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

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.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.Ethylene biosynthesis starts from the conversion of S-adenosyl-L-methione (SAM) into 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS). ACC can then be converted to either 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by ACC N-Malonyl transferase, or to the end product, ethylene, by 1-aminocyclopropane-1-carboxylate oxidase (ACO) [1] (Figure 1).Analytical protocols for each of these metabolites and enzymes have been developed and are available. The easiest component to measure is the gaseous hormone ethylene. It is formed inside cells, diffuses through the tissue and eventually out of the fruit into the surrounding atmosphere. Therefore multiple readings can be made without destructive sampling of the tissue. Typically, measurements are performed by gas chromatography (GC) [2,3] or more recen