Extracellular root protease: Proteases in solution

After one-week, sterile seedlings (n = 8 for each treatment per plant) of similar height and root length were transferred from the Phytatrays® into a pre-autoclaved hydroponic growth system. The plants were firstly placed into a 1.5 ml Eppendorf tube with the bottom removed. This was then placed into the top of a 50 cm³ polypropylene centrifuge tube containing nutrient solution and then into a larger sterile box. Nutrient solution was injected into each centrifuge tube via silicone tubing connected to a 0.22-μm filter located outside the box. The nutrient solution in the centrifuge tube was continually aerated by passing 0.22-μm filtered air into the solution via silicone tubing located outside the box. An air outlet from the centrifuge tube was via silicon tubing with a hydrophobic 0.22-μm filter (Supporting information, Fig. S1). Weekly, nutrient solutions were removed from the hydroponic system through a 0.22-μm filter and protease activity measured. Fresh nutrient solution was then injected into each centrifuge tube through a 0.22-μm filter. Nutrient solutions were changed weekly to ensure nutrients were never limited and provide a weekly time series of protease activity over the seedlings growth. A negative control consisted of nutrient solution with no plant present. All work was carried out in a sterile, laminar flow cabinet. After four weeks of growth, under the constant conditions outlined previously, the experiment was stopped. The roots and shoots were separated, the fresh weight recorded, then oven dried at 80 °C for 24 h after which the dry weight was recorded.

To ensure that the system was sterile, an open Petri-dish with nutrient agar was placed at the bottom of the hydroponic system. At the end of the experiment, nutrient solution was plated onto nutrient agar. If no microbial growth was observed after one week at 37 °C, the system was considered sterile.