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In higher plants, stomata form pores on leaf surfaces that regulate both uptake of CO2 for photosynthesis and loss of water vapour during transpiration. The plant hormone ABA regulates stomatal pore size by mediating turgor changes in the guard cell pair surrounding the pore. These changes involve alterations in ion channel activities, which represent the final effectors of these processes9,10,11. Recently, S1P was shown to stimulate stomatal closure and Ca2+ mobilization in Commelina communis guard cells4. In addition, ABA promotion of stomatal closure in this species was partially attenuated by dl-threo-dihydrosphingosine (dl-threo-DHS)4, a competitive inhibitor of mammalian SphKs12. Nevertheless, little is known about the cellular mechanisms that regulate S1P action in plant cells13,14.

We used dl-threo-DHS and N,N-dimethylsphingosine (DMS), another potent inhibitor of mammalian SphKs12, to investigate whether SphK might be involved in ABA-mediated changes in stomatal apertures in A. thaliana. dl-threo-DHS or DMS alone did not affect stomatal apertures (Fig. 1a, d). However, in epidermal peels that had been pre-treated with dl-threo-DHS or DMS, the effect of ABA on both stomatal opening and stomatal closure was significantly attenuated (Fig. 1a, d). Because inhibition of stomatal opening by ABA involves the inhibition of inwardly rectifying K+ channels10,11, we next assessed the effect of DMS on inward K+ currents in A. thaliana guard cell protoplasts (GCPs)6,15. ABA inhibition of inward K+ currents was abolished by pre-treating GCPs with DMS (Fig. 1b, c). Because ABA also promotes stomatal closure by activating slow anion channels6,11,16, we next tested whether the effect of ABA on slow anion channels was altered by DMS. ABA activation of slow anion channel activity was partially inhibited by pre-treating GCPs with DMS (Fig. 1e, f). Together, these results suggest that ABA regulation of stomatal apertures and guard cell ion channel activities is transduced, at least in part, through SphK.

Figure 1: Effect of SphK inhibitors on ABA regulation of stomatal apertures and guard cell ion channel activities.
figure 1

a, Apertures after a 2.5 h pre-treatment in the dark in 20 µM dl-threo-DHS or 5 µM DMS (vehicle, dimethylsulphoxide; final concentration, ≤0.08%), and a further 2 h light treatment with or without 20 µM ABA. b, Representative whole-cell recordings of K+ currents from wild-type GCPs pre-treated with 5 µM DMS for 30 min, and subsequently treated for 1.5 h with or without 50 µM ABA; 50 µM ABA was also included in bath solution. c, Current–voltage curves of time-activated whole-cell K+ currents in wild-type GCPs; n = 31, 9, 21 and 16 cells for control, DMS, ABA and DMS + ABA treatments, respectively. d, Apertures after a 2.5 h light pre-treatment in 20 µM dl-threo-DHS or 5 µM DMS, and a further 3 h light treatment with or without 20 µM ABA. e, Representative whole-cell recordings of slow anion currents from wild-type GCPs treated as in b, except that 50 µM ABA was also included in the pipette solution16 in ABA and DMS + ABA treatments. f, Current–voltage curves of steady-state whole-cell anion currents in wild-type GCPs; n = 31, 13, 24 and 14 cells for control, DMS, ABA and DMS + ABA treatments, respectively.

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SphK activity has yet to be demonstrated in plants, although this enzyme has been characterized in animals1 and yeast17. A related kinase activity able to phosphorylate d-erythro-dihydrosphingosine (d-erythro-DHS), which is structurally similar to d-erythro-sphingosine (Sph) except for the absence of the trans-4,5 double bond, has been reported in Zea mays18. A recombinant long-chain sphingoid base kinase (AtLCBK1) from A. thaliana was also shown to phosphorylate d-erythro-DHS19. Here, we show by direct assays of sphingoid base phosphorylation20 that SphK activity is present in lysates prepared from leaves, mesophyll cell protoplasts (MCPs), or GCPs of A. thaliana (Fig. 2a). We found that Sph, a low abundance, naturally occurring sphingoid base isomer in A. thaliana leaves21, was the best substrate among various exogenous sphingoid bases tested (Fig. 2b). Of the known inhibitors of mammalian SphKs, dl-threo-DHS was a substrate for A. thaliana SphK, whereas DMS was not (Fig. 2b). Furthermore, we found that DMS inhibited the activity of A. thaliana SphK in a concentration-dependent manner (Fig. 2c). Although a higher concentration of DMS was required for inhibition in vitro, such differences between the in vitro and in vivo concentrations are similar to those reported for other inhibitors used. For example, the phospholipase C (PLC) inhibitor U-73122 required an 80-fold higher concentration than was effective in vivo to inhibit guard cell PLC activity in vitro22. These results, together with those from Fig. 1, support the involvement of SphK in the regulation of guard cell responses to ABA.

Figure 2: Effect of ABA on SphK activity.
figure 2

a, Thin layer chromatography (TLC) analysis of [32P]S1P formed by lysates from leaves (25 µg), MCPs (25 µg), GCPs (3 µg) and recombinant human SphK1 (hSphK1) with 50 µM Sph as substrate (top panel). Without Sph added in the in vitro assay, no S1P was formed. The bottom panel shows the specific activity of SphK. b, Phosphorylation of various sphingoid bases (50 µM) by leaf lysate. c, Effect of various concentrations of DMS on SphK activity in leaf lysate with 5 µM Sph as substrate. d, Stimulation of [3H]S1P formation in vivo by 50 µM ABA in GCPs. The figure shows data representative of one of three separate experiments. e, Stimulation of [3H]S1P formation in vivo by 50 µM ABA in MCPs. Values are the mean ± s.e.m. from four independent experiments and data were compared using the Student's t-test at the 95% confidence level. f, SphK activity assayed in vitro from protein extracts of MCPs that had been treated with 50 µM ABA for various time in vivo.

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We next tested the ability of ABA to modulate S1P levels in vivo by measuring the formation of [3H]S1P from [3H]Sph. Treatment of GCPs and MCPs with ABA resulted in a rapid and transient increase in [3H]S1P formation, reaching a maximum after 2 min of ABA treatment (Fig. 2d, e). As expected, pre-treatment of MCPs with DMS blocked the ABA-induced increases in [3H]S1P formation (data not shown). We next assayed SphK activity in vitro from protein extracts of MCPs that had been treated with ABA for various times in vivo. We found that stimulation of MCPs with ABA rapidly and transiently increased SphK activity (Fig. 2f), with a time course similar to that of ABA-increased [3H]S1P formation in vivo (Fig. 2d, e). Collectively, these results provide evidence that ABA increases the activity of SphK, leading to the production of S1P in plants. To investigate further the nature of the interaction between ABA and SphK, we assayed SphK stimulation by ABA in cell-free systems derived from leaves, MCPs or GCPs. We found that ABA increased the phosphorylation of both exogenously supplied Sph and an endogenous long-chain sphingoid base (see Supplementary Information). These results, together with those of Fig. 2, suggest that the ABA receptor, signal transduction system9,10,11 and ABA-regulated SphK activity might exist in a tightly associated complex that survives the extraction process required for the in vitro assay.

In animal cells, an extracellular action of S1P by way of heterotrimeric G-protein-coupled receptors (GPCRs) for S1P is well established1,2,3. Recently, we used two independent A. thaliana transferred DNA (T-DNA) knockout lines, gpa1-1 and gpa1-2 (ref. 5), to provide evidence that GPA1 is involved in ABA signalling in A. thaliana guard cells6. Therefore, we examined whether S1P signals in guard cells can be transduced through GPA1, which in turn regulates K+ and slow anion channel activities and stomatal apertures6. We found that basal levels of SphK activity were similar in leaf and GCP lysates of wild-type and gpa1 null mutant plants (data not shown). In wild-type plants, S1P inhibited the opening of closed stomata to a similar extent as ABA (Fig. 3a). In addition, S1P promoted the closure of opened stomata (Fig. 3d), in agreement with previous observations in C. communis guard cells4. By contrast, in gpa1 null mutants, S1P neither inhibited stomatal opening nor promoted stomatal closure (Fig. 3a, d), although stomatal closure (Fig. 3d) in these genotypes occurs normally in response to ABA6. These results, together with those of Fig. 1, suggest that GPA1 functions downstream of SphK and S1P in ABA signal transduction pathways that lead to changes in stomatal apertures in A. thaliana. Studies using animal cells have shown that d-erythro-dihydrosphingosine-1-phosphate (dihydro-S1P), which is structurally similar to S1P except for the absence of the trans-4,5 double bond, is a ligand for S1P receptors and activates the same downstream responses as S1P23. However, in contrast to S1P, dihydro-S1P neither promoted stomatal closure (Fig. 3d), as previously reported for C. communis guard cells4, nor inhibited stomatal opening (Fig. 3a) in wild-type and gpa1 null mutant plants.

Figure 3: Effects of S1P on stomatal apertures and guard cell ion channel activities in wild-type and G-protein α-subunit (Gα) null mutant gpa1-1 and gpa1-2 plants.
figure 3

a, Apertures after a 2.5 h pre-treatment in the dark, and a further 2 h light treatment with or without 20 µM ABA, 10 µM S1P or 10 µM dihydro-S1P. b, Representative whole-cell recordings of K+ currents from wild-type, gpa1-1 and gpa1-2 GCPs. Control recordings were obtained 10 min after achieving the whole-cell configuration. GCPs were subsequently challenged with 10 µM S1P, and recordings were acquired again after 20 min. c, Average time-activated whole-cell K+ currents at -219 mV; n = 18, 10 and 7 cells for wild-type, gpa1-1 and gpa1-2, respectively. d, Apertures after a 2.5 h light pre-treatment, and a further 3 h light treatment with or without 20 µM ABA, 10 µM S1P or 10 µM dihydro-S1P. e, Representative whole-cell recordings of slow anion currents from wild-type, gpa1-1 and gpa1-2 GCPs. GCPs were pre-treated with 10 µM S1P for 30 min; 10 µM S1P was also added to bath solution. f, Average steady-state whole-cell anion currents at -145 mV; n = 25, 24 and 23 cells, and n = 29, 29 and 23 cells for wild-type, gpa1-1 and gpa1-2 treated with or without S1P, respectively.

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We next tested the ability of S1P to modulate guard cell ion channel activities in wild-type and gpa1 null mutant plants. In mammalian cells, S1P activates muscarinic receptor-coupled inwardly rectifying K+ channels, probably by way of the S1P3 GPCR24, and Ca2+-dependent K+ channels25. By contrast, our data show that S1P inhibits the inward K+ channels in GCPs from wild-type plants. Importantly, this inhibitory effect is dependent on heterotrimeric G proteins, as evident from the lack of S1P inhibition of inward K+ channels in gpa1 null mutants (Fig. 3b, c). We next examined the effect of S1P on slow anion channels, which are activated by ABA, in part through heterotrimeric G proteins6. We found that S1P stimulated slow anion currents in guard cells of wild-type plants, but not in gpa1 null mutant lines (Fig. 3e, f). This result is consistent with the absence of S1P stimulation of stomatal closure in these mutant lines (Fig. 3d). Together, these results highlight the central role of GPA1 in mediating the diametrically opposing effects of S1P on guard cell inward K+ channels and slow anion channels.

In this study, we have provided a biochemical basis for how S1P is generated in guard cells in response to ABA, and have elucidated the underlying mechanisms whereby the S1P signal is transduced within the cellular context to mediate changes in guard cell turgor (Fig. 4). Future work may allow the GPA1-dependent pathways of S1P action (Fig. 4) to be linked with other known ABA signalling components, such as reactive oxygen species, cytosolic pH and levels of cytosolic Ca2+ (refs 9, 10–11). Furthermore, it will be important to elucidate the molecular mechanisms by which S1P affects GPA1. In animal cells, S1P and dihydro-S1P act as extracellular ligands for the S1P receptor family1,23. In guard cells, S1P signal transduction by way of a GPA1-coupled S1P-like receptor is less likely, because dihydro-S1P did not affect stomatal responses (Fig. 3a, d). Moreover, the putative GPCR of A. thaliana, GCR17,8,26,27, bears no sequence homology to any of the conserved S1P receptors. Therefore, the S1P signal in guard cells may be transduced either through a structurally unique GPCR, by a direct interaction of S1P with GPA1, or through unidentified proteins that function to input signals to heterotrimeric G proteins independently of GPCRs. This latter possibility is lent credence by recent evidence in mammalian cells that AGS (activator of G-protein signalling) proteins can activate heterotrimeric-G-protein signalling pathways independently of GPCRs7,8,28, and might help to explain the enigmatic intracellular action of S1P in mammalian cell growth, given that proteins serving as intracellular targets of S1P have not yet been identified1,2,23. Future work is likely to reveal novel mechanisms of S1P signalling through heterotrimeric G proteins in diverse eukaryotes.

Figure 4: Model of ABA activation of S1P signalling in A. thaliana guard cells.
figure 4

The model integrates only the results presented here and previous data on gpa1 knockout plants6. The other signalling elements known to operate in these cells9–11 are omitted from this S1P-related model for the sake of clarity. S1P formed by ABA activation of SphK may function as a ligand for an unknown receptor (R), or may interact directly with GPA1 to inhibit and activate plasma membrane inwardly rectifying K+ channels and slow anion channels, respectively, resulting in inhibition of stomatal opening and promotion of stomatal closure. Major points that remain to be clarified by further experiments are indicated by question marks. Because ABA activates stomatal closure and slow anion channels in gpa1 knockout lines but S1P does not, the solid line is added to represent an S1P-independent mechanism of ABA action on stomatal closure, the precise nature of which requires further investigation. Possible interactions between the illustrated components and the diverse array of other intermediates known to be involved in guard cell ABA signalling9–11 remain to be elucidated.

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Methods

Preparation of GCPs and MCPs

GCPs were isolated as described6,15. MCPs were isolated by modification of a method described previously15 (see Supplementary Methods).

Stomatal bioassay and electrophysiology

Stomatal aperture bioassays and whole-cell current recordings were conducted as described6, with minor modifications (see Supplementary Methods).

Preparation of lysates from leaves, MCPs and GCPs

All procedures were conducted at 4 °C. Rosette leaves of 4- to 5-week-old plants6,15 were homogenized with SphK buffer20 containing 0.6% (w/v) insoluble polyvinylpyrrolidone (Sigma) and sand ( - 50 +70 Mesh, Sigma). The homogenate was then centrifuged at 10,000g for 10 min. The supernatant was recovered, frozen with liquid N2, and stored at -80 °C. Lysates from GCPs and MCPs were prepared by collecting and resuspending protoplasts in 400 µl of SphK buffer20. Protoplasts were homogenized and centrifuged at 10,000g for 10 min. The supernatants were then frozen with liquid N2, and stored at -80 °C.

SphK assays

Aliquots of the MCP suspension (100 µl, approximately 7.5 × 105 protoplasts) were incubated at room temperature for 5 min, and treated with or without 50 µM ABA prepared as described15. After the appropriate incubation time, each sample was homogenized in a glass/Teflon homogenizer with 300 µl of SphK buffer20, and centrifuged at 10,000g for 10 min. The supernatants were then frozen with liquid N2, and stored at -80 °C. SphK activity was assayed by providing [γ-32P]ATP and Sph (see Supplementary Methods) as substrates to the lysates, and measuring radiolabelled S1P formation20. Values are the mean ± s.e.m. from three to five independent experiments, and data were compared using the Student's t-test at the 95% confidence level.

Measurement of [3H]S1P formation

Intracellular levels of [3H]S1P were measured by modification of a method described previously29. Aliquots of MCP and GCP suspensions (200 µl, approximately 5 × 105 protoplasts) were incubated at 25 °C for 10 min, before the addition of 30 nM [3H]Sph (20 Ci mmol-1) and 0.2 mg ml-1 fatty-acid-free BSA for 1 min (approximately 300,000 d.p.m. per assay). Reactions were started by the addition of 50 µM ABA prepared as described15. After the appropriate incubation time, reactions were terminated by the addition of 500 µl of chloroform/methanol/concentrated HCl (100:200:1, v/v/v) and 40 µl of 2 M HCl. After vortexing, 250 µl of chloroform and 250 µl of 2 M KCl were added. The samples were mixed thoroughly, followed by centrifugation at 2,000g for 5 min. The resultant organic phases were collected, concentrated under a stream of N2, and spotted onto Silica gel 60 thin-layer chromatography (TLC) plates. Samples were run in a 1-butanol/acetic acid/water (3:1:1, v/v/v) solvent system, and Sph and S1P identified with ninhydrin. After scraping off the spots corresponding to [3H]S1P, radioactivity was measured by liquid scintillation counting (Optiphase Hisafe 3, Wallac).