Obtaining Mutant Pollen for Phenotypic Analysis and Pollen Tube Dual Staining

Abstract Development of pollen, the male gametophyte of flowering plants, is tightly controlled by dynamic changes in gene expression. Recent research to clarify the molecular aspects of pollen development has revealed the involvement of several transcription factors in the induction of gene expression. However, limited information is available about the factors involved in the negative regulation of gene expression to eliminate unnecessary transcripts during pollen development. In this study, we revealed that AtNOT1 is an essential protein for proper pollen development and germination capacity. AtNOT1 is a scaffold protein of the AtCCR4–NOT complex, which includes multiple components related to mRNA turnover control in Arabidopsis. Phenotypic analysis using atnot1 heterozygote mutant pollen showed that the mature mutant pollen failed to germinate and also revealed abnormal localization of nuclei and a specific protein at the tricellular pollen stage. Furthermore, transcriptome analysis of atnot1 heterozygote mutant pollen showed that the downregulation of a large number of transcripts, along with the upregulation of specific transcripts required for pollen tube germination by AtNOT1 during late microgametogenesis, is important for proper pollen development and germination. Overall, our findings provide new insights into the negative regulation of gene expression during pollen development, by showing the severely defective phonotype of atnot1 heterozygote mutant pollen.

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Male fertility in Arabidopsis requires active DNA demethylation of genes that control pollen tube function

Nature Communications ◽

10.1038/s41467-020-20606-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Souraya Khouider ◽

Filipe Borges ◽

Chantal LeBlanc ◽

Alexander Ungru ◽

Arp Schnittger ◽

...

Keyword(s):

Pollen Tube ◽

Male Fertility ◽

Essential Role ◽

Dna Demethylation ◽

Dna Glycosylases ◽

Active Dna Demethylation ◽

Molecular Determinants ◽

Tube Function ◽

Mutant Pollen

AbstractActive DNA demethylation is required for sexual reproduction in plants but the molecular determinants underlying this epigenetic control are not known. Here, we show in Arabidopsis thaliana that the DNA glycosylases DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1) act semi-redundantly in the vegetative cell of pollen to demethylate DNA and ensure proper pollen tube progression. Moreover, we identify six pollen-specific genes with increased DNA methylation as well as reduced expression in dme and dme;ros1. We further show that for four of these genes, reinstalling their expression individually in mutant pollen is sufficient to improve male fertility. Our findings demonstrate an essential role of active DNA demethylation in regulating genes involved in pollen function.

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En face dual fluorescence staining of fatty streaks allows observation of initiation and progression of atherosclerotic lesions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140695 ◽

1995 ◽

Vol 53 ◽

pp. 864-865

Author(s):

Anne M. Klinkner ◽

Crystal R. Waites ◽

Peter J. Bugelski ◽

William D. Kerns

Keyword(s):

Fluorescent Dyes ◽

Nile Red ◽

Dimethyl Formamide ◽

High Cholesterol Diet ◽

Methods Development ◽

Atherosclerotic Lesions ◽

En Face ◽

Biochemical Evaluation ◽

Stock Solutions ◽

Dual Staining

A primary effort in the understanding of the progression of atherosclerotic disease has been methods development for visualization of the atherosclerotic plaque. We introduce a new method for the qualitative analysis of lipids in atherosclerotic fatty streaks which also retains those lipids for biochemical evaluation. An original aspect of the process is the ability to view an entire fatty streak en face, selectively stained for specific lipid classes within the lesion.New Zealand white rabbits were fed a high cholesterol diet(0.15%-0.3% for 14 wks). The aorta was removed and fixed in Carson's phosphate buffered formaldehyde followed by dual staining in the fluorescent dyes Nile red and filipin. Stock solutions of nile red(0.5mg/ml acetone) and filipin(2.5mg/ml dimethyl formamide) were prepared and kept at -20°C; all subsequent steps were at RT. 0.5cm × 1.0cm pieces of aorta were trimmed and adventitia removed. The pieces were then washed 3×15 min in PBS w/o CaMg, soaked in Nile red(NR)/filipin(Fl) stain(100(il NR stock + 200μl Fl stock in 10 ml PBS for 30 min, washed in PBS 3×30 min, rinsed with distilled water, mounted(Crystal Mount, Biomedia) and coverslipped and viewed by fluorescence microscopy.

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Protein S mRNA in Patients with Protein S Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653862 ◽

1995 ◽

Vol 73 (05) ◽

pp. 746-749 ◽

Cited By ~ 5

Author(s):

E Sacchi ◽

M Pinotti ◽

G Marchetti ◽

G Merati ◽

L Tagliabue ◽

...

Keyword(s):

Gene Polymorphism ◽

Total Protein ◽

Restriction Analysis ◽

Protein S ◽

Type I ◽

Protein S Deficiency ◽

Phenotypic Analysis ◽

S Deficiency ◽

Deficiency Type ◽

S Genes

SummaryA protein S gene polymorphism, detectable by restriction analysis (BstXI) of amplified exonic sequences (exon 15), was studied in seven Italian families with protein S deficiency. In the 17 individuals heterozygous for the polymorphism the study was extended to platelet mRNA through reverse transcription, amplification and densitometric analysis. mRNA produced by the putative defective protein S genes was absent in three families and reduced to a different extent (as expressed by altered allelic ratios) in four families. The allelic ratios helped to distinguish total protein S deficiency (type I) from free protein S deficiency (type IIa) in families with equivocal phenotypes. This study indicates that the study of platelet mRNA, in association with phenotypic analysis based upon protein S assays in plasma, helps to classify patients with protein S deficiency.

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