Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha.

Gibberellins (GAs) are key phytohormones that regulate growth, development, and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthesis intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here, we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently exhibited altered thallus growth allometry, delayed initiation of gametogenesis, and abnormal morphology of gamete-bearing structures (gametangiophores). By chemical treatments and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, we confirmed that these phenotypes were caused by the deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but were co-opted independently to regulate responses to light quality change in different plant lineages during the past 450 million years of evolution.

Transcription of the Antisense Long Non-Coding RNA, SUPPRESSOR OF FEMINIZATION, Represses Expression of the Female-Promoting Gene FEMALE GAMETOPHYTE MYB in the Liverwort Marchantia polymorpha.

Sexual differentiation is a fundamental process in the life cycles of land plants, ensuring successful sexual reproduction and thereby contributing to species diversity and survival. In the dioicous liverwort Marchantia polymorpha, this process is governed by an autosomal sex-differentiation locus comprising FEMALE GAMETOPHYTE MYB (FGMYB), a female-promoting gene, and SUPPRESSOR OF FEMINIZATION (SUF), an antisense strand-encoded long non-coding RNA (lncRNA). SUF is specifically transcribed in male plants and suppresses the expression of FGMYB, leading to male differentiation. However, the molecular mechanisms underlying this process remain elusive. Here, we show that SUF acts through its transcription to suppress FGMYB expression. Transgene complementation analysis using CRISPR/Cas9D10A-based large-deletion mutants identified a genomic region sufficient for the sex differentiation switch function in the FGMYB-SUF locus. Inserting a transcriptional terminator sequence into the SUF-transcribed region resulted in the loss of SUF function and allowed expression of FGMYB in genetically male plants, leading to conversion of the sex phenotype from male to female. Partial deletions of SUF had no obvious impact on its function. Replacement of the FGMYB sequence with that of an unrelated gene did not affect the ability of SUF transcription to suppress sense-strand expression. Taken together, our findings suggest that the process of SUF transcription, rather than the resulting transcripts, is required for controlling sex differentiation in M. polymorpha.

Thermospermine Is an Evolutionarily Ancestral Phytohormone Required for Organ Development and Stress Responses in Marchantia Polymorpha.

Thermospermine suppresses auxin-inducible xylem differentiation, whereas its structural isomer, spermine, is involved in stress responses in angiosperms. The thermospermine synthase, ACAULIS5 (ACL5), is conserved from algae to land plants, but its physiological functions remain elusive in non-vascular plants. Here, we focused on MpACL5, a gene in the liverwort Marchantia polymorpha, that rescued the dwarf phenotype of the acl5 mutant in Arabidopsis. In the Mpacl5 mutants generated by genome editing, severe growth retardation was observed in the vegetative organ, thallus, and the sexual reproductive organ, gametangiophore. The mutant gametangiophores exhibited remarkable morphological defects such as short stalks, fasciation and indeterminate growth. Two gametangiophores fused together, and new gametangiophores were often initiated from the old ones. Furthermore, Mpacl5 showed altered responses to heat and salt stresses. Given the absence of spermine in bryophytes, these results suggest that thermospermine has a dual primordial function in organ development and stress responses in M. polymorpha. The stress response function may have eventually been assigned to spermine during land plant evolution.

Three-Dimensional Morphological Analysis Revealed the Cell Patterning Bases for the Sexual Dimorphism Development in the Liverwort Marchantia polymorpha.

In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana, those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator MpFGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha.

A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort.

Plant life cycles alternate between haploid gametophytes and diploid sporophytes. While regulatory factors determining male and female sexual morphologies have been identified for sporophytic reproductive organs, such as stamens and pistils of angiosperms, those regulating sex-specific traits in the haploid gametophytes that produce male and female gametes and hence are central to plant sexual reproduction are poorly understood. Here, we identified a MYB-type transcription factor, MpFGMYB, as a key regulator of female sexual differentiation in the haploid-dominant dioicous liverwort, Marchantia polymorpha MpFGMYB is specifically expressed in females and its loss resulted in female-to-male sex conversion. Strikingly, MpFGMYB expression is suppressed in males by a cis-acting antisense gene SUF at the same locus, and loss-of-function suf mutations resulted in male-to-female sex conversion. Thus, the bidirectional transcription module at the MpFGMYB/SUF locus acts as a toggle between female and male sexual differentiation in M. polymorpha gametophytes. Arabidopsis thaliana MpFGMYB orthologs are known to be expressed in embryo sacs and promote their development. Thus, phylogenetically related MYB transcription factors regulate female gametophyte development across land plants.

The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum).

KEY MESSAGE: The seedless mutant tn-1 in chili pepper is caused by a mutation in CaCKI1 (CA12g21620), which encodes histidine kinase involving female gametophyte development. An amino acid insertion in the receiver domain of CaCKI1 may be the mutation responsible for tn-1. Seedlessness is a desirable trait in fruit crops because the removal of seeds is a troublesome step for consumers and processing industries. However, little knowledge is available to develop seedless chili peppers. In a previous study, a chili pepper mutant tn-1, which stably produces seedless fruits, was isolated. In this study, we report characterization of tn-1 and identification of the causative gene. Although pollen germination was normal, confocal laser microscopy observations revealed deficiency in embryo sac development in tn-1. By marker analysis, the tn-1 locus was narrowed down to a 313 kb region on chromosome 12. Further analysis combined with mapping-by-sequencing identified CA12g21620, which encodes histidine kinase as a candidate gene. Phylogenetic analysis revealed CA12g21620 was the homolog of Arabidopsis CKI1 (Cytokinin Independent 1), which plays an important role in female gametophyte development, and CA12g21620 was designated as CaCKI1. Sequence analysis revealed that tn-1 has a 3-bp insertion in the 6th exon resulting in one lysine (K) residue insertion in receiver domain of CaCKI1, and the sequence nearby the insertion is widely conserved among CKI1 orthologs in various plants. This suggested that one K residue insertion may reduce the phosphorylation relay downstream of CaCKI1 and impair normal development of female gametophyte, resulting in seedless fruits production in tn-1. Furthermore, we demonstrated that virus-induced gene silencing of CaCKI1 reduced normally developed female gametophyte in chili pepper. This study describes the significant role of CaCKI1 in seed development in chili pepper and the possibility of developing seedless cultivars using its mutation.

Diminished Auxin Signaling Triggers Cellular Reprogramming by Inducing a Regeneration Factor in the Liverwort Marchantia polymorpha.

Regeneration in land plants is accompanied by the establishment of new stem cells, which often involves reactivation of the cell division potential in differentiated cells. The phytohormone auxin plays pivotal roles in this process. In bryophytes, regeneration is enhanced by the removal of the apex and repressed by exogenously applied auxin, which has long been proposed as a form of apical dominance. However, the molecular basis behind these observations remains unexplored. Here, we demonstrate that in the liverwort Marchantia polymorpha, the level of endogenous auxin is transiently decreased in the cut surface of decapitated explants, and identify by transcriptome analysis a key transcription factor gene, LOW-AUXIN RESPONSIVE (MpLAXR), which is induced upon auxin reduction. Loss of MpLAXR function resulted in delayed cell cycle reactivation, and transient expression of MpLAXR was sufficient to overcome the inhibition of regeneration by exogenously applied auxin. Furthermore, ectopic expression of MpLAXR caused cell proliferation in normally quiescent tissues. Together, these data indicate that decapitation causes a reduction of auxin level at the cut surface, where, in response, MpLAXR is up-regulated to trigger cellular reprogramming. MpLAXR is an ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION 1/DORNRÖSCHEN, which has dual functions as a shoot regeneration factor and a regulator of axillary meristem initiation, the latter of which requires a low auxin level. Thus, our findings provide insights into stem cell regulation as well as apical dominance establishment in land plants.

MarpolBase Expression: A Web-Based, Comprehensive Platform for Visualization and Analysis of Transcriptomes in the Liverwort Marchantia polymorpha.

The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha and promote our understanding of the evolution of land plants.

Regulation of the Poly(A) Status of Mitochondrial mRNA by Poly(A)-Specific Ribonuclease Is Conserved among Land Plants.

Regulation of the stability and the quality of mitochondrial RNA is essential for the maintenance of mitochondrial and cellular functions in eukaryotes. We have previously reported that the eukaryotic poly(A)-specific ribonuclease (PARN) and the prokaryotic poly(A) polymerase encoded by AHG2 and AGS1, respectively, coordinately regulate the poly(A) status and the stability of mitochondrial mRNA in Arabidopsis. Mitochondrial function of PARN has not been reported in any other eukaryotes. To know how much this PARN-based mitochondrial mRNA regulation is conserved among plants, we studied the AHG2 and AGS1 counterparts of the liverwort, Marchantia polymorpha, a member of basal land plant lineage. We found that M. polymorpha has one ortholog each for AHG2 and AGS1, named MpAHG2 and MpAGS1, respectively. Their Citrine-fused proteins were detected in mitochondria of the liverwort. Molecular genetic analysis showed that MpAHG2 is essential and functionally interacts with MpAGS1 as observed in Arabidopsis. A recombinant MpAHG2 protein had a deadenylase activity in vitro. Overexpression of MpAGS1 and the reduced expression of MpAHG2 caused an accumulation of polyadenylated Mpcox1 mRNA. Furthermore, MpAHG2 suppressed Arabidopsis ahg2-1 mutant phenotype. These results suggest that the PARN-based mitochondrial mRNA regulatory system is conserved in land plants.

Loss of CG Methylation in Marchantia polymorpha Causes Disorganization of Cell Division and Reveals Unique DNA Methylation Regulatory Mechanisms of Non-CG Methylation.

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha ortholog of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9) system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Even though numerous TEs were up-regulated, non-CG methylation was generally highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.

The µ Subunit of Arabidopsis Adaptor Protein-2 Is Involved in Effector-Triggered Immunity Mediated by Membrane-Localized Resistance Proteins.

Endocytosis has been suggested to be important in the cellular processes of plant immune responses. However, our understanding of its role during effector-triggered immunity (ETI) is still limited. We have previously shown that plant endocytosis, especially clathrin-coated vesicle formation at the plasma membrane, is mediated by the adaptor protein-2 (AP-2) complex and that loss of the µ subunit of AP-2 (AP2M) affects plant growth and floral organ development. Here, we report that AP2M is required for full-strength ETI mediated by the disease resistance (R) genes RPM1 and RPS2 in Arabidopsis. Reduced ETI was observed in an ap2m mutant plant, measured by growth of Pseudomonas syringae pv. tomato DC3000 strains carrying the corresponding effector genes avrRpm1 or avrRpt2 and by hypersensitive cell death response and defense gene expression triggered by these strains. In contrast, RPS4-mediated ETI and its associated immune responses were not affected by the ap2m mutation. While RPM1 and RPS2 are localized to the plasma membrane, RPS4 is localized to the cytoplasm and nucleus. Our results suggest that AP2M is involved in ETI mediated by plasma membrane-localized R proteins, possibly by mediating endocytosis of the immune receptor complex components from the plasma membrane.

Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development.

The adaptor protein-2 (AP-2) complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. The homologous genes of AP-2 subunits are present in the genomes of plants; however, their identities and roles in endocytic pathways are not clearly defined in plants. Here, we reveal the molecular composition of the AP-2 complex of Arabidopsis thaliana and its dynamics on the plasma membrane. We identified all of the α-, ß-, σ-, and µ-subunits of the AP-2 complex and detected a weak interaction of the AP-2 complex with clathrin heavy chain. The µ-subunit protein fused to green fluorescent protein (AP2M-GFP) was localized to the plasma membrane and to the cytoplasm. Live-cell imaging using a variable-angle epifluorescence microscope revealed that AP2M-GFP transiently forms punctate structures on the plasma membrane. Homozygous ap2m mutant plants exhibited abnormal floral structures, including reduced stamen elongation and delayed anther dehiscence, which led to a failure of pollination and a subsequent reduction of fertility. Our study provides a molecular basis for understanding AP-2-dependent endocytic pathways in plants and their roles in floral organ development and plant reproduction.

Conserved CKI1-mediated signaling is required for female germline specification in Marchantia polymorpha.

In land plants, gametes derive from a small number of dedicated haploid cells.1 In angiosperms, one central cell and one egg cell are differentiated in the embryo sac as female gametes for double fertilization, while in non-flowering plants, only one egg cell is generated in the female sexual organ, called the archegonium.2,3 The central cell specification of Arabidopsis thaliana is controlled by the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1), which is a two-component signaling (TCS) activator sharing downstream regulatory components with the cytokinin signaling pathway.4,5,6,7 Our phylogenetic analysis suggested that CKI1 orthologs broadly exist in land plants. However, the role of CKI1 in non-flowering plants remains unclear. Here, we found that the sole CKI1 ortholog in the liverwort Marchantia polymorpha, MpCKI1, which functions through conserved downstream TCS components, regulates the female germline specification for egg cell development in the archegonium. In M. polymorpha, the archegonium develops three-dimensionally from a single cell accumulating MpBONOBO (MpBNB), a master regulator for germline initiation and differentiation.8 We visualized female germline specification by capturing the distribution pattern of MpBNB in discrete stages of early archegonium development, and found that MpBNB accumulation is restricted to female germline cells. MpCKI1 is required for the proper MpBNB accumulation in the female germline, and is critical for the asymmetric cell divisions that specify the female germline cells. These results suggest that CKI1-mediated TCS originated during early land plant evolution and participates in female germ cell specification in deeply diverged plant lineages.

A non-canonical BZR/BES transcription factor regulates the development of haploid reproductive organs in Marchantia polymorpha.

Gametogenesis, which is essential to the sexual reproductive system, has drastically changed during plant evolution. Bryophytes, lycophytes and ferns develop reproductive organs called gametangia-antheridia and archegonia for sperm and egg production, respectively. However, the molecular mechanism of early gametangium development remains unclear. Here we identified a 'non-canonical' type of BZR/BES transcription factor, MpBZR3, as a regulator of gametangium development in a model bryophyte, Marchantia polymorpha. Interestingly, overexpression of MpBZR3 induced ectopic gametangia. Genetic analysis revealed that MpBZR3 promotes the early phase of antheridium development in male plants. By contrast, MpBZR3 is required for the late phase of archegonium development in female plants. We demonstrate that MpBZR3 is necessary for the successful development of both antheridia and archegonia but functions in a different manner between the two sexes. Together, the functional specialization of this 'non-canonical' type of BZR/BES member may have contributed to the evolution of reproductive systems.

Circadian clock does not play an essential role in daylength measurement for growth-phase transition in Marchantia polymorpha.

Daylength is perceived as a seasonal cue to induce growth-phase transition at a proper time of a year. The core of the mechanism of daylength measurement in angiosperms lies in the circadian clock-controlled expression of regulators of growth-phase transition. However, the roles of the circadian clock in daylength measurement in basal land plants remain largely unknown. In this study, we investigated the contribution of circadian clock to daylength measurement in a basal land plant, the liverwort Marchantia polymorpha. In M. polymorpha, transition from vegetative to reproductive phase under long-day conditions results in differentiation of sexual branches called gametangiophores which harbor gametangia. First, we showed that a widely used wild-type accession Takaragaike-1 is an obligate long-day plant with a critical daylength of about 10 hours and requires multiple long days. Then, we compared the timing of gametangiophore formation between wild type and circadian clock mutants in long-day and short-day conditions. Mutations in two clock genes, MpTIMING OF CAB EXPRESSION 1 and MpPSEUDO-RESPONSE REGULATOR, had no significant effects on the timing of gametangiophore formation. In addition, when M. polymorpha plants were treated with a chemical which lengthens circadian period, there was no significant effect on the timing of gametangiophore formation, either. We next observed the timing of gametangiophore formation under various non-24-h light/dark cycles to examine the effect of phase alteration in circadian rhythms. The results suggest that daylength measurement in M. polymorpha is based on the relative amount of light and darkness within a cycle rather than the intrinsic rhythms generated by circadian clock. Our findings suggest that M. polymorpha has a daylength measurement system which is different from that of angiosperms centered on the circadian clock function.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes.

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A. thaliana and the single LRL/DROP protein MpLRL in M. polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A. thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M. polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.

Peroxisomes are involved in biotin biosynthesis in Aspergillus and Arabidopsis.

Among the eukaryotes only plants and a number of fungi are able to synthesize biotin. Although initial events leading to the biosynthesis of biotin remain largely unknown, the final steps are known to occur in the mitochondria. Here we deleted the Aopex5 and Aopex7 genes encoding the receptors for peroxisomal targeting signals PTS1 and PTS2, respectively, in the filamentous fungus Aspergillus oryzae. In addition to exhibiting defects in the peroxisomal targeting of either PTS1 or PTS2 proteins, the deletion strains also displayed growth defects on minimal medium containing oleic acid as the sole carbon source. Unexpectedly, these peroxisomal transport-deficient strains also exhibited growth defects on minimal medium containing glucose as the sole carbon source that were remediated by the addition of biotin and its precursors, including 7-keto-8-aminopelargonic acid (KAPA). Genome database searches in fungi and plants revealed that BioF protein/KAPA synthase, one of the biotin biosynthetic enzymes, has a PTS1 sequence at the C terminus. Fungal ΔbioF strains expressing the fungal and plant BioF proteins lacking PTS1 still exhibited growth defects in the absence of biotin, indicating that peroxisomal targeting of KAPA synthase is crucial for the biotin biosynthesis. Furthermore, in the plant Arabidopsis thaliana, AtBioF localized to the peroxisomes through recognition of its PTS1 sequence, suggesting involvement of peroxisomes in biotin biosynthesis in plants. Taken together we demonstrate a novel role for peroxisomes in biotin biosynthesis and suggest the presence of as yet unidentified peroxisomal proteins that function in the earlier steps of biotin biosynthesis.

Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null mice.

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic ß-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic ß-cells.

Possible molecular mechanisms of persistent pollen tube growth without de novo transcription.

The vegetative cell nucleus proceeds ahead of a pair of sperm cells located beneath the pollen tube tip during germination. The tip-localized vegetative nucleus had been considered to play a pivotal role in the control of directional pollen tube growth and double fertilization. However, we recently reported the female-targeting behavior of pollen tubes from mutant plants, of which the vegetative nucleus and sperm nuclei were artificially immotile. We showed that the apical region of the mutant pollen tubes became physiologically enucleated after the first callose plug formation, indicating the autonomously growing nature of pollen tubes without the vegetative nucleus and sperm cells. Thus, in this study, we further analyzed another Arabidopsis thaliana mutant producing physiologically enucleated pollen tubes and discussed the mechanism by which a pollen tube can grow without de novo transcription from the vegetative nucleus. We propose several possible molecular mechanisms for persistent pollen tube growth, such as the contribution of transcripts before and immediately after germination and the use of persistent transcripts, which may be important for a competitive race among pollen tubes.