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UMR 1332 Biologie du Fruit et Pathologie

Team : Fruit organogenesis and endoreduplication

Director : Christian Chevalier, DR2 INRA, Deputy Director of the UMR1332 BFP,  (33)557122693, christian.chevalier@inra.fr

Introduction and research objectives :

The "Fruit organogenesis and endoreduplication" (OrFE) team is interested in the developmental biology of fleshy fruits, with the aim of gaining a better understanding of the mechanisms involved in the development of reproductive organs.  Through the study of floral induction to early fruit development, we aim at contributing to the improvement of fruit quality, using tomato and strawberry fruits as a model fruit and an application fruit respectively. The OrFE team is interested in the characterization of genetic, physiological, cytological and molecular events involved in the processes of fruiting and early fruit development that contribute to the development of the final  fruit size and quality.
The study of the mechanisms involved in control of cell size and the final size of the fruit is a historical thematic focus of the team, ensuring its reputation and international positioning. Studies on strawberry fruit on the determinism of flower induction and developmental and genetic determinants contributing to the quality of the fruit are highly recognized both internationally and nationally, and benefits from a strong integration into the strawberry chain production.
The approaches used in the team to study these problems involve a combination of complementary approaches including quantitative and association genetics (strawberry), functional genomics (strawberry and tomato), and cytology (tomato).

The fruit is a specialized organ that provides a suitable environment for seed maturation and dispersal. In tomato (Solanum lycopersicum Mill.), the early fruit development program unfolds broadly into three phases. Phase I results in the maturation of the ovary and the "decision" to form a fruit (fruit set) or to abort. Phase II corresponds mainly to a period of intense mitotic activity at all ovary tissues. Phase III is essentially characterized by cell expansion and a substantial increase in the fruit volume, resulting in a fruit able to commit to the ripening process.
The development of primary characters for the quality of the fruit (such as its size, its mass and composition in primary and secondary metabolites) is determined early during development. The final size of the fruit depends on (i) the formation of patterns of development that can be determined very early in flower initiation, (ii) the number of cells within the fruit, set at the end of the phase of cell divisions that occur before and after fertilization and (iii) cell size, determined primarily during the cell expansion stage of fruit development during which accumulate citric and malic acids, which are essential components of the taste with sugars. In tomato, the increase in the size of the fruit cells can achieve dramatic levels of hundreds or even thousands of times the initial size of a cell. This variation in the size of the cells is accompanied by an increased level of nuclear ploidy. This polyploidy results from the ability of cells to modify their conventional cell cycle into an altered cycle where DNA synthesis takes place independently in the absence of mitosis. This partial cell cycle is called the endoreduplication cycle. Cell division, cell expansion and endoreduplication are under the control of complex interactions between external factors to the plant (environmental influences) and internal signals (developmental and hormonal regulation; photosynthetic carbon allocation). The genes encoding regulators of cell division such as integrators of hormonal signals, transcription factors or proteins of the cell cycle are likely candidates whose functional study may help determine the molecular mechanisms controlling the size of the fruit.

In a socio-economic context of importance for the Aquitaine Region and with an applied objective of controlling production (yield and fruit quality), The OrFE team is developing a research topic on the genetic determinism of floral induction in strawberry. The diploid strawberry (Fragaria vesca) stands out as a model for functional analyses in Rosaceae. This gender also offers a model for the implementation of generic knowledge obtained from the diploid strawberry, into the octoploid grown strawberry (Fragaria × ananassa). The species Fragaria vesca can be transformed by Agrobacterium (more easily than other species of Rosaceae). Many resources are currently available for which the strawberry sub-group of the OrFE team has contributed and continues to expand: the complete genome sequence of the diploid strawberry; collections of "wild" (diploid to octoploid) strawberry and genotypes of wood strawberry (F. vesca, 2x); the development of a collection of EMS TILLING mutants; the technique of genetic transformation being acquired for functional genomic studies.

Lines of research :

I - Functional characterization of genes associated with regulation of fruit organogenesis

As part of this theme, we focus on two candidate genes: (1) the INHIBITOR ACTIVITY OF MERISTEM (IMA) gene, a negative regulator of meristem activity involved in the genetic and development programs of ovules and fruit in tomato, at the interface of hormone signaling pathways; (2) the FW2.2 gene associated with the major QTL governing fruit size in tomato, for which the function and impact on fruit growth remain enigmatic.

INHIBITOR OF MERISTEM ACTIVITY (IMA) :

- Molecular analysis of the interaction mechanism of IMA with its target proteins: evidence of the CSN5A subunit of the COP9 signalosome as an interactant detected by two-hybrid; analysis of the function of IMA on the state of the CULLIN1 subunit neddylation.

- Analysis of the role of IMA in hormone signaling in conjunction with the meristematic activity, integration of hormonal signals and proteolytic degradation of effectors.

- Analysis of the genetic control of the termination of floral meristem through relationships between IMA and AGAMOUS, WUSCHEL and KNUCKLES meristem and flower identity genes.

IMA 2014
FW2.2 :

- Confirmation of the subcellular localization of the protein FW2.2 (fused to EYFP) by transient expression in seedlings of tobacco and Arabidopsis;

- Generation of overexpressors FW2.2 Arabidopsis amount size reduction of all organs including pods; alteration phenotype of cell size in the leaf parenchyma with malformation of the stomata (division problems meristemoids cells).

- Creation of a two-hybrid bank split-ubiquitin for the interactants specific search with a membrane protein; Screening of the current bank.

- Search for the identification of a putative function for FW2.2as an ion carrier: growth overexpressors on media enriched in ions; electrophysiological analyzes in Xenopus oocytes.

FW2.2

II - Study of the contribution of cell division and endoreduplication in fruit growth

The process of endoreduplication (DNA synthesis in the absence of mitosis) is found in all angiosperms and in multiple cell types. Nevertheless, its physiological role remains largely unknown. The role of endoreduplication as an inducer of cell expansion is commonly proposed since a positive correlation between cell size and organ size and endoreduplication has been frequently reported in many plant species. The team is interested in elucidating the role of endoreduplication in fleshy fruits, especially tomato. We are particularly interested in its role as an inducer of cell expansion, but also its potential relationship with the control of gene expression.

Endoreduplication

The OrFE team works contributed (1) to demonstrate the role of endoreduplication in the growth of cells and organs according to the “karyoplasmic ratio” theory: increasing ploidy level influences the final size of cells which tend to adjust their cytoplasmic volume according to the nuclear DNA content; (2) to demonstrate for the first time in plants, the contribution of endoreduplication in the control of transcriptional activities.

Endo cell 2014

III - Functional characterization of genes associated with cell cycle regulation and the endocycle

The team is studying the role of cell cycle regulators in the control of endoreduplication. These regulators are associated with the inhibition of CDK / cyclin complex activities, resulting from (i) a change in the phosphorylation state of CDKs (role of WEE1); (ii) the availability of cyclin subunits controlled by proteolytic degradation (role CSS52); (iii) and the presence of specific inhibitors of CDKs (role of KRPs).

WEE1 Kinase:

- Involvement of the WEE1 kinase in the endoreduplication process: functional analyses of loss of function mutants (transgenic plants harboring the construction Pro35S: WEE1as) showed a decrease in fruit size, cell size and the levels of endoreduplication; the overexpression of WEE1 in a synchronized culture of tobacco BY2 cells results in shortening the G2 phase.

CDK Inhibitors, the KRPs:

- Functional Analysis of the structural domains of SlKRP1 protein, study of protein-protein interactions (and yeast two-hybrid BiFC), co-localization of protein partners in plant cells.

- Functional analysis of gain-of-function mutants (transgenic plants harboring the construction ProPEPC2: KRP1OE, mesocarp-specific overexpression of tomato KRP1) with decreased levels of endoreduplication, without affecting the cell size and the size of the fruit; decoupling the cell size and induces endoreduplication in the fruit.

APC activators (CCS52A and CCS52B:

- Cloning of and tomato CCS52A and CCS52B genes: CCS52B specific expression in mitosis, expression of CCS52A during endoreduplication;

- Functional analysis of transgenic loss-of-function plants: reduction in ploidy levels in Pro35S:CCS52AAS plants with smaller fruit size; large fruit phenotype for Pro35S:CCS52BAS plants due to complementary endogenous overexpression of CSS52A inducing an increase in ploidy.

- Functional analysis of transgenic gain-of-function plants: increased levels of endoreduplication in plants overexpressing CCS52A (Pro35S:CCS52AOE), slightly smaller fruit size than WT but accelerating growth at the end of fruit development by increasing cell size and the levels of endoreduplication.

- Functional analysis of transgenic gain-of-function plants: increased levels of endoreduplication in plants overexpressing CCS52A in a fruit- and cell expansion-specific manner  (ProPEPC2:CCS52AOE), large fruit size with increased levels of endoreduplication; no phenotypic effect for the overexpression of CCS52B (ProPEPC2: CCS52BOE) outside of its natural expression window (phase of cell divisions).

Cycle Endocycle

IV - Analysis of genetic and molecular determinism of flower induction and fruit quality in strawberry

The objective of this theme is to acquire fundamental knowledge on the genetic, molecular and physiological factors governing flowering and specifically flower induction in strawberry.
Flowering is a major event that affects many agronomic traits, particularly yield. Indeed in plants, the yield for fruit / seed is conditioned by the potential of flowering at the time of floral initiation. An original approach to understanding floral initiation is the study of continuous flowering which is the ability of a plant to initiate and bloom throughout the growing season and not just during short days for strawberry (perpetual flowering versus once flowering). Our studies use approaches of (i) comparative genomics between the diploid strawberry (wild strawberry) and the octoploid (cultivated) strawberry, (ii) QTL research related to perpetual flowering and (iii) functional analysis of candidate genes.

4.1 Analysis of genetic and molecular determinism of flower induction:

The objective is to acquire basic knowledge about the genetic, molecular and physiological factors governing flowering and specifically flower induction. This knowledge will enable the development of tools for professionals to move towards a strawberry production over a longer period of time while avoiding the production peaks.
Our research aims at analyzing the role of genes involved in flowering and continuous flowering  (major criterion for the productivity of plants) in the diploid strawberry and octoploid strawberry: KSN repressor gene (ortholog of Terminal Flower1-TFL1) and activator genes, FT1, FT2 and FT3 orthologous to teh Flowering Locus T (FT) gene.
A second line of research is to analyze the genetic determinants involved in the competition between reproductive organs and vegetative propagation by runnering in strawberry.

4.2 Analysis of the early stages of development and genetic determinants of fruit quality:

Through a detailed metabolic phenotyping and analysis of gene expression at a large scale of genetic collections and a segregating population available in the laboratory, different targets have been identified for many of the traits of interest. These major targets are: (1) antioxidants from the secondary metabolism, including anthocyanins which play a role in the color of the fruit, as well as vitamin C; (2) the sensory aspects: taste (sugars, organic acids), visual (brightness), and firmness.
Analysis of genetic determinants for fruit quality call on the following approaches: (1) identification of QTL associated with the characters studied; (2) identification of candidate genes through transcriptome analysis; (3) functional validation (transient and stable transformation) of candidates identified in strawberry (processing technology acquired and implemented within the OrFE team).

Fraisier 2014