Starch is an essential polymer of life. It is a major storage polysaccharide for plant cell and is an important component of a long list of commercial products used for human and animal diets but also for other human purposes (industry, pharmacy, sustainable development…). It may in close future become a sustainable resource for the production of bio-based innovative materials.
Initially, starch is a storage polysaccharide synthesized by plants to accumulate carbon and energy generated by photosynthesis during the day. It accumulates in plastids in the form of huge, dense, water-insoluble, partially crystalline granules. It is composed of both amylose (20-30%) and amylopectin (70-80%) that are the two main components of the starch granule (other minor components such as proteins, lipids and phosphates could also be found in starch, at different levels depending on botanical origin). Both polymers are composed of glucose residues linked by α(1→4) and α(1→6) O-glycosidic bonds. Amylose and amylopectin can be distinguished by their respective structures. Amylose is essentially linear with less than 1% of α(1→6) linkages while amylopectin is a moderately branched polymer (5 to 6 % of α(1→6) branches) organized in the form of clusters.
Throughout the synthesis process, these two polymers are intimately mixed into the starch granule, which is organized as regularly alternating amorphous and semi-crystalline growth rings. These structures develop in plastids under the control of numerous enzymes. Each enzymatic activity is controlled by several genetically independent isoforms. Starch granule organization is a consequence of alternating concentric amorphous and crystalline layers (Yamaguchi et al, 1979). In the model proposed by Jenkins et al (1994) the structure of these semi-crystalline lamellae matches that of the amylopectin cluster with a repetition distance of 9 nm as estimated by X-ray diffraction analysis (Jenkins et al, 1993 ; Donald et al, 2001). In consequence, the crystalline lamellae are essentially made of the short linear glucans of amylopectin while branches (α(1→6) linkages) are mostly found in the amorphous lamellae. The implication of amylose in crystallites formation remains undetermined. However, although the presence of amylopectin is absolutely required for starch granule formation, it is not the case for amylose.
From a metabolic point of view, starch biosynthesis can be artificially divided in four main steps : substrate activation (ADPglucose), glucans elongation, branching and maturation (see Supplementary material 2). One may include a fifth step linked to the priming of the synthesis of macromolecules or the initial event of starch granule formation (both processes may be distinct form one another).
The understanding of starch metabolism was significantly improved since functional genomics has emerged for plants after the sequencing and annotation of the nuclear genomes of several species and the production of huge collections of tagged mutants. This is true for both the synthesis and the degradation. However, it is still difficult to connect in a simple and comprehensive manner the differences of composition observed at the macromolecular level (known to be genetically controlled) with the crystalline structures and the granular organization of the starch granule. Therefore, each level of structure must be taken into account to evaluate the role of each macromolecule on the structural organization of the granule and to understand the mechanisms of biosynthesis and degradation. Moreover, despite the high amount of data accumulated to date, an integrated model implying the different enzymes of both synthesis and degradation in relation to the carbon metabolism is still lacking. It must be stressed that such form of carbon-storage molecule, highly condensed but easily available for metabolization, has never been produced in vitro. This may be due to the lack of understanding of the priming mechanism especially this of amylopectin. This may be also a consequence of the lack of understanding of the way and the kinetic parameters of amylose and amylopectin association. The numerous parameters and enzymes involved in these processes are difficult to identify and therefore difficult to assess according to their importance in the pathway and therefore to reproduce in vitro (see the Third project).
To date, there is no data or strong hypothesis about the physicochemical mechanism responsible for the apparition of such condensed and organized molecule that is far away from the gels usually obtained after in vitro synthesis. Starch biosynthesis may not be simplified as a simple scheme that implies in a first instance the synthesis of the molecule and in a second instance, its aggregation with other molecules to produce the crystallites whose association would give rise to the starch granule. In addition, it must be emphasized that no soluble macromolecule can be isolated during the in vivo synthesis of starch and that starch granules still under synthesis display the same physicochemical properties (expected for their size) as the completed ones.
The objective of the research activity carried out in the group of Plant glycobiology is to increase our understanding of the synthesis and degradation of starch but also to strengthen our knowledge about :
- The priming mechanism that leads to the synthesis of the starch forming-polysaccharides (amylose and amylopectin)
- The process of starch granule formation
- The genetic control of granule number and size in plastid.
- The cross-talk between starch metabolism and plastid division
- The regulation of starch metabolism and its connection to plant carbon metabolism
To this end we make use of a functional genomic approach based on the analysis of Arabidopsis thaliana mutant lines but also on the production by crosses, and the analysis of combined mutant lines (double, triple or quadruple mutants).
Moreover, because of a strong part dealing with the transcriptomic analysis of mutants defective for one or several genes of the starch pathway and data mining of already collected transcriptomic analyses, we would like to evidence the input of “new” genes currently not considered to be involved in the pathway. By the way, we might be able to discover essential genes for the regulation of the pathway and to determine the connection of this pathway to the carbon metabolism in the plant.
Finally, we develop an approach that consists in in vitro synthesis of amylopectin to understand the molecular mechanisms that lead to the formation of this complex polysaccharide. This is achieved through the development of a simplified acellular system involving the main enzymatic activities of biosynthesis : elongation, branching, debranching/maturation. We make use of available commercial enzymes of bacterial origin. Our choice seems relevant since mathematical models indicate that, if debranching is indispensable for the formation of the crystal structure of amylopectin, it seems unnecessary to have a particular specificity of the enzyme to perform this work. Nevertheless, we also use plant recombinant enzymes, especially from maize, because they remain active in vitro (conversely to Debranching Enzyme of A. thaliana). The knowledge of amylopectin synthesis process is a prerequisite for an oriented and controlled modification in planta to meet different industrial applications.
The Plant Glycobiology Team has a secondary project named Structural study of the Effector recognition by the Type III secretion system in Sinorhizobium fredii and Salmonella enterica. Type Three Secretion System (T3SS) is used by many bacterial pathogens to inject virulence factors directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among all plant and animal pathogens suggesting a common mechanism of recognition and secretion for effectors. One the contrary none common motifs are yet known for T3SS effectors whereas they are recognized by conserved motifs in TTSS. Our recent work on the structural analysis of SopB an effector from Salmonella suggest for the first time that an oligomerization analog to the organization of the secretion machinery can promote the specificity and efficiency of substrate recognition.We perform systematic structure/function analysis of Rhizobium effectors to identify common structural motives and their recognition by cytoplasmic conserved molecules of T3SS with the aim to define molecular mechanisms of substrate recognition by T3SS in the very first steps of their secretion process.
Skills and know-how
The group has developed skills and expertise since 15 years in :
- Genetics and functional genomics of Arabidopsis thaliana and Chlamydomonas reinhardtii (selection of mutant lines, screening and crosses). Recently we have developed skills in wheat and potato handling
- Enzymology (in vitro assays, zymograms, in vitro synthesis of glucans, etc.)
- Molecular biology (cloning, heterologous expression)
- Biochemistry (purification of native or recombinant proteins, structural characterization of starch, in vitro synthesis of polysaccharides).
- Structural analysis of proteins (crystallogenesis, RX diffraction, SAXE, etc.)
- More recently, we have developed transcriptomics analysis and we have established a collaborative network with groups specialized in transcriptomics study.