<p>Ethylene as a phytohormone affects many aspects of plant growth and development. To fully understand how ethylene functions in plants, it is essential to measure ethylene production over time and under different conditions. With this goal in mind, we developed a biological assay based on root hair length to screen large numbers of seedlings to determine their relative production of ethylene. This was coupled with a biophysical assay based on the specific detection of ethylene using near infra-red laser spectroscopy coupled to photoacoustic detection. With this sensitive method we examined the effects of light, relative headspace per seedling, and time of ethylene accumulation on rates of ethylene biosynthesis in Arabidopsis (Col). Our results indicate that Col rates of ethylene emission are positively affected by factors such as light but negatively regulated by a sealed environment. Interestingly, a 24h confinement in a sealed environment triggers typical ethylene dependent responses at the molecular level indicating that a decrease in ethylene emission may be the product of an activation of the signalling pathway. Taken together our data suggest that negative feedback regulation of ethylene biosynthesis relies on specific components of the ethylene signalling pathway and may involve a transcriptional regulation. Our observation opens fresh directions to explore the underlying mechanisms by which plants alter ethylene biosynthesis to regulate growth and development and highlight the importance of standardizing various factors when evaluating rates of ethylene biosynthesis.</p>