Following meiosis in maize Plants do not set aside a germ-cell lineage from early development as animals do, but instead generate germ cells on demand. Nelms and Walbot, working in maize, took advantage of a size differential between somatic and developing germ cells in the anthers at the top of the maize plant to isolate individual germ cells during the meiotic progression to pollen development. They used single-cell RNA sequencing to study changes in the transcriptome through meiosis. These studies revealed increasing specialization as meiosis progressed, with a reorganization of the transcriptome in a transition during the leptotene stage of meiosis. Science , this issue p. 52
We present a sensitive approach to predict genes expressed selectively in specific cell types, by searching publicly available expression data for genes with a similar expression profile to known cell-specific markers. Our method, CellMapper, strongly outperforms previous computational algorithms to predict cell type-specific expression, especially for rare and difficult-to-isolate cell types. Furthermore, CellMapper makes accurate predictions for human brain cell types that have never been isolated, and can be rapidly applied to diverse cell types from many tissues. We demonstrate a clinically relevant application to prioritize candidate genes in disease susceptibility loci identified by GWAS.
Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. Pollen actively transcribes its haploid genome, providing phenotypic diversity even among pollen grains from a single plant. In this study, we used allele-specific RNA sequencing of single pollen precursors to follow the shift to haploid expression in maize pollen. We observed widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I, driven by active new transcription from the haploid genome. Genes showed evidence of increased purifying selection if they were expressed after (but not before) pollen mitosis I. This work establishes the timing during which haploid selection may act in pollen.
Although DNA methylation primarily represses TEs, it also represses select genes that are methylated in plant body tissues but demethylated by DNA glycosylases (DNGs) in endosperm or pollen. Activity of either one of two DNGs, MDR1 or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen segregating mutations in both genes, we identified 58 candidate DNG target genes that account for 11.1% of the wild-type transcriptome but are silent or barely detectable in the plant body (sporophyte). They are unusual in their tendency to lack introns but even more so in their having TE-like methylation in their CDS. The majority have predicted functions in cell wall modification, and they likely support the rapid tip growth characteristic of pollen tubes. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with potential for extremely high expression in pollen but constitutive silencing elsewhere.
Endosome transport by transcytosis is the primary mechanism by which proteins and other large cargo traverse epithelial barriers in normal tissue. Transcytosis is also essential for establishing and maintaining membrane polarity in epithelia and other polarized cells. To identify novel components of this pathway, we conducted a high-throughput RNA interference screen for factors necessary for the bidirectional transcytosis of IgG by the Fcγ receptor FcRn. This screen identified 23 genes whose suppression resulted in a reproducible decrease in FcRn-mediated transcytosis. Pulse-chase kinetic transport assays on four of the top-ranking genes (EXOC2, EXOC7, PARD6B, and LEPROT) revealed distinct effects on the apical and basolateral recycling and transcytotic pathways, demonstrating that these pathways are genetically separable. We also found a strong dependence on PARD6B for apical, but not basolateral, recycling, implicating this cell polarity gene in assembly or maintenance of the apical endosomal system. This dataset yields insights into how vesicular transport is adapted to the specialized functions of differentiated cell types and opens new research avenues into epithelial trafficking.
ABSTRACT Single-cell RNA-sequencing (scRNA-seq) can provide invaluable insight into cell development, cell type identification, and plant evolution. However, the resilience of the cell wall makes it difficult to dissociate plant tissues and release individual cells for single-cell analysis. Here, we show that plant organs can be rapidly and quantitatively dissociated into cells if fixed prior to enzymatic digestion. Fixation enables digestion at high temperatures at which enzymatic activity is optimal and stabilizes the plant cell cytoplasm, rendering cells resistant to mechanical shear force while maintaining high quality RNA. This protocol, FX-Cell, releases four to ten-fold more recoverable cells than optimized protoplasting methods applied to maize anthers or root tips with no cell type biases and can be readily applied to a variety of plant taxa and tissues with no optimization. FX-Cell and scRNA-seq analysis were applied to maize anthers for which 95% of the cells were dispersed and provided suitable scRNA-seq data for the identification of anther cell types with marker genes and well-understood biological functions, including rare meiocytes (∼1% anther cells). In addition, the scRNA-seq data provided putative marker genes and gene ontology information for the identification of unknown cell types. FX-Cell also preserves the morphology of the isolated cells, permitting cell type identification without staining. Ultimately, FX-Cell can be applied to a range of plant species and tissues with minimal to no optimization paving the way for plant scRNA-seq analyses in non-model taxa and tissues.
Abstract Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase 1–3 . Pollen actively transcribes its haploid genome 4 , providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (d n /d s ) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.
