Defining the developmental program leading to meiosis in maize

Abstract

In multicellular organisms, the entry into meiosis is a complex process characterized by increasing levels of meiotic specialization. We used single-cell RNA-sequencing to reconstruct the developmental program into meiosis in maize. We observed a smooth continuum of expression stages leading up to meiosis, followed by a sharp reorganization of the transcriptome in early meiotic prophase. This latter transcriptional shift was dramatic, with 26.7% of expressed genes changing by 2 fold or more, and occurred just prior to a proposed cell cycle checkpoint. Changes in cell physiology accompanied the nuclear events of meiosis, including a decrease in protein translation capacity and increase in membrane-bound organelles. We further identified differences in gene expression between the mitotic and meiotic cell cycles. Our results uncover a multi-step pathway into meiosis and highlight the power of single cell RNA-seq to define developmental transitions.Meiosis is a pivotal event in the life cycle of sexually reproducing organisms, reducing the chromosome number in half and creating new allele combinations through recombination. The mechanisms that regulate meiotic entry in plants are not well understood 1,2 , but are of great importance to crop breeding and agricultural yield 3 .In maize anthers, archesporial (AR) cells are the first cell type in the lineage dedicated to a meiotic fate, acting as the developmental switch between somatic and germinal growth 4 . After a ~3 day period of transit amplifying mitotic divisions, AR cells become pollen mother cells (PMCs) and then enter meiotic prophase. There are substantial changes in cell morphology 4 and gene expression 5-8 during pre-meiotic and early meiotic development, but the cellular intermediates that arise during this process are not well-defined.Here, we applied single-cell RNA-sequencing (scRNA-seq) to characterize the developmental program leading up to meiosis in maize with high resolution. We introduce a quantitative framework, 'pseudotime velocity', to infer developmental transitions based on periods of relatively rapid gene expression change, and apply this framework to identify intermediates from our data. These results provide a road-map for reconstructing plant developmental pathways with scRNA-seq. Single-cell RNA-seq of pre-meiotic and early meiotic cellsMaize male germinal cells form within immature anthers, centrally located in each of four anther lobes (Fig. 1A). Anthers expand in size predictably during early development; consequently, anther length can be used as a reliable, continuous staging system that is correlated with both developmental events and organ age 4 . We established methods to isolate single pre-meiotic and meiotic cells from

Single-Cell RNA Sequencing Resolves Molecular Relationships Among Individual Plant Cells

“…Recently, draft manuscripts have been posted online describing additional scRNA-seq results from maize male germinal cells (144 cells; Nelms and Walbot, 2018) and from Arabidopsis roots (3121 cells [Jean-Baptiste et al, 2019] and 4043 cells [Shulse et al, 2018]). It is likely that new technical advances in plant cell isolation and single-cell sequencing methods will emerge to enable single-cell transcriptomics to be applied to more plant organs and species in the future.…”

Conservation and Divergence in the Meiocyte sRNAomes of Arabidopsis, Soybean, and Cucumber

“…During meiosis, chromosomes become increasingly condensed, which may obstruct transcription; in turn, this might lower the necessity for the miRNA machinery. This hypothesis is supported by a recent single-cell sequencing study in maize (Zea mays) meiocytes, which revealed a two-step transcriptome reorganization at early prophase before chromosome condensation (Nelms and Walbot, 2019). During this reorganization, there was downregulation of RNA-mediated translation and gene silencing processes, including those mediated by Ago18a, an AGO1 paralog specifically enriched in the tapetum and meiocytes during meiosis .…”

Actual time-series of single-cell transcriptomics reveals circadian clocks as key regulators of cell differentiation in Arabidopsis

“…To circumvent such low spatiotemporal resolution of bulk analysis, which can be attributed to the crude averaging of various cell types, we performed time-series of scRNA-seq in the VISUAL. Making protoplasts and/or nuclear isolation is the conventional method to perform scRNA-seq in plants [21][22][23] ; however, the protoplast isolation is timeconsuming and potentially stress-inducing, it is not suitable for time-series analysis of the circadian clocks. We therefore obtained total RNA from single cells using glass capillaries in vivo context 24 ( Supplementary Fig.…”