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FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

2012-10-17Journal article Open access
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dc.contributor.authorBorges, Filipe
dc.contributor.authorGardner, Rui
dc.contributor.authorLopes, Telma
dc.contributor.authorCalarco, Joseph P
dc.contributor.authorBoavida, Leonor C
dc.contributor.authorSlotkin, R
dc.contributor.authorMartienssen, Robert A
dc.contributor.authorBecker, Jörg D
dc.date.accessioned2016-08-18T11:03:52Z
dc.date.available2016-08-18T11:03:52Z
dc.date.issued2012-10-17
dc.description.abstractBackground: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.pt_PT
dc.description.sponsorshipNSERC graduate student fellowship; Fred C. Gloeckner Foundation grant.pt_PT
dc.identifier.citationBorges et al. : FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods 2012 8 :44pt_PT
dc.identifier.doi10.1186/1746-4811-8-44pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.7/688
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherBioMed Centralpt_PT
dc.relationGENETIC AND EPIGENETIC MECHANISMS REGULATING SPERM CELL FUNCTIONS DURING DOUBLE FERTILIZATION IN FLOWERING PLANTS
dc.relation.publisherversionhttps://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-8-44pt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectPollenpt_PT
dc.subjectMicrosporept_PT
dc.subjectSperm cellpt_PT
dc.subjectVegetative nucleuspt_PT
dc.subjectPlant germlinept_PT
dc.subjectArabidopsispt_PT
dc.subjectFluorescence-activated cell sortingpt_PT
dc.titleFACS-based purification of Arabidopsis microspores, sperm cells and vegetative nucleipt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleGENETIC AND EPIGENETIC MECHANISMS REGULATING SPERM CELL FUNCTIONS DURING DOUBLE FERTILIZATION IN FLOWERING PLANTS
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FAGR-GPL%2F103778%2F2008/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FBIA-BCM%2F103787%2F2008/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT//SFRH%2FBD%2F48761%2F2008/PT
oaire.citation.endPage8pt_PT
oaire.citation.issue1pt_PT
oaire.citation.startPage1pt_PT
oaire.citation.titlePlant Methodspt_PT
oaire.citation.volume8pt_PT
oaire.fundingStream3599-PPCDT
oaire.fundingStream3599-PPCDT
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
relation.isProjectOfPublicationa11ecff8-6b1c-4365-815e-bb1502381044
relation.isProjectOfPublication0bd97f4e-e163-40b9-a2df-d42173887119
relation.isProjectOfPublication9d14cd7d-7c39-45d3-ae58-3932370a639a
relation.isProjectOfPublication.latestForDiscovery9d14cd7d-7c39-45d3-ae58-3932370a639a

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