Involvement of sphingosine kinase in plant cell signalling

Abstract

In mammalian cells sphingosine-1-phosphate (S1P) is a well-established messenger molecule that participates in a wide range of signalling pathways. The objective of the work reported here was to investigate the extent to which phosphorylated long-chain sphingoid bases, such as sphingosine-1-phosphate and phytosphingosine-1-phosphate (phytoS1P) are used in plant cell signalling. To do this, we manipulated Arabidopsis genes capable of metabolizing these messenger molecules. We show that Sphingosine kinase1 (SPHK1) encodes an enzyme that phosphorylates sphingosine, phytosphingosine and other sphingoid long-chain bases. The stomata of SPHK1-KD Arabidopsis plants were less sensitive, whereas the stomata of SPHK1-OE plants were more sensitive, than wild type to ABA. The rate of germination of SPHK1-KD was enhanced, whereas the converse was true for SPHK1-OE seed. Reducing expression of either the putative Arabidopsis S1P phosphatase (SPPASE) or the DPL1 gene, which encodes an enzyme with S1P lyase activity, individually, had no effect on guard-cell ABA signalling; however, stomatal responses to ABA in SPPASEDPL1 RNAi plants were compromised. Reducing the expression of DPL1 had no effect on germination; however, germination of SPPASE RNAi seeds was more sensitive to applied ABA. We also found evidence that expression of SPHK1 and SPPASE were coordinately regulated, and discuss how this might contribute to robustness in guard-cell signalling. In summary, our data establish SPHK1 as a component in two separate plant signalling systems, opening the possibility that phosphorylated long-chain sphingoid bases such as S1P and phytoS1P are ubiquitous messengers in plants.

Figure 1

Phosphorylation activity of putative Arabidopsis sphingosine kinases (SPHKs). Human embryonic kidney 293 (HEK 293) cells were transfected with the indicated plasmids, and phosphorylation activity was measured in cell lysates with sphingosine added with Triton X-100 (a) or as a complex with BSA (b). HEK 293 cells were transfected with plasmid containing At4g21540, and phosphorylation activity was measured in cell lysates with sphingolipid added under the conditions indicated ± 1 M KCl (c). HEK 293 cells were transfected with plasmid containing At4g21540 (d). Cell lysate proteins were separated by SDS-PAGE and were immunoblotted with anti-V5 antibody to show expression of the recombinant proteins (inset). Data are means ± SE of three independent experiments, each performed in duplicate. hSPHK1: human sphingosine kinase 1 (accession number AAF73423).

Figure 2

Characterization of SPHK1 T-DNA insertion Arabidopsis lines. (a) Diagram showing the positions of T-DNAs in the two independent insertion lines. Quantitative RT-PCR analysis of SPHK1 gene transcript levels in the T-DNA insertion lines compared with Col-0 control (b). Phosphorylation activity in extracts from different subcellular fractions of Col-0, SPHK1-OE and SPHK1-KD Arabidopsis plants using 50 μM sphingosine complexed with BSA as the substrate in the presence of 1 M KCl. Data are means ± SD of two independent experiments, each performed in duplicate (c).

Figure 3

SPHK1 has a role in two ABA-signalling pathways. Comparison of SPHK1 T-DNA insertion lines with Col-0 in an ABA-induced promotion of stomatal closure assay (a) and (b). Epidermal strips were incubated under opening conditions, and then stomatal apertures were measured after subsequent treatment with or without ABA for 2.5 h. Values are means ± SE: (a) n = 120; (b) n = 80. Comparison of SPHK1 T-DNA insertion lines with Col-0 in an ABA inhibition of stomatal opening assay (c). Stomatal apertures were measured after 3 h of incubation with or without ABA. Values are means ± SE (n = 120). Statistical analyses were performed by Student’s t-test: **P < 0.001. The experiments were repeated three times, except for 3C, which was repeated twice. Germination of SPHK1 T-DNA insertion lines compared with Col-0 (d). The experiment was repeated three times. Percentage radicle emergence 1 day after imbibition: the probability of germination was significantly different between lines [generalized linear model (GLM), deviance = 87.86, degrees of freedom (df) = 2, P < 0.0001] and treatments (GLM, deviance = 25.36, df = 2, P < 0.0001).

Figure 4

RNAi silencing of SPPASE and DPL1 in Arabidopsis plants. Quantitative RT-PCR analysis of SPPASE and DPL1 transcript levels in single (a and b) and double SPPASE DPL1 RNAi transgenic plants (c). The experiment was repeated three times.

Figure 5

Characterization of SPPASE DPL1 RNAi plants. Comparison of double mutant SPPASE DPL1 RNAi lines with Col-0 in an inhibition of stomatal opening assay. Apertures were measured after 3 h of incubation with or without ABA (a). Comparison of double mutant SPPASE DPL1 RNAi lines with Col-0 in an ABA-induced promotion of stomatal closure assay. Epidermal strips were pre-treated under opening conditions, and then stomatal apertures were measured after subsequent treatment ± ABA for 2.5 h (b). Values are means ± SE (n = 120). Statistical analyses were performed by Student’s t-test: *P < 0.05; **P < 0.001. Germination of single RNAi lines compared with Col-0 (c). The experiment was repeated three times. The percentage radicle emergence 3 days after imbibition: the probability of germination was significantly different between lines [generalized linear model (GLM), deviance = 100.1, degrees of freedom (df) = 4, P < 0.0001] and treatments (GLM, deviance = 198.8, df = 2; P < 0.0001). There was a significant interaction between line and treatment (GLM, deviance = 25.94, df = 8, P < 0.005), because the treatment had different effects depending on which line it acted upon; the germination reduction that is caused by ABA is greater in SPPASE RNAi-3 and SPPASE RNAi-4 than in the other lines.

Figure 6

Altered SPPASE expression in SPHK1 T-DNA insertion lines. Quantitative RT-PCR analysis of SPHK1, SPPASE and DPL1 transcript levels in SPHK1-OE and SPHK1-KD plants. The experiment was repeated three times.