Elucidating Key Transcriptional Regulatory Programs Associated With Endosperm Proliferation and Cell Differentiation in Maize and Sorghum Using Time-Series Gene Expression Data

Abstract

The endosperm, a filial structure formed from double fertilization in angiosperms, is vital for supporting embryo development and seedling germination. It acts as an absorptive storage organ, accumulating carbohydrates and proteins in cereal grains, constituting an essential source of human nutrition, animal feed, and renewable resources. The transcriptional regulatory programs required for endosperm development are not fully understood. This dissertation utilized temporal RNA-sequencing (RNA-seq) of an extensive developmental time-series from maize endosperm and sorghum kernel and the assay for transposase-accessible chromatin with sequencing (ATAC-seq) in maize endosperm to characterize the gene transcriptional programs associated with early cell proliferation, the differentiation of the basal endosperm transfer layer (BETL), and storage protein expression in maize and sorghum endosperm. Using a time-series RNA-seq and ATAC-seq across maize endosperm development from 6 Days After Pollination (DAP) (cellular proliferation) to 30 DAP (maturation), I identified differentially expressed gene (DEG) sets that are associated with two key transcriptional transitions at 12 to 15 DAP and at 18-22 DAP. Two sets of differential Accessible Chromatin Regions (dACRs) were characterized by comparing 8 and 22 DAP ATAC-seq data and genes associated with these dACRs were identified by mapping dACRs to its closest transcription start sites of genes. Analysis of the expression patterns of the dACR-associated genes showed that increased accessibility at 8 or 22 DAP was correlated with increased expression of the genes. This supports the hypothesis that the regulatory sequences within the detected dACRs constitute distinct sets of cis-regulatory modules (CRMs) driving differential expression of the genes during development. A clustering of the dACR-associated genes based on their expression patterns identified distinct sets of DNA-binding motifs for genes associated with each set of dACRs. These included MYB TF-binding motifs among 8-DAP dACR-associated gene sets, and HSF, PLATZ, NAC TF-binding sites among 22-DAP dACR genes. Further genome wide analysis of these TF gene families and their presumptive binding sites helped define putative sets of target genes with distinct patterns of expression and functions. For early development, using previously published data, I hypothesize a role for the MYB TF networks in regulation of endosperm cell proliferation and differentiation, and that the MYB TF genes themselves are regulated by a set of MYBR TFs. For later stages of endosperm development, I hypothesize that multiple sets of HSF, PLATZ, NAC TF-regulated networks are responsible for accumulation of storage products and programmed cell death. To begin to understand the gene regulatory processes involved in sorghum endosperm development, I utilized a developmental time series of sorghum kernel from 0-12 DAP to identify gene co-expression modules using weighted gene co-expression network analysis (WGCNA). A subset of these modules were shown to be enriched in MYBR, bZIP and NAC TF gene families and their corresponding DNA-binding motifs. The expression patterns of the MYBR (8-10 DAP) and bZIP/NAC (12 DAP in sorghum and 15-30 DAP in maize) co-expression modules along with the available data from maize and sorghum supported a role for these TF-regulated networks in early and late endosperm development in sorghum. These observations were supported further using two approaches based on co-expression analysis of the maize-sorghum orthologs expressed in both the kernel developmental time series in sorghum and an integrated endosperm time series in maize spanning 0 to 30 DAP. These analyses supported a role for a MYBR-regulated network in BETL development and a bZIP-NAC-regulated network in regulation of kafirin gene expression in sorghum. The results described here offer valuable insights into the gene-regulatory processes underpinning endosperm development in maize, including early cellular proliferation, BETL differentiation and storage protein gene regulation. Furthermore, my results suggest that BETL differentiation and storage protein gene expression are likely regulated by a set of conserved genes in sorghum and maize, providing a valuable resource for the understanding the conservation of endosperm development across cereal species.