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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.