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. 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

核内复制（Endoreduplication）指细胞在不经历细胞分裂的前提下，通过多轮完整基因组复制以提升DNA含量的过程，是高等植物核内多倍性（endopolyploidy）的主要来源。核内复制在植物生长与发育中发挥关键作用，且与各细胞类型特有的特异性转录程序激活密切相关，进而决定细胞的身份属性。在植物中，核内复制广泛存在于众多器官和细胞类型中，尤其集中于具有农业经济价值的组织，例如高倍性的番茄肉质果实果皮（pericarp）。本研究以番茄果皮为模型系统，探究果实生长过程中与核内复制进程相关的转录组特征。研究证实基因的表达水平全局随倍性水平发生缩放，并鉴定出特定发育阶段下不同倍性水平间的差异表达基因集。研究发现，非核内复制细胞的核心特征为细胞分裂状态与角质层合成，而核内复制细胞的主要特征为随时间快速动态变化的代谢活性。通过将本数据集与公开可得的时空特异性果皮表达数据相结合，我们构建了可描述果皮内倍性水平分布的图谱。基于转录组的预测结果通过定量果皮组织内的倍性水平得到了验证。该原位倍性定量实验揭示了果实早期发育过程中核内复制的动态进程及其细胞层特异性。综上，本研究阐明了番茄果皮中核内复制、细胞分化与基因表达模式之间的复杂关联。为探究番茄果实发育过程中与核内复制相关的分子异质性，我们采集了开花后天数（DPA）为6、9和12天的果实果皮样本。选择上述时间点的原因在于，它们对应果实快速膨大的旺盛时期：此时果实生长速率较高，且倍性水平发生显著变化——具体表现为2C和4C细胞核占比下降，同时出现8C以上的倍性水平（16C、32C和64C）。我们采用荧光激活细胞核分选（FANS）技术，根据DNA含量从果皮样本中分离收集细胞核。在每个时间点，我们均获得了4个各含10万个细胞核的细胞群体，对应占比超过85%的主要倍性类别：6DPA样本的群体为2C、4C、8C和16C，9DPA与12DPA样本的群体为4C、8C、16C和32C。随后我们提取这些细胞核的总RNA，并通过RNA-seq进行测序。