This protocol allows plant-wide real time imaging of the activity of the plant systemic signaling system through monitoring the dynamics of the calcium and apoplastic glutamate in response to wounding. This plant-wide real-time imaging method provides a robust tool to understand the dynamics of the rapid and long distance signals in plants, combining a high spatial temporal resolution and ease of use. This protocol offers the potential to provide a new insight into the spatial and temporal characteristics of the system calcium signaling in both biotic and abiotic stress responses in other plants species.
Demonstrating the procedure will be Takuya Uemura, a post doctoral fellow from my laboratory. Begin by turning on the motorized fluorescent stereo microscope, equipped with a 1X objective lens and a sCMOS camera. Configure the device settings to irradiate with excitation light centered on 470 nanometers, selected using a filter that transmits light between 450 and 490 nanometers.
Acquire emission light using a filter that transmits between 510 and 560 nanometers. Remove the lid from the dish containing the plant and place it under the objective lens. Check the fluorescent signal from the plant.
Then wait for approximately 30 minutes in the dark until the plants are adapted to the new environmental conditions. Adjust the focus and magnification to see the whole plant in the field of view. Then set the acquisition parameters to detect the fluorescent signals using the microscopes imaging software.
Set the recording time to 11 minutes. Image for five minutes prior to starting the experiment to acclimate the plant to the blue light irradiation from the microscope, then start recording. To determine the average baseline fluorescence record at least 10 frames before wounding or glutamate application.
For real-time imaging of wound induced cytosolic calcium ion and apoplastic glutamate concentration changes, cut the petiole or the middle region of leaf L1 with scissors. For real-time imaging of glutamate triggered cytosolic calcium changes, cut approximately one millimeter from the tip of leaf L1 across the main vein with scissors. After at least 20 minutes apply 10 microliters of 100 millimolar glutamate to the leaf's cut surface.
For fluorescence intensity analysis over time, define a region of interest at the place where fluorescence intensity is to be analyzed. Define two ROIs for the velocity calculation of the calcium wave. In the imaging software click on time measurement, define and circle.
Measure the distance between the two regions by clicking on annotations and measurement, length and simple line. Measure the raw florescence values in each ROI over time by clicking on measure, then export the raw data to spreadsheet software, to convert the fluorescent signal into numbers at each time point. Determine the baseline fluorescence value, which is divined as F zero, by calculating the average F over the first 10 frames in the recorded data, then normalize the F data as described in the text manuscript.
For calcium velocity wave analysis, define a significant signal rise point above the pre-stimulated values as representing detection of a calcium increase in each ROI. Calculate the time difference in the calcium increase between the two ROIs and the distance between them to determine the velocities of any calcium wave. Propagation of a wound-triggered change in the concentrations of the cytosolic calcium ion and apoplastic glutamate is shown here.
Cutting the petiole of a leaf in plants expressing GCaMP-3 led to a significant increase in calcium. It was induced locally and then spread throughout the vasculature. The signal was rapidly propagated to neighboring leaves within a few minutes.
Upon cutting a leaf and plants expressing basic chitinase I glue sniffer, a rapid apoplastic glutamate increase was observed around the cut region. Within a few minutes, the signal also propagated through the vasculature. For real-time imaging of calcium signal propagation triggered by the application of glutamate, the edge of a leaf in plants expressing CCaMP-3 was cut.
This caused a local cytosolic calcium ion concentration increase, but the signal disappeared within a few minutes. After approximately 10 minutes, glutamate was applied to the cut surface, causing a rapid local increase in the concentration of cytosolic calcium and then propagation of this signal to distal leaves. To measure the changes inside a solid calcium concentration induced by wounding in the systemic leaf, the time course change of GCaMP-3 signal intensity was measured in two regions of interest.
Apoplastic glutamate concentration changes in response to mechanical damage were measured as well. The glutamate signature exhibited a single peak at approximately 100 seconds after wounding. This experiment should be conducted under temperature and humidity controlled conditions because are elevated by changes in this environmental conditions.
This protocol offers a potential to provide insights into the molecular mechanisms underlying long distance signaling through using mutants that the defective and putative elements of the lab signaling system.
