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Published in final edited form as: Plant J. 2008 Jun 10;55(6):1000–1009. doi: 10.1111/j.1365-313X.2008.03569.x

Molecular Cloning and Characterization of OsCDase, a Ceramidase Enzyme from Rice

Mickael O Pata 1, Bill X Wu 2, Jacek Bielawski 2, Tou Cheu Xiong 1,3, Yusuf A Hannun 2, Carl K-Y Ng 1
PMCID: PMC3072431  NIHMSID: NIHMS283703  PMID: 18547394

Summary

Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, we know much less about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase following heterologous expression in the yeast double knockout mutant, Δypc1Δydc1, which lacks the yeast ceramidases, YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics with an optimum activity ranging from pH 5.7 to 6.0. OsCDase activity was enhanced in the presence of Ca2+, Mg2+, Mn2+, Zn2+ but inhibited in the presence of Fe2+. OsCDase appears to use ceramide instead of phytoceramide as a substrate. Sub-cellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants..

Keywords: Sphingolipid, ceramide, ceramidase, reverse ceramidase activity

Introduction

Sphingolipids are ubiquitous components of eukaryotic cells where they form key structural components of membranes, and function as important mediators of physiological responses from apoptosis to cell survival and proliferation (Pyne and Phye. 2000; Dickson and Lester, 2002; Hannun and Obeid, 2002, 2008; Spiegel and Milstien, 2003; Futerman and Hannun, 2004). Sphingolipid metabolism has been intensely investigated in animal cells and yeast, and much is known about the different metabolic intermediates and the enzymes responsible for catalyzing their formation (Pyne and Phye. 2000; Dickson and Lester, 2002; Hannun and Obeid, 2002, 2008; Spiegel and Milstien, 2003; Futerman and Hannun, 2004). In yeast, sphingolipid metabolites like dihydrosphingosine and phytosphingosine have been shown to increase the ability of yeast to tolerate and grow under heat-stress conditions (Dickson et al., 1997; Jenkins et al., 1997; Mao et al., 1999). Sphingosine-1-phosphate has been shown in animal cells to be important in mediating cellular survival and proliferation, whereas ceramide has been implicated in mediating programmed cell death (Hannun and Obeid, 2002, 2008; Spiegel and Milstien, 2003; Futerman and Hannun, 2004). This has led to the suggestion that the dynamic balance between levels of sphingolipid metabolites within cells function to regulate cell fate although the situation is likely to be highly complex due to the inter-convertibility of sphingolipid metabolites (Futerman and Hannun, 2004; Hannun and Obeid, 2008). This is particularly true for ceramide and sphingosine/sphingosine-1-phosphate because sphingosine is formed by the action of ceramidases, placing ceramide as a central mediator of sphingolipid-based signalling systems in cells (Hannun and Obeid, 2002, 2008).

In contrast to the situation in animal cells and in yeast, we know much less about the roles of sphingolipid metabolites in regulating cellular processes. Plant membranes are made up of significant amounts of sphingolipids, and it has been estimated that 7 to 26% of plant membrane lipids are sphingolipids (Lynch, 1993, Schmid nad Ohlrogge, 1996). The first information on plant sphingolipids was mainly obtained from analyzing the lipid composition of seeds (Carter et al., 1958; Ito et al., 1985) although there are indications that sphingolipids may be important cellular mediators in plants. It was reported that glucosylceramides (cerebrosides A and C) from wheat grain could induce fruiting in Schizophyllum commune, a fungus that causes wood decay (Kawai et al., 1986; Kawai, 1989), and the same glucosylceramides from the pathogenic rice fungus, Magnaporthe grisea are elicitors of hypersensitive cell death and phytolexin accumulation in rice plants (Koga et al., 1998). More recently, it was reported that sphingosine-1-phosphate is a calcium-mobilizing messenger active in the responses of stomatal guard cells to the drought hormone, abscisic acid (Ng et al., 2001) and that the effects of sphingosine-1-phosphate is dependent on heterotrimeric G-proteins (Coursol et al., 2003). Progress in the understanding of sphingolipid metabolism in plants has been aided by the characterization of Arabidopsis mutants affected in sphingolipid metabolism (Chen et al., 2006; Tsegaye et al., 2007; Shi et al., 2007; Dietrich et al., 2008) and from the development of mass spectrometric methods for the analysis of plant sphingolipids (Markham et al., 2006; Teng et al., 2008). Mutant analyses in Arabidopsis have revealed important roles for sphingolipid metabolites in mediating sensitivity towards the mycotoxin, fumonisin B1 (Tsegaye et al., 2007), and sphingolipid metabolites have been shown to be important regulators of gametophytic and sporophytic development (Imamura et al., 2007; Dietrich et al., 2008; Teng et al., 2008), thereby affecting fertility.

Ceramide has been shown to induce programmed cell death in Arabidopsis cells (Liang et al., 2003; Townley et al., 2005), suggesting that ceramide may be an important regulator of plant development. In order to further elucidate the role of ceramide in plants, it is important to understand the way in which ceramide is metabolized in plant cells. Ceramidases are enzymes responsible for metabolizing ceramide to sphingosine by cleaving the N-acyl linkage between the sphingoid base and the fatty acid. There are three different types of ceramidases, and they have been classified as acid, neutral and alkaline according to their pH optima for activity (Mao et al., 2002).

Here we report the cloning of a plant ceramidase from rice. We demonstrate that OsCDase is a bona fide ceramidase that appears to use ceramide instead of phytoceramide as a substrate. We also used a lipidomic approach to show that OsCDase exhibits reverse ceramidase activity, resulting in the formation of phytoceramides with very long chain fatty acids. Sub-cellular localization showed that OsCDase-DsRed2 protein fusion is targeted to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of sphingolipid metabolism in plants.

Results

Cloning, phylogenetic, and expression analysis of OsCDase

We used the Blast program to search the rice genome database for sequences bearing similarities to the human neutral ceramidase gene, ASAH2 (GenBank Accession No.: NP_063946) and identified a single copy gene on Chromosome 1 designated Os01g43520. The predicted amino acid sequence of the putative rice ceramidase (OsCDase) has a 42% sequence identity and 59% sequence similarity to ASAH2, suggesting that OsCDase may be a neutral ceramidase. We then used RT-PCR and 5’- and 3’-RACE PCR to obtain the full-length transcript for OsCDase. OsCDase is made up of 10 exons (Figure 1A). Interestingly the 5’-UTR of OsCDase contains a 777 bp intron although the Ensembl Exon report on Gramene (http://www.gramene.org) predicted an intron of 255 bp. In silico analysis of the 5’-UTR of OsCDase using Regulatory RNA Motifs and Elements Finder (RegRNA; http://regrna.mbc.nctu.edu.tw/index.html) showed the presence of a 90 bp (position -32 to -122) internal ribosomal entry site (IRES) element. Phylogenetic analysis of neutral ceramidases from various organisms indicates that OsCDase is clustered with the putative ceramidases from Arabidopsis thaliana and the neutral ceramidase homologue of Dictyostelium discoideum (Figure 1B). ClustalW alignment of the amino acid sequence of OsCDase with ceramidases from various organisms (Supplemental Figure 1) showed the presence of the highly conserved hexapeptide sequence, GDVSPN within the larger conserved amidase domain, NXGDVSPNXXC (Figure 1C), important for ceramidase activity (Galadari et al., 2006). Expression analysis indicated that steady-state levels of OsCDase transcripts are expressed throughout the seedling with a higher level of expression in roots compared to shoots (Figure 1D).

Figure 1. Cloning, sequence analysis and expression of rice ceramidase (OsCDase) in planta.

Figure 1

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(A) Schematic representation of the sequence organization of OsCDase. The sizes of the 5’-UTR (white), exons (black), and 3’UTR (grey) are indicated by numbers below the boxes. Sizes of introns are shown in italics. (B) Phylogenetic tree of the neutral ceramidases from various organisms (Os: Oryza sativa; At: Arabidopsis thaliana; Dd: Dictyostelium discoideum; Rn: Rattus norvegicus; Mm: Mus musculus; Cf: Canis familiaris; Hs: Homo sapiens; Gg: Gallus gallus; Dp: Drosophila pseudoobscura; Dm: Drosophila melanogaster) were generated using T-Rex (http://www.trex.uqam.ca/). (C) Clustal alignment of the neutral ceramidases from various organisms showing the highly conserved hexapeptide sequence, GDVSPN within the larger conserved amidase domain, NXGDVSPNXXC, important for ceramidase activity. (D) RT-PCR expression analysis of OsCDase in shoot and roots of 2-week old seedlings. Housekeeping control gene: OsEF1α.

Biochemical characterization of OsCDase

We cloned the full-length OsCDase CDS into the yeast expression vector, pYES2/CT under the control of the Gal1 promoter, and the resulting plasmid was transformed into the yeast double knockout mutant, Δypc1Δydc1, which lacks the yeast CDases, YPC1p and YDC1p (Mao et al., 2000). This is important as it allowed us to express recombinant OsCDase and minimize contamination from endogenous yeast CDase activity that may interfere with subsequent activity characterization of OsCDase. Expression of recombinant OsCDase was induced by culturing the transformed yeast in medium containing galactose, and cells were harvested at different time points to determine the optimal time for induction of OsCDase expression. Homogenates were obtained from harvested cells and assayed for CDase activity using NBD-C12-ceramide as a substrate (Figure 2A). CDase activity was detectable 2 h following induction with galactose, peaking at 10–12 h, and decreasing thereafter. We could not detect any CDase activity after induction with galactose in yeast containing the vector alone (Figure 2A). The amount of recombinant OsCDase was determined in western blot using Anti-His antibody, and the amount of recombinant OsCDase peaked at about 12 h (Figure 2B), in agreement with optimal activity at 12 h post-induction (Figure 2A). On the basis of these data, we selected 12 h has the duration of induction for expression of OsCDase for subsequent biochemical characterization. Importantly, these results demonstrate that OsCDase exhibits robust ceramidase activity.

Figure 2.

Figure 2

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Time course of induction of rice ceramidase (OsCDase) expression in the yeast double-knockout strain, Δypc1Δydc1. OsCDase was cloned into the vector, pYES2/CT and the construct was transformed into the yeast double-knockout strain, Δypc1Δydc1, which lacks the yeast CDases. (A) Expression of OsCDase was induced by growth in galactose medium, and at different time points the culture was terminated and whole cell lysates were assayed for CDase activity using D-erythro-C12-NBD-ceramide as substrate. Values represent means from two independent experiments. (B) Western blot of whole cell lysates (10 µg protein per lane) using anti-His antibody at different time points following growth in galactose medium.

We next examined the biochemical properties of OsCDase and determined the effects of different amounts of protein on CDase activity at different times (Figure 3A). The results indicated that the reaction is linear over a period of 120 min (Figure 3A). The reaction catalyzed by OsCDase exhibited classical Michaelis-Menten kinetics (Figure 3B) and showed an apparent Km of 39.34 µM (1.23 mol%) and an apparent Vmax of 0.079 pmol/h/µg protein as determined using the Lineweaver-Burk plot (Figure 3C). OsCDase activity was enhanced in the presence of Mg2+, Ca2+, Mn2+ (from 2.5 to 7.5 mM) and Zn2+ (2.5 to 5 mM) but inhibited in the presence of Fe2+ (Figure 3D). Because ceramide has been shown to induce micromolar increases in cytosolic free calcium in plants (Townley et al., 2005), we examined the effects of micromolar concentrations of Ca2+ on OsCDase activity in vitro. We showed that OsCDase activity was only weakly activated by free calcium ions in the micromolar range and we observed a significant peak activation of OsCDase activity in the presence of 5 mM Ca2+ (t-test, p < 0.01) (Supplemental Figure 2). We observed OsCDase activity over a broad pH range, with an optimum pH from pH 5.7 to 6.0 (Figure 3E).

Figure 3.

Figure 3

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Biochemical characterization of the rice CDase (OsCDase) in the yeast double-knockout strain, Δypc1Δydc1. OsCDase was cloned into the vector, pYES2/CT and the construct was transformed into the yeast double-knockout strain, Δypc1Δydc1, which lacks the yeast CDases. Expression of OsCDase was induced by growth in galactose medium for 12 h. (A) The effect of different amounts of yeast protein on CDase activity at different assay times. For (B) to (E), 50 µg of total yeast protein was used for CDase activity assay and the reaction time was 1 h. (B) Michaelis-Menten representation for OsCDase activity towards increasing concentrations of D-erythro-C12-NBD-ceramide. (C) Lineweaver-Burk plot of OsCDase activity. (D) Effects of increasing concentrations of cations on OsCDase activity. (E) pH optimum determination and the buffers used are described in the ‘Experimental’ section. Values are means ± S.D. of four replicates from two independent experiments.

We also examined the substrate specificity of OsCDase by substituting D-erythro-C12-NBD-ceramide with NBD-C12-phytoceramide. Phytoceramides are the predominant ceramide species in plants, including rice (Ohnishi et al., 1985; Sperling and Heinz, 2003; Dunn et al., 2004; Lynch and Dunn, 2004). Interestingly, our results suggest that phytoceramide is not a substrate for OsCDase (Figure 4A). We also measured endogenous levels of dihydroceramide (Figure 4B) and phytoceramide (Figure 4C) in the yeast double knockout mutant Δypc1Δydc1 expressing OsCDase. Our results suggest that dihydroceramide is unlikely to be a substrate for OsCDase (Figure 4B). Additionally, measurements of endogenous levels of phytoceramide suggests that OsCDase does not use phytoceramide as a substrate (Figure 4C), an observation consistent with the lack of activity when NBD-C12-phytoceramide was presented as a substrate in vitro (Figure 4A). We also measured changes in the endogenous levels of α-OH-phytoceramide and showed that OsCDase does not use α-OH-phytoceramide as a substrate (data not shown). Interestingly, we observed that endogenous levels of phytoceramide with fatty acid chain lengths of C26 and C28 were elevated in Δypc1Δydc1 when expression of OsCDase was induced by growth in galactose. This suggests that OsCDase may exhibit reverse ceramidase activity and catalyze the formation of phytoceramide with very long chain fatty acids (Figure 4C).

Figure 4.

Figure 4

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Substrate utilization by rice CDase (OsCDase). OsCDase was cloned into the vector, pYES2/CT and the construct was transformed into the yeast double-knockout strain, Δypc1Δydc1, which lacks the yeast CDases. (A) Expression of OsCDase in the yeast double-knockout strain, Δypc1Δydc1 was induced by growth in galactose medium for 12 h and whole cell lysates were assayed for CDase activity using D-erythro-NBD-C12-ceramide (C12-NBD-Cer) or NBD-C12-Phytoceramide (C12-NBD-PhytoCer) as substrates. Wild-type yeast expressing YPC1p and YDC1p was used as a control to show that the yeast CDases uses NBD-C12-Phytoceramide as a substrate. Endogenous levels of (B) dihydroceramide and (C) phytoceramide following induction of OsCDase expression in the yeast double-knockout strain, Δypc1Δydc1. Values are means ± S.D. of four replicates from two independent experiments.

Sub-cellular localization of OsCDase

In silico analysis of the amino acid sequence of OsCDase using TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) indicated that OsCDase is a transmembrane protein. Additionally, we used SLP-Local (Sub-cellular Location Predictor based on Local Features of Amino Acid Sequence; http://sunflower.kuicr.kyoto-u.ac.jp/~smatsuda/slplocal.html) to analyse the OsCDase sequence, and OsCDase is predicted to be localized to the secretory pathway. In order to determine the sub-cellular localization of OsCDase in planta, we cloned the full-length OsCDase CDS into the expression vector, pGDR (Goodin et al, 2000) containing the red fluorescent protein, DsRed2 as the reporter driven by the constitutive CaMV35S promoter from cauliflower mosaic virus for transient expression in onion epidermal cells following biolistic transformation. To determine the sub-cellular localization of DsRed2-OsCDase, we co-transformed onion epidermal cells with green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER-GFP) or Golgi (ST-GFP). We observed co-localization of DsRed2-OsCDase with ER-GFP (Figure 5A) and ST-GFP (Figure 5B), indicating that OsCDase is localized to the ER and Golgi, and that these organelles are sites of ceramide metabolism in planta.

Figure 5.

Figure 5

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Sub-cellular localization of OsCDase fused to the red fluorescent protein (DsRed2) following biolistic transformation of onion epidermal cells. OsCDase was cloned into the vector, pGDR and the plasmid was used for biolistic transformation of onion epidermal cells. ER-GFP (ER-targeted/retained Green Fluorescent Protein) and ST-GFP (Golgi-targeted Green Fluorescent Protein) were used for co-localization studies. Co-localization is visualized in yellow (and indicated with arrows) in the ‘Merge’ panels. Localisation of the nucleus is shown in the transmitted light micrograph. Images were acquired using confocal laser scanning microscopy and are representative of 30 to 40 transformed cells. Scale bar = 50 µm.

Discussion

We have cloned a ceramidase from rice (OsCDase) and sequence analysis showed that it bears similarities to neutral ceramidases from a variety of organisms (Figure 1 and Supplemental Figure 1). Interestingly, we observed the presence of an intron of 777 bp within the 5’-UTR of OsCDase. This is in contrast to the predicted intron within the 5’-UTR of 255 bp. Introns in plant genes are characterized by a strong nucleotide bias towards T proximal to the AG intron acceptor site and that there is an A/T bias throughout the intron relative to the adjacent exon. It has been proposed that such nucleotide biases are required for efficient intron recognition and splicing by the spliceosome (Lorkovic et al., 2000). However, the situation is less clear for introns within the 5’-UTR of plant genes as there appears to be no nucleotide bias to distinguish introns from exon sequences (Chung et al., 2006). The lack of a nucleotide bias for introns in 5’-UTRs appears to hold true for human genes as well (Eden and Brunak, 2004). This may account for the discrepancy between the predicted size of the intron with the results we have obtained from using 5’-RACE PCR. The significance of the long intron within the 5’-UTR of OsCDase is unclear although evidence to date suggests that the presence of 5’-UTR introns may enhance gene expression and that the length of the intron may also influence the level of gene expression in plants (Rose, 2000, 2004; Chung et al., 2006).

In silico analysis of the 5’-UTR of OsCDase showed the presence of a 90 bp (position -32 to -122) internal ribosomal entry site (IRES) element. This is interesting as IRES elements have been found in the 5’-UTRs of mRNAs of proteins involved in cellular proliferation and apoptosis (Holcik et al., 2000). IRES elements were first discovered in piconavirus mRNAs where they function to initiate translation of uncapped viral mRNAs (Pelletier and Sonenberg, 1998). It has been proposed that IRES-dependent (and m7G cap-independent) translation of mRNA may be an important regulatory point for eukaryotic cells to fine-tune their responses to stress through IRES-dependent translation of survival or pro-apoptotic factors (Holcik et al., 2000). Given the recent observations that ceramide can initiate programmed cell death in plant cells (Liang et al., 2003; Townley et al., 2005), the identification of an IRES element in the 5’-UTR of OsCDase is interesting, and suggests that OsCDase may be subject to translational regulation as part of the programmed cell death machinery in plant cells.

In order to determine the biochemical characteristics of OsCDase, we expressed recombinant OsCDase in the yeast double knockout mutant, Δypc1Δydc1, which lacks the yeast CDases, YPC1p and YDC1p (Mao et al., 2000). Our results showed that OsCDase is indeed a bona fide ceramidase and it exhibits typical Michealis-Menten kinetics. OsCDase activity was enhanced in the presence of Ca2+, Mg2+, Mn2+ and Zn2+ but inhibited in the presence of Fe2+ (Figure 3D). We showed that OsCDase activity was significantly activated in the presence of 5 mM Ca2+ (Supplemental Figure 2) whereas Lynch (2000) reported a 100% increase in CDase activity in the presence of 1mM Ca2+. We also tested the effects of micromolar concentrations of Ca2+ on OsCDase activity and showed that OsCDase activity was only weakly activated by micromolar concentrations of Ca2+ (Supplemental Figure 2). It is unlikely that the activity of the ER/Golgi-localised OsCDase is influenced by changes in cytosolic-free calcium concentrations and our observation of significant activation of OsCDase activity in the presence of 5 mM Ca2+ is consistent with the ER being an important intracellular calcium store. The divalent ion-dependent enhancement of CDase activity is in agreement with studies showing that Ca2+, Mg2+, Mn2+ also enhanced the activity of the human neutral CDase (Galadari et al., 2006). Interestingly, while Zn2+ (2.5 to 5 mM) appeared to enhance the activity of OsCDase, the same concentrations inhibited the activity of the human neutral CDase (Galadari et al., 2006). Additionally, the activity of the Dictyostelium discoideum neutral CDase homologue does not appear to be affected by the presence of the following divalent ions, Ca2+, Mg2+, Mn2+, and Zn2+ (Monjusho et al., 2003). The observation that OsCDase exhibits a pH optimum of 5.7 to 6.0 is interesting as it bears sequence similarities to neutral ceramidases. The observation of an acidic pH-dependency of enzymes of the neutral ceramidase family has been reported in Dictyostelium discoideum where it was demonstrated that the neutral CDase homologue exhibited a pH optimum of 3 (Monjusho et al., 2003). Lynch (2000) also reported that CDase activity from plant membrane fractions exhibited an acidic pH dependency of between 5.2 to 5.6. This appears to be in agreement with our observation that OsCDase exhibited an acidic pH optimum of between pH 5.7 to 6.0. The slight acidic pH dependency of OsCDase may explain the observation that it is phylogenetically clustered with the CDase from Dictyostelium discoideum (Figure 1B). Further work on putative members of the neutral ceramidase family from other plants, for example, the 3 candidates from Arabidopsis, is likely to provide greater insights into the acidic pH-dependency of the neutral ceramidase family.

Analysis of substrate utilization by OsCDase revealed a substrate preference for ceramide and not phytoceramide (Figure 4A). This is interesting as phytoceramide is a major ceramide species in fungi and plants (Ohnishi et al., 1985; Sperling and Heinz, 2003; Dunn et al., 2004; Lynch and Dunn, 2004). The substrate preference of sphingolipid metabolizing enzymes from plants for metabolites of low abundance is not without precedence. For example, Coursol and co-workers showed that sphingosine kinase from Arabidopsis exhibited a substrate preference for sphingosine, which is of low abundance compared to dihydrosphingosine and phytosphingosine, both of which are predominant sphingolipid metabolites in plants (Coursol et al., 2003, 2005). Analysis of yeast ceramide species following expression of OsCDase in the yeast double knockout mutant, Δypc1Δydc1 also showed that both dihydroceramide and phytoceramide are unlikely to be substrates for OsCDase (Figs. 4B & 4C). Reverse ceramidase activity has been reported in members of the neutral ceramidase family (Okino et al., 1998; Kita et al., 2000; El Bawab et al., 2001; Wu et al., 2007). Analyses of yeast sphingolipids following induction of OsCDase expression in the double knockout mutant, Δypc1Δydc1 showed elevated levels of phytoceramide with fatty acid chain lengths of C26 and C28 (Figure 4C). This observation points to the possibility that OsCDase may exhibit reverse ceramidase activity, leading to elevated levels of these 2 phytoceramide species.

Neutral ceramidases have been shown to be localized to the plasma membrane, endosome-like organelle, mitochondria and endoplasmic reticulum/Golgi compartments (El Bawab et al., 2000; Mitsutake et al., 2001; Yoshimura et al., 2004). Additionally, neutral ceramidases may also be secreted (Romiti et al., 2000). We showed that OsCDase is localized to the endoplasmic reticulum and Golgi in planta, suggesting that the ER/Golgi compartments are sites of ceramide metabolism in plants. This observation is consistent with in silico prediction of OsCDase as a transmembrane protein localized to the secretory pathway.

In conclusion, we have cloned a bona fide plant ceramidase from rice and showed that it exhibited classical Michaelis-Menten kinetics with an optimum activity ranging from pH 5.7 to 6.0. OsCDase activity was enhanced in the presence of Ca2+, Mg2+, Mn2+ and Zn2+ but inhibited in the presence of Fe2+. Expression analysis indicated that steady-state levels of OsCDase transcripts are expressed throughout the seedling with a higher level of expression in roots compared to shoots. Sub-cellular localization showed that OsCDase is localized to the ER/Golgi compartments, suggesting that these organelles are sites of sphingolipid metabolism in planta. Given the observations that ceramide is an important sphingolipid metabolite involved in the regulation of programmed cell death in plants (Liang et al., 2003; Townley et al., 2005), our characterization of the rice ceramidase is an important first step in understanding how ceramide is metabolized and provide the basis for further investigations into how sphingolipid metabolism is co-ordinated with developmental processes in plants.

Experimental procedures

Plant growth

Rice (Oryza sativa ssp japonica cv. Nipponbare) seeds were germinated in full-strength Murashige and Skoog media (Sigma, UK) at pH 5.8 in a plant growth cabinet (Snijders Microclima 1750, Snijders, Netherlands) under the following conditions: PPFD: 1000 µmol/m2/s, 12h light/12h darkness with a day temperature of 28°C and a night temperature of 24°C under 85% relative humidity.

cDNA cloning and determination of 5’- and 3’-untranslated regions (UTRs)

Total RNA was isolated from 2-week old seedlings using the RNeasy® Mini Kit (Qiagen, UK) according to manufacturer’s instructions. 500 ng of DNAse-treated (Turbo DNA-free™ Kit, Ambion, USA) total RNA was used for cDNA synthesis using Accuscript™ High-fidelity RT-PCR system (Stratagene, UK). Three µl of the product was used for PCR amplification of the full-length with PfuUltra HF DNA polymerase (Stratagene, UK) using the following primers:

  • forward primer: 5’-ATGGAGGCTTCATCTTGGTTGTG-3’

  • reverse primer: 5’-ACGCACCGCGAAAGCACGAGA-3’

Cloning of the 5’- and 3’-UTRs was achieved using the SMART-RACE cDNA amplification kit (Clontech) according to manufacturer’s instructions and using the following gene specific primers.

  • Gene-specific primer for 5’-RACE: 5’-GAAGCTCCCCATACCGACCAG-3’

  • Gene-specific primer for 3’-RACE: 5’-CAAGGCCCGCAAGATTGAGTTC-3’

Full-length coding sequence (CDS) and 5’- and 3’-UTRs were cloned into pGEM T-Easy vector (Promega, UK) before sequencing (Eurofins MWG GmbH, Germany).

Expression of OsCDase in yeast

Full-length OsCDase CDS was sub-cloned into the yeast expression vector, pYES2/CT (Invitrogen, UK) and transformed into the yeast double knockout mutant, Δypc1Δydc1, which lacks the yeast ceramidases YPC1p and YDC1p (Mao et al., 2000) using the lithium acetate method as previously described (31). Transformed yeast cultures were grown in synthetic minimal media enriched in dextrose and complemented by a selective uracil-deficient dropout supplement (Clontech, USA), and cell lysates were isolated as previously described (Galadari et al., 2006).

OsCDase enzyme assays and biochemical characterization

Enzyme activity was measured using D-erythro-C12-NBD-ceramide (Avanti Polar Lipids, USA) or NBD-C12-phytoceramide (Lipidomics Core Facility, MUSC, USA) at a final concentration of 100 µM (3.13 mol%) in a 100 mM Phosphate buffer, pH 5.7, containing 0.2% Triton-X-100 and 5 mM MgCl2 final in a total volume of 100 µl. For determining pH optima, the substrate was dissolved in the following buffers: pH 3.91–5.33, 100 mM acetate buffer; pH 5.04–6.66, 100 mM Phosphate buffer; pH 7.1–8.24, 100 mM Tris buffer. The enzymatic reaction was continued for 1 hr at 37°C, and the reaction was terminated by addition of chloroform/methanol (1:1, v/v). The mixture was left at room temperature for 5 min, after which it was centrifuged for 5 min at 5500 rpm (Accuspin microR, Fisher, USA), and the organic phase was taken and dried using a SpeedVac (Savant, USA) evaporator. Lipids were dissolved in 15 µl of chloroform/methanol (1:1, v/v), and spotted on to TLC plates (Partisil LK6D, Whatman, USA). The NBD-C12-fatty acid products were separated from the substrate by developing the plate in chloroform/methanol/25% ammonium hydroxide (60:20:0.5, by vol.) solvent system. The TLC plate was scanned using a PhosphorImager (Storm 860, USA) system set at blue fluorescence mode. The NBD-fatty acid products were identified by comparison with NBD-C12-fatty acid standards and were quantified using ImageQuant™ software. The specific activity was determined using standard curves with known concentrations of D-erythro-C12-NBD-ceramide.

Analysis of ceramides by mass spectrometry

Accumulated ceramides were analyzed by MS using reverse-phase, high-performance liquid chromatography (HPLC) coupled to an electrospray-triple quadrupole mass spectrometer, operating at positive ionization, multiple reaction monitoring (MRM) mode. Mass separations were performed using a ThermoFinnigan TSQ 7000 mass spectrometer (Foster City, CA) according to published methodology (Bielawski et al., 2006).

Sub-cellular localization of OsCDase

Full-length OsCDase CDS was cloned downstream of the red fluorescent protein, DsRed2 under the control of the constitutive 35S CaMV promoter in the vector, pGDR (kindly provided by Dr. Michael Goodin, University of Kentucky, USA) (Goodin et al., 2000). The vector was sequenced to ensure that OsCDase has been cloned in-frame to the DsRed2 reporter gene. Biolistic transformation of onion (Allium cepa) epidermal cells was done using 1 µg of plasmid DNA precipitated onto 100 µg of 1 µm gold particles using CaCl2 and spermidine. DNA-coated gold particles were washed with ethanol and resuspended in ethanol for transformation of onion epidermal cells using a biolistic particle delivery system (Bio-Rad PDS-1000/He, Bio-Rad Laboratories, USA) with a pressure set at 1100 psi. After an overnight incubation, the localization was observed using confocal laser scanning microscopy using a Leica TCS-SP2 confocal microscope (Leica Systems, UK). Transmitted light micrographs were obtained with a transmitted light detector. Co-localization was performed with green fluorescent protein targeted to the ER (plasmid, pGFP:C-domain; kindly provided by Dr. Wendy Boss, North Carolina State University, USA) (Persson et al., 2002) or Golgi (plasmid, pVKH18-EN6::ST-GFP; kindly provided by Dr. Chris Hawes, Oxford Brookes University, UK) (Saint-Jore et al., 2002).

Supplementary Material

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suppl fig2

Acknowledgements

This work is funded by a Science Foundation Ireland grant BR/04/B0581 (to C.K.-Y.N.), a National Institute of Health (NIH) grant CA87584 (to Y.A.H.), a Irish Research Council for Science, Engineering and Technology Embark Postdoctoral Fellowship (to T.C.X.), and parts of the research was conducted in a facility constructed with support from the NIH Grant C06 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources.

The nucleotide sequence of OsCDase has been submitted to GenBank with the accession number EU422991.

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Supplementary Materials

suppl fig1
NIHMS283703-supplement-suppl_fig1.pdf (82.6KB, pdf)
suppl fig2
NIHMS283703-supplement-suppl_fig2.pdf (42.9KB, pdf)

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