Isolation of living apical and basal cell lineages of early proembryos for transcriptome analysis

Abstract

Plant zygotes usually undergo asymmetrical cell division, giving rise to the formation of two daughter cells with distinct developmental cell fate. The small apical cell will develop into the major part of embryo proper, whereas the larger basal cell will divide to form a transient suspensor. Thus, the apical and basal cell lineages are an excellent model to study cell fate determination in relation to zygote polarity. However, the molecular mechanism underlying the differentiation of two distinct cell lineages is not yet understood, possibly due to the technique limitations. Previously, we have established a protocol for isolating apical cell and basal cell for cDNA library construction in tobacco. However, the method for isolating tiny Arabidopsis embryos has long been considered much more difficult. Here, we present a detailed protocol for isolating early Arabidopsis proembryos and separating apical and basal cell lineages of proembryos, which allow us to establish cell lineage-specific transcriptomes of early proembryos.

Detection of Embryonic Suspensor Cell Death by Whole-Mount TUNEL Assay in Tobacco

“…To date, isolation of tiny early embryos still remains technically challenging. Only a few studies reported the methods for the isolation of early embryos using either laser-capture microdissection (LCM) [ 19 ] or manual isolation [ 19 , 20 ]. However, the LCM equipment is not commonly available, and not suitable for isolating living early embryos.…”

“…In addition, the key hand-made tools are easy to assemble according to the introduction ( Figure 2 ). This basic setup has been proven to be efficient and reliable in different plants [ 18 , 20 ]. Compared with the other available methods, this procedure offers several advantages: (i) the spatial-temporal characters of suspensor PCD could be quickly detected within 5–6 h, (ii) it can be easily adopted by other researchers due to the simple setup, (iii) it requires affordable equipment for the basic setup, and (iv) this method could also be useful for rapid detection of cell death of abortive embryos.…”

Genetic activity during early plant embryogenesis

“…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 ].…”

The Winner Takes It All: Auxin—The Main Player during Plant Embryogenesis

“…In A. thaliana after the first division of a zygote, auxin is transported toward the apical cell due to polar localization of PIN7 proteins [55]. The apical cell Cells 2020, 9, 606 4 of 16 elongates slightly in the direction of auxin flow [56][57][58][59], however progressive auxin accumulation [60] would break default transverse alignment of microtubules and promote longitudinal division in this way. Interestingly, anisotropic growth of the apical and basal cells seems to be necessary for correct embryogenesis.…”