Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation.

As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA-Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to the study of RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.

Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp.

In angiosperms, the ovary wall resumes growth after pollination through a balanced combination of cell division and cell expansion. The quantitative pattern of these events remains poorly known in fleshy fruits such as tomato (Solanum spp.), in which dramatic growth of the pericarp occurs together with endoreduplication. Here, this pattern is reported at the level of each of the cell layers or groups of cell layers composing the pericarp, except for vascular bundles. Overall, cell division and cell expansion occurred at similar rates for 9 days post anthesis (DPA), with very specific patterns according to the layers. Subsequently, only cell expansion continued for up to 3-4 more weeks. New cell layers in the pericarp originated from periclinal cell divisions in the two sub-epidermal cell layers. The shortest doubling times for cell number and for cell volume were both detected early, at 4 DPA, in epicarp and mesocarp respectively, and were both found to be close to 14 h. Endoreduplication started before anthesis in pericarp and was stimulated at fruit set. It is proposed that cell division, endoreduplication, and cell expansion are triggered simultaneously in specific cell layers by the same signals issuing from pollination and fertilization, which contribute to the fastest relative fruit growth early after fruit set.

Fruit growth-related genes in tomato.

Tomato (Solanum lycopersicum Mill.) represents a model species for all fleshy fruits due to its biological cycle and the availability of numerous genetic and molecular resources. Its importance in human nutrition has made it one of the most valuable worldwide commodities. Tomato fruit size results from the combination of cell number and cell size, which are determined by both cell division and expansion. As fruit growth is mainly driven by cell expansion, cells from the (fleshy) pericarp tissue become highly polyploid according to the endoreduplication process, reaching a DNA content rarely encountered in other plant species (between 2C and 512C). Both cell division and cell expansion are under the control of complex interactions between hormone signalling and carbon partitioning, which establish crucial determinants of the quality of ripe fruit, such as the final size, weight, and shape, and organoleptic and nutritional traits. This review describes the genes known to contribute to fruit growth in tomato.