A medium-throughput protease assay for screening green plant material

Authors: Arther, Christina; Molodchenko, Andrew; Panke-Buisse, Kevin

Submitted to: Frontiers in Sustainable Food Systems
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2024
Publication Date: 1/15/2025
Citation: Arther, C.M., Molodchenko, A.S., Panke-Buisse, K. 2025. A medium-throughput protease assay for screening green plant material. Frontiers in Sustainable Food Systems. https://doi.org/10.3389/fsufs.2024.1496189.
DOI: https://doi.org/10.3389/fsufs.2024.1496189

Interpretive Summary: Forages are a component of dairy rations that support nutrition and sustainability goals for the industry. Alfalfa is a particularly beneficial perennial forage due to its high protein content but alfalfa protein is also highly degradable and its nutritional value declines sharply after harvest. Proteases, enzymes that break down proteins, are responsible for the reduction in alfalfa post-harvest protein quality. Reducing protease activity, and thus improving alfalfa quality, may be accomplished through several strategies including plant breeding, management, and treatment with additives. However, challenges related to measuring protease activity accurately and efficiently have limited progress in reducing post-harvest protein losses. Here we present an optimized method for scalable, accurate profiling of protease activity in alfalfa samples.

Technical Abstract: After harvest, aboveground plant material is subject to stress-induced senescence, which serves to liberate nutrients for other parts of the plant. However, for flowers, vegetables and forages, this process can severely impact quality. Proteases, in particular, are responsible for post-harvest protein degradation and contribute to cellular breakdown. The quality of certain crops is particularly affected by the actions of proteases after harvest including broccoli and alfalfa. Alfalfa is a high-quality forage and a perennial source of protein for dairy cows. Its nutritional value and suitability for sustainable agricultural production are also driving interest in the possible use of alfalfa for human consumption, however the issue of rapid protein degradation after harvest needs to be addressed before the protein can be extracted for human use on a large scale. We have optimized a cost-effective (< $1 per sample), medium throughput fluorescence intensity-based protease activity assay specifically for aboveground plant tissue that balances precision and throughput, the latter of which has been a barrier to further experiments. This method will allow higher throughput phenotyping of plant protease activity than is currently feasible: one person can process approximately 125 samples per day. We have used this method in alfalfa to assess protease activity during wilting at pH 7.4 and pH 6.3.