A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.
Identifieur interne : 000592 ( Main/Exploration ); précédent : 000591; suivant : 000593A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.
Auteurs : Fabien Baldacci-Cresp [France, Belgique] ; Corentin Spriet [France] ; Laure Twyffels [Belgique] ; Anne-Sophie Blervacq [France] ; Godfrey Neutelings [France] ; Marie Baucher [Belgique] ; Simon Hawkins [France]Source :
- The Plant journal : for cell and molecular biology [ 1365-313X ] ; 2020.
Abstract
One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.
DOI: 10.1111/tpj.14675
PubMed: 31917878
Affiliations:
- Belgique, France
- Hauts-de-France, Nord-Pas-de-Calais, Région de Bruxelles-Capitale
- Bruxelles, Lille
- Université libre de Bruxelles
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Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Lille</settlement></placeName></affiliation><affiliation wicri:level="4"><nlm:affiliation>Laboratoire de Biotechnologie Végétale (LBV), Université libre de Bruxelles, B-6041, Gosselies, Belgium.</nlm:affiliation><country xml:lang="fr">Belgique</country><wicri:regionArea>Laboratoire de Biotechnologie Végétale (LBV), Université libre de Bruxelles, B-6041, Gosselies</wicri:regionArea><orgName type="university">Université libre de Bruxelles</orgName><placeName><settlement type="city">Bruxelles</settlement><region type="region" nuts="2">Région de Bruxelles-Capitale</region></placeName></affiliation></author><author><name sortKey="Spriet, Corentin" sort="Spriet, Corentin" uniqKey="Spriet C" first="Corentin" last="Spriet">Corentin Spriet</name><affiliation wicri:level="3"><nlm:affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Lille</settlement></placeName></affiliation><affiliation wicri:level="3"><nlm:affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), TISBio, Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), TISBio, Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Lille</settlement></placeName></affiliation></author><author><name sortKey="Twyffels, Laure" sort="Twyffels, Laure" uniqKey="Twyffels L" first="Laure" last="Twyffels">Laure Twyffels</name><affiliation wicri:level="4"><nlm:affiliation>Center for Microscopy and Molecular Imaging (CMMI), Université libre de Bruxelles, B-6041, Gosselies, Belgium.</nlm:affiliation><country xml:lang="fr">Belgique</country><wicri:regionArea>Center for Microscopy and Molecular Imaging (CMMI), Université libre de Bruxelles, B-6041, Gosselies</wicri:regionArea><orgName type="university">Université libre de Bruxelles</orgName><placeName><settlement type="city">Bruxelles</settlement><region type="region" nuts="2">Région de Bruxelles-Capitale</region></placeName></affiliation></author><author><name sortKey="Blervacq, Anne Sophie" sort="Blervacq, Anne Sophie" uniqKey="Blervacq A" first="Anne-Sophie" last="Blervacq">Anne-Sophie Blervacq</name><affiliation wicri:level="3"><nlm:affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Lille</settlement></placeName></affiliation></author><author><name sortKey="Neutelings, Godfrey" sort="Neutelings, Godfrey" uniqKey="Neutelings G" first="Godfrey" last="Neutelings">Godfrey Neutelings</name><affiliation wicri:level="3"><nlm:affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Lille</settlement></placeName></affiliation></author><author><name sortKey="Baucher, Marie" sort="Baucher, Marie" uniqKey="Baucher M" first="Marie" last="Baucher">Marie Baucher</name><affiliation wicri:level="4"><nlm:affiliation>Laboratoire de Biotechnologie Végétale (LBV), Université libre de Bruxelles, B-6041, Gosselies, Belgium.</nlm:affiliation><country xml:lang="fr">Belgique</country><wicri:regionArea>Laboratoire de Biotechnologie Végétale (LBV), Université libre de Bruxelles, B-6041, Gosselies</wicri:regionArea><orgName type="university">Université libre de Bruxelles</orgName><placeName><settlement type="city">Bruxelles</settlement><region type="region" nuts="2">Région de Bruxelles-Capitale</region></placeName></affiliation></author><author><name sortKey="Hawkins, Simon" sort="Hawkins, Simon" uniqKey="Hawkins S" first="Simon" last="Hawkins">Simon Hawkins</name><affiliation wicri:level="3"><nlm:affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille</wicri:regionArea><placeName><region type="region" nuts="2">Hauts-de-France</region><region type="old region" nuts="2">Nord-Pas-de-Calais</region><settlement type="city">Lille</settlement></placeName></affiliation></author></analytic><series><title level="j">The Plant journal : for cell and molecular biology</title><idno type="eISSN">1365-313X</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.</div></front></TEI><pubmed><MedlineCitation Status="In-Process" Owner="NLM"><PMID Version="1">31917878</PMID><DateRevised><Year>2020</Year><Month>07</Month><Day>21</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1365-313X</ISSN><JournalIssue CitedMedium="Internet"><Volume>102</Volume><Issue>5</Issue><PubDate><Year>2020</Year><Month>06</Month></PubDate></JournalIssue><Title>The Plant journal : for cell and molecular biology</Title><ISOAbbreviation>Plant J</ISOAbbreviation></Journal><ArticleTitle>A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.</ArticleTitle><Pagination><MedlinePgn>1074-1089</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1111/tpj.14675</ELocationID><Abstract><AbstractText>One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.</AbstractText><CopyrightInformation>© 2020 The Authors The Plant Journal © 2020 John Wiley & Sons Ltd.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Baldacci-Cresp</LastName><ForeName>Fabien</ForeName><Initials>F</Initials><Identifier Source="ORCID">0000-0002-7402-2638</Identifier><AffiliationInfo><Affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratoire de Biotechnologie Végétale (LBV), Université libre de Bruxelles, B-6041, Gosselies, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Spriet</LastName><ForeName>Corentin</ForeName><Initials>C</Initials><Identifier Source="ORCID">0000-0002-5805-3426</Identifier><AffiliationInfo><Affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), TISBio, Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Twyffels</LastName><ForeName>Laure</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Center for Microscopy and Molecular Imaging (CMMI), Université libre de Bruxelles, B-6041, Gosselies, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Blervacq</LastName><ForeName>Anne-Sophie</ForeName><Initials>AS</Initials><Identifier Source="ORCID">0000-0001-8517-1236</Identifier><AffiliationInfo><Affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Neutelings</LastName><ForeName>Godfrey</ForeName><Initials>G</Initials><Identifier Source="ORCID">0000-0002-4017-6639</Identifier><AffiliationInfo><Affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Baucher</LastName><ForeName>Marie</ForeName><Initials>M</Initials><Identifier Source="ORCID">0000-0002-2404-8573</Identifier><AffiliationInfo><Affiliation>Laboratoire de Biotechnologie Végétale (LBV), Université libre de Bruxelles, B-6041, Gosselies, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hawkins</LastName><ForeName>Simon</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>UMR 8576 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille, Centre national de la recherche scientifique (CNRS), F-59000, Lille, France.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>02</Month><Day>18</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Plant J</MedlineTA><NlmUniqueID>9207397</NlmUniqueID><ISSNLinking>0960-7412</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">cell wall</Keyword><Keyword MajorTopicYN="Y">confocal microscopy</Keyword><Keyword MajorTopicYN="Y">lignin</Keyword><Keyword MajorTopicYN="Y">safranin</Keyword><Keyword MajorTopicYN="Y">technical advance</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2019</Year><Month>10</Month><Day>23</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2019</Year><Month>12</Month><Day>17</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2019</Year><Month>12</Month><Day>19</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2020</Year><Month>1</Month><Day>10</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>1</Month><Day>10</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2020</Year><Month>1</Month><Day>10</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">31917878</ArticleId><ArticleId IdType="doi">10.1111/tpj.14675</ArticleId></ArticleIdList><ReferenceList><Title>References</Title><Reference><Citation>Agarwal, U.P. 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Bot. 69, 1849-1859.</Citation></Reference></ReferenceList></PubmedData></pubmed><affiliations><list><country><li>Belgique</li><li>France</li></country><region><li>Hauts-de-France</li><li>Nord-Pas-de-Calais</li><li>Région de Bruxelles-Capitale</li></region><settlement><li>Bruxelles</li><li>Lille</li></settlement><orgName><li>Université libre de Bruxelles</li></orgName></list><tree><country name="France"><region name="Hauts-de-France"><name sortKey="Baldacci Cresp, Fabien" sort="Baldacci Cresp, Fabien" uniqKey="Baldacci Cresp F" first="Fabien" last="Baldacci-Cresp">Fabien Baldacci-Cresp</name></region><name sortKey="Blervacq, Anne Sophie" sort="Blervacq, Anne Sophie" uniqKey="Blervacq A" first="Anne-Sophie" last="Blervacq">Anne-Sophie Blervacq</name><name sortKey="Hawkins, Simon" sort="Hawkins, Simon" uniqKey="Hawkins S" first="Simon" last="Hawkins">Simon Hawkins</name><name sortKey="Neutelings, Godfrey" sort="Neutelings, Godfrey" uniqKey="Neutelings G" first="Godfrey" last="Neutelings">Godfrey Neutelings</name><name sortKey="Spriet, Corentin" sort="Spriet, Corentin" uniqKey="Spriet C" first="Corentin" last="Spriet">Corentin Spriet</name><name sortKey="Spriet, Corentin" sort="Spriet, Corentin" uniqKey="Spriet C" first="Corentin" last="Spriet">Corentin Spriet</name></country><country name="Belgique"><region name="Région de Bruxelles-Capitale"><name sortKey="Baldacci Cresp, Fabien" sort="Baldacci Cresp, Fabien" uniqKey="Baldacci Cresp F" first="Fabien" last="Baldacci-Cresp">Fabien Baldacci-Cresp</name></region><name sortKey="Baucher, Marie" sort="Baucher, Marie" uniqKey="Baucher M" first="Marie" last="Baucher">Marie Baucher</name><name sortKey="Twyffels, Laure" sort="Twyffels, Laure" uniqKey="Twyffels L" first="Laure" last="Twyffels">Laure Twyffels</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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