Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.     

Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development.     

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes ( AtLCB2a and AtLCB2b ) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.     

The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.     

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.     

Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis

Sphingolipids are structural components of endomembranes and function through their metabolites as bioactive regulators of cellular processes such as programmed cell death. A characteristic feature of plant sphingolipids is their high content of trihydroxy long-chain bases (LCBs) that are produced by the LCB C-4 hydroxylase. To determine the functional significance of trihydroxy LCBs in plants, T-DNA double mutants and RNA interference suppression lines were generated for the two Arabidopsis thaliana LCB C-4 hydroxylase genes Sphingoid Base Hydroxylase1 (SBH1) and SBH2. These plants displayed reductions in growth that were dependent on the content of trihydroxy LCBs in sphingolipids. Double sbh1 sbh2 mutants, which completely lacked trihydroxy LCBs, were severely dwarfed, did not progress from vegetative to reproductive growth, and had enhanced expression of programmed cell death associated-genes. Furthermore, the total content of sphingolipids on a dry weight basis increased as the relative amounts of trihydroxy LCBs decreased. In trihydroxy LCB-null mutants, sphingolipid content was approximately 2.5-fold higher than that in wild-type plants. Increases in sphingolipid content resulted from the accumulation of molecular species with C16 fatty acids rather than with very-long-chain fatty acids, which are more commonly enriched in plant sphingolipids, and were accompanied by decreases in amounts of C16-containing species of chloroplast lipids. Overall, these results indicate that trihydroxy LCB synthesis plays a central role in maintaining growth and mediating the total content and fatty acid composition of sphingolipids in plants.     

Degradation of sphingoid long-chain base 1-phosphates (LCB-1Ps): functional characterization and expression of AtDPL1 encoding LCB-1P lyase involved in the dehydration stress response in Arabidopsis

Sphingoid long-chain base (LCB) 1-phosphates are degradated by LCB 1-phosphate lyase to C(16) fatty aldehydes and phosphoethanolamine. Here, we confirmed that the At1g27980 gene product, AtDPL1, is a functional LCB-1-phosphate lyase. Expression of green fluorescent protein fusion products in suspension-cultured Arabidopsis cells showed that AtDPL1 is located to the endoplasmic reticulum. The rates of fresh weight decreases of dpl1-1 and dpl1-2 mutants were significantly slower than those of the wild-type plants. This ability to limit their transpiration reflected the leaf temperature of the mutant plants more than that of wild-type plants, suggesting that AtDPL1 plays a role in dehydration stress.     

Synthesis and degradation of long-chain base phosphates affect fumonisin B<Subscript>1</Subscript>-induced cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Fumonisin B₁ (FB₁), an inducer of cell death, disrupts sphingolipid metabolism; large accumulations of de novo synthesized free long-chain bases (LCBs) are observed. However, it remains unclear whether tolerance to FB₁ toxicity in plants is connected with preventing the accumulation of free LCBs through their phosphorylation. Here a workflow for the extraction, detection and quantification of LCB phosphates (LCBPs) in Arabidopsis thaliana was developed. We studied the effect of expression of genes for three enzymes involved in the synthesis and degradation of LCBPs, LCB kinase (LCBK1), LCBP phosphatase (SPP1) and lyase (DPL1) on FB₁-induced cell death. As expected, large accumulations of saturated free LCBs, dihydrosphingosine and phytosphingosine, were observed in the FB₁-treated leaves. On the other hand, a high level of sphingenine phosphate was found in the FB₁-treated leaves even though free sphingenine was found in low amounts in these leaves. In comparison of WT and spp1 plants, the LCBP/LCB ratio is likely to be correlated with the degree of FB₁-induced cell death determined by trypan blue staining. The FB₁-treated leaves in dpl1 plants showed severe cell death and the elevation of free LCBs and LCBPs. LCBK1-OX and -KD plants showed resistance and sensitivity to FB₁, respectively, whereas free LCB and LCBP levels in FB₁-treated LCBK1-OX and -KD plants were moderately different to those in FB₁-treated WT plants. Overall, the findings described here suggest that LCBP/LCB homeostasis is an important topic that participates in the tolerance of plant cells to FB₁.     

Characterization of an Arabidopsis cDNA encoding a subunit of serine palmitoyltransferase, the initial enzyme in sphingolipid biosynthesis.

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis. In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT. The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids. The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding. Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT. Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis. Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum. AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants.     

Characterization of sphingolipid long-chain base kinase in Arabidopsis thaliana

Sphingolipid long-chain base (LCB) kinase catalyses the phosphorylation of sphingolipid LCB to form LCB 1-phosphate. Based on sequence identity to a murine sphingosine kinase (murine SPHK1a), we isolated and characterized a LCB kinase-like cDNA in Arabidopsis thaliana. The deduced amino acid sequence of the homologous cDNA shows several regions that are highly conserved in LCB kinases from mouse, yeast, human and Caenorhabditis elegans. These regions are not similar to those of other known kinase families. For a functional identification, the homologous cDNA from A. thaliana was expressed in Escherichia coli, and LCB kinase activity was measured. The recombinant AtLcbk1 protein was found to utilize ATP and sphinganine. These results indicate the first identification of a gene coding for a LCB kinase in plants.     

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.     

Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici

Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization.     

Sphingolipid Δ8 unsaturation is important for glucosylceramide biosynthesis and low-temperature performance in Arabidopsis

Plants contain a large diversity of sphingolipid structures, arising in part from C4 hydroxylation and Δ4 and Δ8 desaturation of the component long-chain bases (LCBs). Typically, 85-90% of sphingolipid LCBs in Arabidopsis leaves contain a cis or transΔ8 double bond produced by sphingoid LCB Δ8 desaturase (SLD). To understand the metabolic and physiological significance of Δ8 unsaturation, studies were performed using mutants of the Arabidopsis SLD genes AtSLD1 and AtSLD2. Our studies revealed that both genes are constitutively expressed, the corresponding polypeptides are ER-localized, and expression of these genes in Saccharomyces cerevisiae yields mixtures of cis/transΔ8 desaturation products, predominantly as trans isomers. Consistent in part with the higher expression of AtSLD1 in Arabidopsis plants, AtSLD1 T-DNA mutants showed large reductions in Δ8 unsaturated LCBs in all organs examined, whereas AtSLD2 mutants showed little change in LCB unsaturation. Double mutants of AtSLD1 and AtSLD2 showed no detectable LCB Δ8 unsaturation. Comprehensive analysis of sphingolipids in rosettes of these mutants revealed a 50% reduction in glucosylceramide levels and a corresponding increase in glycosylinositolphosphoceramides that were restored by complementation with a wild-type copy of AtSLD1. Double sld1 sld2 mutants lacked apparent growth phenotypes under optimal conditions, but displayed altered responses to certain stresses, including prolonged exposure to low temperatures. These results are consistent with a role for LCB Δ8 unsaturation in selective channeling of ceramides for the synthesis of complex sphingolipids and the physiological performance of Arabidopsis.     

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death （PCD）. However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 r_esistant11-1 （/br11-1）, which fails to generate reactive oxygen intermediates （ROIs）, is incapable of initiating PCD when the mutant is challenged by fumonisin B l （FB0, a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 （LCB 1） subunit of serine palmitoyltransferase （SPT）, which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FBl. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine- 1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.     

Signaling role of fructose mediated by FINS1/FBP in Arabidopsis thaliana

Sugars are evolutionarily conserved signaling molecules that regulate the growth and development of both unicellular and multicellular organisms. As sugar-producing photosynthetic organisms, plants utilize glucose as one of their major signaling molecules. However, the details of other sugar signaling molecules and their regulatory factors have remained elusive, due to the complexity of the metabolite and hormone interactions that control physiological and developmental programs in plants. We combined information from a gain-of-function cell-based screen and a loss-of-function reverse-genetic analysis to demonstrate that fructose acts as a signaling molecule in Arabidopsis thaliana. Fructose signaling induced seedling developmental arrest and interacted with plant stress hormone signaling in a manner similar to that of glucose. For fructose signaling responses, the plant glucose sensor HEXOKINASE1 (HXK1) was dispensable, while FRUCTOSE INSENSITIVE1 (FINS1), a putative FRUCTOSE-1,6-BISPHOSPHATASE, played a crucial role. Interestingly, FINS1 function in fructose signaling appeared to be independent of its catalytic activity in sugar metabolism. Genetic analysis further indicated that FINS1-dependent fructose signaling may act downstream of the abscisic acid pathway, in spite of the fact that HXK1-dependent glucose signaling works upstream of hormone synthesis. Our findings revealed that multiple layers of controls by fructose, glucose, and abscisic acid finely tune the plant autotrophic transition and modulate early seedling establishment after seed germination.     

Lcb4p is a key regulator of ceramide synthesis from exogenous long chain sphingoid base in Saccharomyces cerevisiae

Long chain sphingoid bases (LCBs) and their phosphates (LCBPs) are not only important intermediates in ceramide biosynthesis but also signaling molecules in the yeast, Saccharomyces cerevisiae. Their cellular levels, which control multiple cellular events in response to external and intrinsic signals, are tightly regulated by coordinated action of metabolic enzymes such as LCB kinase and LCBP phosphatase. However, little is known about the mechanisms by which the two enzymes generate biosynthetic or signaling outputs. It has been shown that the LCBP phosphatase, Lcb3p, is required for efficient ceramide synthesis from exogenous LCB. Here we present direct evidence that the major LCB kinase, Lcb4p, but not the minor kinase, Lcb5p, regulates synthesis of ceramide from exogenously added LCB. Surprisingly, our biochemical evidence suggests that the LCBP used for ceramide synthesis must be generated on the membrane. Our data show that Lcb4p is tightly associated with membranes and is localized to the endoplasmic reticulum where it can work in concert with Lcb3p. These results raise the conceptually attractive possibility that membrane-associated and cytosolic Lcb4p play distinct roles to differentially generate biosynthetic and signaling pools of LCBP.     

Accumulation of phosphorylated sphingoid long chain bases results in cell growth inhibition in Saccharomyces cerevisiae

Sphingolipid metabolites in mammals can function as signaling molecules with cell-specific functions. In Saccharomyces cerevisiae, phosphorylated long chain bases, such as dihydrosphingosine 1-phosphate and phytosphingosine 1-phosphate, have also been implicated in stress responses. To further explore the biological roles of these molecules, we created disruption mutants for LCB4, LCB5, DPL1, YSR2, YSR3, and SUR2. LCB4 and LCB5 encode kinases that phosphorylate long chain bases. DPL1 and YSR2/YSR3 are involved in degradation of the phosphorylated long chain bases. SUR2 catalyzes conversion of dihydrosphingosine to phytosphingosine. We adapted an HPLC method to measure intracellular concentrations of the phosphorylated long chain bases. Double mutants of dpl1 and ysr2 were inviable, whereas dpl1 ysr2 lcb4 triple mutants were viable. Further, growth inhibition associated with accumulated phosphorylated long chain bases was observed in the triple mutant dpl1 ysr2 lcb4 overexpressing LCB4 or LCB5. These results indicate that phosphorylated long chain bases can inhibit cell growth. Mutants defective in both YSR2 and SUR2, which accumulated dihydrosphingosine 1-phosphate only, grew poorly. The phenotypes of the ysr2 sur2 mutants were suppressed by overexpression of DPL1. Our results clearly show that elevated levels of phosphorylated long chain bases have an antiproliferative effect in yeast.     

Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis

Sphingolipids are important signaling molecules involved in various cellular activities. De novo sphingolipid synthesis is initiated by a rate-limiting enzyme, serine palmitoyltransferase (SPT), a heterodimer consisting of LONG-CHAIN BASE1 (LCB1) and LCB2 subunits. A mutation in the Arabidopsis thaliana LCB1 gene, lcb1-1, was found to cause embryo lethality. However, the underpinning molecular and cellular mechanisms remain largely unclear. Here, we report the identification of the fumonisin B(1) resistant11-2 (fbr11-2) mutant, an allele of lcb1-1. The fbr11-2 mutation, most likely an allele stronger than lcb1-1, was transmitted only through female gametophytes and caused the formation of abortive microspores. During the second pollen mitosis, fbr11-2 initiated apoptotic cell death in binucleated microspores characteristic of nuclear DNA fragmentation, followed by cytoplasm shrinkage and organelle degeneration at the trinucleated stage. In addition, a double mutant with T-DNA insertions in two homologous LCB2 genes showed a phenotype similar to fbr11-2. Consistent with these observations, the FBR11/LCB1 expression was confined in microspores during microgametogenesis. These results suggest that SPT-modulated programmed cell death plays an important role in the regulation of male gametophyte development.