Cloning and phylogenetic analysis of OsCERK

Rice harbors a single gene on chromosome 2 (at locus Os02g0656200) predicted to encode a ceramide kinase. The predicted amino acid sequence of this putative rice ceramide kinase (OsCERK) has 48% sequence identity and 64% sequence similarity to the Arabidopsis ceramide kinase called ACCELERATED CELL DEATH 5 (ACD5 [6]) and 27.2% sequence identity and 40.8% sequence similarity to CERK from humans (hCERK, Genbank accession number NP_073603 [8]. The full length OsCERK is made up of 13 exons, 104 bp 5′-UTR and 447 bp 3′-UTR (Figure 1A). An ORF within the OsCERK gene encodes a polypeptide of 607 amino acid residues, with a predicted molecular mass of 67.29 KDa and a pI of 6.19.

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Figure 1. Sequence analysis and expression of rice ceramide kinase (OsCERK) in planta.

(A) Schematic representation of the sequence organization of OsCERK. The sizes of the 5′-UTR (white), exons (black) and 3′-UTR (grey) are indicated by numbers below the boxes. The sizes of introns are shown above. (B) Phylogenetic analysis of CERKs and the related enzymes sphingosine kinase (SPK)s, and diacylglycerol kinase (DAGK)s. The architecture of each gene was obtained from SMART using the architecture analysis function, and conserved domains (letters a–d) are indicated as follows: a, Pleckstrin homology domain (PH domain; accession number SM00233); b, Diacylglycerol kinase catalytic domain (DAGKc; SMART accession number SM00046); c, segments of low compositional complexity determined by the program; d, diacylgycerol kinase catalytic domain (DAGK_cat; Pfam accession number PF00781). Detailed sequences information is described in Materials and methods. AtCERK is ACD5. (C) CLUSTAL W alignment of CERK from various organisms showing the highly conserved CXXXCXXC motif. (D) RT-PCR expression analysis of OsCERK in leaves, stem and roots of 2-week-old seedlings. β-actin was used as a control. (E) CERK activity of rice extracts. Crude plant extracts made from leaves and roots of rice, respectively, were incubated at 40°C for 30 min as described in Materials and methods. The reaction mixture (1 µl) was resolved on a TLC plate. Purified OsCERK recombinant protein was used as the positive control (+CK). For the negative control (−CK) no extract was added. (F) Comparison of recombinant OsCERK and rACD5. The recombinant protein (1.0 µg) was added in 100 µl reaction system, and incubated at 40°C for 30 min. –CK, no recombinant protein was added. Arrows in (E and F) indicated the formed product C6-NBD-ceramide-1-phosphate (C6-NBD-Cer-1P).

https://doi.org/10.1371/journal.pone.0018079.g001

Database searches indicated that proteins with homology to CERK are ubiquitous in multicellular organisms including vertebrates, invertebrates and plants. OsCERK clusters with the ceramide kinase of Arabidopsis thaliana and possible ceramide kinase orthologues in Populus trichocarpa and Vitis vinifera (Figure 1B). Moreover, putative CERKs clearly cluster into vertebrate, invertebrate and plant lineages, respectively. CLUSTAL W alignment of the amino acid sequences of OsCERK with putative CERKs from various organisms showed that the predicted OsCERK protein has a conserved CERK-specific motif CXXXCXXC that is critical for the enzymatic activity of hCERK [12] (Figure 1C), and five conserved domains (C1–C5) previously identified in human CERK (Figure S1). Gaps in the alignment between OsCERK (or ACD5) and hCERK due to sequence divergence may correspond to regions important for substrate recognition and regulatory mechanisms specific to plants and animals, respectively. A search using the SMART online bioinformatics tool revealed a diacyl glycerol kinase (DAGK) domain in residues 139–357 of OsCERK and in 164–366 of ACD5 as reported before [6].

Steady-state levels of OsCERK transcripts were highest in leaves, followed by the stems, with almost no expression in roots when compared with leaves (Figure 1D). Fresh rice leaf extracts harbored CERK enzyme activity, phosphorylating C6-NBD-ceramide to give C6-NBD-ceramide-1-phosphate, as did recombinant OsCERK and rACD5, respectively (Figure 1E, F). Almost no CERK activity was detected in the root extract (Figure 1E), which is consistent with the low levels of OsCERK transcripts (Figure 1D).

Biochemical characterization of recombinant OsCERK

Purified His-tagged OsCERK had an optimum reaction temperature of 40°C and an optimum pH value of 7, respectively (Figure 2A, B). With synthetic C6-NBD-ceramide as substrate, the reaction catalyzed by OsCERK showed classical Michaelis-Menten kinetics (Figure 2C, D), with an apparent K_m of 4.1 µM and an apparent V_max of 4.74 nmole⁻¹ min⁻¹ mg⁻¹ protein for D-erythro-C6-ceramide (Figure 2E). The K_m and V_max for ATP was 35.36 µM and 2.58 nmole⁻¹ min⁻¹ mg⁻¹ protein, respectively (Figure 2F). To identify the role of divalent ions for CERK activity, the effect of Ca²⁺ and Mg²⁺ on OsCERK activity was determined with C6-NBD-ceramide as substrate. In the absence of Mg²⁺, Ca²⁺ at <200 µM produced no change in CERK activity (Figure 2G), though the activity was increased at 2 mM. In the absence of Ca²⁺, the activity of CERK was increased by Mg²⁺ in a concentration-dependent manner (Figure 2H), which closely resembled the properties of hCERK [8]. Moreover, the addition of Ca²⁺ did not boost the reaction rate when 15 mM MgCl₂ was present in the reaction system (data not shown). Thus, unlike ACD5 [6] and hCERK [8], which can be activated by Ca²⁺, OsCERK activity was mainly activated by Mg²⁺, and to a smaller extent by Ca²⁺.

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Figure 2. Biochemical characterization of the recombinant rice ceramide kinase.

(A) Effect of different temperatures on CERK activity. (B) Effect of different pH values on CERK activity. (C) and (D) Michaelis-Menten representation for OsCERK activity with increasing concentrations of D-erythro-C6-NBD-ceramide and ATP, respectively. (E) and (F) Lineweaver-Burk plot of OsCERK activity. (G) Effect of calcium on ceramide kinase activity; for this experiment, ceramide phosphorylation was determined in the absence of MgCl₂. (H) Effect of magnesium on ceramide kinase activity; for this experiment, the effect on activity by MgCl₂ was measured in the absence of CaCl₂. For (A)–(H), 1 µg purified recombinant OsCERK was used for OsCERK activity assay. The reaction time was 30 min. Values are means ± standard deviation (SD) of four replicates from two independent experiments.

https://doi.org/10.1371/journal.pone.0018079.g002

Complementation of ceramide kinase-deficient acd5 mutant by OsCERK

To assess the in planta function of OsCERK, we characterized T3 homozygous lines of the Arabidopsis acd5 mutant harboring a cDNA of OsCERK. Several phenotypes of acd5 were complemented by OsCERK, including spontaneous cell death and elevated PR1 transcript levels late in development (Figure 3 A–D), and enhanced disease susceptibility to virulent Pseudomonas syringae strain DG3 of younger (21 day old) plants that lack cell death (Figure 3E). Thus, the OsCERK transgene complemented multiple phenotypes of acd5.

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Figure 3. Complementation of Arabidopsis acd5 mutant plants by OsCERK.

(A) Comparison of early development between acd5 mutant plants expressing OsCERK and nontransgenic plants. Note that no phenotype differences were observed in 25-day-old transgenic acd5 plants expressing OsCERK (acd5+OsCERK) and empty vector when compared with wild-type and acd5 plants. (B) Phenotypes of 48-day-old plant leaves (upper part) as shown in (A). Cell death was determined by trypan blue staining on the indicated days in leaf tissue (lower part). Note that acd5 and transgenic acd5 plants expressing EV showed obviously spontaneous cell death in both leaves and stained tissues; in contrast, acd5 plants carrying OsCERK showed a completely normal phenotype, just as wild-type plants. This experiment was repeated twice with similar results. Each time 9–12 leaves from at least 6 plants were used for trypan blue staining and over 85% showed full complementation. (C) Phenotypes of 55-day-old plants as shown in (A). Arrowheads indicate the cell death phenotype of acd5 and transformed plants carrying the empty vector in leaves and stems. Note the lack of cell death regions in the transformed acd5 plants carrying OsCERK. At least ten independent lines were obtained. More than 100 plants were observed and phenotypic complementation was 95% in all homozygous lines. (D) Steady-state levels of PR1 gene transcripts in wild-type and transgenic plants at different times. Tubulin indicates the loading control. This experiment was repeated twice with equivalent results. (E) Growth of P. syringae virulent strain DG3 in the indicated genotypes after infection at OD600 = 0.001. Lesion-free plants were inoculated with bacteria at 3 wk of age. The mean value of the growth of bacteria in six leaves is indicated in each case. Letters indicate that values are different using Student-Newman-Keuls test (P<0.05). Bars indicate standard deviations. This experiment was repeated three times with similar results. (F) CERK activity assay of plant extract in the indicated plants. Equal amounts of protein were extracted from 50-day-old leaves as described in Materials and Methods. Arrow indicates the formed product C6-1-³²P. Purified ACD5 recombinant protein was used as the positive control (+CK). For negative control (−CK) no isotope-labeled ATP was added. This experiment was repeated twice with equivalent results. (G) Amount of C6-1-³²P quantified from panel (F).

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Importantly, in plant extracts, CERK activity was partially restored in acd5+OsCERK plants (Figure 3F, G). We noticed that an extra band appeared with all plant extracts that was more intense with acd5+OsCERK plant extracts. We speculate that OsCERK overexpression activates other lipid kinases and/or the C6 ceramide substrate was incorporated into larger lipids. Taken together, the acd5 phenotypes were rescued by the OsCERK transgene.

Effects of site-directed mutations in a conserved C-rich motif on OsCERK activity and function

To discern the role of the CXXXCXXC motif conserved among all CERK species, we individually mutated each of the three cysteine residues in OsCERK, and assayed CERK activity using purified recombinant enzymes. The three mutant proteins displayed 25% (C461A), 64% (C458A) or 74% (C454A) residual activity, respectively (Figure 4A, B).

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Figure 4. Effects of mutations in highly conserved motif on OsCERK activity and function.

(A) Effects of mutations in the highly conserved CXXXCXXC motif on OsCERK activity. Site directed mutagenesis of OsCERK was performed as described in Materials and Methods. Equal quantities of protein were added into the 100 µl reaction system, and 1 µl of reaction mix was resolved on a TLC plate after 30 min incubation at 40°C. Purified recombinant ACD5 protein (rACD5) was used as a positive control. Arrowhead indicates the product of C6-NBD-Cer-1P. W.T. means recombinant OsCERK. (B) Relative OsCERK activity quantified from same samples as in panel (A). The values on the vertical axis, were normalized to wild-type (W.T.) rOsCERK activity at 100%. Note that the three variant proteins showed decreased activity, with the C461A variant showing the least activity. Bars represent the mean ± SD of two independent experiments. (C) Western blots with Flag antibody show the stable production of OsCERK and its modified variants in yeast. This experiment was repeated twice with equivalent results. (D) Effects of OsCERK and modified versions of OsCERK on yeast growth. Note the dramatic growth inhibition in yeast expressing OsCERK. This experiment was repeated four times with equivalent results. (E) Expression level of OsCERK and PR1 in acd5 and acd5 transgenic plants. Two different lines in each site-mutated OsCERK were used. Note that the plant materials of line acd5+C458A2 were taken from 26-day-old seedlings as shown in (F), the other transgenic lines and acd5 were taken from 36-day-old plants as shown in (F). (F) Comparison of phenotype between acd5 mutant plants expressing mutated OsCERK and non-transgenic acd5 plants. At least 50 plants from 2 independent lines were used for phenotype identification in each site-mutated OsCERK plants. Arrows indicate cell death lesions.

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Because yeast lack a clear ceramide kinase homolog [8], it seemed possible this would be a good surrogate system to assess the effect of mutations on OsCERK function. Indeed, FLAG-tagged wild-type OsCERK expressed under galactose control dramatically suppressed yeast growth (Figure 4C, D), presumably due to a lipid imbalance conferred by OsCERK. In contrast, two mutated versions (C461A and C458A) still accumulated in yeast (Figure 4C), but had a reduced effect on growth (Figure 4D). We also attempted to assess the effects of the C454A variant on yeast growth, but the protein failed to accumulate. Based on the effects of mutation of C461 or C458, the conserved CXXXCXXC motif is important for OsCERK activity and impacts its function.

To test the consequences of disrupting the CXXXCXXC motif in planta, we transformed the site-directed OsCERK mutants into acd5. More than 5 independent lines for each site-directed mutation of OsCERK were obtained. At least 50 plants from each of 2 independent lines were used for phenotypic analysis of transgenic acd5 harboring each site-mutated OsCERK. Lines with the same construct showed similar phenotypes; representative lines are shown in Figure 4. As shown in Figure 4E, the OsCERK gene was stably expressed in transgenic acd5 plants; no OsCERK expression was detected in acd5 control plants. Importantly, acd5 plants transformed with OsCERK harboring mutations in the conserved motif still showed some spontaneous cell death and elevated PR1 expression late in development (Figure 4F, 36 d). Similar to acd5, leaves taken from transgenic plants prior to spontaneous cell death showed no PR1 expression (Figure 4E line acd5+C458A2).

Interestingly, the onset and extent of cell death in acd5 carrying C461A OsCERK at 36 days and thereafter was similar to that seen with acd5 plants. However, acd5 carrying C454A or C458A OsCERK, respectively, showed less cell death than acd5 at 36 days. Thus, the mutated versions of OsCERK showed varying degrees of defects in complementing the acd5 cell death phenotype. Base on all above data, the highly conserved CXXXCXXC motif and particularly C461 is vital for OsCERK activity and function.

Effects of CERK inhibitors on OsCERK activity and cell death

In order to discern whether OsCERK activity is required for cell viability, we investigated the effect of reducing OsCERK activity with recently commercialized CERK inhibitors, such as N,N-dimethylsphingosine (DMS), which partially inhibits hCERK activity [8], and the ceramide kinase inhibitor K1 (K1), a specific noncompetitive CERK inhibitor [33]. As shown in Figure 5, OsCERK activity was inhibited by K1 and DMS in a concentration dependent manner, and K1 showed stronger inhibition than DMS (Figure 5A, B, C), which is consistent with the inhibitory effect on hCERK. OsCERK activity decreased by about 70% in 100 µM DMS and 50 µM K1. K1 caused greater than 90% inhibition of OsCERK activity at 200 µM concentration, demonstrating that K1 can almost completely abolish OsCERK activity.

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Figure 5. Inhibition assay and effects of site-mutation on OsCERK activity.

(A) Effect of ceramide kinase inhibitors K1 and DMS on OsCERK activity. The activity of OsCERK was assayed as described in Materials and Methods using TLC and scanned for NBD fluorescence. Arrowhead indicates the formed product C6-NBD-Cer-1P. K1 and DMS were used at different concentrations. Purified OsCERK recombinant protein was used as a positive control (+CK). EtOH (0.5%) and MeOH (0.5%) indicated solvent controls for K1 and DMS, respectively. (B) and (C) Quantification of data from (A). Bars indicate means ± SD of two independent experiments. (D) Effect of the ceramide kinase inhibitor K1 on rice protoplasts. Protoplasts were isolated from 10-day-old rice seedling and treated with K1 (0.5 µM and 5 µM) for the indicated times under light. (E) Effect of ceramide kinase inhibitor K1 on Arabidopsis protoplasts. Protoplasts were isolated from 18-day-old wild-type and acd5 mutant plants and treated with 5 µM K1 for 24 h under light. The percentage of surviving cells in (D, E) was estimated by FDA staining. These experiments were repeated three times with similar results. Letters indicate that values differed in Fisher's protected least significant difference (PLSD) test, a post hoc multiple t test (P<0.05). Error bars indicate standard deviations. Control treatment was with 0.15% ethanol (the solvent in which K1 was dissolved).

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To examine the effect of K1 on cell viability, rice and Arabidopsis protoplasts were treated with K1. In rice protoplasts treated with different concentrations of K1, cell death occurred in a dose- and time-dependent manner (Figure 5D). In Arabidopsis protoplasts, K1 induced cell death much less in acd5 than in wild-type plants (Figure 5E). These data are consistent with K1 specifically affecting CERK activity, which is crucial for the maintenance of cell viability.

Characterization of ceramide-induced PCD in rice

To assess whether ceramides, the substrates of OsCERK, can induce cell death in rice, we isolated rice protoplasts and treated them with ceramides. We used short-chain ceramides because of the difficulty in ensuring that longer chain ceramides efficiently enter plant cells. In animal cells, short chain ceramides are converted into longer ceramides in vivo [34]; it seems likely that similar events occur in plants. As shown in Figure S2, both C2- and C6-ceramides induced cell death in a time-dependent manner, whereas C2- and C6-dihydroceramide, the biosynthetic precursors of ceramides, had almost no effect at the same concentration as ceramides, similar to what has been reported in animal and Arabidopsis cells [2], [6]. C6-ceramide and C2 similarly induced rice protoplast cell death. However, the concentration of C6 that induced cell death was 2 times higher than that of C2 (Figure S2A, B), suggesting that it is more difficult for C6 than C2 to enter protoplasts. Cell death appeared to be programmed, since 12 h after treatment with C6-ceramide, a clear DNA ladder was seen (Figure S2D) and nuclear fragmentation characteristic of apoptotic cells was seen by TUNEL staining (Figure S2C) [32], [35].

Cell death induced by C6 ceramide in rice involved new protein synthesis and protein phosphorylation. This was evidenced by the inhibition of each process using cycloheximide or the serine/threonine kinase inhibitor K252a, respectively, which suppressed cell death to varying extents (Figure 6A, B). Treatment with the inhibitors alone at the concentrations used did not affect viability of protoplasts. Additionally, as was found in Arabidopsis, the presence of C2-1-phosphate partially blocked cell death induced by C2 ceramide in rice (Figure 6C), supporting the idea that the balance between ceramides and their phosphorylated derivatives modulates cell death in plants.

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Figure 6. Effects of ceramide related inhibitors on cell death induction.

(A) Effects of cycloheximide (CHX) on C6 ceramide-induced cell death. (B) Effects of K252a on C6 ceramide-induced cell death. (C) Effects of C2 ceramide-1-phosphate on C2 ceramide induced cell death. Protoplasts were treated with less than 0.15% solvent or the indicated reagent. Percentage of surviving cells was estimated by FDA staining. These assays were repeated three times with similar results. Letters indicate that values are different using Fisher's PLSD test (P<0.05). Bars show standard deviations.

https://doi.org/10.1371/journal.pone.0018079.g006

As in Arabidopsis, rice protoplasts showed functional changes in mitochondria after ceramide treatment. C6 ceramide, but not C6-DHC, induced a time-dependent loss in mitochondrial membrane potential (Δψ_m), as assessed by flow cytometry using DiOC₆(3) staining (Figure 7). Protoplasts treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), as a positive control, showed dramatic Δψ_m loss after 30 min treatment. The results of treatment with C2 were similar to C6 (data not shown). Although Δψ_m loss was seen in rice and Arabidopsis after ceramide treatment [32], the timing of Δψ_m loss in rice did not precede cell death onset as was previously observed in Arabidopsis [32]. Thus, there may be differences in the relative importance of Δψ_m loss in rice and Arabidopsis.

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Figure 7. Ceramide induced programmed cell death is associated with Δψ_m loss in rice protoplasts.

Mitochondrial membrane potential Δψ_m loss detected by flow cytometry. Protoplasts were cultured under light for the indicated times with 0.1% ethanol (solvent control), 100 µM C6, 100 µM C6-DHC and 100 µM protonophore CCCP (positive control), respectively. Δψ_m was analyzed by flow cytometry using the fluorescent probe DiOC₆(3). Results are from a single analysis, representative of three independent experiments that showed similar results. M1, region of high Δψ_m. The percentages in each panel indicate the proportion of the cells with high Δψ_m.

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Novel biochemical properties of OsCERK

OsCERK activity is stimulated by Mg²⁺, but only modestly Ca²⁺. This contrasts with the properties of human CERK, which shows Ca²⁺ dependence and is stimulated by Mg²⁺ in the micromolar concentration range [8]. The differences in the biochemical properties of the human and rice enzymes might be linked to the domains present in hCERK versus OsCERK and/or their respective structures. hCERK contains a calcium/calmodulin binding motif of the 1-8-14 type B [8] that may modulate calcium/calmodulin binding to CERKs and control their function; this motif is absent from the plant enzymes. Stimulation of CERK activity in plants may need a higher concentration calcium than the human enzyme; indeed, ACD5 activity was significantly stimulated by 100 mM Ca²⁺ [6]. OsCERK was most active in the neutral pH range from 6.5 to 7.5, with optimal activity at pH 7.0. This pH dependence is quite similar to hCERK and is different from ACD5 (optimal pH 8.2) [6]. The K_m with D-erythro-C6-NBD-ceramide was 4.1 µM, much lower than the previous reported K_m values for CERK from other organisms. The K_m for ATP, 36.35 µM was similar to hCERK. The activity may vary with substrate, substrate delivery [36], reaction system and detection methods of activity. Notably, OsCERK has a temperature optimum of 40°C, higher than ACD5 or hCERK. This high temperature optimum may be related to fact that rice can grow at higher temperatures.

The CXXXCXXC motif is important for OsCERK activity and function

The highly conserved CXXXCXXC motif of CERK, between the C4 and C5 domains, is essential for hCERK enzyme activity and cellular localization [12]. However, the role of the cysteine motif for the full in vivo function(s) of hCERK has not been reported. Our site-directed mutagenesis results indicate that each of the three cysteines contributes to the enzyme activity of OsCERK, with the mutation C461A that removes the third cysteine showing the strongest effect. In planta, each of the three cysteines tested were important, since none of the site-directed mutants could fully rescue of the cell death phenotype of acd5 plants. In the surrogate yeast system, we also confirmed that C458A and C461A did not affect yeast growth the way the wild-type protein did. Thus, this conserved C-rich motif is also important for the in planta function of OsCERK.

In hCERK, the first cysteine in the motif is most important for its enzymatic activity [12], whereas in OsCERK, mutation of the third cysteine had the most dramatic effect on activity and in planta function. The reason for this difference is not clear. Whether the motif also impacts the localization of OsCERK in plants awaits further determination. Interestingly, an identical cysteine motif has been characterized in the radical S-adenosylmethionine super family of enzymes, where the motif provides a metal binding site that is essential for activity [37]. Whether CERK is also regulated by metal coordination will require further research.

Ceramides affect cell viability and Δψ_m in rice

CERK phosphorylates ceramide to yield C1P, a molecule with interesting signaling properties [38]. Ceramides, as the substrates of CERK, are closely involved in PCD, and affect many cellular events in animals [2]. Ceramide also activates programmed cell death in Arabidopsis [6] and rice. As was observed in Arabidopsis [6], ceramide-induced cell death in rice is also suppressed by the presence of C1P. This suppressive effect could result from direct competition of C1P with ceramide in affecting the same cellular target or through modulation by ceramide phosphate of signaling. We favor the latter hypothesis, since protein phosphorylation, a modification often associated with signal transduction, is important for ceramide-induced cell death in rice and C1P has signaling roles in animals.

In Arabidopsis, a mitochondrial permeability transition, just like in animal apoptosis, is one of the early events in PCD induced by C2-ceramide [32]. In rice protoplasts Δψ_m loss was also observed during PCD induced by short-chain ceramides, however, the membrane potential loss appears simultaneously with PCD, suggesting that in rice Δψ_m loss may not be essential for PCD induced by ceramide.

In conclusion, a ceramide kinase from rice was cloned and characterized. The ceramide kinase inhibitors, which were characterized on CERK from animals, almost completely blocked OsCERK activity and induced cell death in both rice and Arabidopsis. By site- directed mutagenesis of the conserved CXXXCXXC motif in organisms, three highly conserved cysteines were shown to be vital for CERK activity and function. Since CERK is one of the key enzymes in sphingolipid metabolism, our data offers important information for understanding the conserved sphingolipid functions in animals and plants. It will be interesting to pursue the further role of CERK in plant developmental processes during environmental stress.

Plant growth and plant infections

Rice (Oryza sativa ssp. japonica cv. Nipponbare) seeds were planted in soil after two days of soaking in water and grown in a climate incubator under the following conditions: photo flux density, 225 µmol m⁻² s⁻¹, 16 h light/8 h dark, with a day temperature of 28°C under 80% relative humidity and a night temperature of 26°C under 60% relative humidity. Arabidopsis thaliana plants (ecotype Columbia) were grown in a 16 h light/8 h dark cycle at 22°C under 60% relative humidity. Pseudomonas syringae pv. maculicola strain DG3 (virulent) was inoculated as described previously [31], [39].

Full length cDNA cloning and RT-PCR analysis

Total RNA was isolated from 2-week-old seedlings using Trizol Reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). About 1 µg total RNA was used for cDNA synthesis using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, EU). Aliquots (1 µl) of the product were used for PCR amplification using Pfu high-fidelity DNA polymerase (Tiangen, Beijing, China), with the forward primer 5′-ATGGAAGGCGGCGGCGAGGCGCTCT-3′ and reverse primer 5′ -CTACATGCCTGCTCTTTCCGGTGGC-3′. Cloning of the 5′- and 3′-UTRs was accomplished using a SMART RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) according to manufacturer's instructions with the following gene specific primers: for 3′RACE: NGSP 5′-ATGGGTCCTGCGCGATATGACTT-3′, GSP 5′- GTTTCACCAACTGTGCGCTATGCTG-3′; for 5′RACE: GSP 5′-GATGACCAACATGAAGAACTCACTT-3′, NGSP 5′-ACCTGGTGAAGGGGCTGGAAGGA-3′. The full-length coding sequences were cloned into the pGEM-T vector (Tiangen) before sequencing (Invitrogen). The 5′- and 3′-UTRs were cloned into the pCR2.1 vector (Invitrogen) before sequencing.

For RT-PCR analysis, total RNA samples and Reverse-Transcription were done as described above. The cDNA contents in the different reverse transcription mixtures were normalized by amplifying the transcripts of β-actin using the primer 5′-CCATTGGTGCTGAGCGTT-3′ and 5′-CCCGCAGCTTCCATTCCTA-3′ in rice. Evaluation of the transcript levels of OsCERK in the different tissue was carried out using the normalized cDNA mixtures and the primer 5′-TGGCTCAACGGATGCTATT-3′ and 5′-ACGACCCTTGGACCAGACC-3′. For Arabidopsis expression analysis, the primer 5′-GTCACTTGCTGCCTTCGTTT-3′ and 5′-CTTCTTTCGTGCTCATTTTGC-3′ were for tubulin amplification as an internal control, the PR1 expression was amplified using the primer 5′-GTGCTCTTGTTCTTCCCTCG-3′ and 5′-AGCCTTCTCGCTAACCCACA-3′.

Mutagenesis of OsCERK

Site directed mutagenesis of OsCERK was performed using the following primers with Fast mutagenesis system (Trans, Beijing, China): C454A (5′-GACATCGGAAAACCATCTGCCGAACAAATTG-3′,5′-GCGATGGTTTTCCGATGTCTATTTTGATGGTA-3′), C458A (5′-CCATCTGCCGAACAAATGCTTTAATATGCAA-3′, 5′-GCATTTGTTCGGCAGATGGTTTTCCGATGTC-3′), C461A (5′- GAACAAATTGTTTAATAGCCAAAGGAACTTC-3′,5′-GCTATTAAACAATTTGTTCGGCAGATGGTTT-3′). The construct of pET28b: OsCERK was used as template for PCR amplification. All constructs were transferred to BL21 (DE3) and expressed using the conditions described below.

Plasmid construction of yeast expression, spot assay and western blots

The wild-type and site-mutated coding regions of OsCERK were introduced into Sal I and Xba I digested pYES2+Flag expression vector (reconstructed from pYES2 vector). In the spot assay, yeast cells grown in SC-glucose medium for 1 day were adjusted to A600 of 0.1. Five microliters of five-fold serial dilutions of each yeast culture was spotted on to SC-glucose or SC-galactose medium and incubated for 2 days (glucose medium) or 3 days (galactose medium) at 30°C. For western blots, yeast cells cultured in liquid galactose medium were collected by centrifugation at 0 h and 15 h, respectively. Cell lysates were prepared using acid-washed glass beads (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined with a Protein assay kit (Bio-Rad) using IgG as the standard. Proteins (10–20 µg) were separated by SDS-PAGE (10%, v/v). Separated proteins were electrophoretically transferred to a nitrocellulose filter membrane (0.45 µm, Bio-Rad). Nonspecific binding was blocked with 5% (w/v) skim milk in TBST buffer (25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.1% (w/v) Tween 20) for 1 h at room temperature. N-terminal Flag- fused proteins were detected using anti-Flag antibody (1∶2000 dilutions, Sigma-Aldrich, St. Louis, MO, USA) and developed using the enhanced chemiluminescence kit (ECL, Rockford, Thermo Scientific). The chemiluminescence was finally detected with photographic film (Kodak).

Phylogenetic analysis

Based on analysis of sequence alignment described in Methods S1, the phylogenetic tree and executed bootstrap test were calculated and decided by the Neighbor-Joining method from MEGA 4.0 [40].

Expression of OsCERK and ACD5 in Escherichia coli and purification of recombinant protein

The full-length OsCERK coding sequence was subcloned into the E. coli expression vector pET28b (Novagen) and transformed into BL21 (DE3) with the forward primer 5′- CGGGATCCGATGGAAGGCGGCGGCGAGGCGCTCT-3′ (BamH I site) and reverse primer 5′ -TCACTCGAGCTACACCTCCGGTCCTGACGCAAAG-3′ (Xho I site). The transformed E.coli cultures were grown in LB medium for large-scale expression. Cells were grown at 37°C with shaking in LB medium. When the turbidity (A600) reached 0.5, IPTG (Merck) was added to 0.5 mM and growth was continued at 16°C overnight, then the cultures were harvested at 4°C. Lysates were analyzed by Western blots using anti-His tag antibody (Tiangen, Beijing, China). His-CERK was purified on Ni-NTA His-Bind resin (Novagen, USA) from 100 ml bacterial cultures, as described in the product manual. After purification, protein concentration was assayed with Easy Protein Quantitative Kit (Trans, Beijing, China). Purified enzyme was stored at −20°C after the addition of sterile glycerol to 50% (V/V). For ACD5, the coding region of acd5 was introduced into pET28b (Novagen) with forward primer 5′-CGGGATCCGATGGAGGAAGGTCGTGACGACGAGT-3′ (BamH I site) and reverse primer 5′ -TCACTCGAGTTATATCTCTGGACCAGATGCGAAC-3′ (Xho I site), and the expression system and conditions was same as OsCERK.

OsCERK activity assay and biochemical characterizations

CERK activity was measured as previously described with modification [41]. Briefly, mixed micelles containing 1.25 mM (0.125%) Triton X-100, 0.2 mM cardiolipin (Sigma-Aldrich, St Louis, USA) and 50 µM D-erythro-C6-NBD-ceramide (Avanti Polar lipids, Alabaster, AL, USA), were prepared by drying lipids from ethanol stocks and then sonicating in reaction buffer [20 mM Hepes (pH 7.4), 10 mM KCl, 15 mM MgCl₂ and 1 mM dithiothreitol (DTT)]. The micelles were diluted fivefold with reaction buffer, and 1 mM ATP (Amresco) was added. The 100 µl reactions were started with the addition of 1.0 µg His-CERK and reacted at 40°C for 30 min in the dark, and then transferred to ice. The reaction mixture (1 µl) was spotted directly onto silica TLC plates (Gel60 F254: Merck, Darmstadt, Germany). The products were separated from the substrate by developing the plate in a solvent system comprising butanol/acetic acid/water (3∶1∶1, by volume). Fluorescence was detected with Typhoon Trio⁺ scanner (GE Healthcare) in blue fluorescence mode. The C6-NBD-ceramide-1-phosphate products were identified by comparison with C6-NBD-ceramide (Avanti Polar lipids, Alabaster, AL, USA) and quantified using IQTL software (GE Healthcare). The specific activity was determined using standard curves with known concentrations of D-erythro-C6-NBD-ceramide. To test the effect of pH on the reaction rates, a series of reaction buffers containing 10 mM KCl, 15 mM MgCl₂ and 1 mM dithiothreitol were prepared with 50 mM citrate (pH 4.5, 5.5 or 6.0), 50 mM Hepes-HCl (pH 6.5 or 7.0) or 50 mM Tris-HCl (pH 7.5, 8.0, 8.5 or 9.0). The K_m value was determined using the Lineweaver-Burk plot method.

For OsCERK activity of plant extracts, crude plant extracts were prepared as described previously [6]. The protein concentration was determined with Easy Protein Quantitative Kit (Trans, Beijing, China). CERK activity of rice lysates using C6-NBD-ceramide as a substrate was done with crude lysates using isotope-labeling method with modification [42]. Briefly, 50 µl of leaf lysates (60 to 120 µg of protein) in a total volume of 100 µl was reacted at 40°C for 30 min in the dark and terminated by adding 600 µl chloroform∶methonal (1∶1) and 265 µl of 1 M KCl in 20 mM MOPS buffer (pH 7.2). The reaction mix was analyzed by TLC developed with H₂O∶acetic acid∶methanol∶acetone∶chloroform (5∶10∶15∶20∶50). The TLC plates were autoradiographed for 1–2 h in a phosphor imager cassette and scanned on a STORM 860 (GE Healthcare).

Molecular complementation of acd5 mutant

The full length OsCERK coding sequence was subcloned into pRT104 vector [43] with the forward primer 5′-TCACTCGAGATGGAAGGCGGCGGCGAGGCGCTCT-3′ (Xho I site) and the reverse primer 5′-CGGGATCCCTACACCTCCGGTCCTGACGCAAAG-3′ (BamH I site). The plasmid pRT104∶OsCERK was digested with Pst I, and cloned into the same sites of pCAMBIA1305.1 vector (CAMBIA). For OsCERK mutant constructs (C454A, C458A, C461A), the mutated constructs for prokaryotic expression were cut with BamH I and Xho I, and the site-mutated OsERK regions were introduced into BamH I and Sal I digested pCAMBIA1300′ vector (the 35S promoter and NOS terminal were introduced into pCAMBIA1300). The obtained wild-type and mutant OsCERK constructs were transformed into Agrobacterium tumefaciens strain EHA105, which was used for transformation of acd5 mutant plants by the floral dip method [44]. The transformed progeny were selected using 1/2 MS plates (solidified with 0.8% tissue culture agar) supplemented with 25 µg/ml hygromycin (Newprobe, Beijing, China). After selfing the T0 plants, lines homozygous for the transgene were chosen for further study.

Visualization of dead cells

Fresh tissue was boiled in lactophenol (10 ml of lactic acid, 10 ml of glycerol, 10 ml of liquid phenol, and 10 ml H₂O) containing 10 mg trypan blue for 30 S and boiled in 95% ethanol∶lacophenol (2∶1) for 1 min, and then cleared in 50% ethanol. Photographs were taken with a fluorescence microscope (Axio Imager A1, Carl Zeiss).

Protoplast isolation and treatments

Rice protoplasts were isolated from 10-day-old seedling tissues as described previously with modifications [45]. Detailed method of this experiment is described in Methods S1. Protoplasts were isolated from leaves of 18-day-old Arabidopsis wild-type and acd5 plants as described previously [6], [46].

N-acetyl-D-erythro-sphingosine (C2-ceramide), N-hexanoyl-D-sphingosine (C6-ceramide), N-acetyl-D-erythro-dihydrosphingosine (C2-dihydroceramide) and N-hexanoyl-D-erythro-dihydrosphingosine (C6-dihydroceramide) were purchased from Matreya (Pleasant GAP, PA, USA), and dissolved in ethanol. C2 ceramide-1-phosphate (C2-1-P) and N, N-dimethylsphingosine (DMS) were from Avanti Polar Lipids (Alabaster, AL, USA), and dissolved in ethanol. Cycloheximide (CHX: Sigma) and K252a (Enzo life science), were dissolved in DMSO (Sigma). Ceramide kinase inhibitor K1 (Calbiochem, LaJolla, CA, USA) was dissolved in ethanol. During treatments, the final solvent concentration was never more than 0.15%.

Statistical analyses were performed using Statview statistical package 5.0.1 (SAS Institute, Cary, North Carolina, USA) for Macintosh. Fisher's protected least significant difference (PLSD), a post hoc multiple t-test, was used. Results were considered significant when P<0.05. Data are presented as means ± SD (standard deviation). All experiments were repeated at least three times.

Measurement of the mitochondrial membrane potential (Δψ_m) by flow cytometry

To measure Δψ_m, the mitochondrial probe 3,3-Dihexyloxacarbocyanine iodide [DiOC₆(3), Molecular Probes, Eugene Oregon, USA] was used as described by [32]. Briefly, 5×10⁵ protoplasts were incubated with DiOC₆ (3) (5 nM) for 10 min in culture medium in the dark, at room temperature. As a control for Δψ_m loss, protoplasts were incubated 100 µM protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma-Aldrich, ST. Louis, USA) for indicated times before flow analysis. The protoplasts were washed once with W5 medium, collected by centrifugation at 800 rpm/min for 3 min. Dyed protoplasts were then stored in the dark for analysis by flow cytometry (BD FACS Calibur) using excitation with a single 488 nm argon laser. At least 30000 events were collected per sample. Data acquisition and analysis were performed using the CellQuest software (BD FACS Calibur, Becton Dickinson) and Win MDI2.9 (Scripps Research Institute, San Diego, CA).

Accession numbers

The GenBank accession number for the nucleotide sequence of OsCERK is FJ765452. Other sequence data of the analyzed CERK proteins in this article can be found in the GenBank data library described in Methods S1.