Sterol Glucosyltransferases Tailor Polysaccharide Accumulation in Arabidopsis Seed Coat Epidermal Cells

The conjugation of sterols with a Glc moiety is catalyzed by sterol glucosyltransferases (SGTs). A portion of the resulting steryl glucosides (SG) are then esterified with a long-chain fatty acid to form acyl-SG (ASG). SG and ASG are prevalent components of plant cellular membranes and influence their organization and functional properties. Mutant analysis had previously inferred that two Arabidopsis SGTs, UGT80A2 and UGT80B1/TT15, could have specialized roles in the production of SG in seeds, despite an overlap in their enzymatic activity. Here, we establish new roles for both enzymes in the accumulation of polysaccharides in seed coat epidermal cells (SCEs). The rhamnogalacturonan-I (RG-I) content of the inner layer of seed mucilage was higher in ugt80A2, whereas RG-I accumulation was lower in mutants of UGT80B1, with double mutant phenotypes indicating that UGT80A2 acts independently from UGT80B1. In contrast, an additive phenotype was observed in double mutants for increased galactoglucomannan (GGM) content. Double mutants also exhibited increased polymer density within the inner mucilage layer. In contrast, cell wall defects were only observed in mutants defective for UGT80B1, while more mucilage cellulose was only observed when UGT80A2 was mutated. The generation of a range of phenotypic effects, simultaneously within a single cell type, demonstrates that the adjustment of the SG and ASG composition of cellular membranes by UGT80A2 and UGT80B1 tailors polysaccharide accumulation in Arabidopsis seeds.

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Pectin Modification in Seed Coat Mucilage by In Vivo Expression of Rhamnogalacturonan-I- and Homogalacturonan-Degrading Enzymes

Plant and Cell Physiology ◽

10.1093/pcp/pcab077 ◽

2021 ◽

Author(s):

Robert McGee ◽

Gillian H Dean ◽

Di Wu ◽

Yuelin Zhang ◽

Shawn D Mansfield ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Wall ◽

Seed Coat ◽

Epidermal Cells ◽

Wall Structure ◽

Rhamnogalacturonan I ◽

Degrading Enzymes ◽

Developing Seed ◽

Extracellular Matrix Components

Abstract The cell wall is essential for plant survival. Determining the relationship between cell wall structure and function using mutant analysis or overexpressing cell wall-modifying enzymes has been challenging due to the complexity of the cell wall and the appearance of secondary, compensatory effects when individual polymers are modified. In addition, viability of the plants can be severely impacted by wall modification. A useful model system for studying structure-function relationships among extracellular matrix components are the seed coat epidermal cells of Arabidopsis thaliana. These cells synthesize relatively simple, easily-accessible, pectin-rich mucilage that is not essential for plant viability. In this study, we expressed enzymes predicted to modify polysaccharide components of mucilage in the apoplast of seed coat epidermal cells and explored their impacts on mucilage. The seed coat epidermal-specific promoter TESTA ABUNDANT2 (TBA2) was used to drive expression of these enzymes to avoid adverse effects in other parts of the plant. Mature transgenic seeds expressing Rhamnogalacturonate lyase A (RglA) or Rhamnogalacturonate lyase B (RglB) that degrade the pectin rhamnogalacturonan-I (RG-I), a major component of mucilage, had greatly reduced mucilage capsules surrounding the seeds and concomitant decreases in the monosaccharides that comprise the RG-I backbone. Degradation of the minor mucilage component homogalacturonan (HG) using the HG-degrading enzymes Pectin Lyase A (PLA) or ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2) resulted in developing seed coat epidermal cells with disrupted cell-cell adhesion and signs of early cell death. These results demonstrate the feasibility of manipulating the seed coat epidermal cell extracellular matrix using a targeted genetic engineering approach.

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Cell-type-specific profiling of loaded miRNAs from Caenorhabditis elegans reveals spatial and temporal flexibility in Argonaute loading

Nature Communications ◽

10.1038/s41467-021-22503-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher A. Brosnan ◽

Alexander J. Palmer ◽

Steven Zuryn

Keyword(s):

Caenorhabditis Elegans ◽

Regulatory Networks ◽

Regulatory Mechanisms ◽

Cell Type ◽

A Genome ◽

Temporal Flexibility ◽

Small Regulatory Rnas ◽

Cell Type Specific ◽

Single Cell Type ◽

Regulated Gene Expression

AbstractMulticellularity has coincided with the evolution of microRNAs (miRNAs), small regulatory RNAs that are integrated into cellular differentiation and homeostatic gene-regulatory networks. However, the regulatory mechanisms underpinning miRNA activity have remained largely obscured because of the precise, and thus difficult to access, cellular contexts under which they operate. To resolve these, we have generated a genome-wide map of active miRNAs in Caenorhabditis elegans by revealing cell-type-specific patterns of miRNAs loaded into Argonaute (AGO) silencing complexes. Epitope-labelled AGO proteins were selectively expressed and immunoprecipitated from three distinct tissue types and associated miRNAs sequenced. In addition to providing information on biological function, we define adaptable miRNA:AGO interactions with single-cell-type and AGO-specific resolution. We demonstrate spatial and temporal dynamicism, flexibility of miRNA loading, and suggest miRNA regulatory mechanisms via AGO selectivity in different tissues and during ageing. Additionally, we resolve widespread changes in AGO-regulated gene expression by analysing translatomes specifically in neurons.

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Author Correction: iLoF: An intelligent Lab on Fiber Approach for Human Cancer Single-Cell Type Identification

Scientific Reports ◽

10.1038/s41598-020-67916-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Joana S. Paiva ◽

Pedro A. S. Jorge ◽

Rita S. R. Ribeiro ◽

Meritxell Balmaña ◽

Diana Campos ◽

...

Keyword(s):

Single Cell ◽

Human Cancer ◽

Cell Type ◽

Single Cell Type

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Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type

Naunyn-Schmiedeberg s Archives of Pharmacology ◽

10.1007/bf00169379 ◽

1995 ◽

Vol 352 (5) ◽

pp. 469-476 ◽

Cited By ~ 22

Author(s):

Martina Schmidt ◽

Christine Bienek ◽

Chris J. van Koppen ◽

Martin C. Michel ◽

Karl H. Jakobs

Keyword(s):

Single Cell ◽

Acetylcholine Receptors ◽

Calcium Signalling ◽

Muscarinic Acetylcholine Receptors ◽

Cell Type ◽

Muscarinic Acetylcholine ◽

Single Cell Type

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The Immunohistochemical Characterisation of an SV40-immortalised Human Corneal Epithelial Cell Line

Alternatives to Laboratory Animals ◽

10.1177/026119290303100407 ◽

2003 ◽

Vol 31 (4) ◽

pp. 409-417 ◽

Cited By ~ 7

Author(s):

Anne Huhtala ◽

Sami K. Nurmi ◽

Hanna Tähti ◽

Lotta Salminen ◽

Päivi Alajuuma ◽

...

Keyword(s):

Epithelial Cells ◽

Single Cell ◽

Cell Line ◽

Cell Cultures ◽

Corneal Epithelium ◽

Culture Medium ◽

Corneal Epithelial Cell ◽

Cell Type ◽

Corneal Epithelial ◽

Single Cell Type

Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.

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A Workflow in Single Cell-Type Metabolomics: From Data Pre-Processing and Statistical Analysis to Biological Insights

OMICS-Based Approaches in Plant Biotechnology ◽

10.1002/9781119509967.ch6 ◽

2019 ◽

pp. 105-127

Author(s):

Biswapriya B. Misra

Keyword(s):

Statistical Analysis ◽

Single Cell ◽

Cell Type ◽

Single Cell Type

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Species Having C4 Single-Cell-Type Photosynthesis in the Chenopodiaceae Family Evolved a Photosynthetic Phosphoenolpyruvate Carboxylase Like That of Kranz-Type C4 Species

PLANT PHYSIOLOGY ◽

10.1104/pp.106.085829 ◽

2006 ◽

Vol 142 (2) ◽

pp. 673-684 ◽

Cited By ~ 27

Author(s):

María Valeria Lara ◽

Simon D.X. Chuong ◽

Hossein Akhani ◽

Carlos Santiago Andreo ◽

Gerald E. Edwards

Keyword(s):

Single Cell ◽

Phosphoenolpyruvate Carboxylase ◽

Cell Type ◽

C4 Species ◽

Single Cell Type

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Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00416.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. G592-G605 ◽

Cited By ~ 13

Author(s):

Kunihiro Kishida ◽

Sarah C. Pearce ◽

Shiyan Yu ◽

Nan Gao ◽

Ronaldo P. Ferraris

Keyword(s):

Stem Cells ◽

Cell Types ◽

Paneth Cells ◽

Nutrient Sensing ◽

Intestinal Stem Cells ◽

Intestinal Cell ◽

Cell Type ◽

Extended Lifespan ◽

Small Intestinal ◽

Single Cell Type

Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown; hence we hypothesized that progenitor stem cells (intestinal stem cells, ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet, or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth, or goblet, but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators and of glucose concentrations in the medium but required fructose absorption and metabolism. More mature enterocyte- and goblet-enriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing. NEW & NOTEWORTHY Small intestinal stem cells differentiate into several cell types transiently populating the villi. We used specialized organoid cultures each comprised of a single cell type to demonstrate that 1) differentiation seems required for nutrient sensing, 2) secretory goblet and Paneth cells along with enterocytes sense fructose, suggesting that sensing is acquired after differentiation is triggered but before divergence between absorptive and secretory lineages, and 3) forcibly dedifferentiated enterocytes exhibit fructose sensing and lifespan extension.

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