How does Cell-to-Cell Communication regulate Fruit Growth ? – 3C-FruitGrowth

In order to understand the role of FW2.2 in the regulation of fruit growth, Task 1 aims at characterizing tomato plants modified as to overexpress FW2.2 (OE) as well as loss-of-function (KO) plants, by using morphometric, cytological and histological approaches to study fruit and pericarp size, size and number of cells in the pericarp. We will then investigate the impact of disruption of FW2.2 expression at the molecular level by analyzing genome-wide transcriptional changes occurring in fruits of OE/KO plants.
Task 2 aims at investigating the cellular function of FW2.2 in cell-to-cell signaling via PDs, by confocal imaging approaches on fruit tomato pericarp sections expressing FW2.2 fused to YFP, in order to study its localization and cellular distribution at the PD. Co-labeling with the PD will be achieved using aniline blue, a callose-labeling dye to reveal the PDs. We will use immunofluorescence approaches on pericarp sections with anti-YFP, anti-callose and anti-PDCB1 (PD marker) antibodies. We will assess the impact of altered expression of FW2.2 (OE/KO) on cell-to-cell movement of cytosolic GFP as a control or of a cell cycle inhibitor, KRP fused to GFP, after co -transformation by biolistics. We will quantify the level of callose deposition at PD in the pericarp of wild-type and mutant (OE/KO) fruits, by staining with aniline blue and immunostaining. In order to identify protein partners of FW2.2, the membrane yeast two-hybrid split-ubiquitin system (MYTH) will be used to target known proteins localized to PD, but also to target cell cycle regulators. In parallel, we will use an untargeted in planta approach to identify new FW2.2 interactors by co-immunoprecipitation.
Task 3 aims at establishing the link between the transcriptional regulation of FW2.2 and its role in organ growth, using genetics and molecular approaches. By QTL and eQTL mapping approaches in a segregating tomato population, the genetic architecture of polymorphisms in the promoter region of FW2.2 and its expression, and those of co-regulated genes will be determined in order to link this network of genes to variation in fruit size. We will detail the spatial and temporal expression of FW2.2 by studying GUS reporter lines of the native Ailsa Craig FW2.2 promoter (cultivated line of S. lycopersicum with the «large fruit allele«) and the ancestor of the tomato S. pimpinellifolium («small fruit« allele). QTL information will be used to generate mutated promoters that will be tested in planta for their effect on FW2.2 expression and the consequence on fruit growth.

To carry out the functional analysis of the FW2.2 gene (Task 1), “gain-of-function” plants in the T2 generation for the overexpression of the gene were obtained: 35S::FW2.2, as well as the “loss -of function” for the “silencing” of the gene (by RNAi and CRISPR-Cas9): 35S::RNAi-FW2.2 and CR-fw2.2. The phenotyping of the plants obtained was initiated, and made it possible to show that the overexpression lines 35S::FW2.2 were affected in their vegetative development, displaying a lower plant height, whereas the loss of function lines 35S::RNAi-FW2.2 did not exhibit any phenotype related to whole plant growth. In the 35S::FW2.2 lines, we observed a reduction in mean leaf area in all plants (from 33% to 42%), while in the loss of function plants 35S::RNAi-FW2.2 and CR-fw2.2, no clear phenotype was observed, which is consistent with the absence of FW2.2 expression in the leaves. A significant reduction of 19.6% in average fruit weight was observed in a single 35S::FW2.2-2 line, while no effect was observed in loss-of-function plants. Compared to WT, fruits of 35S::FW2.2 and 35S::RNAi-FW2.2 plants showed no change in pericarp thickness, while the pericarp of CR-fw2.2 fruits was thinner.
In summary, although FW2.2 expression levels were significantly altered in loss- and gain-of-function plants, these alterations did not result in profound defects on plant growth and development, at the except for a decrease in leaf area when FW2.2 was overexpressed. Only a slight effect on fruit weight was reported. This lack of a direct correlation between the level of expression of FW2.2 and fruit size is unexpected, and suggests that the mechanisms by which FW2.2 exerts its effects on fruit growth are likely more subtle, requiring a very specific mode of expression.
To further study the subcellular localization of FW2.2 (Task 2), we used transgenic lines overexpressing FW2.2 fused to EYFP (35S::FW2.2-EYFP). By confocal imaging, we were able to show that FW2.2 is localized to the plasma membrane of root cells, fruit pericarp and tomato leaves. This localization is done at the level of punctures at the cell periphery, suggesting that FW2.2 is enriched at the level of microdomains. Staining with aniline blue to reveal callose deposition at the PDs thus showed perfect co-localization with the FW2.2-EYFP signal, thus confirming the localization of FW2.2 at the PDs. We then quantified the PD index to measure the enrichment of FW2.2 at PD. The PD index of FW2.2-EYFP is greater than 1.7, in the roots and the pericarp of the fruits, thus demonstrating that FW2.2 is indeed enriched at PD.

Having confirmed the localization of FW2.2 at PD, we will evaluate the impact of FW2.2 expression modification (OE/KO) on the functionality of the PD by measuring the callose deposition that regulates the opening of the PDs, and on the cell-to-cell movement of marker molecules.
The identification of protein partners of FW2.2 will be undertaken to determine the protein network of interaction of FW2.2 at the level of the PD.
The establishment of the link between the transcriptional regulation of FW2.2 and its role in the control of growth by genetic and molecular approaches will be initiated in the second half of the project. The analysis of the spatio-temporal expression of FW2.2 will be quickly carried out by obtaining different transgenic plants expressing the following GUS-GFP reporter constructs: pFW2.2-Slyc::GUS- GFP, pFW2.2-Spen::GUS-GFP, pFW2.2-Spim::GUS-GFP, respectively containing the promoter of the FW2.2 gene from Solanum lycopersicum var. Ailsa Craig (corresponding to the “large fruit” allele), Solanum pennelli (wild tomato, ancestor of S. lycopersicum, corresponding to the “small fruit” allele) and Solanum pimpinellifolium (wild tomato, ancestor of S. lycopersicum , corresponding to the “small fruit” allele) to control the coupled expression of the GUS and GFP genes.

Arthur Beauchet, Frédéric Gévaudant, Nathalie Gonzalez, Christian Chevalier*. (2021). In search of the still unknown function of FW2.2 / CELL NUMBER REGULATOR, a major regulator of fruit size in tomato. Journal of Experimental Botany, 72: 5300-5311 ?10.1093/jxb/erab207) (hal-03239239?.

The FW2.2 gene is associated with the major Quantitative Trait Locus (QTL) governing fruit size in tomato. FW2.2 belongs to a multigene family and encodes a plasma membrane located transmembrane protein of 112 amino acids which acts by negatively controlling cell division during fruit development. To date, the molecular mechanisms of FW2.2 action remain unknown. Especially, how FW2.2 functions to regulate cell cycle and fruit growth and size in not yet understood. Recent data from Partners 1 and 3 unexpectedly showed that FW2.2 associates with plasmodesmata (PD) intercellular channels (unpublished data), suggesting a role of FW2.2 in cell-to-cell trafficking. Data from the literature also establish a link between PD-mediated transport of cell cycle regulators or transcription factors and cell differentiation and organ growth.
In this context, the aim of the 3C-FruitGrowth project is to decipher the molecular mechanisms through which FW2.2 functions to regulate organ size, taking tomato fruit as a model system. This project proposal is based on the original hypothesis that FW2.2 functions at PD and regulates the cell-to-cell movement of key cell cycle regulators. This mechanism would then contribute to control spatially cell division and/or the endoreduplication-driven mechanism of cell expansion, which ultimately influences fruit size. Since FW2.2 belongs to a multigene family, this project also aims at studying the role of CELL NUMBER REGULATOR (CNR) family members in the regulation of growth.
To determine the mode of action of FW2.2 and CNR proteins in plant growth, we aim at answering the following biological questions: (1) How do FW2.2 and CNR genes impact cell division and subsequently organ growth? (2) Are FW2.2 and CNR involved in cell-to-cell signalization at PD? (3) What is the role of the genetic polymorphisms in CNR genes, in genes encoding proteins interacting with FW2.2 and genes related to cell cycle in the variation of fruit size, and what determines their expression? Our integrated research program aims at shedding light on a yet unknown mode of control of organ size, namely the cell-to-cell signalization in the determination of fruit size in tomato.
As a plant-specific protein, FW2.2 is present in agronomically important species, both dicots (e.g. tomato, the most produced fruit worldwide, but also in other fruit species) and monocots (e.g. maize, in which a FW2.2 homologue regulates plant height but also the size of ears). Understanding the molecular mechanisms through which the FW2.2 gene family acts to control fruit size/weight and the inferred impact on fruit quality, will help in breeding new varieties of agronomical interest.