BackgroundMost published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) ‘Hongyang’ draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models.ResultsA second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within ‘Hongyang’ The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned ‘Hort16A’ cDNAs and comparing with the predicted protein models for Red5 and both the original ‘Hongyang’ assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised ‘Hongyang’ annotation, respectively, compared with 90.9% to the Red5 models.ConclusionsOur study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4656-3) contains supplementary material, which is available to authorized users.
BackgroundThere is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation.Results‘Scifresh’ (slow softening) and ‘Royal Gala’ (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. ‘Scifresh’ apples showed reduced loss of firmness and greater dry matter accumulation compared with ‘Royal Gala’ during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in ‘Scifresh’ were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of ‘Scifresh’ showed larger, more angular shaped cells than ‘Royal Gala’, with less airspaces and denser tissue. Stronger cell adhesion in ripe ‘Scifresh’ resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in ‘Royal Gala’. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in ‘Scifresh’.ConclusionsOur results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process.
Preeclampsia (PE) is a common, potentially life-threatening pregnancy syndrome triggered by placental factors released into the maternal circulation, resulting in maternal vascular dysfunction along with activated inflammation and coagulation. Currently there is no screening test for PE. We sought to identify differentially expressed plasma proteins in women who subsequently develop PE that may perform as predictive biomarkers. In seven DIGE experiments, we compared the plasma proteome at 20 wk gestation in women who later developed PE with an appropriate birth weight for gestational age baby (n=27) or a small for gestational age baby (n=12) to healthy controls with uncomplicated pregnancies (n=57). Of the 49 differentially expressed spots associated with PE-appropriate for gestational age, PE-small for gestational age or both (p<0.05, false discovery rate corrected), 39 were identified by LC-MS/MS. Two protein clusters that accurately (>90%) classified women at risk of developing PE were identified. Immunoblots confirmed the overexpression of fibrinogen gamma chain and alpha-1-antichymotrypsin in plasma prior to PE. The proteins identified are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity and acute-phase responses, indicating novel synergism between pathways involved in the pathogenesis of PE. Our findings are remarkably similar to recently identified proteins complexed to high-density lipoprotein and linked to cardiovascular disease.
We describe the design of biomimetic anthrax toxin inhibitors that incorporate lipid microdomains. Cellular membranes are believed to contain microdomains (lipid rafts) that influence processes ranging from signal transduction to microbial pathogenesis. [1][2][3][4] We demonstrate that formation of raftlike membrane microdomains significantly enhances the potency of liposome-based polyvalent anthrax toxin inhibitors; phase separation in the liposomal membrane was used to cluster inhibitory peptides to increase the potency of these inhibitors (Scheme 1). We also synthesized "smart" inhibitors in which phase separation and potency can be modulated actively in response to an external stimulus (Scheme 1).Liposomes represent simple models of cellular membranes and are also attractive scaffolds for the polyvalent display of ligands. [5][6][7][8] We showed previously that liposomes displaying multiple copies of an inhibitory peptide bound the heptameric cell-binding component of anthrax toxin, [PA 63 ] 7 , and prevented it from binding the toxic enzyme lethal factor (LF). Blocking the binding of LF to [PA 63 ] 7 prevented the cytosolic delivery of LF, thereby inhibiting cell death. The density of peptide ligands on the surface of the liposome was optimal past a threshold peptide density that corresponded to the average distance between peptide-binding sites on [PA 63 ] 7 . [8] We hypothesized, therefore, that peptides displayed at suboptimal density could be clustered into "raftlike" membrane microdomains to create regions of optimal ligand density. We demonstrate herein that the concentration of peptides into lipid microdomains facilitates toxin inhibition.Coexisting liquid-ordered and liquid-disordered phases can be formed in membranes containing ternary mixtures of unsaturated lipids, saturated lipids, and cholesterol by increasing the amount of cholesterol in homogeneous model membranes.[4, 9, 10] We made liposomes from a mixture of dioleoylphosphatidylcholine (DOPC), diarachidoylphosphatidylcholine (DAPC), a thiol-reactive lipid (PDP-DPPE), and cholesterol. As the use of giant unilamellar vesicles (GUVs) enables phase separation to be visualized, [4,9] we first made GUVs composed of DOPC, the fluorescent dye Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE, which partitions preferentially into less-ordered liquid domains), DAPC, PDP-DPPE, and cholesterol at molar ratios of 79:1:10:5:5 and 64:1:10:5:20. Although GUVs containing 5 % cholesterol showed a uniform distribution of fluorescence (Figure 1 a, i), those containing 20 % cholesterol showed the presence of dark phase-separated domains (Figure 1 a, ii-iv). This is consistent with the coexistence of liquid-ordered and liquiddisordered domains.Next, we tested the effect of domain formation on the potency of liposome-based anthrax toxin inhibitors. Liposomes composed of DOPC, DAPC, PDP-DPPE, and cholesterol (molar ratios of 80:10:5:5 and 65:10:5:20) were allowed to react with the peptide HTSTYWWLDGAPC, which binds to [PA 63 ] 7 and prevent...
Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.
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