Sections were washed in PBS, and terminal deoxynucleotidyl transferase dUTP nick end labeling was performed with the ApoAlert DNA Fragmentation Assay Kit (Takara) according to the manufacturers instructions
August 12, 2021
Sections were washed in PBS, and terminal deoxynucleotidyl transferase dUTP nick end labeling was performed with the ApoAlert DNA Fragmentation Assay Kit (Takara) according to the manufacturers instructions. of calories and proteins for human nutrition and animal feed as well as a coveted resource for bio-sourced industries. In maize (mutant lead to a miniature kernel phenotype (Sosso et al., 2015). The remaining endosperm interface with maternal tissues (initially the nucellus and later on the pericarp) is the AL, which is not known to contribute to nutrient exchange during seed development (Gontarek and Becraft, 2017). The interface between the endosperm and the embryo is also developmentally dynamic. At 3 to 6 DAP, the embryo is totally surrounded by ESR-type cells. As the embryo expands, it emerges from the ESR, which consequently becomes restricted to the zone surrounding the basal part (suspensor) of the embryo and ultimately disappears together with the suspensor at the end of the early development phase (Opsahl-Ferstad et al., 1997; Giuliani et al., 2002). From Isosakuranetin 8 to 9 DAP, the upper part (embryo proper) forms two new interfaces: (1) at the adaxial side, the embryo is enclosed by a single cell layer, which is called the scutellar aleurone layer (SAL) in barley (in the BETL (Hueros et al., 1999a, 1999b; Cai et al., 2002; Gmez et al., 2002; Gutirrez-Marcos et al., 2004), in the AL (Suzuki et al., 2003), and to in the ESR (Opsahl-Ferstad et al., 1997). Genome-wide gene expression studies at numerous developmental stages of whole kernels and/or hand-dissected endosperm and embryo (Downs et al., 2013; Lu et al., 2013; Chen et al., 2014; Li et al., 2014; Qu et al., 2016; Meng et al., 2018) have been complemented by a recent transcriptomic analysis of laser-capture microdissected cell types and subcompartments of 8-DAP kernels (Zhan et al., 2015). However, even the latter study did not address specifically the transcriptomic profiles of the embryo/endosperm interfaces and did not answer the question of whether the endosperm at the scutellum/endosperm interface is composed of cells with specific transcriptional identities. In this study, we took advantage of the large size of the maize kernel to characterize the genome-wide gene expression profile at embryo/endosperm interfaces at 13 DAP. RNA-seq profiling revealed that endosperm cells in close contact with the embryo scutellum have a distinct transcriptional signature, allowing us to define an Isosakuranetin endosperm zone we named the EAS for endosperm adjacent to scutellum, which is specialized in nutrient transport based on Gene Ontology (GO) enrichment analysis. In situ hybridization shows that the EAS is confined to one to three endosperm cell layers adjacent to the scutellum, whereas kinetic analyses show that the EAS is present when the scutellum emerges at around 9 DAP and persists throughout embryo growth, Isosakuranetin up to 20 DAP. The detection of cell death in the EAS together with the impaired expression of EAS marker genes in an mutant suggest that the EAS is a developmentally dynamic interface influenced by the presence of the neighboring growing embryo. RESULTS RNA-Seq Profiling of 13-DAP Maize Kernel Compartments and Embryo/Endosperm Interfaces To obtain the gene expression patterns of embryo/endosperm interfaces in maize kernels, six (sub)compartments were hand-dissected for transcriptomic analysis (Figure 1; Supplemental Figure 1). The three whole compartments were the maternal tissues excluding the pedicel, which were labeled pericarp (Per), the whole endosperm (End), and the whole embryo (Emb; Figure 1). The subcompartments corresponding to three distinct embryo/endosperm interfaces were the SAL (the single endosperm cell Rabbit Polyclonal to PSEN1 (phospho-Ser357) layer at the adaxial side of the embryo), the apical scutellum (AS; Isosakuranetin corresponding to the embryo tip composed uniquely of scutellum tissues without the embryo axis), and a new region that we named the EAS, corresponding to several layers of endosperm cells in close contact with the scutellum at the abaxial side of the embryo (Figure 1; Supplemental Figure 1). The tissues were collected from kernels of inbred line B73 (used to.