Leagues of their own: sexually dimorphic features of meiotic prophase I

Meiosis is a conserved cell division process that is used by sexually reproducing organisms to generate haploid gametes. Males and females produce different end products of meiosis: eggs (females) and sperm (males). In addition, these unique end products demonstrate sex-specific differences that occur throughout meiosis to produce the final genetic material that is packaged into distinct gametes with unique extracellular morphologies and nuclear sizes. These sexually dimorphic features of meiosis include the meiotic chromosome architecture, in which both the lengths of the chromosomes and the requirement for specific meiotic axis proteins being different between the sexes. Moreover, these changes likely cause sex-specific changes in the recombination landscape with the sex that has the longer chromosomes usually obtaining more crossovers. Additionally, epigenetic regulation of meiosis may contribute to sexually dimorphic recombination landscapes. Here we explore the sexually dimorphic features of both the chromosome axis and crossing over for each stage of meiotic prophase I in Mus musculus, Caenorhabditis elegans, and Arabidopsis thaliana. Furthermore, we consider how sex-specific changes in the meiotic chromosome axes and the epigenetic landscape may function together to regulate crossing over in each sex, indicating that the mechanisms controlling crossing over may be different in oogenesis and spermatogenesis.

     

Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans

The development of nematicides targeting parasitic nematodes of animals and plants requires the identification of biochemical targets not found in host organisms. Recent studies suggest that Caenorhabditis elegans synthesizes phosphocholine through the action of PEAMT (S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferases) that convert phosphoethanolamine into phosphocholine. Here, we examine the function of a PEAMT from C. elegans (gene: pmt-1; protein: PMT-1). Our analysis shows that PMT-1 only catalyses the conversion of phosphoethanolamine into phospho-monomethylethanolamine, which is the first step in the PEAMT pathway. This is in contrast with the multifunctional PEAMT from plants and Plasmodium that perform multiple methylations in the pathway using a single enzyme. Initial velocity and product inhibition studies indicate that PMT-1 uses a random sequential kinetic mechanism and is feedback inhibited by phosphocholine. To examine the effect of abrogating PMT-1 activity in C. elegans, RNAi (RNA interference) experiments demonstrate that pmt-1 is required for worm growth and development and validate PMT-1 as a potential target for inhibition. Moreover, providing pathway metabolites downstream of PMT-1 reverses the RNAi phenotype of pmt-1. Because PMT-1 is not found in mammals, is only distantly related to the plant PEAMT and is conserved in multiple parasitic nematodes of humans, animals and crop plants, inhibitors targeting it may prove valuable in human and veterinary medicine and agriculture.     

Glucosylceramide synthase is essential for alfalfa defensin-mediated growth inhibition but not for pathogenicity of Fusarium graminearum

Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The DeltaFggcs1-null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The DeltaFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild-type PH-1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1-mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host-dependent.     

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.     

Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase

Phytochelatin (PC) synthases are gamma-glutamylcysteine (gamma-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (gamma-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes gamma-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes gamma-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent gamma-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary gamma-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary gamma-Glu-Cys acylation.     

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.     

Apparent Role of Phosphatidylcholine in the Metabolism of Petroselinic Acid in Developing Umbelliferae Endosperm

Studies were conducted to characterize the metabolism of the unusual fatty acid petroselinic acid (18:1cis[delta]6) in developing endosperm of the Umbelliferae species coriander (Coriandrum sativum L.) and carrot (Daucus carota L.). Analyses of fatty acid compositions of glycerolipids of these tissues revealed a dissimilar distribution of petroselinic acid in triacylglycerols (TAG) and the major polar lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Petroselinic acid comprised 70 to 75 mol% of the fatty acids of TAG but only 9 to 20 mol% of the fatty acids of PC and PE. Although such data appeared to suggest that petroselinic acid is at least partially excluded from polar lipids, results of [1-14C]acetate radiolabeling experiments gave a much different picture of the metabolism of this fatty acid. In time-course labeling of carrot endosperm, [1-14C]acetate was rapidly incorporated into PC in high levels. Through 30 min, radiolabel was most concentrated in PC, and of this, 80 to 85% was in the form of petroselinic acid. One explanation for the large disparity in amounts of petroselinic acid in PC as determined by fatty acid mass analyses and 14C radiolabeling is that turnover of these lipids or the fatty acids of these lipids results in relatively low accumulation of petroselinic acid mass. Consistent with this, the kinetics of [1-14C]acetate time-course labeling of carrot endosperm and "pulse-chase" labeling of coriander endosperm suggested a possible flux of fatty acids from PC into TAG. In time-course experiments, radiolabel initially entered PC at the highest rates but accumulated in TAG at later time points. Similarly, in pulse-chase studies, losses in absolute amounts of radioactivity from PC were accompanied by significant increases of radiolabel in TAG. In addition, stereospecific analyses of unlabeled and [1-14C]acetate-labeled PC of coriander endosperm indicated that petroselinic acid can be readily incorporated into both the sn-1 and sn-2 positions of this lipid. Because petroselinic acid is neither synthesized nor further modified on polar lipids, the apparent metabolism of this fatty acid through PC (and possibly through other polar lipids) may define a function of PC in TAG assembly apart from its involvement in fatty acid modification reactions.     

Separation and identification of major plant sphingolipid classes from leaves

Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for approximately 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.