In animals, the sphingolipid metabolite sphingosine-1-phosphate (S1P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S1P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis1,2,3. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor4. Here we report that the enzyme responsible for S1P production, sphingosine kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K+ channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S1P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein α-subunit gene, GPA1 (refs 5, 6, 7–8). Our results implicate heterotrimeric G proteins as downstream elements in the S1P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S1P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.
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.
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.
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.
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.
Preparation of GCPs and MCPs
Stomatal bioassay and electrophysiology
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.
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).
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We thank J. P. Hobson for the recombinant purified hSphK1, X.-Q. Wang, A. Olivera and J.-N. Pierre for technical advice, J. Coursol for statistical analyses, and T. Jacob and C. K.-Y. Ng for critically reading the manuscript. This work was supported by grants from the United States Department of Agriculture (USDA) and the National Science Foundation to S.M.A., from the USDA to S.G., and from the National Institutes of Health to S.S.
The authors declare that they have no competing financial interests.
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Coursol, S., Fan, LM., Stunff, H. et al. Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins. Nature 423, 651–654 (2003). https://doi.org/10.1038/nature01643
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