Connections between sphingosine kinase and phospholipase D in the abscisic acid signaling pathway in Arabidopsis

Abstract

Phosphatidic acid (PA) and phytosphingosine 1-phosphate (phyto-S1P) both are lipid messengers involved in plant response to abscisic acid (ABA). Our previous data indicate that PA binds to sphingosine kinase (SPHK) and increases its phyto-S1P-producing activity. To understand the cellular and physiological functions of the PA-SPHK interaction, we isolated Arabidopsis thaliana SPHK mutants sphk1-1 and sphk2-1 and characterized them, together with phospholipase Dα1 knock-out, pldα1, in plant response to ABA. Compared with wild-type (WT) plants, the SPHK mutants and pldα1 all displayed decreased sensitivity to ABA-promoted stomatal closure. Phyto-S1P promoted stomatal closure in sphk1-1 and sphk2-1, but not in pldα1, whereas PA promoted stomatal closure in sphk1-1, sphk2-1, and pldα1. The ABA activation of PLDα1 in leaves and protoplasts was attenuated in the SPHK mutants, and the ABA activation of SPHK was reduced in pldα1. In response to ABA, the accumulation of long-chain base phosphates was decreased in pldα1, whereas PA production was decreased in SPHK mutants, compared with WT. Collectively, these results indicate that SPHK and PLDα1 act together in ABA response and that SPHK and phyto-S1P act upstream of PLDα1 and PA in mediating the ABA response. PA is involved in the activation of SPHK, and activation of PLDα1 requires SPHK activity. The data suggest that SPHK/phyto-S1P and PLDα1A are co-dependent in amplification of response to ABA, mediating stomatal closure in Arabidopsis.

FIGURE 1.

Isolation of T-DNA insertion lines and expression of two SPHKs in Arabidopsis leaves. A, diagram showing the T-DNA insertion sites in Salk_042034 (sphk1-1) and Salk_000250 (sphk2-1). T-DNA is located in front of the start codons of SPHK1 and SPHK2 separately. Thin lines represent noncoding regions and boxes represent exons. B, PCR genotyping of two T-DNA insertion lines. Presence of the T-DNA band and lack of the SPHK1 or SPHK2 bands indicate that each is a homozygous T-DNA insertion mutant. PCR was conducted using genomic DNA with a pair of gene-specific primers (LP1 + RP1 for SPHK1 and LP2 + RP2 for SPHK2) or a combination of T-DNA left border primer (LBa1) and gene-specific primers (RP1 for SPHK1 and RP2 for SPHK2). C, expression levels of SPHK1 and SPHK2 in WT, SPHK mutants, complementation, and overexpression lines determined by real-time PCR normalized to UBQ10. RNA was extracted from 4-week-old Arabidopsis leaves. The experiment was repeated three times. Values are mean ± S.E. (n = 3) for one representative experiment. D, effect of ABA on SPHK1 and SPHK2 expression measured by real-time PCR normalized to UBQ10. The ABA response gene ABI1 was used as a positive control. RNA was extracted from leaves sprayed with 100 μm ABA with 0.01% Triton X-100. The experiment was repeated three times. Values are mean ± S.E. (n = 3) for one representative experiment.

FIGURE 2.

PA interacts with SPHK and is involved in activation of SPHK in response to ABA. A, quantification of NBD-PA bound to SPHK2 pulled down by anti-FLAG resin beads. Inset represents image of NBD-PA immunoprecipitated with SPHK2 on TLC plate. Control indicates protoplasts (incubated with ABA for 30 min) isolated from WT Arabidospsis. B, SPHK activity assay using NBD-sphingosine as substrate. NBD-S1P was produced by both SPHKs as indicated on the TLC plate. − indicates negative control without addition of protein. C, quantification of NBD-S1P production in protoplasts treated with 50 μm ABA. Protoplasts were isolated from WT, pldα1, sphk1-1, sphk2-1, and SPHK2-OE lines. D, quantification of NBD-S1P production in protoplasts treated with 50 μm PA. The level of NBD-S1P was calculated as the percentage of NBD-S1P over the total NBD-labeled lipids. Values in C and D are mean ± S.E. (n = 3).

FIGURE 3.

PLDα1 and PA mediate the phyto-S1P effect on the signaling pathway in ABA-mediated stomatal closure. A, effect of phyto-S1P on stomatal closure in WT and mutants. The epidermal peels were incubated in stomatal incubation buffer containing 10 μm phyto-S1P or 10 μm phyto-S1P plus 0.1% 1-butanol. Asterisks indicate that the mean value is significantly different from that of the samples treated with phyto-S1P at p < 0.05 based on Student's t test. B, PA (18:1/18:1) induces stomatal closure in WT and mutants. Epidermal peels were treated with 50 μm PA. All values are mean ± S.E. (n = 50) in the stomatal assays.

FIGURE 4.

Activation of PLDα1 by ABA requires SPHK. A, representative image of fluorescent-based assay of PLD activity using NBD-PC-labeled protoplasts treated with 50 μm ABA. B, quantification of ABA-induced PA production in protoplasts isolated from WT, pldα1, sphk1-1, and sphk2-1. Protoplasts were labeled with NBD-PC followed by treatment with ABA. WT control was treated with 0.1% ethanol. C, quantification of phyto-S1P-promoted PA production in protoplasts isolated from WT, pldα1, sphk1-1, and sphk2-1. The level of PA was calculated as the percentage of NBD-PA over the total NBD-labeled lipids. Data in B and C are mean ± S.E. (n = 3) for one representative experiment.

FIGURE 5.

ABA-induced PA changes in Arabidopsis leaves. A, change in total PA content in leaves harvested at different times after spraying with ABA (100 μm). B, comparison of PA molecular species in leaves of WT, mutants, and SPHK2-OE lines treated with ABA for 10 min. The experiment was performed three times. Values in A and B are mean ± S.E. (n = 5).

FIGURE 6.

Alterations of SPHKs change LCBP content and composition in Arabidopsis leaves. A, total LCBP content (mol %) in leaves from 4–5-week-old WT, pldα1, sphk1-1, sphk2-1, and SPHK2-OE5. B, LCBP composition in leaves from 4–5-week-old WT, pldα1, sphk1-1, sphk2-1, and SPHK2-OE5. C, total LCBP content in WT Arabidopsis leaves treated with ABA. 4–5-Week-old Arabidopsis was sprayed with 100 μm ABA with 0.01% Triton X-100 followed by sphingolipid extraction and MS analysis. D, LCBP composition in the leaves treated with 50 μm ABA with 0.01% Triton X-100 for 0–15 min. Data were calculated as molar percentage over the total amount of LCB (sphinganine (d18:0), 8-sphingenine (d18:1), phytosphingosine (t18:0), and 4-hydroxy-8-sphingenine (t18:1)) and LCBP (d18:0-P, d18:1-P, t18:0-P and t18:1-P). The experiment was performed twice and the results were consistent. Values are mean ± S.E. for one experiment (n = 5). Asterisks in B indicate that the mean value is significantly different from that of the WT at p < 0.05, based on Student's t test. Asterisks in C indicate that the mean value is significantly different from that of the 0-min ABA treatment for each Arabidopsis line at p < 0.05. Asterisks in D indicate that the mean value is significantly different from that of the 0 min ABA treatment for each Arabidopsis line at p < 0.05 based on Student's t test.

FIGURE 7.

Altered ABA sensitivity in SPHK mutants and SPHK2 overexpression Arabidopsis. A, addition of ABA (25 μm) induced stomatal closure in WT and mutants lines. COM1 is a complimented line for sphk1-1 and COM2 is a complimented line for sphk2-1. Values are mean ± S.E. (n = 50). Asterisks indicate that the mean value is significantly different from that of the WT treated with ABA at p < 0.05 based on Student's t test. B, root growth of WT, sphk1-1, sphk2-1, and SPHK2-OE lines (OE2 and OE5) on ½ MS medium with 0, 5, or 10 μm ABA. Values are mean ± S.E. (n = 20) for one representative experiment. Asterisks indicate that the mean value is significantly different from that of the WT treated with the same concentration of ABA at p < 0.05 based on Student's t test. C, seed germination rate on ½ MS medium with different concentrations of ABA. Desiccated seeds were germinated on ½ MS with or without ABA and scored 3 days after transfer from 4 °C. About 100 seeds per genotype were scored for each experiment. Values are mean ± S.E. (n = 3). D, seed germination and post-germination growth on ½ MS medium without ABA (left) or with 1 μm ABA at day 6.

FIGURE 8.

Proposed model for the role of SPHK/phyto-S1P and PLDα1/PA in the ABA-mediated stomatal closure signaling pathway. This model depicts the known targets of PLD/PA in the ABA-mediated stomatal closure and other ABA regulators are not included in this model. ABA activates SPHKs through unknown mechanisms and ABA receptors may be involved. The activation of SPHKs produces phyto-S1P that activates PLDα1 to produce PA. PA inhibits ABI1 function but promotes NADPH oxidase to promote ABA-mediated stomatal closure. Meanwhile, PLDα1-produced PA stimulates SPHK activity through a positive loop. Arrows with solid lines indicate established links and arrows with dashed lines denote putative links.