To open or to close: species-specific stomatal responses to simultaneously applied opposing environmental factors

Authors


Summary

  • Plant stomatal responses to single environmental factors are well studied; however, responses to a change in two (or more) factors – a common situation in nature – have been less frequently addressed. We studied the stomatal responses to a simultaneous application of opposing environmental factors in six evolutionarily distant mono- and dicotyledonous herbs representing different life strategies (ruderals, competitors and stress-tolerators) to clarify whether the crosstalk between opening- and closure-inducing pathways leading to stomatal response is universal or species-specific.
  • Custom-made gas exchange devices were used to study the stomatal responses to a simultaneous application of two opposing factors: decreased/increased CO2 concentration and light availability or reduced air humidity.
  • The studied species responded similarly to changes in single environmental factors, but showed species-specific and nonadditive responses to two simultaneously applied opposing factors. The stomata of the ruderals Arabidopsis thaliana and Thellungiella salsuginea (previously Thellungiella halophila) always opened, whereas those of competitor-ruderals either closed in all two-factor combinations (Triticum aestivum), remained relatively unchanged (Nicotiana tabacum) or showed a response dominated by reduced air humidity (Hordeum vulgare).
  • Our results, indicating that in changing environmental conditions species-specific stomatal responses are evident that cannot be predicted from studying one factor at a time, might be interesting for stomatal modellers, too.

Introduction

Stomata, the small pores surrounded by guard cells that regulate plant gas exchange, respond to environmental and endogenous stimuli. In nature, environmental conditions (such as light or air humidity) often fluctuate and guard cells need to integrate multiple signals to ensure appropriate stomatal response for plant survival in an ever-changing environment. The principal mechanisms by which to process these signals are probably conserved in angiosperms (Roelfsema & Hedrich, 2005). However, Mori & Murata (2011) suggested that differences in environmental conditions during the evolution of distinct species might have resulted in the variability of stomatal signalling mechanisms. In trees, when opposing photosynthetic and hydraulic signals apply simultaneously, the hydraulic signal dominates: after the decrease in air humidity stomata close even when CO2 concentration decreases or irradiation increases (Aasamaa & Sõber, 2011). It has been suggested that hydraulic priority is fundamentally important in preventing plant desiccation and is maintained in stomatal control through vascular plant phylogeny (Brodribb & McAdam, 2011). Studies which address the stomatal responses to simultaneous application of opposing stimuli are rare; we did not find any other besides Aasamaa & Sõber (2011). There are more reports concerning stomatal behaviour in response to sequential application of stimuli (Bunce, 1997; Hedrich et al., 2001; Doi & Shimazaki, 2008; Easlon & Richards, 2009). Thus, more information on stomatal responses to a simultaneous change in environmental factors from different species is clearly needed and in this paper, we contribute our results on this topic.

A large proportion of stomatal research at whole-plant, cellular and molecular levels has been obtained from the model plant Arabidopsis thaliana (further Arabidopsis) and its responses to different environmental stimuli are well characterized (Merilo et al., 2013). The stomatal conductance (gs) of Arabidopsis decreases in response to elevated CO2, reduced air humidity and darkness, whereas it increases in response to additional illumination and low CO2. These factors induce stomatal responses through different signalling pathways: light-induced stomatal opening is mediated by: (1) blue light- (BL) and phototropin-involved activation of guard cell H+-ATPases (Shimazaki et al., 2007) and BL-activated inhibition of S-type anion channels (Marten et al., 2007); (2) mesophyll photosynthesis resulting in lower intracellular CO2 concentration (Ci) that in turn deactivates anion channels (Roelfsema et al., 2002). However, stomata opened in response to red light (RL) even when Ci was kept constant, indicating that a change in Ci is not inevitably required for stomatal light response to proceed (Messinger et al., 2006). Stomatal closure in response to elevated CO2 involves carbonic anhydrases in the plasma membrane of guard cells (Hu et al., 2010), the protein kinase OST1 that activates guard cell anion channel SLAC1 (Negi et al., 2008; Vahisalu et al., 2008; Xue et al., 2011; Merilo et al., 2013) and the protein kinase HT1 that acts as a negative regulator in CO2-induced stomatal signalling (Hashimoto et al., 2006). There are, however, important details missing in the CO2/light response: for example, it is unclear whether stomatal response to RL is mediated through signals originating in the mesophyll, either reduction in Ci or something else (Roelfsema et al., 2002; Mott et al., 2008), or in the guard cells (Messinger et al., 2006). Recently, an aqueous signal from mesophyll was found to be important in CO2-induced stomatal opening and closure (Fujita et al., 2013). Furthermore, the presence of at least two CO2 response pathways, one that depends on the presence of photosynthetic electron transport and one that does not, has been suggested (Messinger et al., 2006).

Although the plant hormone abscisic acid (ABA) is involved in stomatal closure in response to drought stress and reduced air humidity (Bunce, 1997; Roelfsema & Hedrich, 2005; Bauer et al., 2013), the existence of an ABA-independent pathway participating in low humidity-induced stomatal response has been suggested (Xie et al., 2006). Furthermore, stomata may also directly respond to leaf water status without relying on ABA production and signalling (see Buckley, 2005; Damour et al., 2010; Peak & Mott, 2011). However, both the metabolically mediated hydro-active feedback response of guard cell osmotic pressure to the water status of epidermal evaporative site (Buckley, 2005) and the stomatal response mechanism based on a near-equilibrium in water potential between the guard cells and the air in the stomatal pore (Peak & Mott, 2011) still embed the potential for ABA to be involved in air humidity-related stomatal behaviour. ABA thus participates in stomatal responses to many different stimuli and during stomatal closure, different signals converge at a few common signalling components (Merilo et al., 2013).

Here, we studied the stomatal responses to two simultaneously applied opposing environmental factors in Arabidopsis, salt cress [Thellungiella salsuginea (halophila), further Thellungiella] – a cold-, salt- and drought-tolerant close relative of Arabidopsis (Amtmann, 2009), tobacco (Nicotiana tabacum, further Nicotiana), common rockrose (Helianthemum nummularium, hereafter Helianthemum) and two cereals, barley (Hordeum vulgare, hereafter Hordeum) and wheat (Triticum aestivum, hereafter Triticum). These species differ in their life strategies to survive and reproduce in their environment. Here, we used the competitor/stress-tolerator/ruderal classification of plant life strategies (sensu Grime, 1977), where ruderals are characterized by short life cycle, high growth rate and large seed production, stress-tolerators by inherently slow growth rate, long-lived organs, sequestration of resources and poor flowering, and competitors by high growth rate, dense foliage and great competitiveness (Grime, 1977). This classification of plant life strategies, although a considerable simplification of reality (Wilson & Lee, 2000), is the most general and comprehensive theory in plant functional ecology. Thellungiella and Arabidopsis are typical ruderals (R); Nicotiana, Hordeum and Triticum represent competitor-ruderal (CR) plant strategy, whereas Helianthemum is a stress-tolerator (S) (Hodgson et al., 1999; Kühn et al., 2004). The species differ also in stomatal morphology (dumbbell-shaped guard cells with flanking subsidiary cells in grasses vs kidney-shaped guard cells of other species) and evolutionary distance: Hordeum and Triticum are monocotyledons, that is, were first to diverge from others; the remaining species are dicotyledons with Nicotiana from the clade asterids (i.e. next to diverge) and Arabidopsis, Thellungiella and Helianthemum from the clade rosids. We studied whether the crosstalk between opening- and closure-inducing pathways leading to stomatal response is universal or species-specific and found, interestingly, that it is species-specific.

Materials and Methods

Plant material and growth conditions

Arabidopsis thaliana (L.) Heynh., Thellungiella salsuginea (Pallas) O.E. Schulz, Nicotiana tabacum (L.) (cv Bel-B) and Helianthemum nummularium (L.) Mill seeds were planted in soil containing 4 : 2 (v : v) peat:vermiculite and grown through a hole in a glass plate covering their pot as described in Kollist et al. (2007). For Thellungiella, we used in our experiments NASC stock N22504 labelled as Thellungiella halophila, which has recently been re-classified as Thellungiella salsuginea (Amtmann, 2009). The genus Thellungiella is now known as Eutrema, so that Thellungiella salsuginea is now Eutrema salsugineum (http://phytozome.net/thellungiella.php#A). In Arabidopsis, two common laboratory accessions, Columbia (Col-0) and Landsberg erecta (Ler) were used. Soil moisture was kept at 60–80% of maximum water capacity. Plants were grown in growth chambers (AR-66LX and AR-22L; Percival Scientific, Perry, IA, USA) at 12 h : 12 h photoperiod, 23°C : 18°C temperature, 150 μmol m−2 s−1 light and 70% relative humidity. For gas-exchange experiments, we used plants with total leaf area between 5 and 25 cm2. This corresponds to 21–30-d-old Arabidopsis, Thellungiella and Nicotiana plants. The growth rate of S species Helianthemum was slower and the plants were 33–40 d old (leaf area 3.5–8 cm2) during the experiments. All experimental plants were in a vegetative growth stage. Seeds were obtained from the European Arabidopsis Stock Centre (www.arabidopsis.info), Prof. William Manning (Nicotiana) and Tartu Botanical Garden (Helianthemum).

The seeds of Hordeum vulgare L. (cv Jyvä) and Triticum aestivum L. (cv KWS Scirocco), common agricultural cultivars of spring barley and wheat, were sown into 1 l pots containing the soil mixture described above. Growth conditions in the plant chambers were as described above. For gas-exchange experiments, we used barley and wheat seedlings at the 2- to 3-leaf stage, with plant age of 10–20 d and total leaf area 8–38 cm2.

Gas exchange devices for stomatal conductance measurements

The eight-chamber whole-plant rapid-response gas exchange measurement device was described previously (Kollist et al., 2007). This device can be used to measure plants with total leaf area < 25 cm2 not taller than 4 cm; out of the six studied species, Arabidopsis, Thellungiella, Nicotiana and Helianthemum were suitable. Plants were inserted into three chambers and the treatments started c. 2 h later, when stomatal conductance (gs) had stabilized. By simultaneously using only three chambers out of eight, we could obtain gas exchange data from every single chamber with 6-min intervals. Standard conditions during the stabilization period were as follows: ambient CO2 (c. 400 ppm), light 150 μmol m−2 s−1, relative air humidity (RH) c. 60% (the corresponding VPD was 1.1–1.3 kPa). In reduced air humidity experiments, RH was 70–80% during the stabilization period and c. 30–40% during treatment, the corresponding VPDs were 0.95–1.1 and 2.0–2.5 kPa, respectively. The temperature within the chambers was 23–25°C. Photographs of plants were taken before the experiment and leaf area was calculated using ImageJ v1.46r (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Stomatal conductance for water vapour and net photosynthetic rate were calculated with a custom written program as described in Kollist et al. (2007).

Hordeum and Triticum plants were measured with a different, single-plant device, which was suitable for larger and taller plants and enabled gas exchange data to be recorded every 2 min. The main body of this flowthrough single-plant gas exchange system was the thermostated gas exchange cuvette formed from two glass cylinders (inner diameter 10 cm, height 13.4 cm) with a water jacket between them. The cuvette was placed on a stand composed of two well-fitted glass plates that form the bottom of the gas exchange cuvette. One of these plates contained perforations for plant stems and was removable. The other glass plate contained gas input and output ports, a temperature sensor, and a fan to ensure high turbulence and uniform gas mixing. Modelling gum was used to ensure an airtight separation of plant parts within the chamber from the rest of the plant. The chamber was hermetically sealed and operated only under slight overpressure of a few millibars to avoid uncontrolled leakage and intake of ambient air. The air flow rate through the chamber was 1.5 l min−1, resulting in a response half-time of c. 20 s. Ambient air passing through a large buffer volume of 25 l was used in most experiments. For elevated CO2 experiments, CO2 was added into the gas stream using a capillary mixer. For reduced CO2 treatments, CO2 was first removed by KOH, then it was added by the mixer in the required concentration and air was rehumidifed again in a closed volume containing thermostated water. The temperature inside the chamber was continuously measured with a negative temperature coefficient thermistor (model -001; RTI Electronics, Anaheim, CA, USA) and was between 23 and 25°C. Leaf temperature determined from leaf energy balance was within ±1°C of the air temperature in the chamber. All tubing and connections were made from Teflon and stainless steel. Four 50 W halogen lamps were used for illumination providing a light intensity of 150 μmol m−2 s−1. The concentrations of CO2 and water vapour in the reference channel (i.e. air entering the measuring cuvette) and measurement channel (air coming out from the cuvette) were measured with an infrared gas analyzer (Li-7000; Li-Cor, Lincoln, NE, USA) and stomatal conductance and net photosynthetic rate were calculated with a custom written program as described in Kollist et al. (2007). Standard conditions during the stabilization period in the single-plant device used for cereals were similar to these in multi-chamber device: ambient CO2 (c. 400 ppm), light 150 μmol m−2 s−1, RH 70–80% in reduced air humidity experiments or c. 60% in other experiments.

Application of changes in environmental factors

After the stabilization of stomatal conductance, two environmental factors – one causing stomatal closure and another opening – were rapidly and simultaneously changed. The closure-inducing changes were darkness (from 150 to 0 μmol m−2 s−1, applied by covering the plant chambers one after another in the multi-chamber device and switching off the lamp in the one-plant device), elevated CO2 (from ambient to c. 800 ppm; CO2 was added into the gas stream using a capillary mixer) and low air humidity (from RH = 70–80% to 30–40%; applied by switching between two thermostats), whereas the opening-inducing changes were an increase in light intensity applied as BL (from 150 to 450 μmol m−2 s−1, applied by turning on blue lamps mounted above chambers) and reduced CO2 (from ambient to c. 50 ppm; CO2 was first removed by KOH, then it was added by the capillary mixer in the required concentration: either 400 ppm during the stabilization period or 50 ppm during treatments). Because BL was applied with total incident radiation increasing three times and net assimilation rate c. 1.5–2 times, both phototropin-mediated and photosynthesis-mediated pathways of light-induced stomatal opening were initiated in our experiments. As a rule, the closure-inducing change was applied 20 s before the opening-inducing change. In separate experiments, stomatal responses to these factors applied singly were also studied. When the experiment was over, some leaves were harvested and leaf mass per area (LMA) was determined as the ratio of leaf dry mass (weight of leaves dried at 70°C for 72 h) to leaf area.

Subsequently, we calculated the changes in stomatal conductance as gs2gs1 (gs1, the pre-treatment stomatal conductance; gs2, the value of stomatal conductance 40 min after application of factor change) (see Fig. 1e). In two-factor experiments, we also defined the predicted response as the sum of stomatal responses to these factors applied as single ones [Predicted response = (gs2gs1)Factor 1 + (gs2gs1)Factor 2]. Stomatal response half-times were obtained in single factor experiments by scaling the whole 40-min stomatal response to a range from 0 to 100% and by calculating the time when 50% of stomatal opening/closure was achieved.

Figure 1.

Time-resolved patterns of Arabidopsis (Col-0) stomatal conductance in response to single-factor changes and to simultaneous changes in two opposing factors. To address light-induced stomatal opening (Light), light intensity was increased from 150 to 450 μmol m−2 s−1; low CO2-induced stomatal opening (Low CO2) was addressed by reducing CO2 concentration from 400 ppm to 40–50 ppm; darkness-induced stomatal closure (Darkn) by reducing light intensity from 150 to 0 μmol m−2 s−1; elevated CO2-induced stomatal closure by increasing CO2 concentration from 400 to 800 ppm; and stomatal closure in response to low air humidity (Red hum) by reducing air humidity from 70–80% to 30–40%. In two-factor combinations, the changes described above were combined. Factors were applied at zero timepoint with the closure-inducing change applied c. 20 s before the opening-inducing change. The gs values used to calculate the predicted responses and the changes in stomatal conductance (gs2gs1) presented in Fig. 2 are shown in (e). Stomatal conductances (= 6–7, ± SEM) are shown in relative units; then the average of the two last pre-treatment gs values was used for normalization (a–d) and in absolute units (e–h).

Results

Stomata of Arabidopsis always open in response to two simultaneously applied opposing factors

In Arabidopsis accession Col-0, simultaneous application of closing and opening factors always resulted in stomatal opening, either slight or considerable (Fig. 1). Similar results were obtained for Ler, another commonly used accession of Arabidopsis (Supporting Information Fig. S1). In reduced air humidity + light and high CO2 + light combinations, the predicted (for details see the 'Materials and Methods', last section, and Fig. 1e) and measured stomatal responses generally coincided (Fig. 2a). Low CO2 concentration applied together with darkness or reduced humidity was predicted to result in slight stomatal closure or almost no response, yet the measured reaction was considerable stomatal opening (Fig. 2a). This indicates that the deprivation of photosynthetic substrate is very important and dominates over other factors in the Arabidopsis stomatal responses. The stomata opened even when darkness was applied 3 or 12 min (instead of usual 20 s) ahead of CO2 withdrawal (Fig. S2). In a separate experiment we followed the stomatal responses of Arabidopsis Col-0 for a longer period and found that even after 3 h, stomatal closure was not detected in two-factor experiments (Fig. 3a,c). In reduced air humidity + low CO2, plants were visibly wilted after 3 h.

Figure 2.

Changes in stomatal conductance (gs2gs1, = 5–7, ± SEM) in response to single factors, in response to simultaneously applied opposing factors, and the predicted responses calculated as sums of responses to single factors for studied species (measured response, black bars; predicted response, grey bars). Positive and negative changes refer to stomatal opening and closure, respectively. For calculation of changes in stomatal conductances (gs2gs1) we used gs1 as the pre-treatment stomatal conductance and gs2 as the value of stomatal conductance 40 min after application of factors. Timepoints for gs2 and gs1 are shown in Fig. 1(e). Predicted response was calculated as the sum of stomatal responses to these factors applied as single ones (predicted response = (gs2gs1)Factor 1 + (gs2gs1)Factor 2). Darkn, darkness; Red hum, reduced air humidity.

Figure 3.

Time-resolved patterns of stomatal conductance of studied species in response to opposing stimuli followed during 3 h. (a) Stomatal response to darkness + low CO2 (from 400 to 40–50 ppm) in Arabidopsis thaliana Col-0, Thellungiella salsuginea and Nicotiana tabacum. Factors were applied at zero timepoint, = 5–7, ± SEM. (b) Stomatal response to darkness (Darkn) + low CO2 in Helianthemum nummularium, Hordeum vulgare and Triticum aestivum (= 5–7, ± SEM). (c) Stomatal response of Arabidopsis thaliana Col-0 to high CO2 (from 400 to 800 ppm) and additional light (from 150 to 450 μmol m−2 s−1), to reduced air humidity (Red hum; from 70–80% to 30–40%) and additional light, and to reduced air humidity and low CO2. Factors were applied at zero timepoint (= 6, ± SEM).

Steady-state stomatal conductance and photosynthesis of Thellungiella salsuginea, Nicotiana tabacum, Helianthemum nummularium, Hordeum vulgare and Triticum aestivum

Steady-state whole-plant gs varied three-fold among the species, with Triticum, Helianthemum and Hordeum showing the highest values and Nicotiana the lowest (Table 1). The high stomatal conductance of grasses was no surprise as dumbbell-shaped stomata have large potential gs (Hetherington & Woodward, 2003; Franks & Farquhar, 2007). The high gs of Helianthemum was enigmatic, because stomatal conductance was positively correlated with photosynthesis (Table 1; Hetherington & Woodward, 2003; Jiang et al., 2006) and biomass production (Jiang et al., 2006; Makino, 2011), but Helianthemum, classified as stress-tolerator (S) species, grows very slowly; this was confirmed also in our experiment (data not shown). Data on leaf structural traits showed that the leaf mass per area (LMA) of Helianthemum was significantly larger compared to other species (Table 1), indicating that its leaves were structurally tough and resistant, and thus durable for longer periods (Onoda et al., 2004). Thus, Helianthemum invested proportionally more biomass into structural components, such as cell walls, and its photosynthetic rate per unit mass was the lowest among studied species (Table 1).

Table 1. Stomatal conductance (gs), area- and mass-based net photosynthetic rates (Anet area and Anet mass, respectively) and leaf mass per area (LMA) of studied species
 gs (mmol m−2 s−1)Anet area (μmol m−2 s−1)Anet mass (μmol g−1 s−1)LMA (g m−2)
  1. Values are averages (±SE), = 6. Different letters denote statistically significant differences (ANOVA and Fisher's LSD test).

Arabidopsis 133.3 (± 9.7) a4.2 (± 0.2) ab0.275 (± 0.014) a15.1 (± 0.95) a
Thellungiella 135.6 (± 15.4) a4.1 (± 0.3) ab0.261 (± 0.019) a15.8 (± 0.32) a
Nicotiana 115.7 (± 9.6) a3.7 (± 0.5) a0.294 (± 0.039) a12.6 (± 0.74) a
Hordeum 268.1 (± 16.7) b6.6 (± 0.3) c0.399 (± 0.018) b16.6 (± 0.92) a
Helianthemum 360.5 (± 24.1) c5.2 (± 0.3) b0.103 (± 0.006) c50.8 (± 3.60) c
Triticum 355.0 (± 53.3) c10.0 (± 0.7) d0.448 (± 0.031) b22.3 (± 0.75) b

Stomata of all studied species responded similarly to single environmental factors

In response to a change in one environmental factor at a time, all studied species were able to open and close stomata similarly to Arabidopsis (Fig. 4a–e, time-courses of absolute gs in Fig. S3). In addition to high gs, the grasses were also capable of extensive and rapid stomatal movements (Fig. 4). This is probably related to their dumbbell-shaped guard cells and stomatal complexes where K+ ions shuttle between the guard and subsidiary cells (Hetherington & Woodward, 2003; Büchsenschütz et al., 2005; Franks & Farquhar, 2007). Accordingly, in cereals the magnitudes of stomatal responses were the highest (Figs 2e,f, 4) and half-times for stomatal opening and for reduced humidity-induced stomatal closure were significantly shorter than for dicots (Fig. 4f). In reduced humidity experiments (Fig. 4e), all of the studied species showed initial ‘wrong-way’ responses (transient stomatal opening) resulting from the mechanical advantage of epidermal cells over guard cells.

Figure 4.

Stomatal responses of Thellungiella salsuginea, Nicotiana tabacum, Helianthemum nummularium, Triticum aestivum and Hordeum vulgare to single-factor changes. Factors were applied at zero timepoint and were darkness (Darkn; light intensity from 150 to 0 μmol m−2 s−1) (a), elevated CO2 (CO2 concentration from 400 to 800 ppm) (b), additional light (light intensity from 150 to 450 μmol m−2 s−1) (c), low CO2 (CO2 concentration from 400 to 40–50 ppm) (d) and reduced air humidity (from 70–80% to 30–40%) (e). Data are shown in relative units (= 5–7, ± SEM); the average of the two last pre-treatment gs values was used for normalization. (f) Stomatal response half-times to different factors. Half-times were obtained by scaling the whole 40-min stomatal response to a range from 0 to 100% and by calculating the time when 50% of stomatal opening/closure was achieved. Different letters denote statistically significant differences within the treatment (ANOVA and Fisher's LSD test).

Stomatal responses to two simultaneously applied environmental factors reveal clear differences among studied species

The stomata of Arabidopsis, a typical ruderal (R) species, always opened in response to two simultaneously applied opposing factors (Fig. 1), whereas in Triticum (competitor-ruderal, CR), stomata always closed in two-factor combinations (Fig. 5; time-courses of absolute gs are shown in Fig. S4). In Hordeum (CR species), the dominant stimulus in two-factor experiments was reduced air humidity (Figs 2f, 5). Even though the predicted stomatal response in two-factor combinations with reduced air humidity was opening, the stomata of Hordeum actually closed as much as in the single-factor humidity response (Fig. 2f). The only evidence of the opening factor's presence was slightly slower closure rate in two-factor experiments (Fig. 5, but see also Fig. 4e), but after 40 min in reduced humidity, Hordeum stomata were similarly closed in single- and two-factor experiments. In darkness + low CO2 and high CO2 + light combinations, Hordeum stomata initially opened but then started to close, with faster closure rate in the latter combination (Figs 3b, 5, S4).

Figure 5.

Time-resolved patterns of Thellungiella salsuginea, Nicotiana tabacum, Helianthemum nummularium, Hordeum vulgare and Triticum aestivum stomatal conductance in response to a simultaneous change in two opposing environmental factors. Factors were: darkness (Darkn) and low CO2 (from 400 ppm to 50–60 ppm) (a), reduced air humidity (Red hum; from 70–80% to 30–40%) and low CO2 (b), high CO2 (from 400 to 800 ppm) and additional light (from 150 to 450 μmol m−2 s−1) (c), and reduced air humidity and additional light (d). Factors were applied at zero timepoint with the closure-inducing change applied c. 20 s before the opening-inducing change. Data are shown in relative units (= 5–7, ± SEM); the average of the two last pre-treatment gs values was used for normalization.

The stomatal responses of Thellungiella, another R species, resembled those of Arabidopsis: the predicted response to applied two-factor combinations was always a closure, but the actual responses ranged from slight to considerable opening (Figs 2b, 3a, 5, S4). Furthermore, the two-factor combinations with low CO2 concentration resulted in the largest stomatal openings in Thellungiella (Fig. 2b). In Nicotiana (CR), the predicted stomatal response in two-factor combinations was always a closure and the largest differences between measured and predicted responses were, as in Arabidopsis, the combinations with CO2 withdrawal (Figs 2c, 5). Although Nicotiana was able to avoid considerable stomatal opening in darkness + low CO2 (Figs 2c, 5a), the opening response was still evident after 3 h in these conditions (Fig. 3a). In Helianthemum (S), gs increased in high CO2 + light and reduced humidity + low CO2 combinations (Figs 2d, 5, S4). In high CO2 + light, some stomatal opening may be beneficial in terms of photosynthetic gain and the two ruderals together with stress-tolerator Helianthemum opted for this. The reduced humidity + low CO2 combination resulted in stomatal opening in all studied species except cereals (Fig. 5b), also pointing at CO2 withdrawal as an important signal for plants. Furthermore, stomatal opening in two-factor experiments with reduced humidity (Fig. 2a–d) suggests that the dominance of hydraulic factors in stomatal control is not always maintained through vascular plant phylogeny, as has been previously suggested (Aasamaa & Sõber, 2011; Brodribb & McAdam, 2011). In response to reduced humidity + light and darkness + low CO2, the stomatal responses of Helianthemum (S) were modest (Figs 2d, 3b, 5).

Discussion

Here, we used six species with different life strategies to study their stomatal responses to various combinations of changes in environmental factors that have opposing effects on gs. Arabidopsis and Thellungiella are typical ruderals, striving to maximize dry matter production as a prerequisite for short lifecycle and high seed production (Grime, 1977). They may thus opt for the risky but potentially profitable behaviour of keeping their stomata open to maintain carbon fixation even in unfavourable conditions. In our two-factor combinations, Arabidopsis and Thellungiella always opened their stomata (Fig. 2a,b). Arabidopsis Col-0 wilted after 3 h in reduced humidity + low CO2, indicating that these plants indeed do not prioritize their hydraulic integrity. Hydraulic dysfunction resulting from excessive water loss through open stomata in conditions when either water supply is limited or evaporative demand is high, represents a serious risk to plant survival. Future experiments will show whether Arabidopsis and Thellungiella open their stomata in other two-factor combinations as well, supporting the hypothesis that ruderals are risk-takers in terms of stomatal behaviour and hydraulic integrity. Together, the stomatal responses of the studied species suggest that plant adaptive life strategy is not always a good indicator of stomatal response in two-factor combinations. Nicotiana, Triticum and Hordeum, irrespective of all being competitor-ruderal species, behaved very differently. Furthermore, some combinations such as reduced humidity + low CO2 resulted in considerable stomatal opening even in the stress-tolerator Helianthemum (Fig. 2d), a species that should prioritize its longevity. Stomatal opening in this situation represents a potential threat to hydraulic integrity and, therefore, to longevity. As even the two cereal species showed differences in responses to opposing factors, their specific stomatal behaviour cannot be explained by guard cell shape and/or close connection between guard and subsidiary cells inherent to cereals. Previously, it was suggested that the characteristic stomatal complexes of monocotyledons may explain the specificity of their stomatal responses (Mori & Murata, 2011). Based on results presented herein, it seems that stomatal responses to opposing factors are relatively species-specific.

Stomata initially opened in darkness + low CO2 in Arabidopsis, Thellungiella and Hordeum, although photosynthesis is impossible in such conditions. Prolonged 3-h experiments showed that this opening was transient in Hordeum, but persisted in Arabidopsis and Thellungiella (Fig. 3a,b). Aasamaa & Sõber (2011) found that in trees, a simultaneous application of darkness and reduced CO2 concentration also resulted in slight to moderate stomatal opening. Perhaps plants are inherently very sensitive to CO2 withdrawal. Atmospheric CO2 levels reached c. 3000 ppm around 400 Mya, when stomata are widely considered to have first evolved (Edwards et al., 1998; Royer, 2006), and since then a long-term general decrease in CO2 concentration has taken place (Royer, 2006). During this time, selective pressures on stomata may have operated towards increasing leaf conductance to CO2 diffusion and increasing water-use efficiency, potentially resulting in high stomatal sensitivity to reduced CO2 concentration. In support of this hypothesis, species from different land plant clades (angiosperms, conifers, ferns and lycophytes) all responded to low CO2 concentration, but only angiosperm stomata closed in response to CO2 enrichment (Brodribb et al., 2009). As examples of different responses to darkness+low CO2, Triticum closed its stomata, although more slowly compared to when only darkness was applied, and Nicotiana and Helianthemum opened just slightly (Figs 3, 4a, 5a). Taken together, in dicotyledonous plants reduced CO2 concentration overwhelmed the closing signal of darkness, although considerable stomatal opening in darkness+low CO2 was evident only in Arabidopsis and Thellungiella. In cereals the dominative effect of reduced CO2 over darkness was temporary, with darkness eventually closing stomata. This happened considerably faster in Triticum than in Hordeum. It is worth mentioning here that when darkness and reduced CO2 concentration are applied sequentially, both responses hold: dark-adapted plants respond to CO2 removal and low CO2-adapted plants respond to darkness (Hedrich et al., 2001; Doi & Shimazaki, 2008; Easlon & Richards, 2009; Fujita et al., 2013). From a slightly different angle, in nature, the simultaneous withdrawal of CO2 and light is an unrealistic signal combination both today and across the evolutionary timescale. It is thus possible that a rational stomatal response cannot be expected in these conditions, particularly considering that darkness response is partially mediated via increased internal CO2 concentration.

Guard-cell signalling is organized as a network, wherein different signal pathways converge at some point, and crosstalk must exist between the response pathways of different co- or counterdirectional signals. Ion transport proteins (anion channels, K+ channels and H+-ATPases) and the kinases/phosphatases involved in their regulation could function as the potential crosstalk points, a suggestion supported by experimental data (Jacob et al., 1999; Marten et al., 2007; Merilo et al., 2013). It has been proposed that in trees the communication between different signal response pathways is weak (Aasamaa & Sõber, 2011). We also found that measured and predicted responses differed, often fundamentally, in two-factor experiments (Fig. 2). However, this does not mean that the crosstalk between opening and closure-inducing pathways is weak. Clearly, the response to two simultaneously applied factors is not a simple sum of responses to these factors applied as single ones; through crosstalk between different pathways, a species-specific nonadditive stomatal response is achieved in two-factor combinations. Further studies are needed to find the mechanistic explanation for the species-specific stomatal responses revealed in our study. Even though there are a few results showing species-related differences in stomatal behaviour, for example the stomata of Vicia faba were found to be insensitive to light and CO2 (Mott et al., 2008), the mechanistic cause is unknown. Still, Mori & Murata (2011) hypothesized that species-specific differences in the contribution of intra- and extracellular ABA receptors may result in distinct stomatal behaviour. We suggest that plasticity in the sensitivity of guard cell membrane transport to messengers such as cytosolic Ca2+ (Ca2+cyt) (Dodd et al., 2010), or in the sensitivity of stomatal conductance or leaf hydraulic conductance to ABA concentration or leaf water potential, could be involved in the species-specificity of stomatal behaviour (Brodribb & McAdam, 2013; Pantin et al., 2013). With regards to Ca2+, Commelina plants that were grown at lower temperatures did not exhibit ABA-induced increases in guard cell Ca2+cyt, whereas growth at higher temperatures resulted in an ABA-triggered Ca2+cyt response; however, ABA-induced stomatal closure was detected at both temperatures (Allan et al., 1994). Generally, it seems that species-specific tuning of stomatal signalling mechanisms in response to environmental factors is clearly a research area where the amount of information is limited.

In conclusion, the stomata of all studied herbaceous plants responded to single environmental factors in similar ways to the well-studied Arabidopsis Col-0 accession. However, when two opposing factors were applied simultaneously, the stomatal responses of the studied species were fundamentally different: in Arabidopsis and Thellungiella, stomata always opened; in Hordeum, the response to reduced air humidity dominated over other responses; whereas in Triticum, stomata always closed in two-factor combinations. The priority of hydraulic factors for Hordeum and the clear stomatal closure of Triticum in inconsistent conditions could offer an additional explanation for the extensive spread and diversification of grasses during global aridification 30–45 Mya – a phenomenon generally explained by the faster-acting and more transpiration-effective stomata of grasses (Hetherington & Woodward, 2003). Thus, although studies on Arabidopsis provide crucial information on stomatal signalling and behaviour, important differences exist in the stomatal responses of different species in naturally realistic conditions, when environmental factors change simultaneously. Stomata control water flow through the soil–plant–atmosphere continuum and models of stomatal conductance are of great importance for predicting changes in global carbon and freshwater cycles, and overall future climate scenarios. In stomatal modelling, an important objective is to integrate the complex influences of environmental factors and effective molecules (ABA, H2O2) in the models (Damour et al., 2010). Our results indicate that in changing environmental conditions, different factors are not independent of each other in affecting stomata and stomatal responses are species-specific. Taken together, the crosstalk between stomatal closing and opening pathways is highly species-specific and detailed studies addressing the mechanistic explanation of this phenomenon are needed. Furthermore, our results indicate that in stomatal models both interactions between environmental factors and species-specific physiological determinism of stomatal behaviour need to be considered.

Acknowledgements

We thank Professor Meelis Pärtel for scientific discussions concerning plant functional types, Dr Bakhtier Rasulov for valuable advice, Mikk Tagel from Farm Plant Eesti for kindly providing wheat and barley seeds, Professor William Manning for tobacco seeds and Kersti Tambets from Tartu Botanical Garden for common rockrose seeds. This work was supported by Estonian Ministry of Science and Education (ESF9208, IUT2-21), European Regional Fund (Center of Excellence in Environmental Adaptation) and European Social Fund (Mobilitas Top Researchers grant MTT9).

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