abscisic-acid and sphingosine-kinase

Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N',N'-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

Topics: Abscisic Acid; Analysis of Variance; Fluorescent Dyes; Hydrogen-Ion Concentration; Lysophospholipids; Microscopy, Confocal; Nitric Oxide; Phosphotransferases (Alcohol Group Acceptor); Pisum sativum; Plant Epidermis; Plant Growth Regulators; Plant Stomata; Signal Transduction; Sphingosine; Time Factors

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.

Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Enzyme Activation; Gene Knockdown Techniques; Phosphatidic Acids; Phospholipase D; Phosphotransferases (Alcohol Group Acceptor); Plant Stomata; Signal Transduction; Sphingosine

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.

Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Cells, Cultured; Cloning, Molecular; DNA, Bacterial; Gene Expression Regulation, Plant; Germination; Humans; Lysophospholipids; Membrane Proteins; Mutagenesis, Insertional; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Plant Stomata; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Plant; Signal Transduction; Sphingosine; Substrate Specificity

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 apoptosis. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. 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 alpha-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.

Topics: Abscisic Acid; Anions; Arabidopsis; Enzyme Activation; Heterotrimeric GTP-Binding Proteins; Ion Channel Gating; Ion Channels; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Potassium; Signal Transduction; Sphingolipids; Sphingosine