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

Abstract

BackgroundThe 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.ResultsThis 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.ConclusionsThis 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.

Targeted Systems Biology Profiling of Tomato Fruit Reveals Coordination of the Yang Cycle and a Distinct Regulation of Ethylene Biosynthesis during Postclimacteric Ripening

“…Fruit ethylene production was quantified after 1 h incubation according to the procedure described by Bulens et al (2011). Fruit respiration rate was quantified by measuring CO 2 production simultaneously with ethylene production by gas chromatography (CompactGC, Interscience).…”

“…Fruit respiration rate was quantified by measuring CO 2 production simultaneously with ethylene production by gas chromatography (CompactGC, Interscience). ACC was extracted and quantified by the Lizada and Yang (1979) method recently optimized by Bulens et al (2011). MACC was obtained after acidic hydrolysis according to Hoffman et al (1982) and also recently updated by Bulens et al (2011).…”

“…ACC was extracted and quantified by the Lizada and Yang (1979) method recently optimized by Bulens et al (2011). MACC was obtained after acidic hydrolysis according to Hoffman et al (1982) and also recently updated by Bulens et al (2011). SAM was extracted and quantified by capillary electrophoresis (P/ ACE MDQ, Beckman Coulter) with UV detection according to Van de Poel et al (2010).…”

“…ACO was extracted and in vitro activity determined according to Ververidis and John (1991) updated by Bulens et al (2011). The extraction buffer consisted of a 100-mM Tris buffer, pH 8.0, instead of the described 400-mM 3-(N-morpholino)propanesulfonic acid buffer, pH 7.2.…”

Effects of Anti-Ethylene Treatments on Ethylene Production and Antioxidant Activities in Cut Spray Carnation

“…ACC was assayed essentially as described by Bulens et al (2011). Briefly, 0.4 ml of 10 mM HgCl 2 was added to 1.4 ml of extract in a 9 ml vial, which was immediately sealed with a serum cap.…”