indicates that OSPK2 may not function as a histidine kinase in a phosphorelay manner, but rather play roles by other mechanism, probably through Ser/Thr kinase activity. The expression of theOSPK2 gene was investigated by RT-PCR method under different conditions. We found that this gene was apparently induced by wounding and PEG treatment, but not significantly affected by salt and ABA treatments. The differential expression of theOSPK2 gene may reflect its roles in mediating different abiotic stress responses, consistent with our previous studies on tobacco ethylene receptors.