Chloroplast And Starch DIVision Analysis : identification of the molecular cross talk
Description: Our research group at UGSF has shown that, in the unicellular green alga Ostreococcus tauri, the division of the unique starch granule coincides with that of the unique plastid. We have postulated that this coordination was required to ensure the presence of priming molecules for starch synthesis and preserve the continuity of starch metabolism. The preexistence of such starch-like primers seemed mandatory in O. tauri because it was impossible to completely remove starch of this organism even after the cells were left for several days in the darkness. Although the situation is different in higher plants (because of the apparent absence of control of starch grains distribution in the daughter structures after plastid division), the question of the control of starch initiation during plastid division remains unsolved. The presence of dense, water-insoluble semi-crystalline starch granules might create an overwhelming problem during the plastid splitting by the constriction ring. Moreover, at UGSF we have also demonstrated the primary function of the SS4 protein in the initiation of starch synthesis in Arabidopsis thaliana. In addition, an EzrA-like coiled-coil motif was evidenced in the N-terminal region of SS4. EzrA is a bacterial regulator that controls the localization of the division ring formed, amongst others, by the FtsZ protein. This suggests that SS4 could be involved in the process that synchronizes plastid division and the seeding of the starch granules in higher plants. The existence of such a relationship is reinforced by our own observations showing that KO mutants or over-expressing lines of the plant FtsZ1 have only one plastid per cell containing starch granules of altered size and shape. Several publications describe that the alteration of plastid division modifies the size and shape of the starch grain in potato and rice. These results suggest that a coregulation exists between starch metabolism and specific factors of plastid division. However, the rationale and the precise underlying molecular mechanism of such cross-talk are still unknown. In this project, we first propose to analyze starch accumulation, structure, size and granule number per plastid in mutants impaired in plastid division. The link between plastid division and starch initiation will be evidenced by genetics approaches based on the combination of mutations (between ss4- or ss3- and ftsZX or arc6- mutants) and by searching for SS4 interacting partners. The identification of SS4 partners is ongoing by a wide untargeted Y2H approach (ULTimate Y2H yeast two-hybrid technology developed by Hybrigenics) using SS4 as bait. We will confirm the interactions by a classical 1-by-1 Y2H approach and other techniques such as coimmunoprecipitation. Protein complexes will be localized in leaves or roots by BiFC. The dynamics of FtsZ, ARC and SS4 protein localization will be investigated in both leaves and roots. Finally we will elucidate the function of SS4 in the priming process of starch synthesis by (i) the determination of the kinetic parameters of SS4 and the structural characterization of its products and (ii) the expression, in the ss4- null mutant, of different recombinant forms of SS4 deleted for the coiled-coil domain or specifically altered at the two highly conserved catalytic sites of the enzyme.
Funder: Agence Nationale de la Recherche
Amount: €291,000
Funding type: research
Funder scheme: BLANC
Start date: 2011-11-01
Investigator: Christophe D'HULST
Institution: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE NORD-PAS-DE-CALAIS ET PICARDIE
Funded outputs count: 4
Funder award ID: ANR-11-BSV6-0003
OpenAlex ID: G6184615597