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Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis

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Abstract
Background: The foundations for ethylene research were laid many years ago by researchers such as Lizada, Yang and Hoffman. Nowadays, most of the methods developed by them are still being used. Technological developments since then have led to small but significant improvements, contributing to a more efficient workflow. Despite this, many of these improvements have never been properly documented. Results: This article provides an updated, integrated set of protocols suitable for the assembly of a complete picture of ethylene biosynthesis, including the measurement of ethylene itself. The original protocols for the metabolites 1-aminocyclopropane-1-carboxylic acid and 1-(malonylamino)cyclopropane-1-carboxylic acid have been updated and downscaled, while protocols to determine in vitro activities of the key enzymes 1-aminocyclopropane-1- carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase have been optimised for efficiency, repeatability and accuracy. All the protocols described were optimised for apple fruit, but have been proven to be suitable for the analysis of tomato fruit as well. Conclusions: This work collates an integrated set of detailed protocols for the measurement of components of the ethylene biosynthetic pathway, starting from well-established methods. These protocols have been optimised for smaller sample volumes, increased efficiency, repeatability and accuracy. The detailed protocol allows other scientists to rapidly implement these methods in their own laboratories in a consistent and efficient way.
Keywords
ACC, Ethylene, ACS, ACO, MACC, ADENOSYL-L-METHIONINE, 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID, CAPILLARY-ELECTROPHORESIS, MASS-SPECTROMETRY, IDENTIFICATION, CHROMATOGRAPHY

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Citation

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MLA
Bulens, Inge et al. “Protocol: An Updated Integrated Methodology for Analysis of Metabolites and Enzyme Activities of Ethylene Biosynthesis.” PLANT METHODS 7 (2011): n. pag. Print.
APA
Bulens, I., Van de Poel, B., Hertog, M. L., De Proft, M. P., Geeraerd, A. H., & Nicolaï, B. M. (2011). Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis. PLANT METHODS, 7.
Chicago author-date
Bulens, Inge, Bram Van de Poel, Maarten LATM Hertog, Maurice P De Proft, Annemie H Geeraerd, and Bart M Nicolaï. 2011. “Protocol: An Updated Integrated Methodology for Analysis of Metabolites and Enzyme Activities of Ethylene Biosynthesis.” Plant Methods 7.
Chicago author-date (all authors)
Bulens, Inge, Bram Van de Poel, Maarten LATM Hertog, Maurice P De Proft, Annemie H Geeraerd, and Bart M Nicolaï. 2011. “Protocol: An Updated Integrated Methodology for Analysis of Metabolites and Enzyme Activities of Ethylene Biosynthesis.” Plant Methods 7.
Vancouver
1.
Bulens I, Van de Poel B, Hertog ML, De Proft MP, Geeraerd AH, Nicolaï BM. Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis. PLANT METHODS. 2011;7.
IEEE
[1]
I. Bulens, B. Van de Poel, M. L. Hertog, M. P. De Proft, A. H. Geeraerd, and B. M. Nicolaï, “Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis,” PLANT METHODS, vol. 7, 2011.
@article{5820311,
  abstract     = {Background: The foundations for ethylene research were laid many years ago by researchers such as Lizada, Yang and Hoffman. Nowadays, most of the methods developed by them are still being used. Technological developments since then have led to small but significant improvements, contributing to a more efficient workflow. Despite this, many of these improvements have never been properly documented. 
Results: This article provides an updated, integrated set of protocols suitable for the assembly of a complete picture of ethylene biosynthesis, including the measurement of ethylene itself. The original protocols for the metabolites 1-aminocyclopropane-1-carboxylic acid and 1-(malonylamino)cyclopropane-1-carboxylic acid have been updated and downscaled, while protocols to determine in vitro activities of the key enzymes 1-aminocyclopropane-1- carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase have been optimised for efficiency, repeatability and accuracy. All the protocols described were optimised for apple fruit, but have been proven to be suitable for the analysis of tomato fruit as well. 
Conclusions: This work collates an integrated set of detailed protocols for the measurement of components of the ethylene biosynthetic pathway, starting from well-established methods. These protocols have been optimised for smaller sample volumes, increased efficiency, repeatability and accuracy. The detailed protocol allows other scientists to rapidly implement these methods in their own laboratories in a consistent and efficient way.},
  articleno    = {17},
  author       = {Bulens, Inge and Van de Poel, Bram and Hertog, Maarten LATM and De Proft, Maurice P and Geeraerd, Annemie H and Nicolaï, Bart M},
  issn         = {1746-4811},
  journal      = {PLANT METHODS},
  keywords     = {ACC,Ethylene,ACS,ACO,MACC,ADENOSYL-L-METHIONINE,1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID,CAPILLARY-ELECTROPHORESIS,MASS-SPECTROMETRY,IDENTIFICATION,CHROMATOGRAPHY},
  language     = {eng},
  pages        = {10},
  title        = {Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis},
  url          = {http://dx.doi.org/10.1186/1746-4811-7-17},
  volume       = {7},
  year         = {2011},
}

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