Deciphering the Molecular Functions of Sterols in Cellulose Biosynthesis

Sterols play vital roles in plant growth and development, as components of membranes and as precursors to steroid hormones. Analysis of Arabidopsis mutants indicates that sterol composition is crucial for cellulose biosynthesis. Sterols are widespread in the plasma membrane (PM), suggesting a possible link between sterols and the multimeric cellulose synthase complex. In one possible scenario, molecular interactions in sterol-rich PM microdomains or another form of sterol-dependent membrane scaffolding may be critical for maintaining the correct subcellular localization, structural integrity and/or activity of the cellulose synthase machinery. Another possible link may be through steryl glucosides, which could act as primers for the attachment of glucose monomers during the synthesis of β−(1 → 4) glucan chains that form the cellulose microfibrils. This mini-review examines genetic and biochemical data supporting the link between sterols and cellulose biosynthesis in cell wall formation and explores potential approaches to elucidate the mechanism of this association

Bioorthogonal click chemistry for fluorescence imaging of choline phospholipids in plants

Citation: Paper, J. M., Mukherjee, T., & Schrick, K. (2018). Bioorthogonal click chemistry for fluorescence imaging of choline phospholipids in plants. Plant Methods, 14(1). https://doi.org/10.1186/s13007-018-0299-2Background: Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants. We established that propargylcholine is readily taken up by roots, and that its incorporation is not detrimental to plant growth. After plant tissue is harvested and fixed, a click-chemistry reaction covalently links the alkyne group of propargylcholine to a fluorescently-tagged azide, resulting in specific labeling of choline phospholipids. Results:  Uptake of propargylcholine, followed by click chemistry with fluorescein or Alexa Fluor 594 azide was used to visualize choline phospholipids in cells of root, leaf, stem, silique and seed tissues from Arabidopsis thaliana. Colocalization with various subcellular markers indicated coinciding fluorescent signals in cell membranes, such as the tonoplast and the ER. Among different cell types in the leaf epidermis, guard cells displayed strong labeling. Mass spectrometry-based lipidomic analysis of the various plant tissues revealed that incorporation of propargylcholine was strongest in roots with approximately 50% of total choline phospholipids being labeled, but it was also incorporated in the other tissues including seeds. Phospholipid profiling confirmed that, in each tissue analyzed, incorporation of the bioortholog had little impact on the pool of choline plus choline-like phospholipids or other lipid species. Conclusion:  We developed and validated a click-chemistry based method for fluorescence imaging of choline phospholipids using a bioortholog of choline, propargylcholine, in various cell-types and tissues from Arabidopsis. This click-chemistry method provides a direct way to metabolically tag and visualize specific lipid molecules in plant cells. This work paves the way for future studies addressing in situ localization of specific lipids in plants

An annotated bibliography of available materials for teaching the new grammar in secondary schools

Call number: LD2668 .R4 1967 S

Structural and physical investigations of novel germanium compounds: Aryloxides, nanomaterials, and photolysis of oligogermanes

The work described in this dissertation will explore the synthesis and characterization of novel germanium containing compounds in order to gain a better understanding of the organometallic chemistry of germanium. These compounds include germanium bisamides, aryloxogermylenes, polyfunctional aryloxides such as calix[n]arenes and binaphthoxogermanium compounds, and oligogermanes containing up to four germanium atoms.We have found that the germanium bisamides can be trapped using the germylene trapping agent benzil and we have fully characterized those products. The germanium bisamides can also be used as starting materials to synthesize germanium aryloxides via protonolysis of a phenol that contains one or more phenolic groups. Using this method we have prepared the germanium(IV) aryloxides [Ge(OC6H3Ph2-2,6)2(R)(I)] (R = But or Me) where the R = Me derivative was then converted to the triaryloxo species [Ge(OC6H3Ph2-2,6)3(Me)] upon reaction of the iodine containing compound with an extra equivalent of 2,6-diphenylphenol. Using polyfunctional phenols, we prepared and characterized the germanium(II) calix[5]arene complex {calix[5]arene}2Ge2(OSiMe3)4(OH)2, the calix[6]arene complex [(C6H3)6(CH2)6(OSiMe2Ph)6], and the binaphthoxogermanium(II) complex (S,S)-[Ge{OC20H10(OSiMe2Ph)-2'-(SiMe3)2-3,3'}2].Chapter five describes the synthesis of a series of three oligogermanes including a digermane, a trigermane, and a branched neopentyl germane. We have found that these oligogermanes can be used as precursors for the preparation of germanium(0) nanomaterials, and that the size of the resulting nanoparticles correlates with the number of catenated germanium atoms in the precursor compounds. These nanoparticles are fluorescent and the position of the emission maximum is red shifted as the size of the particles increases.Lastly, the sixth chapter will discuss the synthesis, characterization, and photochemistry of a series of six linear oligogermanes. The optical and electronic properties of these compounds were probed using UV/visible spectroscopy and differential pulse voltammetry. The photochemistry of these compounds will be analyzed by photolyzing each compound using UV-C light (280-100 nm) in the presence of acetic acid as a germylene trapping agent. If germylenes :GeR2 are formed, they should be trapped to yield R2Ge(H)OAc. The photolysis products will be characterized by NMR (1H and 13C) spectroscopy, infrared spectroscopy (FTIR), and gas-chromatography mass spectroscopy (GC/MS)

START lipid/sterol-binding domains are amplified in plants and are predominantly associated with homeodomain transcription factors

BACKGROUND: In animals, steroid hormones regulate gene expression by binding to nuclear receptors. Plants lack genes for nuclear receptors, yet genetic evidence from Arabidopsis suggests developmental roles for lipids/sterols analogous to those in animals. In contrast to nuclear receptors, the lipid/sterol-binding StAR-related lipid transfer (START) protein domains are conserved, making them candidates for involvement in both animal and plant lipid/sterol signal transduction. RESULTS: We surveyed putative START domains from the genomes of Arabidopsis, rice, animals, protists and bacteria. START domains are more common in plants than in animals and in plants are primarily found within homeodomain (HD) transcription factors. The largest subfamily of HD-START proteins is characterized by an HD amino-terminal to a plant-specific leucine zipper with an internal loop, whereas in a smaller subfamily the HD precedes a classic leucine zipper. The START domains in plant HD-START proteins are not closely related to those of animals, implying collateral evolution to accommodate organism-specific lipids/sterols. Using crystal structures of mammalian START proteins, we show structural conservation of the mammalian phosphatidylcholine transfer protein (PCTP) START domain in plants, consistent with a common role in lipid transport and metabolism. We also describe putative START-domain proteins from bacteria and unicellular protists. CONCLUSIONS: The majority of START domains in plants belong to a novel class of putative lipid/sterol-binding transcription factors, the HD-START family, which is conserved across the plant kingdom. HD-START proteins are confined to plants, suggesting a mechanism by which lipid/sterol ligands can directly modulate transcription in plants

Lateral flow–based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.Peer Reviewe

Mutations in UDP-Glucose:Sterol Glucosyltransferase in Arabidopsis Cause Transparent Testa Phenotype and Suberization Defect in Seeds

In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis

Replication of cowpox virus in macrophages is dependent on the host range factor p28/N1R

Zoonotic orthopoxvirus infections continue to represent a threat to human health. The disease caused by distinct orthopoxviruses differs in terms of symptoms and severity, which may be explained by the unique repertoire of virus factors that modulate the host’s immune response and cellular machinery. We report here on the construction of recombinant cowpox viruses (CPXV) which either lack the host range factor p28 completely or express truncated variants of p28. We show that p28 is essential for CPXV replication in macrophages of human or mouse origin and that the C-terminal RING finger domain of p28 is necessary to allow CPXV replication in macrophages.Peer Reviewe