3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei

Tirichine, Leïla; Andrey, Philippe; Biot, Eric; Maurin, Yves; Gaudin, Valérie

doi:10.1186/1746-4811-5-11

Cited by 33 publications

(18 citation statements)

References 32 publications

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“…Indirect immunofluorescence was performed on root cells or isolated nuclei from leaves as described previously (Fujimoto et al , 2004; Tirichine et al , 2009). CENH3 was detected with rabbit anti-HTR12 antibody (Talbert et al , 2002) (1:1 000) and GFP was detected with either rat anti-GFP antibody (1:200 Bio Academia 1A5) or rabbit anti-GFP antibody (1:200 Invitrogen A11122).…”

Section: Methodsmentioning

confidence: 99%

Visualization of specific repetitive genomic sequences with fluorescent TALEs inArabidopsis thaliana

Fujimoto

Sugano

Kuwata

et al. 2016

EXBOTJ

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Section: Methodsmentioning

confidence: 99%

Visualization of specific repetitive genomic sequences with fluorescent TALEs inArabidopsis thaliana

Fujimoto

Sugano

Kuwata

et al. 2016

EXBOTJ

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“…In Arabidopsis, whole-mount (WM) techniques are well established for studying the nuclei of different plant tissues (e.g. root meristems, endosperm or leaves) as well, for DNA quantification, nuclei in situ hybridizations, and others (Bauwens et al, 1994;Boisnard-Lorig et al, 2001;Willemse et al, 2008;Tirichine et al, 2009). These methods, coupled with confocal microscopy and computer-aided modelling software, provide detailed qualitative and quantitative information on the nuclear changes occurring in different plant tissues.…”

Section: Introductionmentioning

confidence: 99%

Whole‐mount confocal imaging of nuclei in giant feeding cells induced by root‐knot nematodes in Arabidopsis

Vieira

Engler

2012

New Phytologist

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Summary• Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide.• The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita.• Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression.• The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.

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“…The absence of extensive genome rearrangements in synthetic Arabidopsis autotetraploids was reported also by other researchers. 30 Stem leafs were fixed in 4% formaldehyde (PF) for 40 min in 4 C. Nuclei were isolated according to Tirichine et al 44 with minor modifications. Breathily, leafs were macerated using enzyme mixture containing 2.5% pectolyase (Sigma), 2.5% pectinase (Sigma) and 2.5% cellulase "Onozuka" (Serra) in 37 C for 40 min., washed in 1x PBS buffer (pH 7.4) and transferred to Tris buffer.…”

Section: Preparation Of Plant Materialsmentioning

confidence: 99%

Spatial relationship between chromosomal domains in diploid and autotetraploidArabidopsis thaliananuclei

Sas–Nowosielska

Bernaś

2016

Nucleus

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Polyploids constitute more than 80% of angiosperm plant species. Their DNA content is often further increased by endoreplication, which occurs as a part of cell differentiation. Here, we explore the relationship between 3D chromatin architecture, number of genome copies and their origin in the model plant, Arabidopsis thaliana. Spatial proximity between pericentromeric, interstitial and subtelomeric domains of chromosomes 1 and 4 was quantified over a range of distances. The results indicate that average nuclear volume as well as chromatin density increase with the genome copy number. Similar dependence is observed when association of homologous chromosomes (in 2C/ endopolyploid nuclei) and sister chromatid separation (in endopolyploid nuclei) is studied. Moreover, clusters of chromosomal domains are detectable at the spatial scale above microscopy resolution. Subtelomeric, interstitial and pericentromeric chromosomal domains are affected to different extent by these processes, which are modulated by endopolyploidy. This factor influences fusion of heterochromatin as well. Nonetheless, local chromatin architecture of Arabidopsis thaliana depends mainly on endopolyploidy level, and to lesser extend on polyploidy.

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3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei

Cited by 33 publications

References 32 publications

Visualization of specific repetitive genomic sequences with fluorescent TALEs inArabidopsis thaliana

Visualization of specific repetitive genomic sequences with fluorescent TALEs inArabidopsis thaliana

Whole‐mount confocal imaging of nuclei in giant feeding cells induced by root‐knot nematodes in Arabidopsis

Spatial relationship between chromosomal domains in diploid and autotetraploidArabidopsis thaliananuclei

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