ABSTRACT
Sphingolipid long-chain bases (LCBs) are building blocks for membrane-localized sphingolipids, and are involved in signal transduction pathways in plants. Elevated LCB levels are associated with the induction of programmed cell death and pathogen-derived toxin-induced cell death. Therefore, levels of free LCBs can determine survival of plant cells. To elucidate the contribution of metabolic pathways regulating high LCB levels, we applied the deuterium-labeled LCB D-erythro-sphinganine-d7 (D7-d18:0), the first LCB in sphingolipid biosynthesis, to Arabidopsis leaves and quantified labeled LCBs, LCB phosphates (LCB-Ps), and 14 abundant ceramide (Cer) species over time. We show that LCB D7-d18:0 is rapidly converted into the LCBs d18:0P, t18:0, and t18:0P. Deuterium-labeled ceramides were less abundant, but increased over time, with the highest levels detected for Cer(d18:0/16:0), Cer(d18:0/24:0), Cer(t18:0/16:0), and Cer(t18:0/22:0). A more than 50-fold increase of LCB-P levels after leaf incubation in LCB D7-d18:0 indicated that degradation of LCBs via LCB-Ps is important, and we hypothesized that LCB-P degradation could be a rate-limiting step to reduce high levels of LCBs. To functionally test this hypothesis, we constructed a transgenic line with dihydrosphingosine-1-phosphate lyase 1 (DPL1) under control of an inducible promotor. Higher expression of DPL1 significantly reduced elevated LCB-P and LCB levels induced by Fumonisin B1, and rendered plants more resistant against this fungal toxin. Taken together, we provide quantitative data on the contribution of major enzymatic pathways to reduce high LCB levels, which can trigger cell death. Specifically, we provide functional evidence that DPL1 can be a rate-limiting step in regulating high LCB levels.
ABSTRACT
Sphingolipid long chain bases (LCBs) are building blocks of sphingolipids and can serve as signalling molecules, but also have antimicrobial activity and were effective in reducing growth of a range of human pathogens. In plants, LCBs are linked to cell death processes and the regulation of defence reactions against pathogens, but their role in directly influencing growth of plant-interacting microorganisms has received little attention. Therefore, we tested the major plant LCB phytosphingosine in in vitro tests with the plant pathogenic fungi Verticillium longisporum, Fusarium graminearum and Sclerotinia sclerotiorum, the plant symbiotic fungal endophyte Serendipita indica, the bacterial pathogens Pseudomonas syringae pv. tomato (Pst), Agrobacterium tumefaciens, and the related beneficial strain Rhizobium radiobacter. Phytosphingosine inhibited growth of these organisms at micromolar concentrations. Among the fungal pathogens, S. sclerotiorum was the most, and F. graminearum was the least sensitive. 15.9 µg/mL phytosphingosine effectively killed 95% of the three bacterial species. Plant disease symptoms and growth of Pst were also inhibited by phytosphingosine when co-infiltrated into Arabidopsis leaves, with no visible negative effect on host tissue. Taken together, we demonstrate that the plant LCB phytosphingosine inhibits growth of plant-interacting microorganisms. We discuss the potential of elevated LCB levels to enhance plant pathogen resistance.
