Meiocyte Isolation by INTACT and Meiotic Transcriptome Analysis in Arabidopsis

Lucia Barra,Gaetana Cremona,Carmine Lanzillo,Pasquale Termolino,Riccardo Aiese Cigliano,Maria Federica Consiglio,Clara Conicella,Rosa Paparo

doi:10.3389/fpls.2021.638051

Abstract

Isolation of nuclei tagged in specific cell types (INTACT) is a method developed to isolate cell-type-specific nuclei that are tagged through in vivo biotin labeling of a nuclear targeting fusion (NTF) protein. In our work, INTACT was used to capture nuclei of meiocytes and to generate a meiotic transcriptome in Arabidopsis. Using the promoter of AtDMC1 recombinase to label meiotic nuclei, we generated transgenic plants carrying AtDMC1:NTF along with biotin ligase enzyme (BirA) under the constitutive ACTIN2 (ACT2) promoter. AtDMC1-driven expression of biotin-labeled NTF allowed us to collect nuclei of meiocytes by streptavidin-coated magnetic beads. The nuclear meiotic transcriptome was obtained by RNA-seq using low-quantity input RNA. Transcripts grouped into different categories according to their expression levels were investigated by gene ontology enrichment analysis (GOEA). The most enriched GO term “DNA demethylation” in mid/high-expression classes suggests that this biological process is particularly relevant to meiosis onset. The majority of genes with established roles in meiosis were distributed in the classes of mid/high and high expression. Meiotic transcriptome was compared with public available transcriptomes from other tissues in Arabidopsis. Bioinformatics analysis by expression network identified a core of more than 1,500 genes related to meiosis landmarks.

Highlights

IntroductionMeiosis appears to be influenced by environmental cues (reviewed in De Storme and Geelen, 2014; Si et al, 2015)
Meiosis is a complex process critical to sexual reproduction
In order to isolate meiocyte nuclei by the INTACT method for RNA sequencing (RNA-seq) analysis in Arabidopsis, we generated transgenic material carrying a nuclear targeting fusion (NTF) protein under the meiosis-specific AtDMC1 promoter (Klimyuk and Jones, 1997) along with the biotinilase BirA under a constitutive ACTIN2 (ACT2) promoter

Summary

Introduction

Meiosis appears to be influenced by environmental cues (reviewed in De Storme and Geelen, 2014; Si et al, 2015). Within this context, global climate change is expected to have an impact on crop production with consequences for food security (Parry et al, 1999). To face the new challenges, a fundamental understanding of meiosis is required in model, crop, and non-model plants (Lambing and Heckmann, 2018). Molecular knowledge of plant meiosis has primarily advanced through understanding the function of single genes involved in key steps being benefited by the conserved pathways across model species (reviewed in Mercier et al, 2015).

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Conclusion