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Ethylene, the main hormone regulating ripening in climacteric fruit, is perceived by the ethylene receptors starting a cascade of reactions of the ethylene signal transduction. In order to gain insight in the perception of ethylene during the ripening process, this work presents the transcriptional characterisation of the ethylene receptor (ETR) and CTR (CONSTITUTIVE TRIPPLE RESPONSE) genes of tomato fruit at different developmental stages and postharvest ripening conditions. Our results show that both ETR and CTR genes were differentially expressed when comparing on the vine ripening with off the vine (postharvest ripening) and in response to 1-MCP. Expression of the six ethylene receptors revealed SlETR3 and SlETR4 as the most expressed genes and together with SlETR6 to be the most ethylene-responsive, as they were the fastest to react to changes in conditions such as the 1-MCP application in detached fruit ripening off the vine. A climacteric pattern of expression similar to that of ethylene production itself was observed for most of the receptors and CTR genes during ripening, most strongly during postharvest ripening. All together our results suggest a dedicated regulation of the expression of ETRs and CTRs that allows the plant to precisely control the timing of ripening. ispartof: Postharvest Biology and Technology vol:140 pages:67-75 status: published

El genoma de Cucurbita pepo comprende 263 Mb y 34,240 genes organizados en 20 cromosomas diferentes. Para mejorar nuestra comprensión de la función génica, hemos generado una plataforma de mutantes EMS que consta de 3.751 familias M2 independientes. La calidad de la colección se ha evaluado en base a los resultados de fenotipado y re-secuenciación del genoma completo (WGS). La evaluación fenotípica de toda la plataforma en la etapa de plántula ha demostrado que la tasa de variación para rasgos fácilmente observables es de más del 10%. El porcentaje de familias con plántulas albinas o cloróticas excedió el 3%, similar o mayor a lo encontrado en otras colecciones EMS de cultivos de Cucurbitáceas. Tras evaluar 4 plantas adultas de 300 familias independientes, se encontró que más del 28% de las familias tenían un efecto fenotípico observable sobre diferentes rasgos vegetativos y reproductivos, incluyendo: el vigor de las plantas; el tamaño y la forma de las hojas; la expresión sexual y la determinación del sexo; y la producción y desarrollo de frutos. Dos muestras de ADN genómico, derivadas de 20 plantas de dos familias mutantes, se sometieron a WGS utilizando la metodología NGS con el fin de estimar la densidad, el espectro, la distribución y el impacto de las mutaciones inducidas por EMS. El número de mutaciones EMS en los genomas de las familias L1 y L2 fue de 1.704 y 859, respectivamente, lo que representa una densidad de 11,8 y 6 mutaciones por Mb, respectivamente. Como se esperaba, las mutaciones predominantes inducidas por EMS fueron las transiciones C>T y G>A (80,3% en L1 y 61% en L2), que se encontraron distribuidas al azar a lo largo de los 20 cromosomas de C. pepo. Las mutaciones afectaron principalmente a las regiones intergénicas, pero el 7,9 y el 6% de las mutaciones EMS identificadas en L1 y L2, respectivamente, se ubicaron en el exoma, y el 0,4 y el 0,2% podían tener un impacto moderado o alto en la función génica. Estos resultados han proporcionado información sobre el uso potencial de la plataforma mutante en el descubrimiento de nuevos alelos tanto para la genómica funcional como para la mejora genética de las especies del género Cucurbita. El etileno es el regulador clave de la determinación del sexo en las especies monoicas de la familia Cucurbitaceae. Esta hormona regula la transición floral femenina y la conversión de cada meristemo floral en una flor femenina o masculina, lo que se logra deteniendo el crecimiento del estambre o el carpelo en las primeras etapas del desarrollo de la flor. Muchos de los genes reguladores descubiertos hasta la fecha codifican para enzimas de biosíntesis de etileno, pero se sabe poco sobre la importancia de los componentes de señalización de etileno en el control de este proceso de desarrollo. Un cribado de la plataforma de mutantes EMS para triple respuesta a etileno en plántulas etioladas permitió identificar cuatro mutantes insensibles a etileno dominantes o semidominantes: ein1 (etr1b), ein2 (etr1a), ein3 (etr2b) y EIN4 (etr1a-1). Las cuatro mutaciones alteraron los mecanismos de determinación del sexo. Las mutaciones etr1a y etr2b promovieron la conversión de flores femeninas en flores bisexuales y hermafroditas, y monoecia en andromonoecia, retrasando también la transición floral femenina y reduciendo el número de flores pistiladas por planta. Las mutaciones etr1a-1 y etr1b, por otro lado, fueron capaces de bloquear por completo la transición floral femenina de C. pepo, haciendo que las plantas produjeran flores masculinas indefinidamente (androecia). Ya sea en homocigosis o heterocigosis, las cuatro mutaciones etr alteraron la tasa de crecimiento y la maduración de los pétalos y los carpelos de flores pistiladas, alargando el tiempo requerido para que las flores alcanzaran la antesis, y favoreciendo el desarrollo partenocárpico del fruto. El análisis BSA mediante secuenciación de genoma completo (BSA-Seq) reveló que los genes subyacentes a las cuatro mutaciones etr codifican para receptores de etileno. Se descubrió que etr1a, etr1a-1 y etr1b eran mutaciones de cambio de sentido en el dominio transmembrana de unión a etileno de los receptores CpETR1A y CpETR1B, mientras que etr2b era una mutación de cambio de sentido entre los dominios GAF e histidina quinasa del receptor de etileno CpETR2B. El nivel de insensibilidad al etileno en los mutantes simples y dobles homocigóticos y heterocigotos para las cuatro mutaciones etr estaba determinado por la fuerza de cada alelo mutante, pero también por el número de alelos WT y etr en la planta, lo que indica que los tres genes receptores de etileno no tienen una función totalmente redundante, sino que cooperan en el control de la respuesta de etileno. El nivel de insensibilidad a etileno determinará el fenotipo sexual de la planta, que va desde monoecia en plantas WT sensibles a etileno, pasando por andromonoecia en las plantas parcialmente insensibles a etileno, hasta androecia en las plantas más insensibles a etileno. Se observó que el bloqueo del programa de desarrollo femenino en los mutantes etr1b y etr1a-1 estaba asociado con una falta de activación transcripcional de CpACS11, CpACO2 y CpACS2/7, tres genes de biosíntesis de etileno necesarios para activar el desarrollo floral femenino, pero no cambiaba la expresión de CpWIP1, un factor de transcripción que parece ser necesario para el desarrollo de flores masculinas en otras especies de Cucurbitáceas. Se propone un modelo que integra la biosíntesis de etileno y los genes receptores de etileno en la red genética que regula la determinación del sexo en C. pepo. SUMMARY: The Cucurbita pepo genome comprises 263 Mb and 34,240 gene models organized in 20 different chromosomes. We generated an EMS mutant platform consisting of 3,751 independent M2 families so as to improve our understanding of gene function. The quality of the collection has been evaluated based on phenotyping and whole-genome resequencing (WGS) results. The phenotypic evaluation of the whole platform at the seedling stage has demonstrated that the variation rate for easily-observable traits is more than 10%. The families with albino or chlorotic seedlings exceeded 3% of the platform, similar or higher to that found in other EMS collections of cucurbit crops. When four adult plants from each of 300 independent families were evaluated, more than 28% of apparent mutations were found to be associated with vegetative and reproductive traits, including: plant vigor; leaf size and shape; sex expression and sex determination; and fruit set and development. Two pools of genomic DNA, derived from 20 plants taken from each of two mutant families, were subjected to WGS by using NGS methodology so as to estimate the density, spectrum, distribution and impact of EMS-induced mutation. The number of EMS mutations in the genome of the L1 family was 1,704 and in the L2 one 859, which represents a density of 11.8 and 6 mutations per Mb, respectively. As expected, the predominant EMS-induced mutations were C>T and G>A transitions (80.3% in L1, and 61% L2), that were found to be randomly distributed along the 20 chromosomes of C. pepo. The mutations mostly affected intergenic regions, but 7.9% of the identified EMS mutations in L1 and 6% of those in L2, were located in the exome. 0.4% (L1) and 0.2% (L2) of the identified mutations had a moderate or high putative impact on gene functions. These results provide information regarding the potential usefulness of the obtained mutant platform in the search for novel alleles. This concerns both functional genomics and Cucurbita breeding, by using direct- or reverse-genetic approaches. Ethylene is the key regulator of sex determination in the monoecious species of the family Cucurbitaceae, this hormone determines which individual floral meristem will develop into a female or a male flower. This is achieved by the arrest of stamen or carpel growth in early stages of flower development, and also during the transition between male and female developmental phases. Many of the regulatory genes discovered to date code for ethylene biosynthesis enzymes, but little is known of the importance of ethylene signaling components in the control of this developmental process. A high-throughput screening of the EMS platform for triple ethylene response in etiolated seedlings allowed the identification of four semi-dominant or dominant ethylene-insensitive mutants: ein1 (etr1b), ein2 (etr1a), ein3 (etr2b) and EIN4 (etr1a-1). These four mutations modified sex determination mechanisms. The mutations etr1a and etr2b promoted the conversion of female into bisexual or hermaphrodite ones, and monoecy into andromonoecy. They also delayed the transition to female flowering and reduced the number of pistillate flowers per plant. The mutations etr1a-1 and etr1b, on the other hand, were able to block female flowering transition of C. pepo; this induces plants to produce male flowers indefinitely (androecy). Whether in homozygous or heterozygous conditions, the four etr mutations disrupted the growth rate and maturation of petals and carpels of pistillate flowers, thereby lengthening the time required for flowers to reach anthesis and encouraging the growth rate of ovaries and the parthenocarpic development of fruits. Bulked-segregant analysis coupled to whole-genome sequencing (BSA-Seq) revealed that the genes affected by the four etr mutations encode for ethylene receptor proteins. The etr1a, etr1a-1 and etr1b were found to be missense mutations that resulted in an amino acid substitution in the ethylene-binding transmembrane domain of the ethylene receptors CpETR1A and CpETR1B, while etr2b was a missense mutation that affected to the coiled-coil domain between the GAF and histidine-kinase of the receptor CpETR2B. The level of ethylene insensitivity in homozygous and heterozygous single and double mutants was determined both by the strength of each mutant allele but also by the number of WT and etr alleles in the plant, indicating that the three ethylene receptor genes do not have a redundant function but cooperate in the control of ethylene response. The level of ethylene response in the plant determines its final sex phenotype: monoecy in ethylene-sensitive WT plants; andromonoecy in the partial ethylene-insensitive ones, and androecy in those which are the most highly ethylene insensitive. The blocking of female flowering transition by etr1a-1 and etr1b -the two mutants which have the strongest effect- was found to be associated with a lack of transcriptional activation of the three ethylene biosynthesis genes CpACS11, CpACO2 and CpACS2/7, which are required for female flower development. However, there was no change in the expression of CpWIP1, a transcription factor gene that seems to be required for male flower development in other cucurbit species. A model is proposed here, integrating the ethylene biosynthesis and receptor genes into the genetic network that regulates sex determination in C. pepo.

Ethylene, the plant ripening hormone of climacteric fruit, is perceived by ethylene receptors which is the first step in the complex ethylene signal transduction pathway. Much progress has been made in elucidating the mechanism of this pathway, but there is still a lot to be done in the proteomic quantification of the main proteins involved, particularly during fruit ripening. This work focuses on the mass spectrometry based identification and quantification of the ethylene receptors (ETRs) and the downstream components of the pathway, CTR-like proteins (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2). We used tomato as a model fruit to study changes in protein abundance involved in the ethylene signal transduction during fruit ripening. In order to detect and quantify these low abundant proteins located in the membrane of the endoplasmic reticulum, we developed a workflow comprising sample fractionation and MS analysis using parallel reaction monitoring. This work shows the feasibility of the identification and absolute quantification of all seven ethylene receptors, three out of four CTRs and EIN2 in four ripening stages of tomato. In parallel, gene expression was analyzed through real-time qPCR. Correlation between transcriptomic and proteomic profiles during ripening was only observed for three of the studied proteins, suggesting that the other signaling proteins are likely post-transcriptionally regulated. Based on our quantification results we were able to show that the protein levels of SlETR3 and SlETR4 increased during ripening, probably to control ethylene sensitivity. The other receptors and CTRs showed either stable levels that could sustain, or decreasing levels that could promote fruit ripening. ispartof: FRONTIERS IN PLANT SCIENCE vol:9 ispartof: location:Switzerland status: published

Tomato (Solanum lycopersicum L.) is a crop of high economic and nutritional value produced worldwide. Besides having a high number of vitamins and minerals, tomato fruit is rich in other bioactive components including carotenoids and phenolic compounds. In addition, it is an ideal model system to study fleshy fruit development and ripening thanks to its relative short life cycle, its small genome size and the availability of a large selection of germplasms and mutants. The ripening of tomato, and of climacteric fruit in general, is regulated by the plant hormone ethylene, which also regulates numerous other aspects of plant growth and development including responses to biotic and abiotic stress. Postharvest control of ethylene is of great importance to assure proper ripening conditions and to control fruit quality. Thus, a good understanding of ethylene perception by the fruit is essential to eventually improve postharvest practices. The objective of this thesis is to gain insight into the dynamic changes in the ethylene signal transduction during climacteric ripening of tomato, in order to, in the long term, improve the quality and storability of tomato after harvest. Ethylene is perceived by the ethylene receptors (ETRs) which are the first steps in the complex pathway of the ethylene signal transduction. Receptors, together with the CTR proteins inhibit the action of EIN2, a key regulator of the signalling pathway which activates ethylene-related transcription factors in the nucleus. A gene expression analysis was carried out using RT-qPCR of SlETRs and SlCTRs during tomato fruit ripening on-vine as compared to ripening off-vine either or not treated with the ethylene inhibitor 1-methylcyclopropene (1-MCP). Off-vine postharvest ripening showed a different regulation than on-vine ripening, while treatment with 1-MCP inhibited the expression of some genes more than others. The results suggested that some genes were more ethylene-responsive than others but that cooperatively might control the timing of ripening. To fully understand the underlying regulation and physiological responses, there was a need to complement these transcriptomic results with the quantification of the proteins, which are the real effectors of the reactions. However, there was not yet a well-established way to identify and quantify these proteins, with only some of the receptors being quantified through western blots. Our approach started as an MS discovery analysis in a highly fractionated tomato peptide sample which allowed the identification of 8,588 proteins, one of the largest dataset described for Solanum lycopersicum L. so far. The complete protein dataset was annotated through GO and KEGG databases, mapping the proteins by their cellular location and biological process. Among all the identified proteins, four tomato ethylene receptors, three SlCTRs and SlEIN2 could also be identified. This information was taken as the starting point to create a targeted MS proteomic assay based on the parallel reaction monitoring (PRM) mode. As a result, all seven SlETRs, three out of the four SlCTRs and SlEIN2 could be identified and quantified. Their protein and mRNA levels were quantified in four ripening stages of tomato fruit. Their behaviour revealed that some of the receptors and SlCTRs may be responsible for initiating the ethylene signalling and with that ripening, while others for controlling the progress of ripening. The levels of SlEIN2, both mRNA and proteins, decreased during ripening, which might be related to its proteolysis and translocation to the nucleus upon ethylene binding to the receptors. Finally, we wanted to further investigate the regulation of the ethylene signalling during ripening at different temperatures. For that a large storage experiment was developed in which mature green tomatoes were stored postharvest at three different temperatures and sampled every certain intervals. The analysis performed on the samples were colour, firmness, ethylene and respiration production rate, gene expression and protein quantification of the ethylene signalling elements ETRs, CTRs and EIN2, ACS and ACO enzyme activities and ACC and MACC accumulation. The results helped us to better understand the regulation of the ethylene signalling through an omics approach and to link it with the ethylene biosynthesis pathway. As a conclusion, we have developed a feasible and reproducible assay to identify and quantify ethylene signalling proteins in tomato pericarp, which can be used by other researchers. On the other hand, we have characterised the receptors, SlCTRs and SlEIN2 using a multiomics approach which casts more light on the regulation of the ethylene signal transduction mechanism during climacteric ripening of tomato. nrpages: 184 status: published

Chapter 11; International audience; The ripening of fleshy fruit is a developmentally regulated process unique to plants during which the majority of the sensory quality attributes are elaborated including aroma, flavour, texture and nutritional compounds. In climacteric fruit, the plant hormone ethylene is the key regulator of the ripening process as exemplified by the dramatic inhibition of fruit ripening that results from the down-expression of ACC (1-amino-cyclopropane-1-carboxylic acid) synthase and ACC oxidase genes involved in ethylene biosynthesis. By contrast, the ripening of non-climacteric fruit is not dependent on ethylene but rather on cues of unknown nature though ethylene may contribute at least partly to the control of some aspects of the ripening process. The expression of the ripening-associated genes is regulated by a network of signalling pathways among which ethylene perception and transduction play a primary role. Building on the knowledge gained on the Arabidopsis thaliana model system, the importance of ethylene signalling in fruit ripening has been extensively studied. This chapter summarizes the present knowledge on the role of ethylene in fruit ripening and addresses the molecular mechanisms involved in ethylene perception and responses. It also highlights recent advances and prospects on the means by which the ethylene transduction pathway leads to diversified physiological responses and how ethylene signalling interacts with other hormones to activate the expression of ripening-related genes. While this review mostly refers to the tomato as major model for fruit research, it also gives insight on the ripening process in other fruit species, including nonclimacteric types.

The lace plant, Aponogeton madagascariensis, is an aquatic monocot that forms perforations in its leaves as part of normal leaf development. Perforation formation occurs through developmentally regulated programmed cell death (PCD). The molecular basis of PCD regulation in the lace plant is unknown, however ethylene has been shown to play a significant role. In this study, we examined the role of ethylene receptors during perforation formation. We isolated three lace plant ethylene receptors AmERS1a, AmERS1b and AmERS1c. Using quantitative PCR, we examined their transcript levels at seven stages of leaf development. Through laser-capture microscopy, transcript levels were also determined in cells undergoing PCD and cells not undergoing PCD (NPCD cells). AmERS1a transcript levels were significantly lower in window stage leaves (in which perforation formation and PCD are occurring) as compared to all other leaf developmental stages. AmERS1a and AmERS1c (the most abundant among the three receptors) had the highest transcript levels in mature stage leaves, where PCD is not occurring. Their transcript levels decreased significantly during senescence-associated PCD. AmERS1c had significantly higher transcript levels in NPCD compared to PCD cells. Despite being significantly low in window stage leaves, AmERS1a transcripts were not differentially expressed between PCD and NPCD cells. The results suggested that ethylene receptors negatively regulate ethylene-controlled PCD in the lace plant. A combination of ethylene and receptor levels determines cell fate during perforation formation and leaf senescence. A new model for ethylene emission and receptor expression during lace plant perforation formation and senescence is proposed.

Ethylene signaling is decisive for many plant developmental processes. Among these, control of senescence, abscission and fruit ripening are of fundamental relevance for global agriculture. Consequently, detailed knowledge of the signaling network along with the molecular processes of signal perception and transfer are expected to have high impact on future food production and agriculture. Recent advances in ethylene research have demonstrated that signaling of the plant hormone critically depends on the interaction of the ethylene receptor family with the NRAMP-like membrane protein ETHYLENE INSENSITIVE 2 (EIN2) at the ER membrane, phosphorylation-dependent proteolytic processing of ER-localized EIN2 and subsequent translocation of the cleaved EIN2 C-terminal polypeptide (EIN2-CEND) to the nucleus. EIN2 nuclear transport, but also interaction with the receptors sensing the ethylene signal, both, depend on a nuclear localization signal (NLS) located at the EIN2 C-terminus. Loss of the tight interaction between receptors and EIN2 affects ethylene signaling and impairs plant ethylene responses. Synthetic peptides derived from the NLS sequence interfere with the EIN2–receptor interaction and have utility in controlling plant ethylene responses such as ripening. Here, we report that a synthetic peptide (NOP-1) corresponding to the NLS motif of Arabidopsis EIN2 (aa 1262–1269) efficiently binds to tomato ethylene receptors LeETR4 and NR and delays ripening in the post-harvest phase when applied to the surface of sampled green fruits pre-harvest. In particular, degradation of chlorophylls was delayed by several days, as monitored by optical sensors and confirmed by analytical methods. Similarly, accumulation of β-carotene and lycopene in the fruit pulp after NOP-1 application was delayed, without having impact on the total pigment concentration in the completely ripe fruits. Likewise, the peptide had no negative effects on fruit quality. Our molecular and phenotypic studies reveal that peptide biologicals could contribute to the development of a novel family of ripening inhibitors and innovative ripening control in climacteric fruit.

It has been generally thought that in ethylene-sensitive plants such as carnations, senescence proceeds irreversibly once the tissues have entered the climacteric phase. While pre-climacteric petal tissues have a lower sensitivity to ethylene, these tissues are converted to the climacteric phase at a critical point during flower development. In this study, it is demonstrated that the senescence process initiated by exogenous ethylene is reversible in carnation petals. Petals treated with ethylene for 12h showed sustained inrolling and senescence, while petals treated with ethylene for 10h showed inrolling followed by recovery from inrolling. Reverse transcription–PCR analysis revealed differential expression of genes involved in ethylene biosynthesis and ethylene signalling between 10h and 12h ethylene treatment. Ethylene treatment at or beyond 12h (threshold time) decreased the mRNA levels of the receptor genes (DcETR1, DcERS1, and DcERS2) and DcCTR genes, and increased the ethylene biosynthesis genes DcACS1 and DcACO1. In contrast, ethylene treatment under the threshold time caused a transient decrease in the receptor genes and DcCTR genes, and a transient increase in DcACS1 and DcACO1. Sustained DcACS1 accumulation is correlated with decreases in DcCTR genes and increase in DcEIL3 and indicates that tissues have entered the climacteric phase and that senescence proceeds irreversibly. Inhibition of ACS (1-aminocyclopropane-1-carboxylic acid synthase) prior to 12h ethylene exposure was not able to prevent reduction in transcripts of DcCTR genes, yet suppressed transcript of DcACS1 and DcACO1. This leads to the recovery from inrolling of the petals, indicating that DcACS1 may act as a signalling molecule in senescence of flowers.

In higher plants, copper ions, hydrogen peroxide, and cycloheximide have been recognized as very effective inducers of the transcriptional activity of genes encoding the enzymes of the ethylene biosynthesis pathway. In this report, the transcriptional patterns of genes encoding the 1-aminocyclopropane-1-carboxylate synthases (ACSs), 1-aminocyclopropane-1-carboxylate oxidases (ACOs), ETR1, ETR2, and ERS1 ethylene receptors, phospholipase D (PLD)-alpha1, -alpha2, -gamma1, and -delta, and respiratory burst oxidase homologue (Rboh)-NADPH oxidase-D and -F in response to these inducers in Brassica oleracea etiolated seedlings are shown. ACS1, ACO1, ETR2, PLD-gamma1, and RbohD represent genes whose expression was considerably affected by all of the inducers used. The investigations were performed on the seedlings with (i) ethylene insensitivity and (ii) a reduced level of the PLD-derived phosphatidic acid (PA). The general conclusion is that the expression of ACS1, -3, -4, -5, -7, and -11, ACO1, ETR1, ERS1, and ETR2, PLD-gamma 1, and RbohD and F genes is undoubtedly under the reciprocal cross-talk of the ethylene and PA(PLD) signalling routes; both signals affect it in concerted or opposite ways depending on the gene or the type of stimuli. The results of these studies on broccoli seedlings are in agreement with the hypothesis that PA may directly affect the ethylene signal transduction pathway via an inhibitory effect on CTR1 (constitutive triple response 1) activity.

A large-scale transcriptome analysis has been conducted using microPEACH1.0 microarray on nectarine (Prunus persica L. Batsch) fruit treated with 1-methylcyclopropene (1-MCP). 1-MCP maintained flesh firmness but did not block ethylene biosynthesis. Compared with samples at harvest, only nine genes appeared to be differentially expressed when fruit were sampled immediately after treatment, while a total of 90 targets were up- or down-regulated in untreated fruit. The effect of 1-MCP was confirmed by a direct comparison of transcript profiles in treated and untreated fruit after 24 h of incubation with 106 targets differentially expressed. About 30% of these targets correspond to genes involved in primary metabolism and response processes related to ethylene, auxin, and other hormones. In treated fruit, altered transcript accumulation was detected for some genes with a role in ripening-related events such as softening, colour development, and sugar metabolism. A rapid decrease in flesh firmness and an increase in ethylene production were observed in treated fruit maintained for 48 h in air at 20 degrees C after the end of the incubation period. Microarray comparison of this sample with untreated fruit 24 h after harvest revealed that about 45% of the genes affected by 1-MCP at the end of the incubation period changed their expression during the following 48 h in air. Among these genes, an ethylene receptor (ETR2) and three ethylene-responsive factors (ERF) were present, together with other transcription factors and ethylene-dependent genes involved in quality parameter changes.

Fruitlet abscission of mango is typically very severe, causing considerable production losses worldwide. Consequently, a detailed physiological and molecular characterization of fruitlet abscission in mango is required to describe the onset and time-dependent course of this process. To identify the underlying key mechanisms of abscission, ethephon, an ethylene releasing substance, was applied at two concentrations (600 ppm, 7200 ppm) during the midseason drop stage of mango. The abscission process is triggered by ethylene diffusing to the abscission zone where it binds to specific receptors and thereby activating several key physiological responses at the cellular level. The treatments reduced significantly the capacity of polar auxin transport through the pedicel at one day after treatment and thereafter when compared to untreated pedicels. The transcript levels of the ethylene receptor genes MiETR1 and MiERS1 were significantly upregulated in the pedicel and pericarp at one, two and three days after the ethephon application with 7200 ppm, except for MiETR1 in the pedicel, when compared to untreated fruitlet. In contrast, ethephon applications with 600 ppm did not affect expression levels of MiETR1 in the pedicel and of MiERS1 in the pericarp; however, MiETR1 in the pericarp at day two and MiERS1 in the pedicel at days two and three were significantly upregulated over the controls. Moreover, two novel short versions of the MiERS1 were identified and detected more often in the pedicel of treated than untreated fruitlets at all sampling times. Sucrose concentration in the fruitlet pericarp was significantly reduced to the control at two days after both ethephon treatments. In conclusion, it is postulated that the ethephon-induced abscission process commences with a reduction of the polar auxin transport capacity in the pedicel, followed by an upregulation of ethylene receptors and finally a decrease of the sucrose concentration in the fruitlets.

When exposed to far-red light followed by darkness, wild-type Arabidopsis thaliana seeds fail to germinate or germinate very poorly. We have previously shown that the ethylene receptor ETR1 (ETHYLENE RESPONSE1) inhibits and ETR2 stimulates seed germination of Arabidopsis during salt stress. This function of ETR1 requires the full-length receptor. These roles are independent of ethylene levels and sensitivity and are mainly mediated by a change in abscisic acid (ABA) sensitivity. In the current study we find that etr1-6 and etr1-7 loss-of-function mutant seeds germinate better than wild-type seeds after illumination with far-red light or when germinated in the dark indicating an inhibitory role for ETR1. Surprisingly, this function of ETR1 does not require the receiver domain. No differences between these mutants and wild-type are seen when germination proceeds after treatment with white, blue, green, or red light. Loss of any of the other four ethylene receptor isoforms has no measurable effect on germination after far-red light treatment. An analysis of the transcript abundance for genes encoding ABA and gibberellic acid (GA) metabolic enzymes indicates that etr1-6 mutants may produce more GA and less ABA than wild-type seeds after illumination with far-red light which correlates with the better germination of the mutants. Epistasis analysis suggests that ETR1 may genetically interact with the phytochromes (phy), PHYA and PHYB to control germination and growth. This study shows that of the five ethylene receptor isoforms in Arabidopsis, ETR1 has a unique role in modulating the effects of red and far-red light on plant growth and development.

The ripening of fleshy fruit is a developmentally regulated process unique to plants during which the majority of the sensory quality attributes are elaborated including aroma, flavour, texture and nutritional compounds. In climacteric fruit, the plant hormone ethylene is the key regulator of the ripening process as exemplified by the dramatic inhibition of fruit ripening that results from the down-expression of ACC (1-amino-cyclopropane-1-carboxylic acid) synthase and ACC oxidase genes involved in ethylene biosynthesis. By contrast, the ripening of non-climacteric fruit is not dependent on ethylene but rather on cues of unknown nature though ethylene may contribute at least partly to the control of some aspects of the ripening process. The expression of the ripening-associated genes is regulated by a network of signalling pathways among which ethylene perception and transduction play a primary role. Building on the knowledge gained on the Arabidopsis thaliana model system, the importance of ethylene signalling in fruit ripening has been extensively studied. This chapter summarizes the present knowledge on the role of ethylene in fruit ripening and addresses the molecular mechanisms involved in ethylene perception and responses. It also highlights recent advances and prospects on the means by which the ethylene transduction pathway leads to diversified physiological responses and how ethylene signalling interacts with other hormones to activate the expression of ripening-related genes. While this review mostly refers to the tomato as major model for fruit research, it also gives insight on the ripening process in other fruit species, including nonclimacteric types.

The UDP glucose-flavonoid 3-O-glucoslyltransferase (UFGT) is a key enzyme for biosynthesis and stability of anthocyanin pigments of red grapes. Understanding factors affecting expression of this enzyme is thus important for the control of grape colour. A 1640 bp promoter region of the grapevine ufgt gene was cloned and sequenced. Sequence analysis revealed seven putative ethylene-responsive cis-elements and others related to three major signals known to induce anthocyanin accumulation in plant tissues: light, sugar, and abscisic acid. In order to evaluate the ability of ethylene and other signals to drive expression from the ufgt promoter, we ran transient expression experiments using an anthocyanin-rich grape cell culture, with very low green auto-fluorescence. After biolistic bombardment, the cells were treated with various combinations of the four signals on gfp expression (green fluorescent protein). The comparison of fluorescent light intensity in cells subjected to the various treatments showed that ethylene better stimulates expression of the ufgt promoter in the dark than under light. In addition, results showed that there may be a positive interaction between ethylene and abscisic acid. This system, a promoter of interest driving the gfp expression in cells with low auto-fluorescence, may be a good tool for studies about synergistic or antagonist roles of transcription factors. Moreover, treatment of grape berries with a specific inhibitor of ethylene receptors (1-methylcyclopropene) inhibited ufgt mRNA accumulation. This confirms that the ethylene signal is likely a regulator of grape UFGT expression in a non-climacteric fruit., VITIS - Journal of Grapevine Research, Vol. 48 No. 1 (2009): Vitis

To understand the importance of ethylene receptor genes in the quality of coVee berries three full-length cDNAs corresponding to a putative ethyl- ene receptor gene (ETR1) were isolated from CoVea canephora cDNA libraries. They diVered by their 3UTR and contained a main ORF and a 5UTR short ORF putatively encoding a small polypeptide. The CcETR1 gene, present as a single copy in the C. cane- phora genome, contained Wve introns in the coding region and one in its 5UTR. Alternative splicing can occur in C. canephora and C. pseudozanguebariae, lead- ing to a truncated polypeptide. C. pseudozanguebariae ETR1 transcripts showed various forms of splicing alterations. This gene was equally expressed at all stages of fruit development. A segregation study on an inter-speciWc progeny showed that ETR1 is related to the fructiWcation time, the caVeine content of the green beans, and seed weight. Arabidopsis transformed etio- lated seedlings, which over-expressed CcETR1, dis- played highly reduced gravitropism, but the triple response was observed in an ethylene enriched envi- ronment. These plants behaved like a low-concentra- tion ethylene-insensitive mutant thus conWrming the receptor function of the encoded protein. This gene showed no induction during the climacteric crisis but some linkage with traits related to quality.

Methylcyclopropene (1-MCP) is an antagonist of ethylene for receptor binding sites and the effects of its application differ in rela- tion to a number of factors including genotype and ripening physiology. Peach (Prunus persica L. Batsch cv. 'Summer Rich') and apple (Malus × domestica L. Borkh cv. 'Golden Delicious') fruits were incubated with 1-MCP (1 LL −1 ) for 24 h at 20 ◦ C and respiration rate, ethylene production and fruit firmness, together with ACC synthase, ACC oxidase, ETR1, ERS1, and CTR1 gene expression patterns were assessed throughout the post-treatment phase. 1-MCP was confirmed to be effective in delaying ripening in apples while in peaches only a limited effect of the chemical was observed. A dramatic inhibition of ethylene biosynthesis and ACS gene expression was induced by 1-MCP in apples whereas no marked difference was observed in peaches between the two controls (in air and in sealed jars without 1-MCP) and the treated fruit. In apples, Md-ETR1 and Md-ERS1 gene expression was down-regulated by 1-MCP starting from the end of the treatment, while Md-CTR1 appeared negatively affected by the chemical at a later stage. Transcription of Pp-ETR1, Pp-ERS1 and Pp-CTR1 genes appeared unaffected in 1-MCP treated peaches. Differences in receptor transcript levels between control fruit maintained in air and those enclosed in sealed jars without 1-MCP may be due to an effect of CO2 that rapidly accumulates following incubation of ripening peaches. Results support the hypothesis that the different behaviour of peaches and apples in response to 1-MCP application might be related to differences in terms of ratio, expression patterns and/or turn-over of the ethylene receptors.

The time course of ethylene biosynthesis and perception was investigated in ripening peach fruit (Prunus persica) following treatments with the polyamines putrescine (Pu) and spermidine (Sd), and with aminoethoxyvinylglycine (AVG). Fruit treatments were performed in planta. Ethylene production was measured by gas chromatography, and polyamine content by high-performance liquid chromatography; expression analyses were performed by Northern blot or real-time polymerase chain reaction. Differential increases in the endogenous polyamine pool in the epicarp and mesocarp were induced by treatments; in both cases, ethylene production, fruit softening and abscission were greatly inhibited. The rise in 1-aminocyclopropane-1-carboxylate oxidase (PpACO1) mRNA was counteracted and delayed in polyamine-treated fruit, whereas transcript abundance of ethylene receptors PpETR1 (ethylene receptor 1) and PpERS1 (ethylene sensor 1) was enhanced at harvest. Transcript abundance of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) was transiently reduced in both the epicarp and mesocarp. AVG, here taken as a positive control, exerted highly comparable effects to those of Pu and Sd. Thus, in peach fruit, increasing the endogenous polyamine pool in the epicarp or in the mesocarp strongly interfered, both at a biochemical and at a biomolecular level, with the temporal evolution of the ripening syndrome.

The plant hormone ethylene regulates growth and development as well as stress responses. This review focuses on recent discoveries that support a model for ethylene signal transduction that involves overlapping and non-overlapping roles for members of the ethylene receptor family. The roles of ethylene receptors in regulating plant growth, pathogen responses, and development are discussed. Mechanisms are proposed by which receptors can modulate downstream responses together and independently., The plant hormone ethylene regulates growth and development as well as responses to biotic and abiotic stresses. Over the last few decades, key elements involved in ethylene signal transduction have been identified through genetic approaches, these elements defining a pathway that extends from initial ethylene perception at the endoplasmic reticulum to changes in transcriptional regulation within the nucleus. Here, we present our current understanding of ethylene signal transduction, focusing on recent developments that support a model with overlapping and non-overlapping roles for members of the ethylene receptor family. We consider the evidence supporting this model for sub-functionalization within the receptor family, and then discuss mechanisms by which such a sub-functionalization may occur. To this end, we consider the importance of receptor interactions in modulating their signal output and how such interactions vary in the receptor family. In addition, we consider evidence indicating that ethylene signal output by the receptors involves both phosphorylation-dependent and phosphorylation-independent mechanisms. We conclude with a current model for signalling by the ethylene receptors placed within the overall context of ethylene signal transduction.

Ethylene, a regulator of plant growth and development, is perceived by specific receptors that act as negative regulators of the ethylene response. Five ethylene receptors, i.e., ETR1, ERS1, EIN4, ETR2, and ERS2, are present in Arabidopsis and dominant negative mutants of each that confer ethylene insensitivity have been reported. In contrast, maize contains just two types of ethylene receptors: ZmERS1, encoded by ZmERS1a and ZmERS1b, and ZmETR2, encoded by ZmETR2a and ZmETR2b. In this study, we introduced a Cys to Tyr mutation in the transmembrane domain of ZmERS1b and ZmETR2b that is present in the etr1-1 dominant negative mutant and expressed each protein in Arabidopsis. Mutant Zmers1b and Zmetr2b receptors conferred ethylene insensitivity and Arabidopsis expressing Zmers1b or Zmetr2b were larger and exhibited a delay in leaf senescence characteristic of ethylene insensitive Arabidopsis mutants. Zmers1b and Zmetr2b were dominant and functioned equally well in a hemizygous or homozygous state. Expression of the Zmers1b N-terminal transmembrane domain was sufficient to exert dominance over endogenous Arabidopsis ethylene receptors whereas the Zmetr2b N-terminal domain failed to do so. Neither Zmers1b nor Zmetr2b functioned in the absence of subfamily 1 ethylene receptors, i.e., ETR1 and ERS1. These results suggest that Cys65 in maize ZmERS1b and ZmETR2b plays the same role that it does in Arabidopsis receptors. Moreover, the results demonstrate that the mutant maize ethylene receptors are functionally dependent on subfamily 1 ethylene receptors in Arabidopsis, indicating substantial functional conservation between maize and Arabidopsis ethylene receptors despite their sequence divergence.

Ethylene, the plant ripening hormone of climacteric fruit, is perceived by ethylene receptors which is the first step in the complex ethylene signal transduction pathway. Much progress has been made in elucidating the mechanism of this pathway, but there is still a lot to be done in the proteomic quantification of the main proteins involved, particularly during fruit ripening. This work focuses on the mass spectrometry based identification and quantification of the ethylene receptors (ETRs) and the downstream components of the pathway, CTR-like proteins (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2). We used tomato as a model fruit to study changes in protein abundance involved in the ethylene signal transduction during fruit ripening. In order to detect and quantify these low abundant proteins located in the membrane of the endoplasmic reticulum, we developed a workflow comprising sample fractionation and MS analysis using parallel reaction monitoring. This work shows the feasibility of the identification and absolute quantification of all seven ethylene receptors, three out of four CTRs and EIN2 in four ripening stages of tomato. In parallel, gene expression was analyzed through real-time qPCR. Correlation between transcriptomic and proteomic profiles during ripening was only observed for three of the studied proteins, suggesting that the other signaling proteins are likely post-transcriptionally regulated. Based on our quantification results we were able to show that the protein levels of SlETR3 and SlETR4 increased during ripening, probably to control ethylene sensitivity. The other receptors and CTRs showed either stable levels that could sustain, or decreasing levels that could promote fruit ripening.