In recent years, the effect of heat-induced electrical signalling on plant photosynthetic activity has been demonstrated for many plant species. However, the underlying triggers of the resulting transient inhibition of photosynthesis still remain unknown. To further investigate on this phenomenon, we focused in our present study on soybean (Glycine max L.) on the direct effect of signal transmission in the leaf mesophyll on conductance for CO2 diffusion in the mesophyll (gm) and detected a drastic decline in gm following the electrical signal, whereas the photosynthetic electron transport rate (ETR) was only marginally affected. In accordance with the drop in net photosynthesis (AN), energy dispersive X-ray analysis (EDXA) revealed a shift of K, Mg, O and P on leaf chloroplasts. Control experiments under elevated CO2 conditions proved the transient reduction of AN, ETR, the chloroplast CO2 concentration (Cc) and gm to be independent of the external CO2 regime, whereas the effect of the electrical signal on stomatal conductance for CO2 (gs) turned out much less distinctive. We therefore conclude that the effect of electrical signalling on photosynthesis in soybean is triggered by its immediate effects on gm.
Plants perceive environmental stimuli through a number of signalling pathways, which comprise of short- and long-distance signals of variable duration (Foyer & Noctor 2000; Knight & Knight 2001; Baier et al. 2006; Cutler et al. 2010). Much of our current knowledge is based on chemical signalling, including reactive oxygen species (ROS) waves (Mittler et al. 2011), involved in plant responses to abiotic and biotic stresses, whereas other types of signals such as electrical signals are still poorly understood (Davies 2004; Fromm & Lautner 2007). As electrical signals can travel with high velocity over short to long distances, they are likely candidates for an important role in the immediate plant response to sudden and adverse environmental stimuli (e.g. wounding). Two types of electrical signals can be distinguished in plants: (1) the action potential (AP) with its all-or-nothing character of unchanged amplitude and shape (after passing the threshold of stimulus), depolarizing the cell membranes transiently; and (2) the variation potential (VP) with its variable range of membrane depolarization and retarded repolarization, decreasing in amplitude and speed with distance (Fromm & Lautner 2007). Both types of electrical signals have been described to elicit a number of plant responses. For instance, APs are involved in leaf movements after mechanical stimulation (Fromm & Eschrich 1988a,b) and stimulation of respiration after pollination (Fromm, Hajirezaei & Wilke 1995), whereas VPs have been shown to play a predominant role in the wounding response (Koziolek et al. 2004; Lautner et al. 2005; Grams et al. 2009). However, the effects of stress-induced electrical signals on physiological functions are still poorly understood.
Recent data on heat-induced electrical signalling suggest a strong link with photosynthetic activity (Herde et al. 1999; Koziolek et al. 2004; Lautner et al. 2005; Grams et al. 2009). In these studies, rapid changes of leaf photosynthetic activity after heat stimulation (flaming) have been associated to changes in membrane potential, resulting from electrical signals that originate from the site of heat stimulus. Most remarkably, heat-induced electrical signalling (VP) resulted in transient inhibition of photosynthesis in all cases studied so far (Koziolek et al. 2004; Lautner et al. 2005; Grams et al. 2009), suggesting that this a common response of many plant species. However, the distance of signal transmission varied between a few centimetres, when measured at a single leaf (Grams et al. 2009), and more than 10 cm when measured on leaves other than the heat stimulated (Lautner et al. 2005). Thus, electrical signals that influence photosynthesis can travel through various plant tissues/organs and cover a broad range of distances, perhaps dependent on structural factors associated to growth form (trees, shrubs or herbs). Changes in chloroplast pH (i.e. across thylakoids) and/or in membrane/cell wall conformation have been speculated to result from heat-induced electrical signals and hence impair photosynthesis (Lautner et al. 2005; Grams et al. 2009). Moreover, changes of apoplastic pH can affect electrical signalling as well as photosynthesis and stomatal conductance (gs) directly, as shown by varying the CO2 concentration around bean and tomato leaves (Hedrich et al. 2001). However, the physiological basis of such photosynthetic changes, in particular within the mesophyll, remains still unclear.
Apart from the commonly observed transient impairment of photosynthesis after heat stimulation, the conductivity of stomata was either increased (maize) (Grams et al. 2009), decreased (mimosa) (Koziolek et al. 2004) or not affected at all (poplar) (Lautner et al. 2005). These differences between photosynthetic and stomatal responses to heat suggest independent or differentially regulated processes within the mesophyll and at the sites of stomata, which may be species or growth-form related. Moreover, photosynthetic electron transport and net CO2 fixation responded differently to heat-induced electrical signalling in these studies, the former being less responsive than the later. This result, together with the fact that photosynthesis changes are apparently independent of stomatal movements, leads us to hypothesize that such changes are largely mediated by electrical-induced decrease of leaf mesophyll conductance to CO2 (gm).
gm has been shown to be finite and highly variable, and can change as fast as gs to varying environmental conditions (Flexas et al. 2008; Evans et al. 2009; Galle et al. 2009). Although gm responds dynamically to various abiotic stresses, as shown in a number of studies on different plant species, its regulation is still poorly understood (Flexas et al. 2012). The factors controlling gm have been associated with the mesophyll surface area exposed to the intercellular airspace (Terashima, Miyazawa & Hanba 2001; Tholen et al. 2008), carbonic anhydrase (Gillon & Yakir 2000) and aquaporins (Uehlein et al. 2003, 2008; Flexas et al. 2006), but nothing is known concerning the signalling cascade leading to rapid variations of gm. Nonetheless, adjustment of internal CO2 supply for photosynthesis via the regulation of gm provides plant a high degree of flexibility to cope with stressful conditions and may also be helpful for rapid recovery after relief of stress. The up-to-now observed effects of electrical signals on photosynthesis and the knowledge that gm responds to environmental changes very rapidly (within seconds) suggest that electrical signals can be one of the triggers of gm variations. With regard to this hypothesis, the present study analyses the photosynthetic responses to heat-induced electrical signals, dissecting diffusional (gs, gm) and biochemical (photochemistry) traits of leaf photosynthesis in soybean (Glycine max L). Additionally, we explore whether changes in VPs at the leaf level are associated with changes of specific ions (Mg, K, P, O) around the chloroplast envelope, which could be identified as intermediates in the signalling cascade from electrical signals to gm modifications.
MATERIALS AND METHODS
Growth conditions and measurements of online photosynthetic CO2 discrimination (New Mexico)
Plants were grown from seed in a roof-top greenhouse from March through May with day/night temperatures 27/24 °C, relative humidity 20–40%. Plants were watered daily via a drip system and fertilized three times a week with a commercial fertilizer (Jacks 20-20-20, J.R. Peters, Inc., Allentown, PA, USA).
Online measurements of photosynthetic 13CO2 discrimination from a combined IRGA-Tunable Diode Laser (TDL) system (Flexas et al. 2006; Barbour 2007) were used to calculate gm and chloroplast Co2 concentration (Cc) using the equations of Evans et al. (1986). The TGA-100 (Campbell Scientific, Logan, UT, USA) measures absolute concentrations of 13CO2 and 12CO2 at a frequency of 10 Hz from dry air before and after exposure to a photosynthesizing leaf for calculating the isotopic composition (δ13C) of sampled air with a precision of 0.05–0.09‰. After reaching steady-state gas exchange, an adjacent leaflet was burned and the isotopic composition of the air exiting the leaf chamber was sampled continuously for 16 min before returning to calibration tanks and the air entering the chamber. Calibrations throughout each measurement day were fit with a spline to generate time-specific gains and offsets for each 10 Hz data point between calibrations and the 10 Hz data are presented as 30 s running averages. Measurements of photosynthesis and stomatal conductance were made using IRGAs in a LI-6400 portable gas exchange system (Li-Cor Inc., Lincoln, NE, USA) with the standard 6 cm2 red/blue LED light leaf chamber. CO2 concentrations were converted to partial pressures (atmospheric pressure was approximately 84.3 kPa) for isotopic point-based calculations of gm (Bickford et al. 2009) at a photosynthetically active radiation (PAR) of 500 µmol m−2 s−1, leaf temperature of 25 °C, reference concentration of 700 µmol mol−1 CO2 and a δ13C of approximately −4‰.
Growth conditions and experimental set up (Hamburg, Germany)
Soybean (Glycine max L.) plants were grown in 5 L pots containing horticulture substrate mixed with perlite and vermiculite (1:1:1) inside a growth chamber under controlled conditions (day/night air temperature of 25 °C/21 °C; 12 h at PAR of ca. 500 µmol m−2 s−1; 40–60% humidity). Plants were irrigated every second day and fertilized once a week with half-strength Hoagland's solution. Measurements of electrical signals and gas exchange parameters in leaves were carried out under laboratory conditions inside a Faraday cage on 4 to 6-week-old plants. In all cases, the second youngest, fully developed trifoliate was used for measurements. Electrical signals were induced by flaming one leaflet and measured in a different leaflet of the same trifoliate (Fig. 1). It should be noted that flaming may not have a significant ‘regulating’ role for photosynthesis in nature, though we have chosen this method, because it easily and reproducibly induces VPs in many species (Fromm & Lautner 2007). Simultaneous recordings of electrical and physiological signals in leaves after heat stimulation were carried out on the two lateral leaflets of the trifoliate, as depicted in Fig. 1. The central leaflet was used for the heat stimulus (flaming). To block the electrical signal, ice was placed at the basal part of this leaf before heat stimulation.
Leaf gas-exchange and chlorophyll a fluorescence
Photosynthetic net CO2 assimilation (AN), gs and chlorophyll (Chl) a fluorescence were measured simultaneously with an open infrared gas-exchange analyser system (LI-6400; LI-COR Inc.) equipped with a leaf chamber fluorometer (LI-6400-40, LI-COR Inc.). Recordings of AN, gs and Chl a fluorescence (apparent and maximum fluorescence in the light, Fs and Fm′) at 30 s intervals were started at least 10 min before the heat stimulus and continued until all parameters were restored to pre-stressed levels (at least for 30 min). The experimental set-up for each plant took a minimum 1 h. With regard to the prevailing growth conditions all records were taken at a PAR of 500 µmol m−2 s−1 (provided by the light source of the LI-6400 with 10% blue light) and at 25 °C (fixed block temperature of the LI-6400). The CO2 concentration in the LI-6400 leaf chamber (Ca) was set to 700 µmol CO2 mol−1 air to match outside (laboratory) concentration of CO2 during measurements (‘ambient CO2’) and to minimize CO2 leakage across the gaskets of the LI-6400 leaf chamber. In case of ‘elevated CO2’ measurements, the Ca was set to 2000 µmol CO2 mol−1 air, while also the CO2 concentration around the plant (inside the Faraday cage) was increased to about 2000 µmol CO2 mol−1 air. The relative humidity of the incoming air ranged around 40%. Corrections for leakage of CO2 into and out of the leaf chamber of the LI-6400 have been applied to all gas-exchange data, as described by Flexas et al. (2007a).
From the fluorescence measurements, the actual quantum efficiency of the photosystem II (PS II)-driven electron transport (ΦPSII) was determined according to Genty, Briantais & Baker (1989) as
where Fs is the steady-state fluorescence in the light (PAR 500 µmol m−2 s−1) and Fm′ is the maximum fluorescence obtained with a light-saturating pulse (∼8000 µmol m−2 s−1). As ΦPSII represents the number of electrons transferred per photon absorbed by PSII, the electron transport rate (ETR) can be calculated as
where the term α includes the product of leaf absorptance and the partitioning of absorbed quanta between photosystem I and PSII.
The α (0.47) was determined from the slope of the relationship between ΦPSII and ΦCO2 (i.e. the quantum efficiency of gross CO2 fixation), which was obtained by varying light intensity under non-photorespiratory conditions in an atmosphere containing <1% O2 (Valentini et al. 1995).
From combined gas-exchange and Chl a fluorescence measurements, the gm was estimated according to Harley et al. (1992) as
where Γ* is the O2 photo-compensation point and Rd is day respiration. AN and CO2 concentration of the intercellular airspace (Ci) were obtained from gas-exchange measurements. A value of 45.7 µmol mol−1 for the CO2 compensation point under non-respiratory conditions (Γ*) was calculated from its ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) specificity factor ‘τ’ of 82 (Jordan & Ogren 1981, 1983) and according to Brooks & Farquhar (1985): τ = 0.5O / Γ* (O denotes for the oxygen molar fraction at the oxygenation site). The estimation of Γ* according to the method of Laisk (1977) from measurements of AN − Ci curves under different light intensities (Von Caemmerer 2000) revealed similar values. From these latter curves, the Rd (2.8 µmol CO2 m−2 s−1) was obtained.
Calculated values of gm were used to convert Ci into Cc according to the following equation:
Chlorophyll fluorescence imaging
Spatiotemporal variations of the quantum yield of energy conversion at PSII (ΦPSII) were assessed with a Dual-PAM 100 (Walz, Effeltrich, Germany) on attached leaves of soybean according to Schreiber, Schliwa & Bilger (1986) and Genty et al. (1989). Pre-illuminated leaves (PAR 150 µmol m−2 s−1) were stimulated by heat, and the fluorescence signals were followed continuously while saturating light pulses were applied every 10 s (for at least 30 min).
For intracellular measurements of the membrane potential, a microelectrode was filled with 100 mm KCl and inserted into a mesophyll cell of a leaflet. The reference electrode was immersed together with the cut end of a neighbouring leaflet into artificial pond water (APW, composed of 1.0 mm NaCl, 0.1 mm KCl, 0.1 mm CaCl2 and 1.0 mm MES, adjusted with Tris to pH 6.0). Both electrodes were connected to a differential amplifier (World Precision Instruments, Model Duo 773, Sarasota, FL, USA). After microelectrode insertion, heat stimulation was performed by the flame of a lighter (ca. 1000 °C) for the duration of 1 to 2 s. All measurements were recorded by a chart recorder.
Energy dispersive X-ray analysis (EDXA)
Lateral leaflets were taken 3, 5 and 9 min after heat stimulation of the central leaflet as well as of non-stimulated control plants (four plants per treatment) and immediately frozen in liquid nitrogen. After the following freeze-drying process, the epidermal layer of the samples was cut off and the mesophyll tissue was coated with carbon before being examined by a scanning electron microscope (SEM, S-520 Hitachi, Tokyo, Japan) equipped with an energy dispersive X-ray device (EDX eumex Si(Li)-detector, EUMEX GV, Mainz, Germany). Single-point measurements on chloroplasts were performed at 10 keV excitation energy, which excites a measurement area of <2 µm in diameter Element concentration provided by the analysis data represents the atomic ratio of the analysed ions in percent, rather than absolute quantities.
Typical response curves of electrical membrane potential are compared with net photosynthetic changes in a leaflet of a soybean trifoliate distal to the heat stimulation (i.e. flaming the central leaflet, see Fig 1) to illustrate the short-term responses of photosynthesis and membrane potential of mesophyll cells to heat stimulation (Fig. 2). These heat-induced responses were carried out under ambient and elevated CO2 conditions (Fig. 2a,b). Elevated CO2 concentration was used to alkalize the leaf apoplast, and because it is known to affect gm and AN (Flexas et al. 2007b) as well as electrical signalling (Hedrich et al. 2001). A detailed description of changes in other relevant leaf gas exchange parameters can be found below. First and irrespectively of CO2 conditions, membrane potential hyperpolarized after about 0.5 min and peaked at around 1.5 min, followed by the drop of AN around 1.5 min after the heat stimulus (Fig. 2a,b). The membrane potential hyperpolarized by about 20 mV and remained at this level for another 1 to 2 min, thereafter it decreased again to reach pre-stressed values. According to the distance of about 8 to 9 cm between heat stimulus and signal detection, the induced electrical signal travelled at least at a speed of 3–4 mm s−1. AN started to decline immediately after the membrane potential hyperpolarized, transiently reaching 60% and less than 10% of pre-stressed values after about 2.5 min under ambient CO2 of 700 µmol mol−1 air (Fig. 2a, Table 1) and elevated CO2 of 2000 µmol mol−1 air (Fig. 2b, Table 2), respectively. When the electrical signal levelled off (Fig. 2a,b), AN was transiently restored to ca. 80% of initial values in both CO2 environments (at around 6 min), thereafter decreasing again until AN reached 40 to 50% of initial values (Tables 1 and 2). This second steady state lasted for more than 10 min, while finally AN recovered to initial values after more than 30 min following the heat pulse (data not shown). Both electrical and gas exchange signals were completely blocked (Fig. 2c) when mounting a cooling block between the heat stimulus and the detection systems (see Fig. 1), thus confirming the association of electrical signals and photosynthetic changes.
Table 1. Summary of the major changes (‘local extrema’; see Fig. 3) of AN (µmol CO2 m−2 s−1), ETR (µmol e- m−2 s−1), Ci (µmol CO2 mol−1 air), Cc (µmol CO2 mol−1 air), gm (mol CO2 m−2 s−1) and gs (mol CO2 m−2 s−1) after flaming at 700 µmol CO2 mol−1 air
‘New steady state’
Means and standard deviations of three different measurements/plants are shown. Asterisks indicate values that differ significantly from control values (0 min) within a given parameter at a P value ≤ 0.05. Local extrema are highlighted by underlined values.
ETR, electron transport rate, AN, net photosynthesis; Ci, CO2 concentration of the intercellular airspace; Cc, chloroplast CO2 concentration; gs stomatal conductance; gm mesophyll conductance.
10.9 ± 1.1
6.6 ± 2.7*
7.0 ± 2.8*
9.5 ± 1.2
6.5 ± 3.0*
101.6 ± 2.4
85.5 ± 9.2*
84.7 ± 10.2*
93.0 ± 6.6
87.6 ± 10.7
453 ± 25
566 ± 37*
546 ± 39
468 ± 8
441 ± 15
205 ± 14
160 ± 27*
180 ± 29
201 ± 8
148 ± 34*
0.044 ± 0.002
0.017 ± 0.010*
0.023 ± 0.011*
0.035 ± 0.006
0.024 ± 0.015*
0.049 ± 0.008
0.057 ± 0.005
0.060 ± 0.001*
0.048 ± 0.003
0.027 ± 0.011*
Table 2. Summary of the major changes (‘local extrema’; see Fig. 3) of AN, ETR, Ci, Cc, gm and gs after flaming at 2000 µmol CO2 mol−1 air
Changes of the most relevant leaf gas exchange parameters after heat stimulation are presented in Figs 3 and 4. Figure 3 represents data obtained with a combined fluorimeter − IRGA system (LI-6400) at the University of Hamburg (Germany), while (Fig. 4 and Table 3) show soybean data obtained with a combined IRGA-TDL system at the University of New Mexico (USA). In general, similar changes of leaf gas exchange parameters were observed when using different equipment and methods (Figs 3 & 4), as well as when treated with different CO2 concentrations (Fig. 3). However, amplitudes differed significantly in some parameters between both CO2 environments (Tables 1 and 2). It should be noted that elevated CO2 did not lead to increased AN (Figs 2 & 3) due to the strong reductions in gm and gs, which have been the result of the prevailing high CO2 concentrations (e.g. Flexas et al. 2007b). In parallel with AN, the Cc and the gm dropped markedly after about 1.5 min until they reached a first minimum around 2.5 min (Fig. 3), while the photosynthetic ETR decreased slower and to a minor degree (minimum around 3.5). These parameters were partly restored to initial values after about 5–6 min (second maximum), followed by another decline until a new steady state was established around 10 to 12 min after the heat stimulus (Fig. 3, Tables 1 and 2). In contrast to the parameters directly linked to CO2 diffusion and fixation within the mesophyll (AN, gm, Cc), the response of stomata (i.e. gs) was somewhat delayed (by ca. 1 min), though resulting in an increase of about 25% after 3.5 min (first maximum; Table 1). That is, initially, gs increased while AN, gm, ETR and Cc decreased. Thereafter, gs decreased progressively until a new steady state was reached at around 10 to 12 min, which was similar for all the other parameters (AN, Cc, gm and ETR). All leaf gas exchange parameters remained at this new steady state for the next 10 to 20 min, while being completely restored to initial values after more than 30 min following the heat stimulus (data not shown). Despite some time shift in the extremes of the leaf gas exchange parameters determined via the IRGA-TDL system (Fig. 4), the pattern and amplitudes are very similar to those obtained with the fluorimeter-IRGA system (Fig. 3). Variations of leaf temperature were small throughout the entire experiment, typically ranging within ±1 °C (data not shown; see also supplementary material), and leaf temperature correlated negatively with gs.
Table 3. Summary of the initial major changes (see Fig. 4) of AN (µmol CO2 m−2 s−1), Ci (µmol CO2 mol−1 air), Cc (µmol CO2 mol−1 air), gm (mol CO2 m−2 s−1) and gs (mol CO2 m−2 s−1) after flaming at 700 µmol CO2 mol−1 air, determined via the isotope method (see Materials and Methods section) in another set of plants
Local extremum (AN, gm)
Local extremum (gs)
Means and standard deviations of three different plants are shown (averages of 30 s intervals). Asterisks indicate values that differ significantly from control values (0 min) within a given parameter at a P value ≤ 0.05. Local extrema are highlighted by underlined values. AN, net photosynthesis; Ci, CO2 concentration of the intercellular airspace; Cc, chloroplast CO2 concentration; gs stomatal conductance; gm mesophyll conductance.
19.1 ± 2.8
8.9 ± 1.3*
15.6 ± 2.4
0.06 ± 0.02
0.11 ± 0.04
0.15 ± 0.04*
0.28 ± 0.14
0.06 ± 0.05*
0.16 ± 0.15
319 ± 32
543 ± 47
518 ± 51
239 ± 38
325 ± 172
340 ± 187
With regard to the two different CO2 conditions, changes of AN, Cc, gm and gs were greater under high CO2 (2000 µmol CO2 mol−1 air; Fig. 3b, Table 1) than under ambient (700 µmol CO2 mol−1 air; Fig. 3a, Table 2), whereas changes in ETR were overall minor in both CO2 conditions. Hence, at the new steady state, AN, Cc and gm remained somewhat lower (in % of initial values) under elevated CO2 than under ambient CO2, while gs and ETR hardly differed among both CO2 conditions (Tables 1 and 2).
In order to assess the signal distribution across the leaf, temporal and spatial imaging of photochemistry via Chl a fluorescence was carried out with an Imaging-PAM system (Dual Mini-PAM, Walz GmbH, Effeltrich, Germany) (Fig. 5). From the imaging data on ΦPSII no difference in fluorescence signal transmission between vein and intervein regions could be detected (Fig. 4a), apart from the drop of ΦPSII after 1.5 min and its minimum after 2.5 to 3 min (Fig. 4b), which was similar as observed for ETR with the LI-6400 (Fig. 3, Table 1). However it is worth to note, that differences in signal distribution between vein and intervein regions on the exposed leaf area of ca. 32 × 32 mm may be difficult to detect, due to the high speed of the electrical signal (about 3–4 mm s−1) and the time interval of fluorescence recordings of 10 s.
Cross-sections of leaf mesophyll were prepared and the composition of prevalent ions at the chloroplast envelope was determined using EDXA (Fig. 6). According to the changes in electrical and photosynthetic signals (minima and maxima; Table 1), measurements were taken at 0, 3, 5 and 9 min after the heat stimulus. After 3 min, a rise in K (70%), Mg (35%) and O (25%) could be observed, while P remained unaltered as compared with the initial situation (control). After 5 min, Mg increased further (50%), O and P hardly changed, and K dropped again (30%). Finally, after 9 min, K and O were almost restored to initial values, while Mg dropped by more than 90%, thus approaching ca. −30% of control values (similar as for P).
It is commonly accepted that electrical signals can affect physiological processes in plants. For example, an electrical signal was found to be the underlying mechanism of the systemic proteinase inhibitor induction in wounded tomato plants (Wildon et al. 1992). Herde et al. (1998) analysed the effects of mechanical wounding, heat treatment and electrical current application in tomato plants, indicating that wound-induced changes in membrane potential seem to be dependent on the endogenous level of abscisic acid (ABA) and are involved in regulation of turgor pressure within the plant. With regard to the ionic mechanism of electrical signals, it has been shown that Ca2+ influx as well as Cl- and K+ efflux are involved in action potential formation. To resolve the temporal increase in cytosolic Ca2+ during AP formation Fisahn and co-workers (2004) generated transgenic potato plants that expressed Ca2+ photoprotein apoaequorin, demonstrating that increases in cytosolic Ca2+ levels emerged in a very early phase in the time course of the AP. Concerning soybean plants, the ionic components of the electrical signal still have to be identified in future investigations. However, it can be hypothesized that Ca2+ transients might be involved accordingly in the signal transduction cascade.
As soybean plants have been shown to transmit electrochemical signals induced by blue light and/or wounding (Loreto & Sharkey 1993; Baluska, Mancuso & Volkmann 2006), in the present study the interaction of electrical signals and photosynthesis has been examined in soybean leaves (Fig. 2). Stimulating a leaflet by heat (flaming) caused rapid and consistent electrical signals with amplitudes of around 20 mV that were detectable in neighbouring leaflets, and that were accompanied by profound changes in photosynthesis-related parameters. No such responses were obtained when electrical signalling was blocked through cooling a part of the plant between the sites of heat stimulus and detection (Fig. 2c, see also Fig. 1 for the set up scheme). Consequently, it can be assumed that a direct link between alterations of photosynthetic parameters and electrical signals exists (Fig. 2). This link has been confirmed by other studies on different species, where a transient photosynthetic decline after heat stimulation was always detected (Koziolek et al. 2004; Lautner et al. 2005; Grams et al. 2009). However, the physiological processes that govern these changes in photosynthesis are still poorly understood.
The present study on soybean unravelled a direct effect of electrical signalling on gm and hence on photosynthesis through variations of the mesophyll membrane potential (i.e. VP). In fact, the results of heat-stimulated soybean leaves provide evidence that the drastic decline of gm, which was reduced the most of all determined parameters (>60%, Table 1), and hence the large reduction of chloroplastic CO2 concentration (Cc) were the main limiting factors for AN during the initial phase of heat-induced electrical signalling (Fig. 3). The photosynthetic ETR was only marginally affected (declined maximally by about 15%; Table 1) and can therefore not explain the initial and profound drop of AN (by about 40%; Fig. 3, Table 1). According to that, it seems likely that electrical signals directly modified the CO2 diffusion within the mesophyll (gm) and thereby reduced CO2 availability in the chloroplasts (Cc), which resulted eventually in the drop of AN despite an initial stomatal opening in response to the stimulus. In addition, the transient changes in ion concentrations (i.e. K, Mg, O) around the chloroplast envelope (Fig. 6) may be associated to the changes in the membrane potential (VP) as well as to the alteration of intrinsic diffusion pathways (gm). Such rapid changes of mesophyll (membrane) diffusion in conjunction with shifts of prevalent ions suggest modifications at the chloroplast membrane, which may include modification of membrane porosity or the de-activation of channels/transporters for CO2, among which aquaporins are potential candidates that can be actively involved in conducting CO2 (Uehlein et al. 2003, 2008) and are considered to be pH regulated (Luu & Maurel 2005).
Interestingly, rising the CO2 concentration around the leaves did not affect the course, amplitude or velocity of the perceived electrical signals (Fig. 2), whereas the initial decline of gm, Cc and AN was even more pronounced than under ambient CO2 conditions (Table 1). Changes in gs and ETR were similar under both CO2 conditions (Table 1). From that and with regard to the initial situation (before heat stimulation), it may be concluded that high CO2 impacted mainly on gm and to lesser extent on gs (Fig. 3), which in turn led to the strong transient suppression of photosynthesis (Table 2). Moreover, gm remained also more reduced after having reached the new steady state (Table 1), indicating a lasting effect of elevated CO2 on gm in soybean. Such induced reductions of gm and gs by rising the CO2 concentration around the leaf are commonly observed phenomena (Flexas et al. 2007b; Galle, Haldimann & Feller 2007; Hassiotou et al. 2009). A possible role of transient bicarbonate leakage on gm might also be considered (Tholen & Zhu 2011).
Most remarkably, heat-induced electrical signalling seemed to transiently decouple gs from gm, as gs increased while gm declined and its response was slightly delayed as compared with gm (Figs 3 & 4). However, although leaf diffusion components (gs, gm) seemed to be temporary disturbed or disentangled during the first 3–4 min after heat stimulation, the typically observed co-regulation of gm and gs (e.g. Lauteri et al. 1997; Centritto, Loreto & Chartzoulakis 2003) was re-established thereafter, leading to a coordinated response of both conductances and a new steady state after 10–12 min (Table 1). The initially opposite responses of gs and gm may reflect a situation of imbalance and disturbance within the leaf diffusion system, which was most likely a result of the variations in ion fluxes/gradients and the modified membrane diffusivity as induced by the electrical signal.
In all previous studies, heat stimulation resulted in a transiently marked decrease of AN, while gs responded in different ways or not at all (Koziolek et al. 2004; Lautner et al. 2005; Grams et al. 2009). In soybean however, a transient and partial restoration of AN, gm and Cc has been observed after the initial drop, which thereafter resumed in a closely coordinated joint course with gs (Fig. 3). Therefore, it appears that the re-organization of the leaf intrinsic diffusion system began after the electrical signal levelled off (Fig. 2), thereby partially restoring gm, Cc and AN, approaching a new steady state at a lower level of photosynthetic CO2 fixation and water loss thereafter (Fig. 3). The reason for this new steady state remains unclear, though an involvement of metabolic factors like the hormone ABA might be considered among others. However, changes in leaf ABA are usually not as quick as necessary to explain the transient steady state after around 10 min, and rather appear (and last) in the range of hours after stress imposition (Herde et al. 1999; Davies, Wilkinson & Loveys 2002). Nevertheless, the system perturbations through heat-induced electrical signalling have caused a distinct response in non-stressed leaves towards reduced photosynthesis and water loss, presumably to minimize the risk of rapid dehydration. After about 20 to 30 min, all parameters recovered to the initial (pre-stress) values, indicating that the new steady state after 10–12 min represented (only) a transient phase and all the heat-induced changes were totally reversible. The metabolic processes involved in the response to electrical signalling, in particular with regard to the transiently established new steady state, await further research.
Remarkably, the velocity of the signal transmission between leaflets was very high (3–4 mm s−1) and spanned over a distance of several centimetres, thus being in a similar range like the fast-responding leaflets of Mimosa (Koziolek et al. 2004; Fromm & Lautner 2007), making soybean an interesting model plant for further studies on electrical signalling and the interaction with photosynthetic processes.
Heat-induced electrical signals that travelled with high velocity (3–4 mm s−1) over distances of several centimetres between soybean leaves caused primarily strong perturbations among the intrinsic diffusion components (i.e. gm), which resulted in transiently suppressed photosynthesis. Thus, it can be concluded that electrical signals predominantly and immediately affect gm, which caused a strong reduction of Cc and hence of AN. Impaired photochemistry per se was of minor relevance for the observed drop of AN, as ETR hardly changed. Stomata (i.e. gs) responded slower and opposite to gm during the occurrence of electrical signals, indicating a temporary disturbance of leaf diffusion components (i.e. membranes) that led to the decoupling of both conductances. However, after the electrical signal levelled off (ca. 6 min), the re-coordination of gm and gs was observed, driving a concerted response towards a new steady state at a lower level of photosynthetic CO2 fixation and water loss (ca. 12 min), which was followed by a full recovery after about 30 min. The reason for the transiently established new steady state remains unclear, while eventually all heat-induced changes were fully reversible and restored to initial (pre-stress) levels. Finally, in this study the close link of electrical signalling and modifications of intrinsic diffusion components (mesophyll/chloroplast) in soybean leaves via the immediate and direct effect of electrical signals on gm (and hence on photosynthesis) has been demonstrated for the first time. However, the metabolic processes involved in the transient impairment of gm caused by heat-induced electrical signals await further research.
The authors would like to thank the gardeners at the Institute of Wood Science/vTI Hamburg (Germany) for taking care of the soybean plants. This study was carried out within the framework of the Integrated Action Programme between Spain and Germany co-financed by the Spanish Ministry of Science and Innovation and the DAAD (Project 0811914). The work was also partly financed by the Spanish Ministry of Education and Research (Project BFU2008-1072-E/BFI). A.G. had a post-doctoral fellowship from the SNSF (PA00P3_126259).