Unregulated Sphingolipid Biosynthesis in Gene-Edited Arabidopsis ORM Mutants Results in Nonviable Seeds with Strongly Reduced Oil Content

Orosomucoid-like proteins (ORMs) interact with serine palmitoyltransferase (SPT) to negatively regulate sphingolipid biosynthesis, a reversible process critical for balancing the intracellular sphingolipid levels needed for growth and programmed cell death. Here we show that ORM1 and ORM2 are essential for lifecycle completion in Arabidopsis thaliana. Seeds from orm1 orm2 mutants (generated by crossing CRISPR/Cas9 knockout mutants for each gene) accumulated high levels of ceramide, pointing to Plant Cell Advance Publication. Published on June 11, 2020, doi:10.1105/tpc.20.00015 ©2020 American Society of Plant Biologists. All Rights Reserved 2" " unregulated sphingolipid biosynthesis. orm1 orm2 seeds were nonviable, displayed aberrant embryo development, and had >80% reduced oil content vs. wild-type seeds. This phenotype was mimicked in Arabidopsis seeds expressing the SPT subunit LCB1 lacking its first transmembrane domain, which is critical for ORM-mediated regulation of SPT. We identified a mutant for ORM1 lacking one amino acid (Met51) near its second transmembrane domain that retained its membrane topology. Expressing this allele in the orm2 background yielded plants that did not advance beyond the seedling stage, hyperaccumulated ceramides, and showed altered organellar structures and increased senescence and pathogenesis-related gene expression. These seedlings also showed upregulated expression of genes for sphingolipid catabolic enzymes, pointing to additional mechanisms for maintaining sphingolipid homeostasis. ORM1 lacking Met51 had strongly impaired interactions with LCB1 in yeast (Saccharomyces cerevisiae) model), providing structural clues about regulatory interactions between ORM and SPT.

and were severely wrinkled.This phenotype was observed for ~7% of seeds collected 100" from the F 2 orm1 +/− orm2 +/− plants of orm1 −/− and orm2 −/− crosses, which is consistent 101" with the expected 6.25% Mendelian ratio for the occurrence of homozygous double 102" mutants.The remaining seeds were visually indistinguishable from wild-type seeds 103" (Figure 1E and 1F).Of the seeds in these two populations, dark, wrinkled seeds did not 104" germinate, whereas seeds with normal appearance showed no impairment in 105" germination on solid sucrose-containing medium (Figure 1G and 1H) and soil.Strikingly, 106" free ceramide concentrations in pooled abnormal seeds were ~40-fold higher than those 107" in wild-type seeds and ~8-fold higher than in the normal appearing seed segregants 108" from orm1 +/− orm2 +/− plants (Figure 1I).We also observed a similar seed phenotype in interactions (Han et al., 2019).In these experiments, the segregating seeds from 112"

6" "
Cer profiles of the single mutants were similar to those of the wild type (Figure 189" 7C-7E).By contrast, the orm1 Δ met/ Δ met orm2 +/− mutant had increased amounts of Cer with 190" C16 fatty acids relative to wild type and single mutant plants (Figure 7F).This

191"
phenotype was more accentuated in orm1 Δ met/ Δ met orm2 −/− seedlings, which primarily 192" accumulated Cer species with C16 fatty acids linked to the dihydroxy LCB d18:0 and 193" d18:1 (Figure 7G).Increased amounts of Cer with C22, C24 and C26 fatty acids as well 194" as atypical C18 and C20 fatty acid-containing species were also detected in orm1 Δ met/ Δ met 195" orm2 −/− seedlings relative to wild-type plants and mutants of either ORM gene (Figure 196" 7G).Overall, the primary change in the composition of all sphingolipid classes, 197" especially Cer, hCer and nhGlcCer, in the in orm1 Δ met/ Δ met orm2 −/− seedlings was the 198" change in the total and/or relative amounts of those containing C16 fatty acids bound to 199" dihydroxy LCB, which are derived from the LOH2 ceramide synthase (Figure 7G; with the LCB deoxy-sphinganine (DoxSA), which is derived from the condensation of 204" alanine, rather than serine, to palmitoyl-CoA by SPT (Figure 6I).In addition, the 205" concentration of inositolphosphorylceramides (IPCs), the precursors of GIPCs,

208"
Overall, these findings are consistent with the notion that SPT regulation by the Given that sphingolipids are abundant endomembrane and plasma membrane 217" components that contribute to vesicular trafficking, we used transmission electron 218" microscopy (TEM) to evaluate the subcellular phenotypes associated with enhanced 219" sphingolipid accumulation in 10-day-old orm1 Δ met/ Δ met orm2 −/− seedlings relative to wild-220" type seedlings of the same age.Mesophyll cells from wild-type seedlings showed large

227"
Notably, increased vesicle numbers were observed around the ER network in

229"
membrane vesicles consistent with autophagosomes were detected inside the vacuoles 230" of these cells.Moreover, entire chloroplasts were engulfed and appeared to be in the 231" 10" " process of degradation (Figure 8G and 8H).Despite these large defects, Golgi stacks

249"
consistent with the notion that the induction of LCB catabolism is one route (in addition 250" to ceramide biosynthesis) for the mitigation of unregulated LCB production in the 251" orm1 Δ met/ Δ met orm2 −/− mutant.

254"
The accumulation of ceramides has been linked to the activation of signaling pathways

260"
11" " Supplemental Figure 8E).A similar expression pattern was also observed for the

261"
senescence-related gene SAG13 (Figure 9H).SPT activity.This amino acid is located in the ER luminal domain immediately adjacent

266"
to the second transmembrane domain of ORM1 (Supplemental Figure 9).We

267"
hypothesized that, without this amino acid, the conformation of the second 268" transmembrane domain of ORM1 is altered such that the interaction with LCB1 for the 269" repression of SPT activity is disrupted.To better understand this regulatory mechanism,

270"
we stably expressed the Arabidopsis ORM1 Δ met mutant protein in a S. cerevisiae mutant 271" background in which AtLCB1, AtLCB2, and AtssSPTa replaced the corresponding yeast

273"
assessed in vivo SPT activity by measuring the DoxSA produced when expressing 274" AtLCB1 C144W (Figure 10B).Deoxy-LCBs cannot be phosphorylated/degraded and are

276"
When expressed in this yeast background, wild-type Arabidopsis ORM1 was able to 277" suppress DoxSA production, which is consistent with its function as a negative regulator 278" of SPT activity.By contrast, DoxSA concentrations in ORM1 Δ met -expressing cells were 279" similar to those in vector control cells lacking ORM1, which is consistent with a lack of 280" repressed SPT activity.

281"
ORMs interact with the first transmembrane domain of LCB1 to repress SPT 282" activity in S. cerevisiae (Han et al., 2019), although the structural components of ORM 283" associated with this interaction have not been defined.To test whether ORM1 Δ met 284" physically interacts with AtLCB1, as does wild-type ORM1, we performed co- ELO3, an ER protein that does not interact with SPT.By contrast, only trace amounts of

290" 12" "
This finding indicates that Met51 is critical for the ORM-LCB1 physical interaction 291" to regulate SPT activity.To determine whether the impaired ORM-LCB1 interaction is 292" due to gross or subtle alterations in the secondary structure of ORM induced by the

293"
Met51 deletion, we compared the membrane topology of ORM1 and ORM1 Δ met .We reconstituted Arabidopsis SPT.The analysis showed that the cassettes in the predicted 298" luminal domains were glycosylated while the cassette in the predicted cytosolic domain

303"
Our findings identified the essential role of sphingolipid biosynthetic regulation at 304" the level of SPT for seed viability, which was previously unclear due to the lack of 305" complete knockout mutants for ORM genes in plants.We showed that orm1 −/− orm2 to unchecked sphingolipid biosynthesis.These responses include compromised 318" organellar structures, the induction of catabolic genes to maintain sphingolipid 319" 13" " homeostasis, and clues about the structural requirements of ORM for interaction with 320" LCB1.

321"
Our findings emphasize that the full significance of ORMs to plant viability can 322" only be assessed by complete knockout of the corresponding genes.By contrast,

323"
Arabidopsis ORM-suppressed plants previously generated by RNAi or amiRNA 324" methods were fully viable, although the response to bacterial pathogens was altered in 325" these plants and early senescence was observed with the most extreme suppression of such, it is likely that pollen is able to tolerate unregulated sphingolipid synthesis that 336" results from complete ORM knockout.

337"
The mechanism underlying the loss of seed viability from unregulated SPT 338" activity in orm1 −/− orm2 −/− and orm1 Δ met/ Δ met orm2 −/− mutants likely involves a combination 339" of the functions of sphingolipids as major structural components of the endomembrane 340" and as bioactive mediators of cellular activities such as PCD that lead to aberrant 341" embryo development.As shown in orm1 Δ met/ Δ met orm2 −/− seedlings, strong upregulation of 342" sphingolipid biosynthesis results in large alterations in membrane and organellar

343"
structures in plant cells (Figure 8).These seedlings appear to have defects in ER 344" function, as indicated by the relative reduction in the total content of very long-chain 345" fatty acids in the abnormal seeds from the progeny of orm1 −/− and orm2 −/− crosses and

351"
Among the gene-edited ORM variants identified in our studies was a mutant that 352" contained an in-frame deletion of Met51 combined with a homozygous knockout of 353" ORM2 (orm1 Δ met/ Δ met orm2 −/− ).Seeds from this mutant were viable, in contrast to wild-type seedlings.Cells from the orm1 Δ met/ Δ met orm2 −/− seedlings displayed gross 360" defects in membrane and organellar structures as well as apparent autophagosome-like

361"
structures.The early cell death displayed by the orm1 Δ met/ Δ met orm2 −/− seedlings can be 362" attributed to the activation of PCD pathways, as indicated by the high transcript levels of 363" pathogenesis-and senescence-related genes that have been shown to be activated by 364" the accumulation of LCB and ceramides.

365"
Notably, Met51 is predicted to occur at a position that is adjacent to the second 366" transmembrane domain of ORMs but is not a conserved residue across eukaryotic

368"
Arabidopsis SPT complex, we determined that the ORM1 Met51 mutant has greatly 369" reduced interaction with Arabidopsis LCB1, which is required for ORM-induced 370" suppression of SPT activity.Given that Met51 is not conserved in eukaryotes, it is likely 371" that LCB1 does not directly interact with this residue.Instead, the lack of this amino acid that impedes its regulatory interaction with the first transmembrane domain of LCB1.

374"
The maintenance of the topology of ORM1 ΔMet51 in microsomal membranes was verified

375"
by Endo H digestion studies using the mutant ORM1 protein carrying glycosylation 376" cassettes.To date, no residues or structural features in ORMs have been identified that 377" are associated with their interaction with the LCB1/LCB2 heterodimer of SPT.Our between ORM and LCB1.

382"
The use of gene editing also allowed us to assess the redundancy of ORM1 and

383"
ORM2.Notably, single mutants and progeny from the crosses that genotype as orm1 +/− 384" orm2 +/− had an appearance similar to wild-type plants under normal conditions.

394"
However, we did observe that orm1 +/− orm2 −/− plants have a highly bushed appearance investigation, but it suggests that ORM2 contributes more strongly to meristem function 398" than ORM1, perhaps due to cell-type-specific differences in the expression of the ORM 399" genes or to a non-sphingolipid function of ORM proteins.

400"
Our results also revealed transcriptional mechanisms for maintaining sphingolipid glucosylceramides, which accumulated in orm1 Δ met/ Δ met orm2 −/− seedlings but were 409" 16" " detected at only low concentrations in wild type and ORM1 and ORM2 single mutants.

410"
These findings are consistent with our previous report that LOH2 activity is upregulated 411" in Arabidopsis ORM RNAi plants, presumably as a pathway for reducing cytotoxicity of in the orm1 Δ met/ Δ met orm2 −/− mutant, indicating that the transcriptional regulation of genes

415"
for SPT complex proteins is not a pathway for maintaining sphingolipid homeostasis in

416"
response to deregulated long-chain base biosynthesis.Instead, the expression of genes 417" involved in the catabolism of LCBs increased ~six-to seven-fold (SPHK2 and DPL1) in

418"
this mutant, suggesting that an unknown mechanism is activated in response to 419" increased ceramide and/or LCB levels.

420"
Our overall findings about the metabolic and developmental defects associated supplemented with 1% sucrose and 0.8% agar (pH 5.7) with 16 h light (100µmol/ m -2 s -

456"
For CRISPR/Cas9-mediated gene editing of ORM1 and ORM2, designed target sites 457" (Figure 1A) were fused with a single guide RNA (sgRNA) and expressed under the Table 1).The purified PCR products were digested with BsaI and ligated to the BsaI-462" linearized binary vector pHEE401E.The final CRISPR/Cas9 binary vector was 463" electroporated into Agrobacterium strain GV3101 and then transformed into Arabidopsis

465"
were screened for hygromycin resistance on MS plates containing 25 mg/L hygromycin.

466"
For genotyping, fragments including the target regions of ORM1 and ORM2 were 467" amplified by PCR from the genomic DNA of transgenic plants (primers P5-P8;

470"
analyze for non-transgenic plants, progeny of hygromycin selected and confirmed

473"
Supplemental Table 1) for the lack of the Cas9 gene with the presence of the CRISPR 474" mutation, in the T 2 generation.The plants lacking Cas9 but containing the CRISPR 475" mutation were kept and used for further studies as mutated but non-transgenic lines.

484"
with the pBinGlyRed3-ORM1 construct by the floral dip method (Clough and Bent, 485" 1998).Transformants were selected based on DsRed fluorescence and genotyped.

486"
Mutation was confirmed by sequencing.

497"
Anthers of mature plants were isolated and smeared on a glass slide.The pollen was 498" stained using Alexander staining method (Alexander, 1969) for 1 h at 25°C.Pollen

499"
imaging was performed using the EVOS FL Auto Cell Imaging System.

503"
to 15-day-old Arabidopsis seedlings grown on solid medium were collected from 504" independent plates for each biological replicate.The seedlings were lyophilized and 10-505" 30 mg of tissue was homogenized and extracted with isopropanol/heptane/water 506" (55:20:25 v/v/v).We used one to four mg of plant material for each biological replicate (5:2:5 v/v/v) containing 0.1% formic acid.The sphingolipid species were analyzed using

511"
a Shimadzu Prominence ultra-performance liquid chromatography system and a 4000 512" QTRAP mass spectrometer (AB SCIEX).Data analysis and quantification were 513" performed using the software Analyst 1.5 and Multiquant 2.1 as described (Markham

517"
To quantify the TAG content, lipids were extracted from ~1 mg of seeds using a method 518" based on that of Bligh and Dyer (Bligh and Dyer, 1959).Seeds were ground using a 519" glass rod in 13 × 100-mm glass screw cap tubes with 3 mL methanol:chloroform (2:1 520" v/v).Triheptadecanoin (17:0-TAG) was added to the seeds as an internal standard prior 521" to extraction.After 1 h incubation at 25°C, 1 mL of chloroform and 1.9 mL of water were 522" added.The solution was mixed thoroughly and centrifuged at 400•g for 10 min.The (Supelco Supelclean LC-Si SPE column; Sigma-Aldrich) pre-equilibrated with heptane.

527"
A purified TAG fraction was eluted from the column and converted to fatty acid methyl 528" esters, which were analyzed by gas chromatography as previously described (Zhu et   RNA was extracted from 12 to 15-day-old Arabidopsis seedlings grown on solid MS 534" medium.Each replicate corresponds to pooled seedlings from independent plates.

535"
RNA extraction was performed using an RNeasy Kit (Qiagen) according to the 536" manufacturer's protocol.The isolated RNA (1 µg) was treated with DNase I (Invitrogen).

537"
cDNA conversion was performed with a RevertAid cDNA synthesis kit (Thermo Fisher).

538"
SYBR Green was used as the fluorophore in a qPCR supermix (Qiagen).PP2AA3 and 539" UBIQUITIN (UBQ) were used as internal reference genes.qPCR was performed using 540" a Bio-Rad MyiQ iCycler qPCR instrument.The thermal cycling conditions were an initial 541" step of 95°C for 10 min followed by 45 cycles at 95°C for 15 s, 60°C for 30 s and 72°C

542"
for 30 s. Primers used in this study are listed in Supplemental Table 1.

548"
to post fixation with 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated with 549" ethanol and acetone, and embedded with a Spurr's Embedding Kit.Ultra-thin sections 550" (100 nm) were cut and stained with uranyl acetate and lead citrate.Samples were 551" imaged on a Hitachi H7500 TEM at an accelerating voltage of 80 kV.

559"
Plasmids for the expression of AtLCB1-FLAG, Myc-AtLCB2a, and HA-AtssSPTa in or AtORM1 Δ met .Microsomal membrane proteins were solubilized in 1.5% digitonin at 4°C 571" for 2.5 h and incubated with Flag-beads (Sigma-Aldrich) overnight.The bound proteins

589"
Two-tailed Student's t test was performed to evaluate statistically significant differences 590" compared to the control (wild-type).One-way ANOVA followed by Tukey's test was 591" used to determine the differences among the five genotypes for a given variable.The          (A) AtORM1 met was stably expressed in Saccharomyces cerevisiae with the native SPT complex replaced by the Arabidopsis SPT complex (see Methods).AtLCB1-FLAG, MYC-AtLCB2a, HA-AtssSPTa without or with HA-AtORM1 or HA-AtORM1 met were expressed in yeast strain lcb1 tsc3.5, 10 and 15 µg of microsomal proteins were loaded and analyzed by (4-12%, Invitrogen) and detected with Anti-LCB1 (1:3000) and anti-HA antibodies (Covance).(B) DoxSA levels were determined from cells expressing AtLCB1 C144W and AtLCB2a, HA-AtssSPTa along with vector, HA-AtORM1 wild-type, or HA-AtORM1 met .Shown are the mean ± SD of doxSA levels from six independent colonies for each strain.Asterisks denote significant differences, as determined by two-tailed Student's t test with a significance of p ≤0.001; NS, not significant, n=6.(C) Co-immunoprecipitation of FLAG-tagged AtLCB1 in yeast expressing AtLCB1-FLAG, MYC-AtLCB2a, HA-AtssSPTa, and either HA-AtORM1 or HA-AtORM1 met .Solubilized yeast microsomes were incubated with anti-FLAG beads and protein was eluted with FLAG peptide.Solubilized microsomes (Input), unbound and bound (IP-FLAG) were analyzed by immunoblotting.ELO3, an integral ER membrane protein, was used as a negative control.(D) Topology mapping of AtORM1 met51 .Glycosylated cassettes (GC) were inserted after the indicated amino acids, and the GC-tagged proteins were expressed in yeast.Increased mobility following treatment of microsomes with endoglycosidase H (EndoH) revealed that the GCs at residues 46 and 121 are glycosylated and therefore reside in the lumen of the endoplasmic reticulum.However, the GC at residue 82 is not glycosylated, indicating that residue 82 is located in the cytosol.AtORM1 met51 retains the topology of wild-type ORM1.ORM proteins and LCB1 are integral ER-membrane proteins with multiple transmembrane domains (TMDs).The ORM proteins contain four TMDs, with both termini located in the cytosol, while LCB1 has three TMDs, with its Nterminus located in the ER-Lumen and C-terminus located in the cytosol.LCB1, along with LCB2 and ssSPTa, comprise serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid biosynthesis.TMD1 of LCB1 is required for ORM to SPT (A).Expression of LCB1 without its first transmembrane domain (B) or the complete knockout of ORM1 and 2 (C) results in the loss of SPT regulation.This is characterized by strongly enhanced accumulation of ceramides and selected complex sphingolipids and the loss of seed viability marked by a strong reduction in TAG content.The lack of MET51 before the second transmembrane domain (TMD2) of ORM1 it thought to causes a conformational change that dramatically decreases its interaction with LCB1 for SPT regulation (D).CS, ceramide synthase; LCB, long chain bases; LCB-P, long chain bases-phosphate; CER, ceramide; LCBK, long chain base kinase: DPL1, LCB phosphate lyase; PE, phosphoethanolamine; HXD, hexadecanal.Black arrows indicate de novo sphingolipid biosynthesis and blue arrows indicate catabolic reactions.

ORM1Δ
met Fails to Interact with LCB1 to Suppress SPT Activity264"Our results clearly show that ORM1 lacking Met51 is strongly impaired in repressing 265" readout for in situ SPT activity (Gable et al., 2010; Kimberlin et al. 2016).
-tagged AtLCB1 with solubilized microsomes from yeast 286" cells expressing Myc-AtLCB2a, HA-ssSPTa and HA-ORM1 or HA-ORM1 Δ met .Pull-287" downs of AtLCB1 resulted in co-immunoprecipitation of AtLCB2a and AtORM1, but not 288" into the two predicted ER luminal loops (at amino acids 295" 46 and 121) and into the cytosolic loop between the second and third transmembrane 296" domains (at amino acid 82) and expressed the proteins in S. cerevisiae along with 297" − orm2 −/− ; however, the plants did not advance beyond the seedling stage and had355"strong developmental defects.Like the orm1 −/− orm2 −/− seeds, the orm1 Δ met/ Δ met orm2 −/− 356" seedlings hyperaccumulated ceramides with C16 fatty acids.These seedlings also 357" accumulated aberrant sphingolipids including DoxSA-containing ceramides, GlcCer 358" containing non-hydroxylated fatty acids, and IPCs, all of which were nearly absent from 359" 372"likely produces a conformational change at the second transmembrane domain of ORM 373" 378"findings point to the possible interaction of the first transmembrane domain of LCB1 with 379" 15" " the second transmembrane domain of ORM as the basis for SPT regulation.Additional380"structural studies are required to fully elucidate these potential regulatory interactions 381" 395"and are strongly delayed in flowering (>80 days to flowering) (Figure4D), pointing to a 396" meristem defect(Tantikanjana et al. 2001).This phenotype requires further 397" 401"homeostasis upon the enhanced production of long-chain bases in the orm1 Δ met/ Δ met 402" orm2 −/− mutant.LOH2 and LOH3 (encoding the functionally distinct ceramide synthases 403" LCB kinases) and DPL1 (encoding the last step in long-chain base degradation) were 404" transcriptionally upregulated in the mutant.Notably, upregulating LOH2 expression was 405" associated with the preponderance of ceramides containing C16 fatty acids and 406" dihydroxy long-chain bases (the principal products of LOH2 ceramide synthase activity) 407"in free ceramides and glucosylceramides, including non-hydroxylated 408" chain bases and ceramides (which are metabolized to glucosylceramides) 413" (Kimberlin et al., 2016).No changes were detected in LCB1 or ssSPTa transcript levels 414" 421"with the deregulation of SPT by disrupting ORM genes or removing the first 422" transmembrane domain of LCB1 are schematically summarized in Figure 11.These 423" sphingolipid-related regulatory processes identified in Arabidopsis are likely found in 424" other plant species due to the conservation of sphingolipid metabolic enzymes in the 425" plant kingdom.Still unanswered is how ORM interactions with LCB1 are regulated in 426" response to perturbations in intracellular sphingolipid levels or abiotic and biotic 427"stresses (e.g., bacterial and fungal pathogenesis).Similar to mammalian ORMDLs,428"plant ORMs lack the serine-rich N-terminal extension found in S. cerevisiae ORMs,429"which is phosphorylated or dephosphorylated in response to intracellular sphingolipid 430" levels to mediate ORM-LCB1 interactions (Breslow et al., 2010; Han et al., 2010).431" Mammalian ORMDLs have recently been shown to bind ceramides directly, which 432" affects the interactions of ORMs with LCB1 (Davis et al., 2019).A similar regulatory 433" mechanism might occur in plants.In this regard, we previously speculated that LOH2-434" derived ceramides or glycosphingolipids enriched in dihydroxy LCBs and C16 fatty 435" acids likely provide minimal SPT regulation relative to those containing trihydroxy LCBs 436" and very long-chain fatty acids based on the hyperaccumulation of sphingolipids found 437" in sbh1 sbh2 mutants and LOH2-overexpressing plants (Chen et al., 2008; Luttgeharm 438" et al., 2015a).Still, how ORMs reversibly regulate SPT activity in response to cellular 439" sphingolipid requirements remains an outstanding question in plants.Arabidopsis thaliana Columbia-0 (Col-0) was used as the wild-type reference in this 445" study.Arabidopsis seedlings were grown on Murashige and Skoog (MS) medium 446" standard wide-spectrum fluorescent bulbs type F32/841/ECO 32 watt 449" U6 promoter.The egg cell-specific EC1 promoter was used to drive Cas9 459" expression as previously reported (Wang et al., 2015).In short, BsaI sites were 460" incorporated by PCR into the ORM target sequences (Primers P1-P4; Supplemental 461" silent mutations of the ORM1 gRNA target sequence to 480" mitigate possible editing of the transgene.The cDNA was amplified by overlapping PCR 481" and cloned into the EcoRI and XbaI sites of binary vector pBinGlyRed3 under the 482" control of the native ORM1 promoter 600 bp region upstream of the ORM1 start codon 483"
523"lower organic phase containing total lipids was transferred to a new glass tube and 524" solvent evaporated under a N 2 stream with heating at 40°C.The sample was525"redissolved in 1 mL of heptane and loaded onto a solid phase extraction column 526" 2016).TAG fatty acid content was quantified relative to 17:0 fatty acid methyl ester 530" from the internal standard.

Figure 1 .
Figure 1.The ORM Double Knockout Mutant is Seed Lethal.(A) Schematic representation of CRISPR/Cas9-induced mutations in ORM genes.Gene structures of ORM1 and ORM2; black boxes represent exons.The CRISPR/Cas9 target site is indicated, as well as the nucleotide deletions for each gene in the single mutants.(B) Representative images of 25-day-old wild-type Col-0, orm1 −/− and orm2 −/− plants.(C) Representative images of pollen and anthers (treated with Allexander stain) collected from wild-type Col-0 and orm1 +/− orm2 +/− plants.(D) Viability of pollen determined by counts of ~100 pollen grains from five randomly selected flowers from independent Col-0 and orm1 +/− orm2 +/− plants.Shown are the mean ± SD. (E) Developing seeds in siliques from wild-type Col-0 and orm1 +/− orm2 +/− plants.Shriveled, brown (abnormal) seeds are indicated by arrows.(F) Percentage of shriveled and brown (abnormal) seeds in siliques determined by counts of an average of 200 developing seeds from 10 randomly selected siliques of independent wild-type Col-0 and orm1 +/− orm2 +/− plants.Shown are the mean ± SD.Asterisks denote significant differences, as determined by two-tailed Student's t test with a significance of p ≤0.01.(G) Seeds from wild-type Col-0; seeds from orm1 +/− orm2 +/− were separated and classified into normal and the darker, shriveled seeds as abnormal.Bars=1 mm.(H) Phenotypes of 10-day-old seedlings from wild-type Col-0 seeds, normal and abnormal seeds from orm1 +/− orm2 +/− .Abnormal seeds did not germinate.(I) Ceramide content in seeds from wild-type Col-0, normal and abnormal seeds from orm1 +/− orm2 +/− .Shown are the mean ± SD, n=3.Asterisks indicate significant differences based on one-way ANOVA followed by Tukey's multiple comparisons test, with a significance of (*) P≤0.05 and (***) P≤0.001.

Figure 3 .
Figure 3. Abnormal Seeds from ORM and LCB1ΔTMD1 Mutant Plants Have Altered Embryo Morphology and Reduced Triacylglycerol Concentrations.Morphology of embryos from (A) wild-type seeds and (B-D) abnormal seeds from orm1 +/− orm2 +/− plants showing that the embryo is not fully developed.Embryos were dissected from mature seeds.Bars=200 M. (E) 100 seed weight.Values are the mean ± SD of seeds harvested from 4 independent plants.Asterisks indicate significant difference based on one-way ANOVA followed by Tukey's multiple comparisons test, with a significance of (****) P≤0.0001.NS, not significant.(F) Triacylglycerol (TAG) content in seeds from wild-type Col-0 and normal and abnormal seeds from orm1 +/− orm2 +/− and Atlcb1 +/− expressing LCB1ΔTMD1.Values are the mean ± SD of three independent lipid extractions.Asterisks indicate significant difference based on one-way ANOVA followed by Tukey's multiple comparisons test with a significance of (***) P≤0.001 and (****) P≤0.0001.NS, not significant.(G) Composition of TAG as weight percent of fatty acid in seeds from wild-type Col-0 and normal and abnormal seeds from orm1 +/− orm2 +/− and Atlcb1 +/− expressing LCB1ΔTMD1.Values are the mean ± SD of three independent samples.

Figure 9 .
Figure 9. Expression of Genes Associated with Sphingolipid Homeostasis, Plant Defense Responses, and Senescence are Upregulated in the orm1 met/ met orm2 −/− Mutant.Wild-type, orm1 −/− , orm2 −/− orm1 met/ met orm2 +/− and orm1 met/ met orm2 −/− seedlings (12-day-old plants) were used to examine gene expression by qPCR to monitor genes encoding enzymes in sphingolipid biosynthetic and catabolic pathways: (A) ceramide synthase gene LOH2, (B) ceramide synthase gene LOH3, (C) sphingosine kinase 2 gene SPHK2 and (D) LCB phosphate lyase gene DPL1; and selected pathogenesis-and senescence-related genes: (E) -1,3-glucanase gene PR2, (F) class III peroxidase gene PRXC, (G) flavin monooxygenase gene FMO and (H)senescence-related 13 gene SAG13.PP2AA3 transcript levels were used as a control for the sphingolipid genes and UBIQUITIN for the pathogenesis and senescence-related genes.Specific primers used for this analysis are shown in Supplemental Table1.Gene expression levels are normalized to those in wild-type seedlings.Values are the mean ± SD (n=6-12).Different letters indicate significant difference based on one-way ANOVA followed by Tukey's multiple comparisons test (P≤0.05).

Figure 10 .
Figure 10.AtORM1 met Fails to Regulate SPT Activity and Does Not Interact with LCB1.

Figure 11 .
Figure 11.Model of ORM-Mediated Sphingolipid Biosynthesis in Wild-type Plants and ORM and LCB1 Mutants.