Recombinant Cucumis melo var. cantalupensis Ethylene receptor 1 (ETR1)
Description
Introduction
Cucumis melo var. cantalupensis, commonly known as cantaloupe melon, is an economically important fruit crop worldwide . Ethylene, a gaseous plant hormone, plays a crucial role in various aspects of plant development and ripening, including fruit ripening, senescence, and responses to environmental stresses . Ethylene perception in plants is mediated by a family of ethylene receptors, including Ethylene Receptor 1 (ETR1) . Recombinant ETR1 refers to the ETR1 protein that has been produced using recombinant DNA technology, typically in a heterologous expression system such as E. coli .
Production of Recombinant ETR1
Recombinant ETR1 is produced using in vitro expression systems, commonly E. coli . The process involves cloning the ETR1 gene from Cucumis melo var. cantalupensis into an expression vector, transforming the vector into E. coli cells, and inducing protein expression. The recombinant protein is then purified for downstream applications such as biochemical assays and structural studies .
Role of Ethylene in Melon Development and Ripening
Ethylene is a key regulator of fruit ripening in cantaloupe melons. During ripening, ethylene triggers a cascade of events that lead to changes in fruit color, texture, aroma, and flavor . Studies have shown that the expression of ethylene receptor genes, including ETR1, is developmentally regulated during melon fruit ripening .
Research Findings and Applications
Recombinant ETR1 has several applications in plant research, including:
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Studying Ethylene Signaling Pathways: Recombinant ETR1 can be used in in vitro assays to investigate the biochemical mechanisms of ethylene perception and signaling .
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Investigating Protein-Protein Interactions: Recombinant ETR1 can be used to identify proteins that interact with the ethylene receptor, providing insights into the regulation of ethylene signaling .
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Developing Ethylene-Based Technologies: Understanding the role of ETR1 in ethylene signaling can aid in the development of technologies to manipulate fruit ripening and improve postharvest quality .
Genetic Variability
Genetic studies of Cucumis melo var. cantalupensis have revealed considerable variability in both quantitative and qualitative traits, except for fruit flesh color, weight, and sugar content . The variability is maintained through intercrossing and recombination. High heritability has been observed for fruit shape and color traits after selfing, indicating that these traits are controlled by simple allelic factors .
Impact on Fruit Quality and Shelf Life
The flavor profiles of cantaloupe melons are influenced by maturity at harvest, with mature fruits exhibiting a sweeter and fruitier character compared to immature fruits . Long shelf-life melons may lack some of the fruity flavors and exhibit a musty, earthy character .
Metal Homeostasis
Research has demonstrated the importance of iron and copper homeostasis in Cucumis melo. Studies involving the fefe mutant have highlighted the crosstalk between iron and copper, revealing that simultaneous deficiency of both synergistically upregulates iron uptake gene expression .
Tendril Development
Genetic analysis of the Chiba Tendril-Less (ctl) melon mutant has provided insights into tendril development in Cucumis melo. The ctl mutant lacks tendrils, and the CTL locus has been identified as a TCP transcription factor gene (CmTCP1) that is specifically expressed in wild-type tendrils .
Antioxidant and Antiproliferative Effects
Extracts from melon peels and seeds exhibit high antioxidant activity and have shown antiproliferative effects against human tumor cells. Different extracts of melon have demonstrated iron and copper ions chelating activity, with the hydroethanolic extract of melon peel displaying significant hydroxyl radical scavenging ability .
Tables
| Property | Description |
|---|---|
| Gene | ETR1 |
| Protein Family | Ethylene Receptor |
| Source Organism | Cucumis melo var. cantalupensis |
| Expression System | In vitro E. coli expression system |
| Function | Mediates ethylene perception and signaling, regulates fruit ripening, senescence, and stress responses |
| Applications | Biochemical assays, protein-protein interaction studies, development of ethylene-based technologies |
Product Specs
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Target Background
Q&A
What is ETR1 and what is its role in plant development?
ETR1 (Ethylene receptor 1) is a membrane-associated protein that functions in the ethylene signal transduction pathway in Cucumis melo var. cantalupensis (netted muskmelon). It may act as an ethylene receptor or as a regulator of this pathway . ETR1 belongs to the ethylene receptor family, which is responsible for detecting the plant hormone ethylene and initiating downstream signaling events . The full-length protein consists of 740-741 amino acids with a molecular mass of approximately 82.7 kDa .
The protein plays specific roles not only in fruit ripening but also in early development of melon fruit, with distinct roles in particular fruit tissues at specific developmental stages . The expression patterns of ETR1 vary significantly across different tissues and developmental phases, indicating its differential regulation and importance throughout plant growth and development .
How does ETR1 expression change during fruit development and ripening?
ETR1 exhibits stage- and tissue-specific expression patterns throughout melon fruit development. Northern analysis studies have revealed that in fully enlarged fruit, Cm-ETR1 mRNA levels are very high in seeds and placenta, while relatively lower in other tissues . During the ripening process, the expression of Cm-ETR1 mRNA increases markedly in parallel with climacteric ethylene production .
This expression pattern differs from that of Cm-ERS1 (another ethylene receptor), which shows high expression during fruit enlargement that decreases at the end of this phase . These distinct expression profiles suggest that ETR1 and ERS1 have complementary but different functions during fruit development and ripening.
How does Cm-ETR1 compare to ethylene receptors from other plant species?
Sequence analysis reveals that the open reading frame of Cm-ETR1 cDNA shares more than 67% and 69% identity in nucleotide and amino acid sequences, respectively, with ethylene receptor genes from other plants . It is also 68.6% identical at the nucleotide level and 75.3% identical at the amino acid level to Cm-ERS1, another ethylene receptor in melon .
This high degree of conservation suggests functional similarity across species while allowing for species-specific adaptations. The conservation is particularly evident in the hydrophobic domains involved in ethylene binding and in the histidine kinase domain, reflecting the evolutionary importance of these functional regions.
What methodological approaches are recommended for studying ETR1 function in melon?
To study ETR1 function in melon, researchers should consider a multi-faceted approach:
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Gene expression analysis: RT-PCR and Northern blotting with specific probes can be used to analyze tissue- and stage-specific expression patterns. For ETR1, probes such as RD (response regulator domain) or MET0.4 (sequences coding for the hydrophobic domains) have been successfully employed .
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Protein localization: Immunohistochemistry or fluorescent protein tagging can reveal the subcellular localization of ETR1, which is critical for understanding its function in ethylene perception.
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Mutational analysis: Based on studies in Arabidopsis, specific mutations in ETR1 (such as etr1-1, etr1-2, etr1-3, and etr1-4) cause ethylene insensitivity . Similar mutations could be introduced in melon ETR1 using CRISPR/Cas9 to study their effects on fruit development and ripening.
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Transgenic approaches: Overexpression or silencing of ETR1 in melon can provide insights into its role in fruit development and ripening. Transgenic expression of mutant forms (like etr1-1) can be used to create ethylene-insensitive plants for functional studies .
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Protein-protein interaction studies: Yeast two-hybrid assays or co-immunoprecipitation can identify ETR1-interacting proteins, helping to elucidate the signaling pathway.
What experimental considerations are important when working with recombinant ETR1 protein?
When working with recombinant Cucumis melo ETR1 protein, researchers should consider:
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Storage conditions: Store at -20°C for regular use or -80°C for extended storage. Avoid repeated freezing and thawing as this can compromise protein integrity .
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Working concentration: Optimize protein concentration for specific assays. For most applications, working aliquots can be stored at 4°C for up to one week .
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Buffer composition: Recombinant ETR1 is typically stored in Tris-based buffer with 50% glycerol, optimized for protein stability . When changing buffers for specific assays, gradual dialysis is recommended to maintain protein folding.
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Membrane association: ETR1 is a membrane-associated protein, so detergents or lipid reconstitution may be necessary for functional studies.
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Copper requirement: ETR1 requires copper as a cofactor for ethylene binding. Including copper in binding assays may be essential for functional studies.
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Tag considerations: The presence and type of tags may affect protein function. Consider tag removal using specific proteases if tag interference is a concern .
How can genomic analysis of ETR1 inform functional studies?
Genomic Southern analysis has revealed that there are at least two ethylene receptor homolog genes in the melon genome corresponding to Cm-ETR1 and Cm-ERS1 . Understanding this genomic organization is crucial for:
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Designing specific primers/probes: To distinguish between different receptor genes when studying expression patterns or for gene editing.
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Evolutionary studies: Comparing ETR1 sequences across species can reveal conserved domains critical for function and species-specific adaptations.
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Promoter analysis: Identifying regulatory elements in the promoter regions can explain tissue-specific and developmental stage-specific expression patterns.
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Functional redundancy: Understanding the presence of multiple receptor genes helps explain potential functional redundancy and the need for multiple gene knockouts to observe phenotypes.
A systematic approach to genomic analysis should include:
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Isolation of genomic DNA
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PCR amplification with gene-specific primers
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Southern blotting with specific probes (like XE0.4 or MET0.4)
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Sequence analysis of promoter regions
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Comparative genomics across related species
What is the significance of ETR1 mutations on ethylene sensitivity and fruit ripening?
Studies in Arabidopsis have identified four dominant mutations in ETR1 (etr1-1, etr1-2, etr1-3, and etr1-4) that cause ethylene insensitivity . All these mutations result in single-amino acid replacements in the three putative membrane-spanning hydrophobic domains of the protein. When these mutant genes are transformed into wild-type plants, the transgenic plants lose ethylene sensitivity .
This phenomenon has significant implications for fruit ripening research:
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Controlled ripening: Introduction of dominant ETR1 mutations into climacteric fruits like melon could delay ripening, potentially extending shelf life.
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Mechanistic insights: These mutations provide valuable information about the structural requirements for ethylene binding and signal transduction.
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Tissue-specific effects: Given the differential expression of ETR1 in various tissues, tissue-specific expression of mutant ETR1 could affect specific aspects of fruit development and ripening.
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Cross-species conservation: Studies have shown that the dominant etr1-1 mutation eliminates ethylene sensitivity not only in Arabidopsis but also when transformed into tomato and petunia , suggesting a conserved mechanism that could extend to melon.
How does ETR1 expression regulation differ from other ethylene receptors?
ETR1 exhibits distinct expression regulation compared to other ethylene receptors:
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Ethylene responsiveness: Unlike ERS1, ETR1 expression is not affected by ethylene treatment . This is similar to what has been observed with Arabidopsis ETR1 and tomato eTAE1.
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Developmental regulation: In fully enlarged fruit, ETR1 mRNA levels are very high in seeds and placenta, while ERS1 levels are low in all tissues . During ripening, ETR1 mRNA increases markedly in parallel with climacteric ethylene production.
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Tissue specificity: ETR1 and ERS1 show different tissue-specific expression patterns, suggesting distinct roles in particular fruit tissues at particular developmental stages .
This differential regulation has methodological implications:
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When designing experiments to study ethylene perception, researchers should consider which receptor(s) might be most relevant for their specific developmental stage or tissue of interest.
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The lack of ethylene-induced expression of ETR1 suggests it may play a more constitutive role in ethylene sensing, while ethylene-induced receptors like ERS1 might function more in feedback regulation.
