Osmotic and Salt Stresses Modulate Spontaneous and Glutamate-Induced Action Potentials and Distinguish between Growth and Circumnutation in Helianthus annuus Seedlings

Abstract

Action potentials (APs), i.e., long-distance electrical signals, and circumnutations (CN), i.e., endogenous plant organ movements, are shaped by ion fluxes and content in excitable and motor tissues. The appearance of APs and CN as well as growth parameters in seedlings and 3-week old plants of Helianthus annuus treated with osmotic and salt stress (0-500 mOsm) were studied. Time-lapse photography and extracellular measurements of electrical potential changes were performed. The hypocotyl length was strongly reduced by the osmotic and salt stress. CN intensity declined due to the osmotic but not salt stress. The period of CN in mild salt stress was similar to the control (~164 min) and increased to more than 200 min in osmotic stress. In sunflower seedlings growing in a hydroponic medium, spontaneous APs (SAPs) propagating basipetally and acropetally with a velocity of 12-20 cm min^-1 were observed. The number of SAPs increased 2-3 times (7-10 SAPs 24 h^-1plant^-1) in the mild salt stress (160 mOsm NaCl and KCl), compared to the control and strong salt stress (3-4 SAPs 24 h^-1 plant^-1 in the control and 300 mOsm KCl and NaCl). Glutamate-induced series of APs were inhibited in the strong salt stress-treated seedlings but not at the mild salt stress and osmotic stress. Additionally, in 3-week old plants, the injection of the hypo- or hyperosmotic solution at the base of the sunflower stem evoked series of APs (3-24 APs) transmitted along the stem. It has been shown that osmotic and salt stresses modulate differently hypocotyl growth and CN and have an effect on spontaneous and evoked APs in sunflower seedlings. We suggested that potassium, sodium, and chloride ions at stress concentrations in the nutrient medium modulate sunflower excitability and CN.

Keywords: action potential; circumnutation; electrical transmission; electrophysiology; osmotic potential; plant movement; salt stress; signaling.

Figure 1

Parameters of growth and CN of Helianthus annuus seedlings under osmotic and salt stresses. Bars represent mean ± SE. The number of plants is indicated in parentheses. The data were tested for normality distribution using the Shapiro-Wilk test. The the non-parametric Mann-Whitney U-test for pairwise analysis (A,C) and Tukey test (B) were used to assess the statistical difference between the control plants and each sunflower group growing in different nutrients; p-value ranges are marked by asterisks: ^***p < 0.001,^**p < 0.01,^*p < 0.05. (A) Changes in hypocotyl length, data normally distributed, unequal variance (Levene's test p = 0.000109), Kruskal-Wallis ANOVA (Chi square = 27.39 df = 8 p = 0.0006). (B) Changes in hypocotyl CN intensity. The distance covered by the hypocotyl apex during one cycle was used to calculate the CN rate. CN intensity was the rate of CN divided by hypocotyl length. Data normally distributed, equal variance Levene's test (p = 0.30), one-way ANOVA (SS = 0.2149 df = 8 F = 10.015 p = 0.0000). (C) Changes in hypocotyl CN period. The CN period was the time required by the hypocotyl apex to trace a single CN cycle (time between two subsequent maximum northward bends of the hypocotyl). Data normally distributed, unequal variance (Levene's test p = 0.000053), Kruskal-Wallis ANOVA (Chi square = 24.55 df = 8 p = 0.0018).

Figure 2

Spontaneous action potentials and glutamate induced series of action potentials in Helianthus annuus seedlings under osmotic and salt stresses. (A) Helianthus annuus seedlings, electrode arrangement (1, 2, ref—reference electrode) and site of glutamate injection (i). (B) Example of recordings of spontaneous action potentials during 1 day (also shown in Supplementary Video S2). In the parentheses, the time of SAP appearing. (C) Example of recordings of acropetally and basipetally propagating spontaneous action potentials. (D) Example of recordings of action potential series after glutamate (Glu) injection into the hypocotyl base of seedlings growing under 80 mM KCl (upper) as well as 160 mM NaCl nutrient solutions (lower). Data details are presented in Tables 1, 2.

Figure 3

Osmotically and potassium chloride-induced series of action potentials in 3-week old Helianthus annuus. (A) Helianthus annuus plants, electrode arrangement (1, 2, 3, 4, ref—reference electrode) and site of solution injection (i). (B) Example of recordings of action potential series after D-sorbitol 500 mOsm (500 mM) injection into the stem base. (C) Example of recordings of action potential series after KCl 500 mOsm (250 mM) injection into the stem base. Data details are presented in Table 3.