Introduction

In plants, APs—long-distance electrical signals—are induced by a variety of stimuli, e.g. light, shadow, wounding, touch, temperature, or chemicals (glutamate, potassium chloride) (Dziubinska 2003; Fromm and Lautner 2007; Król et al. 2010; Mousavi et al. 2013; Salvador-Recatala et al. 2014; Salvador-Recatala 2016). They are involved in fast leaf or trap closure, elongation, regulation of respiration and fertilization, and gene expression (Dziubinska 2003; Fromm and Lautner 2007; Król et al. 2010). The involvement of APs in fast leaf and trap closure in sensitive plants Mimosa pudica and Dionaea muscipula is the best-visible and evident effector response in plants (Sibaoka 1991; Krol et al. 2012). Besides the stimuli-induced APs, there are SAPs described 20 years ago by Zawadzki et al. (1995). They appear under lack of external stimuli and their source is unknown. Spontaneous APs (SAPs) have lately been observed in Solanum lycopersicum plants (Macedo et al. 2015) and in Helianthus annuus (Zawadzki et al. 1995), which is also known to exhibit intense and varied endogenous movements named circumnutation (CN). CNs are commonly observed in growing plants (Darwin and Darwin 1880); they vary during different stages of plant growth, and exhibit an ultradian, daily, and circadian rhythm as well as complex geometrical patterns (Buda et al. 2003; Charzewska and Zawadzki 2006). CN is an endogenous movement modulated by multiple stimuli, e.g. light, wounding, touch, temperature, chemicals, and gravity (Buda et al. 2003; Hayashi et al. 2004; Charzewska and Zawadzki 2006; Stolarz et al. 2010). The same stimuli also trigger APs, as mentioned above; therefore, the search for relationships between endogenous CN and excitation in plants is advisable. Our previous studies showed the effect of glutamate and lithium on CN. Glutamate-induced series of APs differ in vigorously and weakly circumnutating sunflower seedlings treated with lithium (Stolarz et al. 2015). Sunflowers with a lithium-induced decrease in the circumnutation intensity had an increased number of APs in Glu-induced series. Recently, we have shown that the ultradian oscillations of the membrane potential (depolarisation and hyperpolarisation) accompany CN as well (Kurenda et al. 2015). Additionally, the same ion channel and proton pump inhibitors modulating excitation in plants modulate CN as well (Millet and Badot 1996; Krol and Trebacz 2000; Krol et al. 2006; Kurenda et al. 2015).

The ability to respond to changing light environmental conditions and light stimuli is a key feature in a photosynthesising organism. Light intensity and photoperiod changes mainly affect not only photosynthesis but also organ movements and excitability (Zawadzki et al. 1995; Stankovic et al. 1998; Buda et al. 2003; Charzewska and Zawadzki 2006). The aim of this study was to investigate the relation between CN (Appendix S1) and SAPs in different light conditions using extracellular electrical measurements and time-lapse photography in H. annuus plants. To our knowledge, the influence of light on plant spontaneous excitation and its relation to CN movement have not been described so far. We have shown for the first time an ultradian rhythm of the long-distance SAPs beside the CN rhythm and SAPs accompanying the irregularity of the CN trajectory in very low light conditions. Additionally, it was shown that the number of SAPs is higher in freely circumnutating sunflower, compared with immobilized plants.

Materials and methods

Experimental plants

The studies were carried out on 20–30-day-old Helianthus annuus L. plants (PNOS, Ożarów Maz. Poland) grown in a vegetation room in pots filled with garden soil. They were watered with tap water and no other treatment was applied. A 16:8-h light:dark (04:00–20:00) photoperiod was maintained. The intensity of white light in the PAR range (Power Star HQT-T400W/DOSRAM GmbH, Munich, Germany) at the level of plant leaves was approximately 70 μmol m−2 s−1. The vegetation room was air-conditioned; the temperature was 24 ± 1 °C and humidity 50–70%. Approximately 20–35-cm-high plants with one or two pairs of developed leaves were taken for the experiments. To examine the relationship between CN and SAPs, freely circumnutating and immobilized sunflowers were used for measurements (Fig. 1a). The stem of the immobilized sunflowers was tied to a wooden pole for prevention of CN.

Fig. 1
figure 1

CN and SAPs in H. annuus. a Example of a H. annuus plant, top and side view (an example of a circumnutating plant is shown in Appendix S1), and electrode arrangement in extracellular SAP measurements. Four Ag/Cl electrodes (1, 2, 3, 4) were inserted across the stem. The reference (ref) electrode was inserted at the base of the stem. An immobilized sunflower—in the lower right corner. b Top view of CN regular (continuous light) and irregular (continuous very low light) stem apex trajectory, examples of approx. 24-h records (Appendix S2). c Examples of basipetally and acropetally transmitted SAPs along the stem

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Circumnutation measurements—time-lapse method

The H. annuus plant was filmed in a Faraday cage with simultaneous electrophysiological measurements. A top view monochromatic camera (Mintron MTV-1368CD, Mintron Enterprise Co. Ltd, Taipei, Taiwan) was used to record the trajectory movement of the stem apex. For filming in darkness, very dim green light with intensity <0.1 μmol m−2 s−1 PAR (25 1.I Pila, Poland) was used. Time-lapse images were recorded at a rate of one frame per minute or five minutes by Gotcha!Multicam software (Prescient System Inc., West Chester, PA, USA). The system was calibrated by a millimetre scale placed at the level of the pot. The time-lapse images were digitized using Circumnutation Tracker (Stolarz et al. 2014). Experimental points (coordinates x, y of the stem apex on the horizontal plane) were determined at 1- or 5-min intervals. The CN length (the distance that the organ apex covers during a single CN cycle) and the CN period (the time that the organ apex needs to trace a single circumnutation cycle) were calculated by Circumnutation Tracker software (Stolarz et al. 2014).

Electrophysiological measurements—extracellular method

The measurements were carried out in the laboratory in a Faraday cage. Changes in the electrical potential were measured with four extracellular Ag/AgCl electrodes (silver wire, 0.2 mm diameter, World Precision Instruments, Sarasota, FL, USA) inserted across the sunflower stem and then interfaced with a multi-channel data acquisition system composed of a differential amplifier (ME-4600 Meilhaus, Germany) and RealView software (Abacom, Germany). During the preparation of the Ag/AgCl electrodes, the silver wire was electrolytically coated with silver chloride. A reference electrode of the same type as the measuring electrodes was placed in the base of the stem. The long-distance SAPs pass by electrodes 1, 2, 3, 4 and the reference, basal electrode; therefore, the reference electrode is a relative reference electrode because they pass through it. The local, apical SAPs pass only by electrodes 1 and 2 and they never reach the reference, basal electrode; hence, the reference electrode in this case is an absolute reference electrode. The frequency of sample recording was 1 Hz. The arrangement of the electrodes on the sunflower is shown in Fig. 1a. The SAP interval, i.e. the time spacing between two subsequent SAPs was calculated.

Experimental variants

The measurements were carried out in three different light conditions: light/dark: 16 h of 25–40 μmol m−2 s−1/8 h darkness, continuous light: 25–40 μmol m−2 s−1, and continuous very low light: 5 μmol m−2 s−1 in PAR range white light (Power Star HQT-T400W/DOSRAM GmbH, Munich, Germany) and a temperature of 24 ± 1 °C. Freely circumnutating sunflowers and those with circumnutation prevented by stem immobilization were investigated.

Data analyses

Ten plants were investigated in each of the variants of the experiments and the results are presented as the mean ± SE. The data were analysed using Statistica ver. 12 software (StatSoft, Inc. 2014). The data set was first tested for normality using Shapiro–Wilk test and homogeneity of variance was assessed with Levene’s test. All data had non-normal distribution and unequal variance; therefore, non-parametric Kruskal–Wallis ANOVA was used. Multiple comparisons of the mean ranks for all groups (pairwise analysis) were used. The level of statistical significance for all tests was set at p < 0.05.

Results

The sunflower plants circumnutated (Appendix S1) in an approx. 2–3 h ultradian rhythm and the CN trajectory had a regular or irregular pattern (Fig. 1b; Appendix S2). Simultaneously, spontaneous electrical changes in a form of a 5–60 mV 1-min-long-lasting SAPs were recorded. A typical graphic example of SAPs transmitted basipetally and acropetally along the sunflower stem is shown in Fig. 1c. The CN length and period as well as the number and transmission direction of SAPs were different in the different light conditions. Vigorous CN or their arrest was observed (Fig. 2) and no SAPs or single acropetally or basipetally transmitted SAPs (Fig. 1c) and even series of APs were recorded. The CN and SAP parameters in light/dark cycles, continuous light (25–40 μmol m−2 s−1) and continuous very low light (5 μmol m−2 s−1) in freely circumnutating and immobilized sunflowers are shown in Table 1 (Supplementary Table 1 and Supplementary Table 2).

Fig. 2
figure 2

Examples of CN in different light conditions in freely circumnutating H. annuus. The plot is an x- or y-coordinate projection on the time course from a top-view recording. a Light/dark: 16 h of 25–40 μmol m−2 s−1/8 h darkness, light-on 04:00 light-off 20:00. b Continuous light: 25–40 μmol m−2 s−1. c Continuous very low light: 5 μmol m−2 s−1

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Table 1 Parameters of circumnutation (CN) and spontaneous action potentials (SAPs) in H. annuus in different light conditions
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Ultradian and daily rhythm of CN and SAPs in light/dark conditions

In light/dark conditions, the ultradian rhythm and daily CN pattern appeared; an example is shown in Fig. 2a. Small daily and large nocturnal CNs were observed (Fig. 3a). In those plants, approx. 9.8 ± 1 (N = 30) SAPs/day/plant were generated (Table 1) mainly during the dark period, which is shown in Fig. 3a. The number of SAPs emerging during 3-h intervals was calculated, and an evident daily rhythm was revealed. The greatest number of SAPs preceded the maximal-length CNs by approx. 3 h. It was found that the interval between SAPs in the light/dark conditions was on average 121 ± 9 min (n = 266) and was nearing the CN period, which was 143 ± 1 min (n = 293) (Table 1). In the immobilized sunflower, the number of SAPs/day/plant was halved significantly (4.6 ± 0.6 (N = 28) SAPs/day/plant) and the SAPs interval was doubled [237 ± 30 min (n = 118)]. In free CN, similar to the immobilized plants, approx. 60–70% of the SAPs were generated in the upper part of the plant (leaves and stem apex) and approx. 25% in the lower part of the plant. Beside acropetally or basipetally propagated SAPs, occasional acropetal–basipetal SAPs were recorded (10–20%, Table 1). The acropetal–basipetal SAPs originated from some place along the stem and propagated from that place acropetally and basipetally simultaneously, presumably through some phloem vessels or in one direction only, and later in the opposite direction along different vessels that were not excited before. Refractoriness would not allow basipetal transmission followed promptly by acropetal transmission (or vice versa). Propagation velocity of basipetally and acropetally propagated SAPs was approx. 8–19 cm min−1 (Supplementary Table 2). It is known that light-off and light-on evoke CN disturbance (Stolarz et al. 2008) and AP induction (Stankovic et al. 1998; Stahlberg et al. 2006; Król et al. 2010); therefore, to exclude this stimulus and ensure constant environmental conditions, we applied continuous light to study the relation between SAPs and CN.

Fig. 3
figure 3

Comparison of the CN length and number of SAPs in H. annuus. The number of SAPs for 3-day extracellular measurements was calculated at 3-h intervals (12:00–15:00, 15:00–18:00, 18:00–21:00…) in ten plants (column). The CN length is the length of the trajectory of CN, the plot is a moving average of a 3-h window from ten plants (line). a Light/dark: 16 h of 25–40 μmol m−2 s−1/8 h darkness, light-on 04:00 light-off 20:00. b Continuous very low light: 5 μmol m−2 s−1

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Vigorous CNs in continuous light but no SAPs

In continuous light (25–40 μmol m−2 s−1), the sunflower circumnutates vigorously in a regular elliptical manner (Fig. 2b; Table 1) but does not generate SAPs. This complete lack of SAPs under continuous light in the presence of vigorous CN indicates that SAP does not play a role in the basic mechanism generating CN and SAPs are not induced by CN at this light intensity. In the immobilized sunflower, SAPs were absent as well. To evoke the SAP phenomenon, continuous very low light was applied in the next experiments.

SAP ultradian rhythm and CN irregularity in continuous very low light conditions

In continuous very low light (5 μmol m−2 s−1), the CNs were weak and irregular as shown in Appendix S3 and Appendix S4 and in Table 1, Figs. 2c, 3b and 4. During the second day, there were many CN disturbances and during the third day CNs were usually arrested. The number of SAPs on average was approx. 4–5 SAPs/24 h/plant during all days of the experiments, even during the third day of the CN arrest. The relation of the CN length vs. the SAP number is shown in Fig. 3b. Examples of an irregular CN trajectory and simultaneous SAP emergence are shown in Fig. 4a–d. Many SAPs emerge during maximum stem bending. The examples of SAP occurrence followed by CN direction changes are shown in Fig. 4e–h and, respectively, in Appendix S5 and Appendix S6. It is noticeable, therefore, that in very low light SAPs accompany the irregular CNs. Another type of stem movement changes is the 2–4-mm stem “torsion” shown in video Appendix S7 and Appendix S8 (marked by a red point) accompanied by SAPs propagating acropetally along the stem. “Torsion” is a sudden twitch against the background of the slow circumnutation movement of the plant.

Fig. 4
figure 4

The CN and appearance of SAPs in H. annuus plants in very low light (5 μmol m−2 s−1). The plots are the x- and y-coordinate projection on the time course (a, c, e, f) or the top view of the CN trajectory (b, d, f, h). Black point marks the moment of SAP appearance. The arrow marks the beginning of CN, dashed line—trajectory of CN after SAP. a–d Examples of weak and irregular CN associated with SAPs passing along the stem motor region. Highly excitable plants are presented with more SAPs than the average SAP number. Examples of circumnutating plants are shown in Appendix S3 and Appendix S4, respectively. e–h Examples of CN direction changes after acropetal SAP passing along the motor region. Examples of circumnutating plants are shown in Appendix S5 and Appendix S6, respectively

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It was found that in very low light conditions in a free-CN sunflower the number of SAPs/day/plant was 4.8 ± 0.6 (N = 29) and the interval between all SAPs was on average 259 ± 22 min (n = 120) and these were similar as in the immobilized sunflower (Table 1). In the freely CN plants, two kinds of series of SAPs were observed: ultradian series of long-distance transmitted SAPs and series of local SAPs. The series consisting of 2–7 SAPs with, on average, 159 ± 8 min (n = 75) intervals between them were recorded and named ultradian series of SAPs (Table 2). Examples of the ultradian SAPs are presented in Fig. 5. The intervals between the SAPs were the same as the CN period, i.e. 171 ± 6 min (n = 152) (Table 1). The ultradian SAPs propagated throughout whole stem; they were registered by electrodes 1, 2, 3, 4 and the reference electrode (Fig. 5). In low-light condition, 73% of APs were generated in the upper part of the sunflower (leaves and stem apex) and transmitted basipetally and 20% of APs were generated in the lower part of the plant and transmitted acropetally. Additionally, during the third day of the experiment, series of locally propagated APs appeared in some plants as shown in Fig. 6 and Table 2. They persisted on average for 66 ± 7 min and consisted of 18 ± 2 SAPs (n = 15 series); there was a correlation between the duration of the series and the number of APs (Fig. 7). During the approx. 1-h-long local series, the sunflower generated SAPs at ca. 3–4-min intervals.

Table 2 Spontaneous excitation in H. annuus under very low light (5 μmol m−2 s−1)
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Fig. 5
figure 5

Ultradian series of long-distance-propagated SAPs in H. annuus plants in very low light (5 μmol m−2 s−1). Examples of traces from an extracellular electrical recording. Electrode (1, 2, 3, 4) arrangement shown in Fig. 1a. Arrow up—acropetally transmitted SAP, arrow down—basipetally transmitted SAP. a, b Ultradian, long-distance-propagated SAPs, c, d Ultradian, long-distance-propagated SAPs and series of local SAPs. Examples of a local series of SAPs are marked by asterisks

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Fig. 6
figure 6

Series of local SAP H. annuus plants emerging during the third day of very low light (5 μmol m−2 s−1). Electrode arrangement shown in Fig. 1a. Arrow up—acropetally transmitted SAP, arrow down—basipetally transmitted SAP

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Fig. 7
figure 7

SAPs and CN in H. annuus in continuous very low light. The relation between the duration of local series and the number of SAPs. In eight plants, 15 series were observed during the third day under very low light

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When transferred back to the vegetation room, plants kept for 3 days in the very low light began flowering after 2 months.

Discussion

SAP-associated light/dark sensing

Active movements in plants are a way of adaptation to the changing light conditions in the environment. The examples include, e.g. the widely spread phenomena of tropism and nastic movements as in the pulvinus-driven leaf movement after illumination or darkening in Samanea, Phaseolus, Oxalis, and Desmodium (Moshelion et al. 2002; Moran 2007). H. annuus is a common sun-tracing plant, which moves its stem apex to follow the relative position of the sun (Vandenbrink et al. 2014) and shows an ultradian, daily and circadian rhythm of CN and CN disturbance after illumination and darkening (Buda et al. 2003; Charzewska and Zawadzki 2006; Stolarz et al. 2008). There are reports that AP is a light/dark-guided signal in plants (Król et al. 2010). Lately, in S. lycopersicum plants the highest number of SAPs was observed in the early morning (Macedo et al. 2015). Shade and darkness-induced APs were reported also in H. annuus (Stankovic et al. 1998; Stahlberg et al. 2006). Here we have checked the effect of light on plant spontaneous APs and their relation to CN. The light conditions applied here reflect the conditions present in the natural environment and imitate the effect of prolonged shading or cloudy days on plants. The 3-day-long very low light conditions applied here constituted well-tolerated stress because after the treatment plants underwent typical vegetative and generative development after the return to the vegetation room. It has been shown here using long-lasting simultaneous time lapse and extracellular recordings that stem movements (CN) and spontaneous excitation (SAPs) in sunflower plants are light dependent (Table 1). The CN study has shown a regular elliptical or irregular CN trajectory pattern (Fig. 1b; Appendix S2). Regular ellipses dominated under continuous light while irregularity was observed under light/dark and very low light conditions. The CN irregularity is illustrated in Appendix S3 and Appendix S4 and Fig. 4a–d, respectively. This regular and irregular pattern is related to the absence of SAPs and presence of many SAPs, respectively. The greatest number of SAPs preceded the maximal-length CNs by 3 h (light/dark, Fig. 3a) and many SAPs appeared at the absence of CNs in very low light (Figs. 3b, 4a, c) and in immobilized plants (Table 1). The daily rhythm of the SAPs number in light/dark condition and the lack of SAPs under continuous light (Table 1) indicate that the light conditions influence the number of SAPs. This is also consistent with the opinion that stronger light decreases excitability (Król et al. 2010). The difference in the number of SAPs in different light conditions (Tables 1, 2) should be a suggestion about the information-encoding role of APs in plants.

Spontaneous action potentials

The circumstances of the occurrence of SAPs and their function are still unclear. It was shown here that SAPs appeared singly or in groups forming ultradian series of long-distance SAPs (Fig. 5) and spontaneous approx. 1-h series of local SAPs (Fig. 6) (Table 2). Here we have shown for the first time the slow approx. 2–4-h ultradian rhythm of SAPs, beside the slow ultradian CN rhythm. Additionally, another series of local SAPs (Fig. 6) at 3–4-min intervals was recorded and they resembled those recorded after glutamate injection (Stolarz et al. 2010, 2015). This confirmed the assumption that there are spontaneous series of APs in plants and that an AP frequency code may exist in plants (Favre et al. 1999; Paszewski et al. 1982). In our study, we observed acropetally or basipetally transmitted SAPs (Fig. 1c). It is probable that SAPs are propagated along the vascular bundle similar to stimuli-induced APs (Dziubińska et al. 2001; Dziubinska 2003; Fromm and Lautner 2007; Salvador-Recatala et al. 2014; Hedrich et al. 2016). Propagation velocity was approx. 10–20 cm min−1, typical for sunflower (Zawadzki et al. 1995). Additionally, occasional acropetal–basipetal SAPs were also recorded. They were similar to SAPs described previously by Zawadzki et al. (1995) and APs evoked by glutamate (Stolarz et al. 2010).

SAPs vs. CN

Zawadzki et al. (1995) found and our present study confirmed the SAP phenomenon, whose physiological function in the sunflower is not determined. Herein, CN changes accompanied by SAPs were revealed. A daily rhythm of SAPs and CNs (Fig. 3a) and the ultradian interval of the SAP and CN period (Table 1) were shown. Additionally, the example in Fig. 4 shows that SAPs accompany CN irregularity and a decrease in the CNs and even changes in the CN trajectory direction from cw to ccw or vice versa. The sunflower stem “torsion” associated with acropetal SAP passing throughout the motor region was presented in the films (Appendix S7 and Appendix S8). The AP-associated elongation/contraction in H. annuus (Stankovic et al. 1998) and the growth rate inhibition in Luffa cylindrica (Shiina and Tazawa 1986) reported previously support the suggestion that the ultradian SAPs passing throughout the motor region of the stem could disturb a growth-dependent CN. Simultaneously, experiments with immobilized plants showed that freely circumnutating plants had more SAPs than the immobilized plants. This showed that CN increased the number of SAPs in the sunflower plants. The experiments with inhibited CN (free-CN and immobilized sunflowers in continuous very low light) demonstrate that CNs are not necessary to appearance of SAPs. On the other hand, sunflower immobilization in light/dark conditions halved the number of SAPs (the number was similar as in the free-CN and immobilized sunflower in continuous very low light); thus, CN could increase the number of SAPs. This suggested that in light/dark conditions CN could evoke APs.

The present investigation shows that external conditions can modulate the number of SAPs in H. annuus plants and there is a complex relation between the CN and SAP phenomena and light conditions. The answers to the question if the CN changes can evoke APs and if APs could evoke CN changes (as an effector response) need a future investigation.

Conclusions

In the present work, it has been shown that there is the ultradian SAP rhythm beside the CN rhythm and the number of SAPs and CN trajectory changes depends on light conditions. In continuous light, no SAPs under vigorous regular CN are observed. In continuous very low light conditions, the irregularity of the CN trajectory is accompanied by SAPs. Presented results showed that SAP appearance and CN changes play a role in adaptation to very low light conditions in H. annuus plants. We believe that in future studies SAPs will be found in other plant species and other stress conditions and that the CN changes could be a helpful phenomenon for future investigations of the role of long-distance electrical signals in plant behaviour.

Author contribution statement

MS designed and carried out the experiments, collected and analysed the results, and wrote the manuscript. HD helped in the analysis of the results and editing the manuscript.