Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1-phosphate in Arabidopsis

Abstract

The role of Delta4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Delta4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Delta4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Delta4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Delta4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Delta4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Delta4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Delta4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides.

Figure 1.

Sphingolipid biosynthesis and the role of the sphingolipid Δ4-desaturase. A, A schematic representation of sphingolipid LCB modification in higher plants. Note that the actual substrates in terms of free LCBs versus N-acylated LCBs are currently undefined, as is the order of desaturation reactions to generate d18:2Δ^4,8. Standard sphingolipid LCB nomenclature is used (e.g. d18:1Δ⁸ indicates that this is a dihydroxy-C₁₈ LCB with a double bond at the Δ⁸ position; t18:0 indicates that this is a trihydroxy-C₁₈ LCB with no double bonds). The sphingolipid class in which different LCBs are enriched is indicated (broken arrows), although it should be noted that other LCBs occur in these classes. Also shown (boxed) is the enzymatic conversion of LCBs to their phosphorylated form LCB-1-P (via LCB kinases), the dephosphorylation of LCB-1-Ps back to LCBs (via LCB-1-P phosphatases), and also the breakdown of LCB-1-Ps via the LCB-1-P lyase to generate phosphoethanolamine and hexadecanal. Free LCBs can be generated either by de novo synthesis or ceramidase-mediated release from sphingolipids (the latter is the predominant route in animals). B, Phylogenetic tree of sphingolipid Δ4-desaturase orthologs from different organisms. Functionally characterized examples are from P. pastoris (AY700778), C. albicans (XM_716863), S. pombe (NM_001022326), H. sapiens (AF002668; MLD), and Drosophila (AF466379; DES-1). Higher plant orthologs from Arabidopsis (NM_116731) and tomato (AF466378) have previously proved intractable to heterologous characterization. An unusual virally encoded ortholog is also included for comparison from Emiliania huxleyi virus 86 (EhV86; AJ890364).

Figure 1.

Sphingolipid biosynthesis and the role of the sphingolipid Δ4-desaturase. A, A schematic representation of sphingolipid LCB modification in higher plants. Note that the actual substrates in terms of free LCBs versus N-acylated LCBs are currently undefined, as is the order of desaturation reactions to generate d18:2Δ^4,8. Standard sphingolipid LCB nomenclature is used (e.g. d18:1Δ⁸ indicates that this is a dihydroxy-C₁₈ LCB with a double bond at the Δ⁸ position; t18:0 indicates that this is a trihydroxy-C₁₈ LCB with no double bonds). The sphingolipid class in which different LCBs are enriched is indicated (broken arrows), although it should be noted that other LCBs occur in these classes. Also shown (boxed) is the enzymatic conversion of LCBs to their phosphorylated form LCB-1-P (via LCB kinases), the dephosphorylation of LCB-1-Ps back to LCBs (via LCB-1-P phosphatases), and also the breakdown of LCB-1-Ps via the LCB-1-P lyase to generate phosphoethanolamine and hexadecanal. Free LCBs can be generated either by de novo synthesis or ceramidase-mediated release from sphingolipids (the latter is the predominant route in animals). B, Phylogenetic tree of sphingolipid Δ4-desaturase orthologs from different organisms. Functionally characterized examples are from P. pastoris (AY700778), C. albicans (XM_716863), S. pombe (NM_001022326), H. sapiens (AF002668; MLD), and Drosophila (AF466379; DES-1). Higher plant orthologs from Arabidopsis (NM_116731) and tomato (AF466378) have previously proved intractable to heterologous characterization. An unusual virally encoded ortholog is also included for comparison from Emiliania huxleyi virus 86 (EhV86; AJ890364).

Figure 2.

Functional characterization of Arabidopsis gene At4g04930 as a sphingolipid Δ4-desaturase. A, Expression of Arabidopsis At4g04930 ORF in a P. pastoris mutant lacking sphingolipid Δ4-desaturase activity restores the synthesis of GlcCer. The P. pastoris mutant disrupted in the endogenous sphingolipid Δ4-desaturase AY700778 was transformed with either empty expression vector or vector containing the Arabidopsis ORF. Sphingolipids were extracted, separated by thin-layer chromatography, and stained by spraying with α-naphthol/sulfuric acid and subsequent heating to 160°C. The presence of GlcCer is clearly visible in P. pastoris mutants complemented with the Arabidopsis ORF (PpΔ4KO + At4g04930) but absent in mutants transformed with the empty vector (PpΔ4KO). For comparison, wild-type P. pastoris cells transformed with the empty vector are also accumulating GlcCer (PpWT). B, GlcCer from P. pastoris cells was isolated and subjected to sphingolipid LCB analysis via deacylation and derivatization with 1-fluoro-2,4-dinitrobenzene. LCBs were fractionated by HPLC and detected by A₃₅₀. GlcCers from wild-type P. pastoris transformed with the empty vector (top trace) and from the P. pastoris sphingolipid Δ4-desaturase mutant transformed with Arabidopsis ORF At4g04930 (bottom trace) both contain Δ4-unsaturated LCBs (predominantly in the form of the C9-methyl-sphinga-4,8-dienine). The P. pastoris mutant transformed with the empty vector does not contain any GlcCers (middle trace).

Figure 3.

Molecular species composition of GlcCer of wild-type (WT) and sphingolipid Δ4-desaturase mutant plants. Sphingolipids were extracted from wild-type Col-0 flowers (A), SALK_107761 flowers (B), wild-type No-0 flowers (C), and wild-type Col-0 leaves (D), and the amount of sphingolipid in the extract was determined by LC-MS/MS as described previously (Markham and Jaworski, 2007). Only GlcCers were found to contain appreciable amounts of d18:2 LCBs (A) with no d18:2 detectable in the other sphingolipid classes (data not shown; Markham et al., 2006). GlcCers containing d18:2 were undetectable (white arrows) in the SALK mutant line (B) and also absent from wild-type Col-0 leaf tissue (D). No d18:2 GlcCers were detected in floral tissues in wild-type No-0 (C) from which the RIKEN transposon mutant was isolated (Supplemental Table S2). Bars show averages (n = 5), and error bars show sd. dw, Dry weight.

Figure 4.

Sphingolipid content of wild-type (WT) and sphingolipid Δ4 desaturase mutant lines. Sphingolipids were extracted from floral tissue (three samples for wild-type Col-0 and six samples for SALK_107761), and the amount of sphingolipid in the extract was determined by LC-MS/MS as described previously (Markham and Jaworski, 2007). Total amounts of each class of sphingolipid were calculated compared with added internal standards. Significant differences (P < 0.05) between the samples were determined by Student's t test and are indicated by stars. dw, Dry weight.

Figure 5.

Phenotypic characterization of mutants for altered drought response. A, The wild type (WT) and the respective insertion mutant were grown to first bolt stage and then water was withheld. As a control, wild-type material was also fully watered. Leaves were harvested from drought-treated and control plants every 2 d for 17 d, and the (fresh weight − dry weight)/fresh weight [(FW-DW)/FW] percentage was calculated. As can be seen, there is no significant difference between the wild-type (Col-0 and No-0) and the mutant (SALK-107761 and RIKEN 15-1202-1) lines in terms of their decrease in fresh weight in response to drought. Each point represents an average (n = 3), and error bars show se. B, The wild type and the respective insertion mutants were assessed for their ability to regulate water loss through their transpiration stream via excision of roots and measurement of total mass as an indicator of water loss. Measurements were taken over a 7-h period, and data are expressed as percentages of the original starting mass (in grams) of the rootless plant. Plant material used was as for A. As can be seen, neither insertion mutant had any significant difference in mass loss. Each point represents an average (n = 3), and error bars show se.

Figure 5.

Phenotypic characterization of mutants for altered drought response. A, The wild type (WT) and the respective insertion mutant were grown to first bolt stage and then water was withheld. As a control, wild-type material was also fully watered. Leaves were harvested from drought-treated and control plants every 2 d for 17 d, and the (fresh weight − dry weight)/fresh weight [(FW-DW)/FW] percentage was calculated. As can be seen, there is no significant difference between the wild-type (Col-0 and No-0) and the mutant (SALK-107761 and RIKEN 15-1202-1) lines in terms of their decrease in fresh weight in response to drought. Each point represents an average (n = 3), and error bars show se. B, The wild type and the respective insertion mutants were assessed for their ability to regulate water loss through their transpiration stream via excision of roots and measurement of total mass as an indicator of water loss. Measurements were taken over a 7-h period, and data are expressed as percentages of the original starting mass (in grams) of the rootless plant. Plant material used was as for A. As can be seen, neither insertion mutant had any significant difference in mass loss. Each point represents an average (n = 3), and error bars show se.

Figure 6.

Stomatal aperture measurements of wild-type (WT) and mutant lines after ABA treatment. Stomatal apertures were measured from wild-type and mutant leaves treated with or without ABA for 2 h. Data are from three independent experiments, and measurements were carried out double blind. Each bar represents an average (n = 3), and error bars show se. Significant differences (P < 0.01; stars) between the treated and untreated samples were determined by Student's t test.

Figure 7.

Pollen viability of mutants. Pollen from wild-type (WT) and insertion mutant flowers was assayed for germination, with percentage germination assessed by microscopic examination. Plant material used was as for Figure 5. No significant difference between the wild type and mutants was observed. Each bar represents an average (n = 3), and error bars show se.