Experiment: Sensitive Mimosa Electrophysiology Using the Venus Fly Trap, we previously introduced you to plant electrophysiology, showing that plants can generate electrical impulses too! We now move to a second exquisite "rapid movement plant" - the sensitive mimosa. Background Note: As you cannot normally buy sensitive mimosa plant seedlings, you have to buy seeds and grow them. Mimosas are famously tricky to germinate, and see our note at the bottom of this page for recommendations on mimosa growth. With its lovely purple flowers and the hypnotic ways the leaves fold when touched, the sensitive mimosa (Mimosa pudica) has enraptured home gardeners and plant physiologists alike for its beauty and its unique behavior. In a healthy mimosa plant, you can observe two "rapid movement" responses to touch. With a light touch brushed along the leaves (calledpinnules), the leaves fold together at points (pulvinules) along the rib (rachis). With a strong touch, the leaves will fold and the branch will drop along the point (pulvinus) where the main branch (petiole) joins the stem. How does such dramatic movement occur? How does the plant even detect the touch to begin with? Hard working scientists have hypothesized that small red mechanoreceptor cells on the underside of the leaves respond to mechanical disturbance. This initiates an electrical impulse (action potential) propagation along the rachis that results in the plant movement behavior we observe. With a strong touch, the action potential travels along the rachis, down the petiole, and all the way to the main joint (pulvinus) via "phloem tubes." The exact nature of this signal propagation is still actively being investigated. But how then do the plants actually move? Since plants do not have muscles like we do, plant movement occurs through hydraulic forces (the flow of water). Plant cells have special large organs called "vacuoles" which are filled with water and can make up 70-80% of the cell volume. Plants thus have developed ways to rapidly move water in and out of the the vacuoles through special transport channels in their cell walls called "aquaporins." These are like ion channels, but instead of permitting ions to flow across membranes, they permit the rapid flow of water. As such, plants capable of rapid movement quickly flush water out of select cells. Such efflux of water shrinks the cell, and the shrinking of multiple cells at once, depending on location in the plant, can cause a mechanical strain that results in rapid movement. What initiates the water movement to begin with? Why, the action potential itself! The movement of ions across the cell membrane, which causes the action potential we observe, also creates the osmotic imbalance that results in water movement The illustration below depicts this process. The action potential begins with an increase in the intracellular calcium levels, which, being positively charged, makes the voltage on inside of the cell more positive. This increase in voltage then opens the voltage sensitive chloride channels, causing chloride to flow out of the cell, making the inside of the plant cell yet even more positive. In response to this chloride efflux, potassium channels then open to permit potassium to also flow out of the plant cell. Potassium being positive, this restores the resting potential and balances the chloride charges. Buuuuuttttt...we now have an ionic situation the plant cell can exploit: an excess of chloride and potassium ion are now outside the cells, or, we have an osmotic imbalance. Through the aquaporins, Water will then "chase" the potassium and chlorine ions, causing the plant cells to lose water rapidly, shrink, and, ultimately, result in the rapid movement of plant structures. After the cells have shrunk, they can be refilled with water again by moving the chloride and potassium ions back into the cells, but this requires energy expenditure, and is a slower process. In mimosas, this takes ~10 minutes, in Venus Fly Traps: ~1-2 days. We will now observe and measure this sensitive mimosa action potential. Join us as we continually expand our plant electrophysiology knowledge! Before you begin, make sure you have the Backyard Brains Spike Recorder and Arduino Programs installed on your computer. The Arduino "Sketch" is what you install on your Arduino circuit board using the Arduino laptop software (your board comes preinstalled if you bought the Arduino from us), and the Backyard Brains Spike Recorder program allows you to visualize and save the data on your computer when doing experiments. Wemade a software video for you to explain this! Spike Recorder Software for Displaying and Saving Data on Computer. Arduino Sketch for Sending Data to Computer Tutorial Video of Experiment Procedure In this experiment, we are going to measure the action potentials generated at the stem/petiole joint of mimosa plants. 1. You will use our Plant SpikerShield Bundle which has all the materials you need (sans plant) to do the experiment. 2. Grow a sensitive mimosa. You will need to start about 3-4 months before you do your experiments to have plants large enough for recording. Some planning is necessary. Our mimosas were grown in pots exposed to ambient light and air and not in a green house. Thus, the research for these experiments was always done during Spring/Summer. Life. Plants. Seasons. Cycles. 3. Place our plant electrode in the manipulator, and position your manipulator such that an inch of free silver wire is close to a stem/petiole joint. 4. Now, carefully wrap the silver wire around the union of the joint. Note that the plant branch will droop (move) as you do this due to the mechanical disturbance of wrapping the wire around the branch. 5. Put the ground wire pin in the...wait for it...ground of the plant pot. 6. Wait about 10 minutes for the plant to recover from its droopping movement. As said before, an advantage of the sensitive mimosa is that, unlike the Venus Fly Trap, it only takes 10 minutes to recover instead of 1-2 days. 7. Place some conductive gel along the silver spiral wrapped around the petiole. Note: we have noticed that placement of excessive conductive gel (perhaps due to ionic shunting?) prevents movement of the branch. Place only a small a small amount of conductive gel along the silver spiral. 8. To get a clean signal we do what we like to call "Plant time", a time where we are devoted to only the Mimosa pudica. How do we do this? Not singing or praying to the plant, but turning off all the lights and unplugging every single power outlet that's in the room, including the internet router if it's near the experiment setup. This will reduce the electromagnetic noise that can interfere with your recording. We recommend to give heads up to people near you by yelling "Plant time!" before unplugging everything. 9. Open our SpikeRecorder software, and in the settings window (click on the gear shaped symbol in the upper left hand of the screen to get there), connect to your USB port by clicking on the plug button. 10. Now, the line on your screen should become flat, and we are likely recording from the plant if we have a proper interface. Press the "Record" button (red button on top right of screen) to begin saving your data as a .wav file. 11. With a plastic probe, tap in one smart motion the leaves of the branch you are recording from. The plant branch should move in a dramatic fashion, and, in the SpikeRecorder software, you should notice a long deflection! Congratulations! you have just recorded an action potential in the mimosa! Such a universal signal that keeps us all functioning. 12. if your action potential is too big, resulting in "flat tops," your gain is too high and you need to reduce it on the SpikerShield. The SpikerShield has gain wheel that can appear counter- intuitive, as counterclockwise movement increases gain but clockwise movementdecreases gain. We have found one quarter gain works well. 13. To analyze the data, such as the duration and amplitude of the plant action potential, you can open your .wav files by clicking the "open button" (looks like three vertical lines) next to the "record button." 14. Now go investigate further the electrophysiology of the Mimosa and make new discoveries! Discussion / Further Work The background text was our best effort at a synthesis of our readings and and most likely contains errors. We are not plant experts (the last time we formally studied botany was in high school). See our references at the end of this page for more detailed information and to make your own analysis. Please do e-mail us if you have corrections/commentary. If you had two Plant SpikerShields stacked, you could possibly measure the conduction velocity along the branch. You would need an electrode wire sufficiently loose and slack though.... Cold stimuli supposedly also affect the mimosa. If cold water is applied to the soil, does this cause action potential propagation and branch movement? We stated above that excessive gel will actually prevent branch movement. Why could this be> Perhaps because conductive gel contains ionic elements and can affect the osmotic pressure? In the osmosis figure, the inside of the cell becomes more positive initially due to the chloride ion efflux. But, in our recordings, which were done outside the cells, we also noticed an increase. It should be in reverse? We should be observing a decrease in potential. Why is this? We are unsure. Now that we have studied the two most famous "rapid movement plants," an obvious next step is to study the electrical impulsos of other "normal" plants that don't have rapid movement properties.