Evidence for the Involvement of Electrical, Calcium and ROS Signaling in the Systemic Regulation of Non-Photochemical Quenching and Photosynthesis

Abstract

In contrast to the function of reactive oxygen species, calcium, hormones and small RNAs in systemic signaling, systemic electrical signaling in plants is poorly studied and understood. Pulse amplitude-modulated Chl fluorescence imaging and surface electrical potential measurements accompanied by pharmacological treatments were employed to study stimuli-induced electrical signals in leaves from a broad range of plant species and in Arabidopsis thaliana mutants. Here we report that rapid electrical signals in response to a local heat stimulus regulate systemic changes in non-photochemical quenching (NPQ) and PSII quantum efficiency. Both stimuli-induced systemic changes in NPQ and photosynthetic capacity as well as electrical signaling depended on calcium channel activity. Use of an Arabidopsis respiratory burst oxidase homolog D (RBOHD) mutant (rbohD) as well as an RBOH inhibitor further suggested a cross-talk between ROS and electrical signaling. Our results suggest that higher plants evolved a complex rapid long-distance calcium-dependent electrical systemic signaling in response to local stimuli that regulates and optimizes the balance between PSII quantum efficiency and excess energy dissipation in the form of heat by means of NPQ.

Keywords: Calcium signaling; Chlorophyll fluorescence; Electrical signals; Photosynthesis; Reactive oxygen species; Systemic acquired acclimation.

Fig. 1

Changes in Chl a fluorescence of a dandelion leaf following heat stimulation. (a) Spatiotemporal changes of NPQ, Y(II) and qP assessed by Chl fluorescence imaging. The arrow indicates the area stimulated for 1 s with a flame-heated metal wire. Time is shown in min:s format (heat stimulation was performed at 0:00). Scale bar = 1 cm. The false color scale represents values of assessed parameters. (b) NPQ, (c) Y(II) and (d) qP changes in stimulated (Heat stress) and non-stimulated (Control) leaves measured by Chl fluorescence imaging at representative areas [shown in (a) as a black circle] approximately 1 cm from the stimulated region or the region touched with the unheated wire in the control measurement, respectively. Representative data of five independent experiments are shown. Persistence of (e) NPQ and (f) Y(II) changes caused by a heat stress. Additional Chl fluorescence measurements were performed, and NPQ and Y(II) values were recorded up to 55 min after stimulation. Values are means ± SEM (n = 5). Asterisks indicate a significant difference in comparison with the control (**P < 0.01, ***P < 0.001; P-values calculated using Tukey’s test).

Fig. 2

Leaf surface electrical potential changes following heat stimulation of the tip of the leaf. (a and b) Effect of lanthanum chloride (LaCl₃) on electrical signal propagation. Representative potentials recorded with two electrodes placed on the same leaf irritated by heat: the proximal electrode on the area not treated directly (Non-treated) and the distal electrode on the area directly treated by solution: (a) without inhibitor (Mock) or (b) with LaCl₃. (c) Comparison of electrical signals in Arabidopsis and dandelion. Representative potentials recorded on Arabidopsis and dandelion leaves. (d) Effect of Arabidopsis RBOHD gene mutation on electrical signal propagation. Representative potentials recorded on rbohD plants vs. the WT. The arrows indicate the moment of heat stimulation of the tip of the leaf for 1 s by the flame of a lighter. Statistical significance of observed changes is shown in Supplementary Fig. S1.

Fig. 3

Changes in Chl a fluorescence of a dandelion leaf treated with a calcium channel blocker (LaCl₃) following heat stimulation. (a) Spatiotemporal changes of NPQ, Y(II) and qP assessed by Chl fluorescence imaging. Arrow indicates the area stimulated for 1 s with a flame-heated metal wire. The black rectangle shows the area treated with LaCl₃. The time is shown in min:s format (heat stimulation was performed at 0:00). Scale bar = 1 cm. The false color scale represents values of assessed parameters. (b) NPQ, (c) Y(II) and (d) qP changes measured by Chl fluorescence imaging at representative areas [shown in (a) as black circles] of treated (Area 2 represented by circle 2) and untreated (Area 1 represented by circle 1) parts of the leaf at a similar distance from the stimulated region. Representative data of five independent experiments are shown. The statistical significance of the observed changes is shown in Supplementary Fig S1.

Fig. 4

Changes in Chl fluorescence of a dandelion leaf treated with a RBOHD inhibitor (DPI) following heat stimulation. (a) Spatiotemporal changes of NPQ, Y(II) and qP assessed by Chl fluorescence imaging. The arrow indicates the area stimulated for 1 s with a flame-heated metal wire. The black rectangle shows the area treated with DPI. Time is shown in min:s format (heat stimulation was performed at 0:00). Scale bar = 1 cm. The false color scale represents values of assessed parameters. (b) NPQ, (c) Y(II) and (d) qP changes measured by Chl fluorescence imaging at representative areas [shown in (a) as black circles] of treated (Area 2 represented by circle 2) and non-treated (Area 1 represented by circle 1) parts of the leaf at a similar distance from the stimulated region. Representative data of five independent experiments are shown.

Fig. 5

Changes of the Chl fluorescence in dandelion leaf treated with a photosynthetic electron transport inhibitor (DCMU). (a) Spatiotemporal changes of NPQ, Y(II) and qP assessed by Chl fluorescence imaging. The arrow indicates the area stimulated for 1 s with a flame-heated metal wire. The black rectangle shows the area treated with DCMU. Time is shown in min:s format (heat stimulation was performed at 0:00). Scale bar = 1 cm. The false color scale represents values of assessed parameters. (b) NPQ, (c) Y(II) and (d) qP changes measured by Chl fluorescence imaging at representative areas shown in (a) as a black circles (Area 1 and Area 2 represented by circles 1 and 2, respectively) on the two sides of the region treated with DCMU. Representative data of five independent experiments are shown.