Ethylene Response Sensor Research Articles

Inhibition of PAL, CHS, and ERS1 in ‘Red d’Anjou’ Pear ( Pyrus communis L.) by 1-MCP

The ethylene antagonist 1-MCP was investigated for its potential impact on the transcription of two key flavonoid biosynthetic (phenylalanine ammonia-lyase, PAL, E.C. 4.3.1.5; and chalcone synthase, CHS, E.C. 2.3.1.74), flavonoid transport (glutathione S-transferase, GST, E.C. 2.5.1.18) and ethylene perception (ethylene response sensor 1, ERS1) transcripts during the postharvest storage and ripening of pear ( Pyrus communis L.). ‘Red d’Anjou’ pear fruit were harvested from Wenatchee, WA, USA, transported to Guelph, Ont., Canada, then treated with 1 μL L −1 1-MCP, and subsequently placed in cold storage (0–1 °C, 90–95% RH) for up to 126 days. After removal, fruit were warmed to room temperature (1 or 7 days) then tissue samples were collected for Northern blot analysis and determination of flavonoid and chlorogenic acid concentration by HPLC. In general, PAL content decreased during storage, with content increasing during the post-storage ripening period in parallel with the increase in respiration rate and ethylene content. In contrast, CHS content decreased dramatically during the 1-week ripening period, while ERS1 remained constant. The expression of PAL, CHS and ERS1 transcripts were all inhibited by 1-MCP. GST transcript abundance decreased during storage, and was largely unaffected by the 1-MCP treatment. The flavonoid concentration remained constant throughout storage and subsequent ripening. However, after the first removal and warming to room temperature, chlorogenic acid concentration increased in the untreated, but not in the 1-MCP-treated fruit. These results suggest that 1-MCP significantly inhibits the transcription of key flavonoid biosynthetic enzymes and ethylene perception proteins, but not the flavonoid transport enzyme. The increase in PAL with the concomitant post-storage decrease of CHS and postharvest decrease of GST suggests a diversion of carbon from flavonoid compounds into chlorogenic acid.

Ethylene receptor transcript accumulation patterns during flower senescence in Oncidium ‘Gower Ramsey’ as affected by exogenous ethylene and pollinia cap dislodgment

The cDNA encoding a putative ethylene response sensor (ERS) type ethylene receptor OgERS1 of Oncidium was cloned and the deduced amino acid sequence of OgERS1 was structurally characterized. Phylogenetic analysis of amino acid sequences of ethylene receptor homologues indicated that OgERS1 was closely related to other members of ethylene receptors from monocotyledonous plants. OgERS1 was expressed abundantly in roots and flower buds, and a lesser extent in pseudobulbs, leaves, and fully opened flowers. OgERS1 mRNA levels in fully opened flowers during the natural senescence increased with days after cutting and reached the maximum in the fifth day of bloom. The accumulation of OgERS1 mRNA began to decrease at the day when ethylene raised abruptly and the flowers started to wilt. The expression of OgERS1 mRNA in pollinia de-capped Oncidium flowers showed an increasing and then decreasing trend toward the late stage of flower senescence as in un-dislodged fully opened flowers, and the overall expression level was decreased throughout the senescence period referred to un-dislodged flowers. Our results concluded that the expression of ethylene receptor OgERS1 is differentially expressed in different organs and the pollinia cap dislodgment alleviated the mRNA levels of ethylene receptor initiated by exogenous ethylene treatments.

Expression of ethylene receptors Dl-ERS1-3 and Dl-ERS2, and ethylene response during flower senescence in Delphinium

To clarify the relationships of flower senescence, especially sepal abscission, and ethylene receptor gene expression in different flower parts, we isolated two cDNAs encoding ethylene receptors Dl-ERS1-3 and Dl-ERS2 from Delphinium flowers. Deduced polypeptides possessed no response regulator domain, indicating that they belong to a family of ethylene response sensor (ERS) ethylene receptors. Dl-ERS1-3 and Dl-ERS2 exhibited constitutive levels during flower senescence. Exogenous ethylene increased transcript levels in sepals, which are influenced by ethylene but not in gynoecia and receptacles, which produce ethylene. It was suggested that expression of ethylene receptor genes under ethylene exposure was differentially regulated in each organ of the flower.

Ethylene insensitive and post-harvest yellowing retardation in mutant ethylene response sensor (boers) gene transformed broccoli (Brassica olercea var. italica)

A mutant broccoli ers (ethylene-response-sensor, boers) gene was obtained through site directed mutagenesis by replacing the isoleucine with phenylanine at the 62th residue. Two plasmids were constructed with this mutant gene regulated by the CaMV 35S promoter together with the nptII (kanamycin resistance gene) coding sequence and hpt (hygromycin resistance gene), respectively, for the pBI-mERSI62F and pSM1H-mERSI62F plasmids. Genetic transformation of the above two constructs via A. tumefaciens has been conducted to evaluate their effects on floret yellowing of harvested broccoli. Over a hundred transformants have been obtained on the selected cotyledon and hypocotyl explants. PCR and Southern analysis demonstrated integration of the transgenes in the transformants. However, through Southern hybridization, we determined that multi-site integration and DNA rearrangements had occurred in most transformants. Morphological and characteristic alternation such as slower plant growth, shorter plant height, easy branching, late bolting, and relative higher mortality in comparison with other transgenes were noted in some transformants. Transgenic lines showing delayed senescence in leaves and floral heads were obtained. The expression of transgene was confirmed by Northern blot analysis. The transformed progenies also showed ethylene insensitivity in seed germination, detached leaves and harvested florets. Nevertheless, in most lines, the yellowing was only delayed 1–2 days.

Heterologous expression of Arabidopsis ERS1 causes delayed senescence in coriander.

The phytohormone ethylene is involved in many developmental processes, including leaf and flower senescence. Ethylene is perceived by plants through receptors that trigger the downstream signal transduction pathway. The mutated ethylene receptor ERS1 (ethylene response sensor) from Arabidopsis is of a dominant negative nature and confers ethylene insensitivity in Arabidopsis. To investigate if the altered ERS1 gene can affect the tissue senescence in heterologous plants, we introduced it into coriander by Agrobacterium-mediated transformation. Transgenic plants were regenerated by cocultivating hypocotyl segments with A. tumefaciens harboring binary vector pCGN1547 that carried the ERS1 gene. The presence and expression of the transgene were confirmed by genomic Southern blot and reverse transcriptase-PCR analyses. Leaf and flower senescence were delayed significantly in the transgenic plants. The ability of the mutated ERS1 gene to confer the ethylene-insensitive phenotype can be exploited for extending the shelf-life of leafy vegetables.

Ethylene production and enhanced transcription of an ethylene receptor gene, ERS1, in Delphinium during abscission of florets

Delphinium spp. L florets are known to undergo severe senescence a few days after harvesting as cut flowers. Using a cDNA library made from mRNA extracted from Delphinium florets 4 d harvesting, we isolated and identified cDNAs encoding ethylene response sensor (ERS)-type ethylene receptors, which contained an open reading frame of 1821 bp encoding a polypeptide of 606 amino acid residues. These were classified into two types with minor differences; Dl-ERS1 ( Delphinium little-strain ERS1) type-1 (DDBJ/EMBL/GenBank AB055429) and Dl-ERS1 type-2 (DDBJ/EMBL/GenBank AB055430). We were able to identify only ERS1 in Delphinium using the cDNA library screening, 3′ RACE (DDBJ/EMBL/GenBank AB106892, AB106893), and genomic PCR (DDBJ/EMBL/GenBank AB106891). Northern blot analysis showed that the transcription of ERS1 gene doubled between 0 and 3 d after harvesting, increased sharply to a maximum at 4 d, and then declined, while abscission of florets increased slowly until 3 d, and then increased rapidly at 4–5 d after cutting. Ethylene evolution increased until 4 d, and then decreased at 6 d after cutting. From these results, we postulate that abscission of florets in Delphinium is caused by the elevated levels of ethylene receptor, ERS1, and that these will be influenced by endogenous ethylene generated after cutting the stem.

Differential Regulation of Genes Encoding Ethylene Biosynthesis Enzymes and Ethylene Response Sensor Ortholog during Ripening and in Response to Wounding in Avocados

We investigated the differential regulation of two 1-aminocyclopropane-1-carboxylate synthase (ACS) genes, one 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and one ethylene response sensor (ERS1) ortholog during ripening and in response to wounding in avocados (Persea americana Mill. `Bacon'). The 1-aminocyclopropane-1-carboxylate (ACC) content, ACS activity and detectable expression of PA-ACS1 mRNA increased and reached a maximum prior to the climacteric peak, whereas ACO activity and the PA-ACO mRNA levels increased markedly only at the upsurge of ripening ethylene. A basal level of PA-ERS1 transcript was detected as from harvest, however, PA-ERS1 transcript was hyper-induced at the climacteric peak of ethylene production. 1-Methylcyclopropene (1-MCP) application at thepreclimacteric and the onset of climacteric stages inhibited the ACS and ACO activities, the transcription of PA-ACS1 and suppressed PA-ACO and PA-ERS1 mRNAs to trace levels. Discontinuation of 1-MCP treatment led to super-induction of PA-ACS1, PA-ACO, and PA-ERS1 transcripts. Wound induced ethylene biosynthesis and wound-induced PA-ACS2 mRNA accumulation were enhanced by 1-MCP, whereas wound-induced PA-ACO mRNA accumulation was unaffected by 1-MCP. These results indicate positive feedback regulation of the PA-ACS1 gene and negative feedback regulation of the PA-ACS2 gene by ethylene, while PA-ACO exhibits positive feedback regulation by ethylene and is also induced by wounding. The hyper-induction of PA-ERS1 mRNA at relatively high concentrations of ethylene may be a mechanism of avocados to regulate the ethylene responsiveness of the tissues by dissipation of the gas.

Ethylene perception by the ERS1 protein in Arabidopsis.

Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.

Ethylene regulation of an ERS1 homolog in mung bean seedlings

ETR1 (ethylene resistant) and ERS1 (ethylene response sensor) have been shown to act as a receptor for ethylene in Arabidopsis. We isolated a full length cDNA clone (VR‐ERS1) encoding the ERS1 homolog from mung bean hypocotyls by screening a cDNA library with a partial cDNA fragment obtained by the reverse transcriptase‐polymerase chain reaction (RT‐PCR) method. VR‐ERS1 is 2 477 bp long and contains a single open reading frame encoding 636 amino acids (Mr=70.9 kDa). The predicted amino acid sequence shares a high degree of identity (72–78%) with other ERS1 homologs from Arabidopsis, tomato and Rumex palustris, and is 39% identical to Arabidopsis ERS2. Genomic Southern blot analysis using a gene‐specific probe suggests the existence of one copy of the VR‐ERS1 gene in the mung bean genome. Northern hybridization indicates that the level of VR‐ERS1 transcript varies in different parts of dark‐ and light‐grown mung bean seedlings, with the greatest amount of the transcript being detected in apical hooks. Results showed that the abundance of the message was highly inducible by ethylene in all tissues examined. However, the magnitudes of ethylene‐induction of VR‐ERS1 were significantly different in each part of mung bean seedlings, with the leaf tissue being most sensitive to ethylene and there being an 8‐fold increase in mRNA level after 6 h ethylene treatment. These results suggest that ethylene positively modulates the expression of its receptor gene in the vegetative tissues of mung bean plants.

Sticky-end PCR: new method for subcloning.

Sticky-end PCR: new method for subcloning.

Ethylene Sensitivity and Response Sensor Expression in Petioles of Rumex Species at Low O2 and High CO2 Concentrations.

Rumex palustris, a flooding-tolerant plant, elongates its petioles in response to complete submergence. This response can be partly mimicked by enhanced ethylene levels and low O2 concentrations. High levels of CO2 do not markedly affect petiole elongation in R. palustris. Experiments with ethylene synthesis and action inhibitors demonstrate that treatment with low O2 concentrations enhances petiole extension by shifting sensitivity to ethylene without changing the rate of ethylene production. The expression level of the R. palustris gene coding for the putative ethylene receptor (RP-ERS1) is up-regulated by 3% O2 and increases after 20 min of exposure to a low concentration of O2, thus preceding the first significant increase in elongation observable after 40 to 50 min. In the flooding-sensitive species Rumex acetosa, submergence results in a different response pattern: petiole growth of the submerged plants is the same as for control plants. Exposure of R. acetosa to enhanced ethylene levels strongly inhibits petiole growth. This inhibitory effect of ethylene on R. acetosa can be reduced by both low levels of O2 and/or high concentrations of CO2.

Ethylene insensitivity conferred by Arabidopsis ERS gene.

ERS (ethylene response sensor), a gene in the Arabidopsis thaliana ethylene hormone-response pathway, was uncovered by cross-hybridization with the Arabidopsis ETR1 gene. The deduced ERS protein has sequence similarity with the amino-terminal domain and putative histidine protein kinase domain of ETR1, but it does not have a receiver domain as found in ETR1. A missense mutation identical to the dominant etr1-4 mutation was introduced into the ERS gene. The altered ERS gene conferred dominant ethylene insensitivity to wild-type Arabidopsis. Double-mutant analysis indicates that ERS acts upstream of the CTR1 protein kinase gene in the ethylene-response pathway.