Circadian rhythms in biologically closed electrical circuits of plants

Abstract

The circadian clock regulates a wide range of electrophysiological and developmental processes in plants. Here, we discuss the direct influence of a circadian clock on biologically closed electrochemical circuits in vivo. The biologically closed electrochemical circuits in the leaves of C. miniata (Kaffir lily), Aloe vera and Mimosa pudica, which regulate their physiology, were analyzed using the charge stimulation method. Plants are able to memorize daytime and nighttime. Even at continuous light or darkness, plants recognize nighttime or daytime and change the input resistance. The circadian clock can be maintained endogenously and has electrochemical oscillators, which can activate ion channels in biologically closed electrochemical circuits. The activation of voltage gated channels depends on the applied voltage, electrical charge, and the speed of transmission of electrical energy from the electrostimulator to plants.

In plants, circadian rhythms are linked to the light–dark cycle. Many of the circadian rhythmic responses to day and night continue in constant light or dark, at least for a period of time.^1,2 The circadian clock is an endogenous oscillator with a period of approximately 24 h; its rhythm is linked to the light–dark cycle. The circadian clock in plants is sensitive to light, which resets the phase of the rhythm. Molecular mechanism underlying circadian clock function is poorly understood, although it is now widely accepted for both plants and animals that it is based on circadian oscillators. The circadian clock was discovered in 1729 by De Mairan³ in his first attempt to resolve experimentally the origin of rhythm in the leaf movements of Mimosa pudica. We also investigated the electrical activity of Mimosa pudica in the day light, at night and in darkness the following day.²

Isolated pulvinar protoplasts are responsive to light signals in vitro.^4-6 In the dark period, the closed inward-directed K⁺ channels of extensor cells are opened within 3 min by blue light. Conversely, the inward-directed K⁺ channels of flexor cells, which are open in the darkness, are closed by blue light. In the light period, however, the situation is more complex. Premature darkness alone is sufficient to close the open channels of extensor protoplasts, but both darkness and a preceding pulse of red light are required to open the closed channels in the flexor protoplasts.^5,6

The main goal of our experiments was to investigate circadian variation of the C. miniata electrical properties.¹ We investigated electrical responses of C. miniata to electrical stimulation during the day in daylight, darkness at night, and the following day in darkness with different timing and voltages. Experimental setup is shown in Figure 1.

Figure 1. Experimental setup.

The electrical discharge of 10 °F capacitor between two Ag/AgCl electrodes in the leaf of C. miniata parallel to the conductive bundles was studied. The difference between the two experiments is the polarity of the electrodes: the positive pole is closer to the base of a leaf and the positive pole is closer to the apex. This presentation immediately shows if the resistance of the leaf remains constant in time and how it varies with applied voltage. One can see that with +/− position of electrodes (“+” to the base and “−” to the apex), the traces are actually slightly curved lines and slope varies with initial voltage U₀. From here we calculated input resistance. In both cases there is a deviation in logarithmic coordinates from the linear predictions in equation. The deviation is rather small with +/− polarity, but very pronounced with −/+ electrodes. This means that the conductance between two electrodes cannot be presented by a single constant resistance. Similar rectification effects were found in Aloe vera, the Venus flytrap and the Mimosa pudica. While using a silicon rectifier Schotky diode NTE583, as a model of a voltage gated channel, we reproduced experimental dependencies of the capacitor discharge in plant tissue.⁷ Figure 2A shows a passive RC equivalent electrical scheme, which is not sensitive to a polarity of applied voltage. Figure 2B shows a simple active circuit, which is sensitive to polarity of applied voltage. We model voltage gated ion channels by rectifying diodes, which have voltammetric characteristics similar to current-voltage dependencies of voltage-gated ion channels.

Figure 2. Electrical equivalent schemes of a capacitor discharge in plant tissue. Abbreviations: C₁, charged capacitor from voltage source U₀; C₂, capacitance; R , resistance; D₁ and D₂ are diodes as models of voltage gated ion channels.

The same electrical discharge was displayed in darkness during the nighttime. The kinetics of the night discharge is significantly slower. This means that leaf resistance strongly increases during the night time.

The biological clock in the Clivia recognizes the daytime, even in darkness. The discharge during the following day and in darkness is very similar to the first day of the experiment. Input resistance in the initial moment of the capacitor discharge was the same as during the day light. During the third day when the lights are on, the results are the same as shown in the first experiment done in the daylight.

There is a difference in kinetics of 10°F capacitor discharge during daytime depending on the polarity of electrodes. This difference is small at low applied voltage of 0.25 V and increases with the increase in the initial voltage of a capacitor. There is a slight difference in kinetics of 10°F capacitor discharge during nighttime depending on polarity of electrodes. There is a difference in kinetics of 10°F capacitor discharge during daytime in darkness. The amplitude of variation is similar to the same in the first day. In all three examples, kinetics of a capacitor discharge depends on polarity of electrodes in a leaf of C. miniata due to electrical anisotropy of the leaf. We were also able to illustrate the plant memory of a “sunset.” Normally, the lights were switched off at 5:00 p.m., however in this experiment they were not switched off at that time. Any time from the morning and during a day until 4:00 p.m., the time dependencies of a 10°F capacitor discharge coincide. At 5:00 p.m. resistance in leaves starts to increase and at 7:00 p.m. it reaches the same parameters as at night even at continuous light. The capacitor discharge is fast during a daytime, but speed of the capacitor discharge decreases after 4:00 p.m. even under continuous light and reaches minimal value at 7:00 p.m. as in the dark during nighttime.

Circadian oscillators are components of the biological clocks that regulate the activities of plants in relation to environmental cycles and provide an internal temporal framework. Darwin⁸ found that leaves in Clivia move periodically: “A long glass filament was fixed to a leaf, and the angle formed by it with the horizon was measured occasionally during three successive days. It fell each morning until between 3:00 and 4:00 p.m., and rose at night. The smallest angle at any time above the horizon was 48°, and the largest 50°; so that it rose only 2° at night; but as this was observed each day, and as similar observations were nightly made on another leaf of a distinct plant, there can be no doubt that the leaves move periodically. The position of the apex when it stood highest was 0.8 of an inch above its lowest point.” The periodical movement of leaves in Clivia has the electrophysiological component. Two hours before the light was turned on, the speed of electrical discharge decreased due to increase of resistance in leaves, which is probably related to the closing of voltage gated channels. These results are very impressive: the biological clock in C. miniata recognizes the approaching of darkness after 4:00 p.m. even under constant light from 6.00 a.m. to 6.00 p.m. The circadian rhythm can be related to the difference in the membrane potentials during the day and night time, which was found in pulvini of different plants.^5,6,9

While in darkness the following day, the plant remembers the time and the rate of discharge drastically increases approaching the rate of the first day. However, the input resistance is the same as during day light in the beginning of the capacitor discharge and increases to night values during the discharge process. That means that the internal clock does change electrical conductance, but alone, without environmental clues (light), it cannot ideally generate the same values of day and night conductance. The plant needs additional environmental information, and then the properties of electrical circuits will be restored to the same conditions. These results demonstrate that the circadian clock can be maintained endogenously, probably involving electrochemical oscillators, which can activate or deactivate ion channels in biologically closed electrochemical circuits.

Glossary

Abbreviations:

C

capacitance

D

diode

R

resistance

U₀

the initial voltage of a capacitor

Volkov AG, Wooten JD, Waite AJ, Brown CR, Markin VS. Circadian rhythms in electrical circuits of Clivia miniata. J Plant Physiol. 2011;168:1753–60. doi: 10.1016/j.jplph.2011.03.012.