Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

This protocol describes an optimized L-012-based chemiluminescence method to detect ROS production in real time upon PAMP elicitation in rice tissues. This method is easy, standardized, and highly reproducible under firmly controlled conditions. Demonstrating this procedure will be On Ying, a PhD student from my lab.

To begin, sterilize the dehusked rice seeds with 70%ethanol for one minute, then with 40%sodium hypochlorite for one hour. Rinse the seeds five times with sterile water to remove residual chlorine. In the rice sheath method, directly plate the seeds in the sterile glass vessel with Murashige and Skoog or MS medium.

To plate the seeds in the leaf disc method, Plate them on MS plates for five to seven days and then transplant them to the growth matrix or soil. Grow the seedlings in a growth room with a 12 hour light, 12 hour dark photo period. Cut the sheath from 10 day old rice seedlings into three millimeter segments with a sharp razor blade or surgical blade for pre-treatment one day before the ROS assay.

Place five sheath segments in an individual well of a 96 well microtiter plate containing 100 microliters of double distilled water for 10 to 12 hours. Cut the leaf discs from four to six week old rice plants using a biopsy punch with a plunger. Always cut the leaf discs from the middle third of the second leaf of the main tiller to reduce data variation.

Next, place one leaf disc in an individual well of a 96 well microtiter plate containing 100 microliters of double distilled water for 10 to 12 hours for pre-treatment. Keep all the leaf discs floating and facing up in the wells of a microtiter plate for water pre-treatment to avoid leaf side associated variation. Prepare elicitation solution by combining 9.4 milliliters of 50 micromolar Tris Hcl, 400 microliters of L012 solution, 100 microliters of horse radish peroxidase, and 100 microliters of Flg22.

Add 200 microliters of elicitation solution to the wells. Start the software and click on the experiments button to create a new protocol or use an existing protocol. Click procedure in the popup to set up the plate and select the wells from the plate to be monitored.

Click start kinetic and set the runtime to 30 minutes or longer, depending on the experimental requirements. Select minimum interval to obtain the readings as frequently as possible. And for integration time, choose one second or longer depending on the signal intensity.

Click on validate, followed by Okay to confirm the settings, then click on detect the new plate in the popup and wait for the software to prompt the load plate dialogue box. Carefully remove the double distilled water from the wells containing the pre-treated tissues, avoiding any tissue damage or desiccation. Use a multi-channel pipette to add 200 microliters of the elicitation solution to the wells containing the tissues.

Place the plate to be tested on the carrier and begin detection. To induce ROS by Flg22, leaf discs and three millimeter long sheaths were used. ROS generation is monitored for 35 minutes.

The bars indicate the means of standard deviations calculated from five technical repeats. In rice, the increase in ROS production was first detected in one to two minute, peaked at 10 to 12 minute and returned to the baseline in around 30 to 35 minute. The total amount of ROS was calculated from the curve.

To obtain the total amount of ROS values, apply the formula to the corresponding data sets to calculate the ROS generated at each time interval, which can be combined by applying the formula sum to calculate the total amount generated. A single hole or two halves of a leaf disc were placed into the wells of a 96 well microtiter plate pretreated with 100 microliters of double distilled water for 10 to 12 hours and then treated with Flg22 for ROS induction. The reading values from the two half disc samples are much higher than that from the whole leaf disc.

On average, the total values from the two half disc samples are almost 1.6 times that from the whole leaf disc, which is proportional to the edge length, not to the area of the samples. This result supports that ROS are mainly generated in cells at the wound site. When attempting with this procedure, operate with tissues gently and do not make extra cuts out which could be a source of a data variation.

This method can be applied to any other ROS-producing physiological processes in plant tissues.