Flow Cytometry and Sorting in Arabidopsis

Sun, Guiling

doi:10.1007/978-1-0716-0880-7_12

Cited by 8 publications

(6 citation statements)

References 138 publications

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“…To assess GFP-AGO1 sub-cellular localization in whole aerial organs, nuclei were purified from leaves, inflorescences, and roots (reference organs) of A1 P:GFP-AGO1(WT), A1 P:GFP-AGO1 mNES (WT), and non-transgenic Arabidopsis, using fluorescence-activated cell sorting (FACS) (Galbraith, 2014 (B) Amino-acid sequence alignment of AGO1 N coil NLS-NES dyads among various plant species. In the consensus NES sequence F-X 2,3 -F-X 2,3 -F-X-F, F represents L, I, V, M, or F and X 2,3 any two or three amino acids.…”

Section: Ago1 Exhibits Pervasive Expo1-dependent Nucleocytosolic Shuttlingmentioning

confidence: 99%

“…DAPI was added and incubation conducted for 5 min at RT before FACS-based nuclear sorting was applied as described in Ref. (Galbraith, 2014) using a BD FACS Aria IIIu Cell Sorter Flow at Cytometry Core Facility -ETH Zurich. After sorting, nuclei were centrifuged for 60 min at 300 g at 4 C, sonicated in a Bioruptor, and reverse cross-linked by 15 min incubation at 50 C and for 30 min at 65 C with NaCl (final concentration 200 mM) and 40 mg of proteinase K. RNA was phenol-extracted, precipitated in sodium acetate, ethanol, and glycogen, washed in 80% ethanol and re-suspended in RNase-free water.…”

Section: Declaration Of Interestsmentioning

confidence: 99%

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Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway

Bologna¹,

Iselin²,

et al. 2018

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Unlike in metazoans, plant microRNAs (miRNAs) undergo stepwise nuclear maturation before engaging cytosolic, sequence-complementary transcripts in association with the silencing effector protein ARGONAUTE1 (AGO1). Since their discovery, how and under which form plant miRNAs translocate to the cytosol has remained unclear, as has their sub-cellular AGO1 loading site(s). Here, we show that the N termini of all plant AGO1s contain a nuclear-localization (NLS) and nuclear-export signal (NES) that, in Arabidopsis thaliana (At), enables AtAGO1 nucleo-cytosolic shuttling in a Leptomycin-B-inhibited manner, diagnostic of CRM1(EXPO1)/NES-dependent nuclear export. Nuclear-only AtAGO1 contains the same 2'O-methylated miRNA cohorts as its nucleo-cytosolic counterpart, but it preferentially interacts with the miRNA loading chaperone HSP90. Furthermore, mature miRNA translocation and miRNA-mediated silencing both require AtAGO1 nucleo-cytosolic shuttling. These findings lead us to propose a substantially revised view of the plant miRNA pathway in which miRNAs are matured, methylated, loaded into AGO1 in the nucleus, and exported to the cytosol as AGO1:miRNA complexes in a CRM1(EXPO1)/NES-dependent manner.

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Section: Ago1 Exhibits Pervasive Expo1-dependent Nucleocytosolic Shuttlingmentioning

confidence: 99%

Section: Declaration Of Interestsmentioning

confidence: 99%

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway

Bologna¹,

Iselin²,

et al. 2018

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“…Illumina re-sequencing reads of all the C. occulta accessions were assembled using SPAdes (v3.13.0) with kmer 77 91 . To estimate the genome size by flow cytometry assay, plant homogenates were prepared as described with modifications 92 . Briefly, four rosette leaves were chopped in Galbraith’s buffer and stained with 4,6-diamidino-2-phenylindole (DAPI, AAT Bioquest, Cat No./ID: 28718903) 93 .…”

Section: Methodsmentioning

confidence: 99%

Common evolutionary trajectory of short life-cycle in Brassicaceae ruderal weeds

Chang

et al. 2023

Nat Commun

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Weed species are detrimental to crop yield. An understanding of how weeds originate and adapt to field environments is needed for successful crop management and reduction of herbicide use. Although early flowering is one of the weed trait syndromes that enable ruderal weeds to overcome frequent disturbances, the underlying genetic basis is poorly understood. Here, we establish Cardamine occulta as a model to study weed ruderality. By genome assembly and QTL mapping, we identify impairment of the vernalization response regulator gene FLC and a subsequent dominant mutation in the blue-light receptor gene CRY2 as genetic drivers for the establishment of short life cycle in ruderal weeds. Population genomics study further suggests that the mutations in these two genes enable individuals to overcome human disturbances through early deposition of seeds into the soil seed bank and quickly dominate local populations, thereby facilitating their spread in East China. Notably, functionally equivalent dominant mutations in CRY2 are shared by another weed species, Rorippa palustris, suggesting a common evolutionary trajectory of early flowering in ruderal weeds in Brassicaceae.

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“…Although proteomic screening methods are helpful in quantifying the expression and variation or modification of proteins at the functional stage, setup of proteomic screening techniques can only control and manage the expression of a partial or limited subset of proteins in the particular cell at a time. Furthermore, advanced techniques such as flow cytometry facilitate the activity of screening of the protein at the resolution of thousands of cells; but this comes at the price of a limited coverage of the proteome (Galbraith 2014). Proteomic data enable detection of functional proteins and their modifications that have a role in the development and progression of disease and development of causal models for cellular signaling, driving translational science at the functional level in crop plants (Quirino et al 2010;Lodha et al 2013;Gong et al 2015).…”

Section: Proteomementioning

confidence: 99%

Augmentation of crop productivity through interventions of omics technologies in India: challenges and opportunities

et al. 2018

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With the continuous increase in the population of developing countries and decline of natural resources, there is an urgent need to qualitatively and quantitatively augment crop productivity by using new tools and technologies for improvement of agriculturally important traits. The new scientific and technological omics-based approaches have enabled us to deal with several issues and challenges faced by modern agricultural system and provided us novel opportunities for ensuring food and nutritional security. Recent developments in sequencing techniques have made available huge amount of genomic and transcriptomic data on model and cultivated crop plants including Arabidopsis thaliana, Oryza sativa, Triticum aestivum etc. The sequencing data along with other data generated through several omics platforms have significantly influenced the disciplines of crop sciences. Gene discovery and expression profiling-based technologies are offering enormous opportunities to the scientific community which can now apply marker-assisted selection technology to assess and enhance diversity in their collected germplasm, introgress essential traits from new sources and investigate genes that control key traits of crop plants. Utilization of omics science and technologies for crop productivity, protection and management has recently been receiving a lot of attention; the majority of the efforts have been put into signifying the possible applications of various omics technologies in crop plant sciences. This article highlights the background of challenges and opportunities for augmentation of crop productivity through interventions of omics technologies in India.

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Flow Cytometry and Sorting in Arabidopsis

Cited by 8 publications

References 138 publications

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway

Common evolutionary trajectory of short life-cycle in Brassicaceae ruderal weeds

Augmentation of crop productivity through interventions of omics technologies in India: challenges and opportunities

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