Meiocyte Isolation by INTACT and Meiotic Transcriptome Analysis in Arabidopsis

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.

Coexpression of MEIOTIC-TOPOISOMERASE VIB-dCas9 with guide RNAs specific to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis

“…CRISPR/dCas-mediated targeting efficiencies could also vary between different cell types. Although U6 snRNAs are expressed in meiocytes ( Yang et al 2011 ; Barra et al 2021 ), and we show that our 3a -specific gRNAs driven by the AtU6-26 promoter are expressed in Arabidopsis closed buds that contain meiotic cells, we cannot rule out that AtU6-26 promoter is less active in meiocytes compared to other cell types resulting in lower-than-expected efficiency of MTOPVIB-dCas9 recruitment to 3a .…”

Fast and Precise: How to Measure Meiotic Crossovers in Arabidopsis

“…Along with super-resolution microscopy techniques, live imaging systems have been recently established to visualize the dynamics of chromosome behavior during meiosis ( Prusicki et al, 2019 ). In the future, advanced genomic approaches, including meiosis-specific transcriptomics, single-cell RNA sequencing, and epigenome maps using INTACT (isolation of nuclei tagged in specific cell types), will provide important genomic information on CO formation that will help elucidate the molecular mechanisms controlling meiotic recombination in plants ( Barra et al, 2021 ). As an efficient biochemical method for identifying new CO modulators, proximity labeling techniques can be adapted to generate protein interactomes for meiotic proteins in Arabidopsis ( Mair and Bergmann, 2022 ).…”