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Team leader : Nathalie Gonzalez (nathalie.gonzalez@inrae.fr)

Introduction and research objectives :

The research objectives of the Team "Flowering, Fruit Development and Environmental Constraints" (formerly "Fruit Organogenesis and Endoreduplication") are to better understand the developmental biology of fleshy fruits by dissecting in particular the genetic, physiological, cellular and molecular mechanisms involved in the establishment of reproductive organs. For this, our three lines of research focus on i) the study of floral induction and the fate of the meristem in strawberry, ii) the study of early fruit development in tomato, iii) the study of response of plants to environmental constraints in connection with the developmental processes studied. The purpose of this work is to help improve the quality traits of the fruit and maintain this quality in the event of stress.

The team's work is based on the complementary skills of its members (quantitative and association genetics, genome editing and functional genomics, phenology and phenotyping, transcriptomics, cyto-histology) and on a large network of national and international collaborations

Lines of research :

I –Meristem fate in strawberry

Strawberry can reproduce sexually via the flowering process and asexually via the production of stolons or runners, elongated stems carrying daughter-plants. Both reproduction modes present agronomical interests: flowering impacts fruit yield, and runnering allows the production of daughter-plants for multiplication of a variety. Fruit yield and daughter-plant yield are in competition. The trade-off between both takes place first in the axillary meristem (AxM) that can become either a new shoot (bearing fruits) or a stolon or runner (bearing daughter-plants), and, secondly, in the shoot terminal meristems (SAM) that can either stay vegetative or go to flower. Hence understanding the mechanisms controlling the meristem fates and so, the balance between flowering and runnering presents not only a fundamental interest but also an agronomical one. 

Our research program integrates physiology, genetics, genomics and functional characterization to decipher at meristem level, the gene network regulating the balance between flowering and runnering. As observed for natural mutations of FvGA20ox4 gene responsible for the runnerless [r] phenotype and FvTFL1 gene responsible for the perpetual flowering [PF] phenotype, playing on these actors influences the plant architecture and so the fruit and daughter-plant yield.

II – Fruit development and fruit growth in tomato

In Tomato (Solanum lycopersicum), after fruit set, two main processes govern fruit growth: cell proliferation and cell expansion associated with a process called endoreduplication, corresponding to an altered cell cycle, resulting in an amplification of genomic DNA that is not followed by cell division (endocycle) and leads to the production of highly polyploid cells.

Our objectives are to dissect these cellular processes and identify the molecular networks regulating these key events in fruit development. For this, we use a multi-scale approach for the study of cell proliferation and endoreduplication, ranging from the regulation of gene expression to growth control, by combining cell biology analyses, global approaches, "-omics" type and functional analyses.

1°- A complex coordination between cell cycle, cell expansion and endoreduplication

We are studying the establishment of cell divisions and how endocycles coordinate with cell growth in the context of a developing tissue, by measuring in situ, ploidy levels and cell size in the growing fruit. To assess the respective role of these processes in the regulation of fruit growth and to identify key regulatory elements, the analyses are also carried out on tomato plants mutated or grown under conditions (hormonal treatments or stress) causing alterations in fruit growth. These data are used to generate tissue-scale mapping to visualize and modelize cell population dynamics as a function of ploidy levels.

2°- Influence of endoreduplication on gene expression

To identify the molecular pathways associated with the progression of endoreduplication and in particular the genes expressed in a ploidy-specific manner, we produce and explore transcriptomic data on nuclei sorted according to their level of ploidy and during fruit development. We are now progressing towards the development of a single nuclei transcriptomic approach which will allow (i) the identification of sub-populations of nuclei for a given ploidy level, impossible to detect until now, and (ii) the reconstruction of the trajectories of cells during fruit development according to the evolution of their transcriptome. Based on the hypothesis that an increase in DNA content during endoreduplication will affect the structure, organization and activity of chromatin, we wish to study in situ and at the genome scale, the changes in this organization and the dynamics of epigenetic marks as a function of ploidy levels to study their role in the regulation of gene expression.

3°- Molecular networks regulating cell proliferation and endoreduplication

We are currently investigating the regulation of cell proliferation, endoreduplication and therefore fruit growth using  functional characterization of candidate genes.
In particular, we are studying the function of the FW2.2 gene, associated with the major QTL controlling fruit weight and encoding a negative regulator of cell division. We are also studying how a GUANYLATE BINDING PROTEIN, the mutation of which alters anisotropic cell expansion, thus  affecting fruit growth, and causing the division of polyploid cells in the mesocarp, controls the exit of endoreduplication (Musseau et al., 2020 ). For this, we use functional genomics and cell biology approaches.

To identify new genes involved in the regulation of endoreduplication or in fruit growth, we are also looking for mutants presenting respectively an alteration in ploidy levels or in total fruit yield, in a collection of Microtom EMS mutants available in the UMR (collection developed by C. Rothan and co-workers.). The genes responsible for these traits of interest for the regulation of fruit production are identified by mapping sequencing and fine mapping. Their functions and modes of action are studied by functional analyses based on the production of tomato plants known as loss- (genome editing) and / or gain-of-function (ectopic expression).

4°. Cuticle and surface of the fruit (ex SURF group)

The work of the former SURF (SURface du Fruit) group focuses on the formation and specialization of the tissues that make the fruit skin. The skin not only contributes to the attractiveness of the fruit for the consumer (shine, color, roughness etc.) but also constitutes the interface between the fruit and its environment (control of water loss, resistance to pathogens etc.). Central questions remain on the formation of the fruit skin: how is it regulated and coordinated with the development of other fruit tissues?  How does its composition and structure influence its properties?

During the early development of the fruit, cells will differentiate, organize into distinct tissues and specialize. In this process, the epidermal cells will play a major role in the protection of the fruit, by establishing a complex lipid barrier, the cuticle, to ensure the integrity of the fruit. This outer layer is responsible for a large number of agronomic properties of the fruit such as surface appearance, preservation or resistance to pathogens (Petit et al., 2017). It must also continuously remodel to allow for fruit growth.

Using a direct genetic approach exploiting a collection of tomato EMS mutants in the miniature variety Micro-Tom, we isolated and then characterized mutants affected at key cuticle biosynthesis genes, such as Slcyp86a69 (Shi et al., 2013), Slgpat6-a (Petit et al., 2016; Philippe et al., 2016), Slcus1 (Girard et al., 2012; Petit et al., 2014), and its regulation, like Slshn2 (Bres et al., submitted). Cytological, molecular, and biochemical analyses conducted on these mutants have already provided us with a better understanding of fruit cuticle development. The establishment of a network of cuticle mutants (double and triple mutants generation) will allow us, in close collaboration with various French and European partners, to deepen our understanding of the regulation of cuticle formation, of the properties of cuticular polyesters in pathogen resistance but also of the interactions between cuticular compounds and other parietal polymers. This last point is the subject of the ANR project COPLAnAR (COrrelative investigations of PLAnt cuticle ARchitecture associated to its functionalities) which starts in 2022.

III – Responses to environmental constraints

1°- In strawberry

In almost every country where strawberry is cultivated, breeding programmes are implemented due to specific environment or cultural techniques and consumer acceptance. The phenotypic plasticity of a genotype (G) is its ability to produce different phenotypes resulting from the complex relationships with the environment (E) in which it is grown, including management decisions.

The objectives of our research are to decipher the genetic and molecular control of plasticity for developmental and fruit traits (e.g. flowering for earliness and period length) by using complementary approach: GWAS, QTL detection, GxE studies, functional genomics.

We selected traits-of-interest subjected to strong GxE interactions that are crucial for both producers (yield, earliness, duration of flowering, plant architecture, powdery mildew resistance) and consumers (several fruit quality traits). We start to demonstrate in cultivated strawberry the strong effect of environment (combination of geographical region and year of trial) and GxE interaction depends on various traits-of-interest measured in multi-environmental conditions.

Research on cultivated strawberries is conducted in collaboration with strawberry professionals including Invenio (https://www.invenio-fl.fr/), a French grower association involved in the experimentation in fruits and vegetables as well as in the breeding of strawberry.

2°- In tomato

Climate change amplifies the effects of abiotic stresses, including drought or high temperatures, that plants can experience, threatening the stability of agricultural production and food security.

Responses to high temperature  stress

The increase in temperatures, especially the occurrence of heat waves, due to climate change, affects fruit production in tomato. As to elucidate how the development of floral organs is affected by heat stress and to identify the associated genetic determinants, we use two complementary approaches: (i) the screening of the collection of MicroTom EMS mutants in order to isolate mutants tolerant to high temperatures, and (ii) the production and comparison of transcriptome data between tolerant and susceptible tomato genotypes grown under standard growing conditions and high temperatures. Analysing these data should make it possible to identify the signalling pathways affected by high temperatures and to select candidate genes which, after a functional validation step, could be used as selection markers for tolerant varieties.

Biostimulants, growth and high temperature stress

The transition towards new sustainable cultivation practices allowing the reduction of phytosanitary products without affecting the yield, represents an important issue in agronomy. In this context, we are studying the effects of the application of biostimulants, defined in particular as products that improve the resilience of plants to stress, to control the response to thermal stress. For this, we set up a multi-scale phenotypic study and mechanistic studies to identify the effects of biostimulants on plant growth (mainly flower and fruit development), and to study changes at the physiological, cellular and molecular levels which may be induced.