Dataset Information

Direct Comparison of the Performance of Commonly Employed In Vivo F-actin Markers (Lifeact-YFP, YFP-mTn and YFP-FABD2) in Tobacco Pollen Tubes.

ABSTRACT: In vivo markers for F-actin organization and dynamics are extensively used to investigate cellular functions of the actin cytoskeleton, which are essential for plant development and pathogen defense. The most widely employed markers are GFP variants fused to F-actin binding domains of mouse talin (GFP-mTn), Arabidopsis fimbrin1 (GFP-FABD2) or yeast Abp140 (Lifeact-GFP). Although numerous reports describing applications of one, or occasionally more, of these markers, are available in the literature, a direct quantitative comparison of the performance of all three markers at different expression levels has been missing. Here, we analyze F-actin organization and growth rate displayed by tobacco pollen tubes expressing YFP-mTn, YFP-FABD2 or Lifeact-YFP at different levels. Results obtained establish that: (1) all markers strongly affect F-actin organization and cell expansion at high expression levels, (2) YFP-mTn and Lifeact-YFP non-invasively label the same F-actin structures (longitudinally oriented filaments in the shank, a subapical fringe) at low expression levels, (3) Lifeact-YFP displays a somewhat lower potential to affect F-actin organization and cell expansion than YFP-mTn, and (4) YFP-FABD2 generally fails to label F-actin structures at the pollen tube tip and affects F-actin organization as well as cell expansion already at lowest expression levels. As pointed out in the discussion, these observations (1) are also meaningful for F-actin labeling in other cell types, which generally respond less sensitively to F-actin perturbation than pollen tubes, (2) help selecting suitable markers for future F-actin labeling experiments, and (3) support the assessment of a substantial amount of published data resulting from such experiments.

SUBMITTER: Montes-Rodriguez A

PROVIDER: S-EPMC5540898 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Direct Comparison of the Performance of Commonly Employed <i>In Vivo</i> F-actin Markers (Lifeact-YFP, YFP-mTn and YFP-FABD2) in Tobacco Pollen Tubes.

Montes-Rodriguez Adriana A Kost Benedikt B

Frontiers in plant science 20170803

<i>In vivo</i> markers for F-actin organization and dynamics are extensively used to investigate cellular functions of the actin cytoskeleton, which are essential for plant development and pathogen defense. The most widely employed markers are GFP variants fused to F-actin binding domains of mouse talin (GFP-mTn), Arabidopsis fimbrin1 (GFP-FABD2) or yeast Abp140 (Lifeact-GFP). Although numerous reports describing applications of one, or occasionally more, of these markers, are available in the l ...[more]

PMID: 28824684

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Project description:BackgroundPollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6).ResultsDe novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization.ConclusionsA major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.

Dataset Information

Direct Comparison of the Performance of Commonly Employed In Vivo F-actin Markers (Lifeact-YFP, YFP-mTn and YFP-FABD2) in Tobacco Pollen Tubes.

Publications

Direct Comparison of the Performance of Commonly Employed <i>In Vivo</i> F-actin Markers (Lifeact-YFP, YFP-mTn and YFP-FABD2) in Tobacco Pollen Tubes.

Similar Datasets

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