Single-cell RNA-seq analysis reveals ploidy-dependent and cell-specific transcriptome changes in Arabidopsis female gametophytes

Background Polyploidy provides new genetic material that facilitates evolutionary novelty, species adaptation, and crop domestication. Polyploidy often leads to an increase in cell or organism size, which may affect transcript abundance or transcriptome size, but the relationship between polyploidy and transcriptome changes remains poorly understood. Plant cells often undergo endoreduplication, confounding the polyploid effect. Results To mitigate these effects, we select female gametic cells that are developmentally stable and void of endoreduplication. Using single-cell RNA sequencing (scRNA-seq) inArabidopsis thalianatetraploid lines and isogenic diploids, we show that transcriptome abundance doubles in the egg cell and increases approximately 1.6-fold in the central cell, consistent with cell size changes. In the central cell of tetraploid plants,DEMETER(DME) is upregulated, which can activatePRC2family membersFIS2andMEA, and may suppress the expression of other genes. Upregulation of cell size regulators in tetraploids, includingTORandOSR2, may increase the size of reproductive cells. In diploids, the order of transcriptome abundance is central cell, synergid cell, and egg cell, consistent with their cell size variation. Remarkably, we uncover new sets of female gametophytic cell-specific transcripts with predicted biological roles; the most abundant transcripts encode families of cysteine-rich peptides, implying roles in cell-cell recognition during double fertilization. Conclusions Transcriptome in single cells doubles in tetraploid plants compared to diploid, while the degree of change and relationship to the cell size depends on cell types. These scRNA-seq resources are free of cross-contamination and are uniquely valuable for advancing plant hybridization, reproductive biology, and polyploid genomics.