Maternal Control of Male-Gamete Delivery in Arabidopsis Involves a Putative GPI-Anchored Protein Encoded by the LORELEI Gene

In Angiosperms, the male gametes are delivered to the female gametes through the maternal reproductive tissue by the pollen tube. Upon arrival, the pollen tube releases the two sperm cells, permitting double fertilization to take place. Although the critical role of the female gametophyte in pollen tube reception has been demonstrated, the underlying mechanisms remain poorly understood. Here, we describe lorelei, an Arabidopsis thaliana mutant impaired in sperm cell release, reminiscent of the feronia/sirène mutant. Pollen tubes reaching lorelei embryo sacs frequently do not rupture but continue to grow in the embryo sac. Furthermore, lorelei embryo sacs continue to attract additional pollen tubes after arrival of the initial pollen tube. The LORELEI gene is expressed in the synergid cells prior to fertilization and encodes a small plant-specific putative glucosylphosphatidylinositol-anchored protein (GAP). These results provide support for the concept of signaling mechanisms at the synergid cell membrane by which the female gametophyte recognizes the arrival of a compatible pollen tube and promotes sperm release. Although GAPs have previously been shown to play critical roles in initiation of fertilization in mammals, flowering plants appear to have independently evolved reproductive mechanisms that use the unique features of these proteins within a similar biological context.     

Identification of new members of Fertilisation Independent Seed Polycomb Group pathway involved in the control of seed development in Arabidopsis thaliana

In higher plants, double fertilisation initiates seed development. One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm. The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole. We report that ontogenesis of the posterior pole in Arabidopsis thaliana involves oriented migration of nuclei in the syncytium. We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED (FIS) class, MEDEA (MEA), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM (FIE). A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia. We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1). The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker.     

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.     

Specificity of L,D-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,D-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldt(fm) from Enterococcus faecium, was previously shown to bypass the D,D-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and beta-lactam antibiotics. Ldt(fm) homologues from Bacillus subtilis (Ldt(Bs)) and E. faecalis (Ldt(fs)) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP(3)) and L-Lys(3)-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys(3)-d-iAsn in E. faecium. Ldt(fs) differed from Ldt(fm) and Ldt(Bs) by its capacity to hydrolyze the L-Lys(3)-D-Ala(4) bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldt(fs), which was also specific for its cognate acyl donor, Ldt(fm) tolerated substitution of L-Lys(3)-D-iAsn by L-Lys(3)-L-Ala-L-Ala. Likewise, Ldt(Bs) tolerated substitution of mesoDAP(3) by L-Lys(3)-D-iAsn and L-Lys(3)-L-Ala-L-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the L,D-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases.     

Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.     

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34⁺ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.Subject terms: Cell biology, Cancer     

Acyl acceptor recognition by Enterococcus faecium l,d‐transpeptidase Ldtfm

In Mycobacterium tuberculosis and ampicillin‐resistant mutants of Enterococcus faecium, the classical target of β‐lactam antibiotics is bypassed by l ,d ‐transpeptidases that form unusual 3 → 3 peptidoglycan cross‐links. β‐lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l ,d ‐transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium l ,d ‐transpeptidase Ldt_fm by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)‐driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldt_fm that were evaluated by site‐directed mutagenesis and development of a cross‐linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldt_fm active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β‐lactams.     

Combined Mass Mapping and Biochemical Characterization of Grape β-Glycosidase-enriched Extract

A beta-glucosidase enzyme activity was enriched from skins of ripe grape berry by cell wall fractionation, hydrophobic interaction and cation-exchange chromatographies. This enriched enzyme extract contained several beta-glycosidase activities hydrolyzing a wide range of synthetic and natural monoglycosides and diglycosides, as well as a beta-fructosidase activity. The enzyme extract was further characterized by two-dimensional gel electrophoresis coupled to peptide mass fingerprinting of eight spots using MALDI-TOF mass spectrometry. No beta-glucosidase but a beta-fructosidase associated to the relevant spot at 66 kDa/pI 5.1 was identified. Taken together all results issued from the biochemical characterization, the substrate specificity and the mass spectrometry-based identification of this enriched enzyme extract, we propose that this protein could be a specific beta-fructosidase isoform associated with a broad spectrum of beta-glycosidase activities in grape berry skin and involved in cell wall modifications which occur during the ripening-induced thickness of the grape.     

A novel class of MYB factors controls sperm-cell formation in plants

In contrast to animals, the plant male germline is established after meiosis in distinctive haploid structures, termed pollen grains. The germline arises by a distinct asymmetric division of the meiotic products . The fates of the resulting vegetative and generative cells are distinct. In contrast to the larger vegetative cell, arrested in the G1 phase of the cell cycle, the smaller generative cell divides once to produce the two male gametes or sperm cells. Sperm cells are delivered to the female gametes by the pollen tube, which develops from the vegetative cell. In spite of recent efforts to understand pollen development , the molecular pathway controlling sperm-cell ontogenesis is unknown. Here, we present the isolation of DUO1, a novel R2R3 MYB gene of Arabidopsis, as the first gene shown to control male gamete formation in plants. DUO1 is specifically expressed in the male germline, and DUO1 protein accumulates in sperm-cell nuclei. Mutations in DUO1 produce a single larger diploid sperm cell unable to perform fertilization. DUO1 appears to be evolutionarily conserved in several plant species and defines a new subfamily of pollen-specific MYB genes.