Ploidy-specific transcriptomes shed light on the heterogeneous identity and metabolism of developing pericarp cells
Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication plays pivotal roles in plant growth and development and is associated with the activation of specific transcriptional programs that are characteristic to each cell type, thereby defining their identity. In plants, endoreduplication is found in numerous organs and cell types and especially in agronomically valuable ones, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. We used the tomato pericarp tissue as a model system to explore the transcriptomes associated with endoreduplication progression during fruit growth. We confirmed that expression globally scales with ploidy level and identified sets of genes differentially expressed when comparing ploidy levels at a specific developmental stage. We found that non-endoreduplicated cells are defined by cell division state and cuticle synthesis while endoreduplicated cells are mainly defined by their metabolic activity changing rapidly over time. By combining this dataset with publicly available spatiotemporal pericarp expression data, we proposed a map describing the distribution of ploidy levels within the pericarp. These transcriptome-based predictions were validated by quantifying ploidy levels within the pericarp tissue. This in situ ploidy quantification revealed the dynamic progression of endoreduplication and its cell layer specificity during early fruit development. In summary, the study sheds light on the complex relationship between endoreduplication, cell differentiation, and gene expression patterns in the tomato pericarp. Overall design: To investigate the molecular heterogeneity related to endoreduplication during tomato fruit development, we used pericarp from fruits at 6, 9 and 12 days post-anthesis (DPA). These timepoints were chosen because they correspond to an intense period of fruit enlargement, due to a high fruit growth rate, and with significant changes in ploidy levels, in particular the appearance of ploidy levels beyond 8C (16C, 32C and 64C), at the expense of 2C and 4C nuclei. We used Fluorescence Activated Nuclei Sorting (FANS) to collect nuclei from the pericarp according to their DNA content. At each timepoint, four populations of 100k nuclei, corresponding to the major ploidy classes (>85% of all nuclei), were obtained, namely 2C, 4C, 8C and 16C for 6DPA and 4C, 8C, 16C and 32C for 9 and 12DPA, their RNA was extracted and sequenced by RNA-seq