Adapting INTACT to analyse cell-type-specific transcriptomes and nucleocytoplasmic mRNA dynamics in the Arabidopsis embryo

Abstract

In the early embryo of vascular plants, the different cell types and stem cells of the seedling are specified as the embryo develops from a zygote towards maturity. How the key steps in cell and tissue specification are instructed by genome-wide transcriptional activity is poorly understood. Progress in defining transcriptional regulation at the genome-wide level in plant embryos has been hampered by difficulties associated with capturing cell-type-specific transcriptomes in this small and inaccessible structure. We recently adapted a two-component genetic nucleus labelling system called INTACT to isolate nuclei from distinct cell types at different stages of Arabidopsis thaliana embryogenesis. We have used these to generate a transcriptomic atlas of embryo development following microarray-based expression profiling. Here, we present a general description of the adapted INTACT procedure, including the two-component labelling system, seed isolation, nuclei preparation and purification, as well as transcriptomic profiling. We also compare nuclear and cellular transcriptomes from the early Arabidopsis embryo to assess nucleocytoplasmic differences and discuss how these differences can be used to infer regulation of gene activity.

Genetic activity during early plant embryogenesis

“…They also share control of many other directly regulated genes that are not TFs and these targets are what actually ‘builds’ the cell. Transcriptomic studies can now measure and compare transcriptomes in individual cell types [ 64 , 65 , 255 ]. Some examples include comparison of egg to zygote transcriptomes [ 256 ], apical compared with basal cell transcriptomes [ 257 ], determination of an embryo epidermal specific transcriptome [ 258 ], and other subregions of the seed/embryo [ 259 ].…”

“…WOX8 and another family member WOX2 , along with WRKY2 are expressed in the egg and zygote, but after the first division, WOX8 is only in the BC along with WOX9 , whereas WOX2 is in the AC [ 62 , 64 , 65 ]. WOX8 and WOX9 are important for BC lineage determination as wox8 wox9 double mutants showed abnormal suspensor development [ 66 ].…”

Research Tools for the Functional Genomics of Plant miRNAs During Zygotic and Somatic Embryogenesis

“…Plant embryogenesis is challenging to analyse due to the small, early embryos that are deeply embedded in the maternal tissues, which often results in RNA contamination from maternal tissue, and due to difficult distinguishing of the cells that are undergoing SE within an explant tissue, protocols, which are specifically dedicated for ZE and SE, have been developed and successfully used for miRNA analysis [ 28 , 38 , 84 , 95 ]. Since the low-input small RNA sequencing (sRNA-seq) method, which can be used to generate the profiles of miRNAs from as little as one to five ng of RNA and INTACT (isolation of nuclei tagged in specific cell types) and FANS (fluorescence-activated nuclei sorting) which permit the isolation of nuclei from cells that are undergoing SE or ZE from the majority of non-embryogenic cells from an explant, it makes a high-throughput analysis during embryogenesis much more accurate [ 28 , 84 , 96 , 97 , 98 , 99 ]. It is worth mentioning the requirement to share the data from NGS during the publication process, which ensures access to big data without performing an experiment.…”

Laser-Assisted Microdissection of Plant Embryos for Transcriptional Profiling

“…Other methods involve the use of transgenic lines allowing the profiling of individual cell and tissue types, such as fluorescence-activated cell or nuclei sorting (FACS, FANS) (3,21,22). Labelling and affinity purification of nuclei (INTACT) (23,24) or polysomes (TRAP) (25,26) also allow the analysis of gene expression at a finer scale. Because all these methods require the expression of transgenes under highly specific embryonic promoters, their use has so far largely been restricted to Arabidopsis.…”