Chloroplast And Starch DIVision Analysis : identification of the molecular cross talk – CaSta DivA

This project will be undertaken through an approach combining biochemistry, molecular biology and genetics. Thus, starch quantity and structure, granule size and number in the mutant lines of Arabidopsis thaliana altered for plastid division will be established. The link between plastid division and the initiation of starch synthesis will be deciphered by a genetic approach based on the combination of mutations dealing with starch metabolism and the plastid division machinery. We will also establish the list of SS4-interacting partners (if any) using a yeast double hybrid techniques and SS4 as bait. Such interactions will be confirmed by other techniques such as coimmunoprecipitation. Protein-complexes will be localized in leaves and roots and the dynamic localization of FtsZ, ARC and SS4 proteins will be established in vivo. Finally we will highlighted the function of SS4 in the process of starch initiation by a double analysis based on i) the study of its catalytic parameters and enzymatic products and ii) the expression in the ss4- null mutant of recombinant forms of SS4 with either deletion of the N-term coiled-coil domain or the targeted modification of the catalytic sites highly conserved throughout the enzymes of the same family.

This project should shed light on the initiation process of starch synthesis in plants. It should decipher the cross talk between plastid division and starch metabolism that occurs in the plastid. Understanding these biological processes is important in a fundamental point of view, but also in term of future applications. Indeed the efficiency of starch transformation by chemical ways strongly depends on granule size. The same applies for extraction yields. Controlling the process of starch initiation will specify how modifying crops upon their use. This work is part of the research projects included in IFMAS, Institut Français des Matériaux Agrosourcés. This Institute funded in the frame of the Investissements d’Avenir (program Instituts d’Excellence en Energies Décarbonées) groups together several public research laboratories and industrial partners with the primary objective to produce bio-plastics from starch thus avoiding the use of petrol derivatives. Starch transformation into plastic depends on its potential to be chemically modified. Such modification is improved by the use of starch granules as small as possible. The objective is then to multiply the number of small starch granules in plants for post-extraction requirement without compromising yields and extraction procedures.

The issues raised in the frame of this project are particularly important since starch defines one of the feedstock largely used in both food and non-food industries. Starch is used for the production of plastics, adhesives, stabilizers, and excipients and can be converted to bioethanol. It is widely used in the food industry for its stabilizing and gelatinizing properties. Controlling the size of the starch granule according to the application would represent a major advantage for the different industries. This project will determine the way for selecting plants that will answer requirements.

Not yet available

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.