Parallel online determination of ethylene release rate by Shaken Parsley cell cultures using a modified RAMOS device

Ethylene is an important plant hormone that controls many physiological processes in plants. Conventional methods for detecting ethylene include gas chromatographs or optical mid-infrared sensors, which are expensive and, in the case of gas chromatographs, are hardly suitable for automated parallelized online measurement. Electrochemical ethylene sensors are cheap but often suffer from poor resolution, baseline drifting, and target gas oxidation. Thus, measuring ethylene at extremely low levels is challenging.

This report demonstrates the integration of electrochemical ethylene sensors into a respiration activity monitoring system (RAMOS) that measures, in addition to the oxygen transfer rate, the ethylene transfer rate in eight parallel shake flasks. A calibration method is presented that is not prone to baseline drifting and considers target gas oxidation at the sensor. In this way, changes in ethylene transfer rate as low as 4 nmol/L/h can be resolved. In confirmatory experiments, the overall accuracy of the method was similar to that of gas chromatography-mass spectrometry (GC/MS) measurements. The RAMOS-based ethylene determination method was exemplified with parsley suspension-cultured cells that were primed for enhanced defense by pretreatment with salicylic acid, methyl jasmonate or 4-chlorosalicylic acid and challenged with the microbial pattern Pep13. Ethylene release into the headspace of the shake flask was observed upon treatment with salicylic acid and methyl jasmonate was further enhanced, in case of salicylic acid and 4-chlorosalicylic acid, upon Pep13 challenge.

A conventional RAMOS device was modified for simultaneous measurement of the ethylene transfer rate in eight parallel shake flasks at nmol/L/h resolution. For the first time electrochemical sensors are used to provide a medium-throughput method for monitoring ethylene release by plants. Currently, this can only be achieved by costly laser-based detection systems and automated gas chromatographs. The new method is particularly suitable for plant cell suspension cultures. However, the method may also be applicable to intact plants, detached leaves or other plant tissues. In addition, the general principle of the technology is likely extendable to other volatiles or gases as well, such as nitric oxide or hydrogen peroxide.