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Laser Microdissection of Woody and Suberized Plant Tissues for RNA-Seq Analysis

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Abstract

An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory mechanism of woody plants. Laser microdissection is one technique that allows isolation of specific, target cells or tissue from a heterogeneous cell population. This technique entails microscopic visualization of the selected tissue and use a laser beam to separate the desired cells from surrounding tissue. Initial identification of these cells is made based on morphology and/or histological staining. Some works have been made in several tissues and plant models. However, there are few studies of laser microdissection application in woody species, particularly, lignified and suberized cells. Moreover, the presence of high level of suberin in cell walls can be a big challenge for the application of this approach. In our study it was developed a technique for tissue isolation, using laser microdissection of four different plant cell types (phellogen, lenticels, cortex and xylem) from woody tissues of cork oak (Quercus suber), followed by RNA extraction and RNA-Seq. We tested several methodologies regarding laser microdissection, cryostat equipments, fixation treatments, duration of single-cells collection and number of isolated cells by laser microdissection and RNA extraction procedures. A simple and efficient protocol for tissue isolation by laser microdissection and RNA purification was obtained, with a final method validation of RNA-Seq analysis. The optimized methodology combining RNA-Seq for expression analysis will contribute to elucidate the molecular pathways associated with different development processes of the xylem and phellem in oaks, including the lenticular channels formation.

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Acknowledgements

The present work was supported by Programme Alentejo 2020 under the scope of Lentidev—A molecular approach to cork porosity (ALT20-03-0145-FEDER-000020) and by Programme PORTUGAL 2020 Partnership Agreement, under the scope of Biodata.pt—Infraestrutura Portuguesa de Dados Biológicos (22231/01/SAICT/2016), through the European Regional Development Fund (ERDF). The authors also acknowledge FCT to Contrato—Programa to L. Marum (CEECINST/00131/2018) and A. Usié (CEECINST/00100/2021). This work was also funded through FCT under the projects UIDB/05183/2020 to Mediterranean Institute for Agriculture, Environment and Development (MED), LA/P/0121/2020 to Global Change and Sustainability Institute (CHANGE), and UID/50017/2020+UIDB/50017/2020+LA/P/0094/2020 to Centre for Environmental and Marine Studies (CESAM).

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Author notes

  1. Ana Ferro & Tiago Capote

    Present address: Center for Genomics and Systems Biology, New York University Abu Dhabi, NYUAD Campus, 129188, Abu Dhabi, United Arab Emirates

  2. Bárbara Correia

    Present address: B-hive Innovations Ltd., Boole Technology Centre, Beevor Street, Lincoln, LN6 7DJ, UK

  3. Esther Menéndez

    Present address: Department of Microbiology and Genetics/CIALE, Universidad de Salamanca, 37007, Salamanca, Spain

Authors and Affiliations

  1. Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908, Beja, Portugal

    Rita Costa Pires, Ana Ferro, Tiago Capote, Ana Usié, Bárbara Correia & Liliana Marum

  2. MED – Mediterranean Institute for Agriculture, Environment and Development, CEBAL – Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo, 7801-908, Beja, Portugal

    Ana Ferro & Tiago Capote

  3. MED – Mediterranean Institute for Agriculture, Environment and Development & CHANGE – Global Change and Sustainability Institute, CEBAL – Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo, 7801-908, Beja, Portugal

    Ana Usié & Liliana Marum

  4. Department of Biology, Centre for Environmental and Marine Studies (CESAM), University of Aveiro, 3810-193, Aveiro, Portugal

    Glória Pinto

  5. MED—Mediterranean Institute for Agriculture, Environment and Development & CHANGE – Global Change and Sustainability Institute, Institute for Advanced Studies and Research (IIFA), University of Évora, Polo da Mitra, Ap. 94, 7006-554, Évora, Portugal

    Esther Menéndez

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  1. Rita Costa Pires
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Contributions

Conceptualization: LM; Methodology: RCP, AF, TC, BC and EM; Formal analysis and investigation: RCP and TC; AU; Writing—original draft preparation: RCP and LM; Writing—review and editing: RCP, AF, AU, GP, EM and LM; Project administration: LM; Supervision: GP and LM.

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Correspondence to Liliana Marum.

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12033_2022_542_MOESM1_ESM.tif

Supplementary file1 Fig. S1 PCR amplification of β-tubulin reference gene in xylem (Xyl) and phellogen (Phel) tissues by reverse transcription-polymerase chain reaction (RT-PCR). Lane M, 1 kb DNA size marker. The PCR products showed a band at position 92 bp related to the amplicon size. (TIF 22133 kb)

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Pires, R.C., Ferro, A., Capote, T. et al. Laser Microdissection of Woody and Suberized Plant Tissues for RNA-Seq Analysis. Mol Biotechnol 65, 419–432 (2023). https://doi.org/10.1007/s12033-022-00542-9

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